DNA nanotechnology-based composite-type gold nanoparticle-immunostimulatory DNA hydrogel for tumor photothermal immunotherapy.

PubMed

Yata, Tomoya; Takahashi, Yuki; Tan, Mengmeng; Nakatsuji, Hirotaka; Ohtsuki, Shozo; Murakami, Tatsuya; Imahori, Hiroshi; Umeki, Yuka; Shiomi, Tomoki; Takakura, Yoshinobu; Nishikawa, Makiya

2017-11-01

Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA polymorphism sensitive impedimetric detection on gold-nanoislands modified electrodes.

PubMed

Bonanni, Alessandra; Pividori, Maria Isabel; del Valle, Manel

2015-05-01

Nanocomposite materials are being increasingly used in biosensing applications as they can significantly improve biosensor performance. Here we report the use of a novel impedimetric genosensor based on gold nanoparticles graphite-epoxy nanocomposite (nanoAu-GEC) for the detection of triple base mutation deletion in a cystic-fibrosis (CF) related human DNA sequence. The developed platform consists of chemisorbing gold nano-islands surrounded by rigid, non-chemisorbing, and conducting graphite-epoxy composite. The ratio of the gold nanoparticles in the composite was carefully optimized by electrochemical and microscopy studies. Such platform allows the very fast and stable thiol immobilization of DNA probes on the gold islands, thus minimizing the steric and electrostatic repulsion among the DNA probes and improving the detection of DNA polymorphism down to 2.25fmol by using electrochemical impedance spectroscopy. These findings are very important in order to develop new and renewable platforms to be used in point-of-care devices for the detection of biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultrasensitive sensing platform for platelet-derived growth factor BB detection based on layered molybdenum selenide-graphene composites and Exonuclease III assisted signal amplification.

PubMed

Huang, Ke-Jing; Shuai, Hong-Lei; Zhang, Ji-Zong

2016-03-15

A highly sensitive and ultrasensitive electrochemical aptasensor for platelet-derived growth factor BB (PDGF-BB) detection is fabricated based on layered molybdenum selenide-graphene (MoSe2-Gr) composites and Exonuclease III (Exo III)-aided signal amplification. MoSe2-Gr is prepared by a simple hydrothermal method and used as a promising sensing platform. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity is limited on the duplex DNAs with more than 4 mismatched terminal bases at 3' ends. Herein, aptamer and complementary DNA (cDNA) sequences are designed with four thymine bases on 3' ends. In the presence of target protein, the aptamer associates with it and facilitates the formation of duplex DNA between cDNA and signal DNA. The duplex DNA then is digested by Exo III and releases cDNA, which hybridizes with signal DNA to perform a new cleavage process. Nevertheless, in the absence of target protein, the aptamer hybridizes with cDNA will inhibit the Exo III-assisted nucleotides cleavage. The signal DNA then hybridizes with capture DNA on the electrode. Subsequently, horse radish peroxidase is fixed on electrode by avidin-biotin reaction and then catalyzes hydrogen peroxide and hydroquinone to produce electrochemical response. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, providing a quantitative measure of target protein with a broad detection range of 0.0001-1 nM and a detection limit of 20 fM. Copyright © 2015 Elsevier B.V. All rights reserved.

A general approach to DNA-programmable atom equivalents.

PubMed

Zhang, Chuan; Macfarlane, Robert J; Young, Kaylie L; Choi, Chung Hang J; Hao, Liangliang; Auyeung, Evelyn; Liu, Guoliang; Zhou, Xiaozhu; Mirkin, Chad A

2013-08-01

Nanoparticles can be combined with nucleic acids to programme the formation of three-dimensional colloidal crystals where the particles' size, shape, composition and position can be independently controlled. However, the diversity of the types of material that can be used is limited by the lack of a general method for preparing the basic DNA-functionalized building blocks needed to bond nanoparticles of different chemical compositions into lattices in a controllable manner. Here we show that by coating nanoparticles protected with aliphatic ligands with an azide-bearing amphiphilic polymer, followed by the coupling of DNA to the polymer using strain-promoted azide-alkyne cycloaddition (also known as copper-free azide-alkyne click chemistry), nanoparticles bearing a high-density shell of nucleic acids can be created regardless of nanoparticle composition. This method provides a route to a virtually endless class of programmable atom equivalents for DNA-based colloidal crystallization.

Cytophotometric and biochemical analyses of DNA in pentaploid and diploid Agave species.

PubMed

Cavallini, A; Natali, L; Cionini, G; Castorena-Sanchez, I

1996-04-01

Nuclear DNA content, chromatin structure, and DNA composition were investigated in four Agave species: two diploid, Agave tequilana Weber and Agave angustifolia Haworth var. marginata Hort., and two pentaploid, Agave fourcroydes Lemaire and Agave sisalana Perrine. It was determined that the genome size of pentaploid species is nearly 2.5 times that of diploid ones. Cytophotometric analyses of chromatin structure were performed following Feulgen or DAPI staining to determine optical density profiles of interphase nuclei. Pentaploid species showed higher frequencies of condensed chromatin (heterochromatin) than diploid species. On the other hand, a lower frequency of A-T rich (DAPI stained) heterochromatin was found in pentaploid species than in diploid ones, indicating that heterochromatin in pentaploid species is made up of sequences with base compositions different from those of diploid species. Since thermal denaturation profiles of extracted DNA showed minor variations in the base composition of the genomes of the four species, it is supposed that, in pentaploid species, the large heterochromatin content is not due to an overrepresentation of G-C repetitive sequences but rather to the condensation of nonrepetitive sequences, such as, for example, redundant gene copies switched off in the polyploid complement. It is suggested that speciation in the genus Agave occurs through point mutations and minor DNA rearrangements, as is also indicated by the relative stability of the karyotype of this genus. Key words : Agave, DNA cytophotometry, DNA melting profiles, chromatin structure, genome size.

Methods and compositions for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

2003-07-22

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

EPA Science Inventory

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

Methods for chromosome-specific staining

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1995-01-01

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogenous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include methods for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes.

Methods for chromosome-specific staining

DOEpatents

Gray, J.W.; Pinkel, D.

1995-09-05

Methods and compositions for chromosome-specific staining are provided. Compositions comprise heterogeneous mixtures of labeled nucleic acid fragments having substantially complementary base sequences to unique sequence regions of the chromosomal DNA for which their associated staining reagent is specific. Methods include ways for making the chromosome-specific staining compositions of the invention, and methods for applying the staining compositions to chromosomes. 3 figs.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

Biologically active nanocomposite of DNA-PbS nanoparticles: A new material for non-volatile memory devices

NASA Astrophysics Data System (ADS)

Murgunde, B. K.; Rabinal, M. K.; Kalasad, M. N.

2018-01-01

Composite films of deoxyribonucleic acid (DNA) and lead sulfide (PbS) nanoparticles are prepared to fabricate biological memory devices. A simple solution based electrografting is developed to deposit large (few cm2) uniform films of DNA:PbS on conducting substrates. The films are studied by X-ray photoelectron spectroscopy, field emission SEM, FTIR and optical spectroscopy to understand their properties. Charge transport measurements are carried out on ITO-DNA:PbS-metal junctions by cyclic voltage scans, electrical bi-stability is observed with ON/OFF ratio more than ∼104 times with good stability and endurance, such performance being rarely reported. The observed results are interpreted in the light of strong electrostatic binding of nanoparticles and DNA stands, which leads doping of Pb atoms into DNA. As a result, these devices exhibit negative differential resistance (NDR) effect due to oxidation of doped metal atoms. These composites can be the potential materials in the development of new generation non-volatile memory devices.

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy.

PubMed

Hu, Hongyan; Huang, Xiangyi; Ren, Jicun

2016-05-01

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA-based nanomaterials because of its direct recognition of natural double-stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single-strand DNA (ssDNA) fluorescent probes and fluorescent probe-double-strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K(a)) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20-mer perfectly matched ssDNA probe and three single-base mismatched ssDNA probes with 146-mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single-base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.

Sequence-dependent response of DNA to torsional stress: a potential biological regulation mechanism.

PubMed

Reymer, Anna; Zakrzewska, Krystyna; Lavery, Richard

2018-02-28

Torsional restraints on DNA change in time and space during the life of the cell and are an integral part of processes such as gene expression, DNA repair and packaging. The mechanical behavior of DNA under torsional stress has been studied on a mesoscopic scale, but little is known concerning its response at the level of individual base pairs and the effects of base pair composition. To answer this question, we have developed a geometrical restraint that can accurately control the total twist of a DNA segment during all-atom molecular dynamics simulations. By applying this restraint to four different DNA oligomers, we are able to show that DNA responds to both under- and overtwisting in a very heterogeneous manner. Certain base pair steps, in specific sequence environments, are able to absorb most of the torsional stress, leaving other steps close to their relaxed conformation. This heterogeneity also affects the local torsional modulus of DNA. These findings suggest that modifying torsional stress on DNA could act as a modulator for protein binding via the heterogeneous changes in local DNA structure.

Sequence-dependent response of DNA to torsional stress: a potential biological regulation mechanism

PubMed Central

Reymer, Anna; Zakrzewska, Krystyna; Lavery, Richard

2018-01-01

Abstract Torsional restraints on DNA change in time and space during the life of the cell and are an integral part of processes such as gene expression, DNA repair and packaging. The mechanical behavior of DNA under torsional stress has been studied on a mesoscopic scale, but little is known concerning its response at the level of individual base pairs and the effects of base pair composition. To answer this question, we have developed a geometrical restraint that can accurately control the total twist of a DNA segment during all-atom molecular dynamics simulations. By applying this restraint to four different DNA oligomers, we are able to show that DNA responds to both under- and overtwisting in a very heterogeneous manner. Certain base pair steps, in specific sequence environments, are able to absorb most of the torsional stress, leaving other steps close to their relaxed conformation. This heterogeneity also affects the local torsional modulus of DNA. These findings suggest that modifying torsional stress on DNA could act as a modulator for protein binding via the heterogeneous changes in local DNA structure. PMID:29267977

Simulations Using Random-Generated DNA and RNA Sequences

ERIC Educational Resources Information Center

Bryce, C. F. A.

1977-01-01

Using a very simple computer program written in BASIC, a very large number of random-generated DNA or RNA sequences are obtained. Students use these sequences to predict complementary sequences and translational products, evaluate base compositions, determine frequencies of particular triplet codons, and suggest possible secondary structures.…

Ultrasensitive Biosensor for the Detection of Vibrio cholerae DNA with Polystyrene-co-acrylic Acid Composite Nanospheres

NASA Astrophysics Data System (ADS)

Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Ling, Tan Ling

2017-08-01

An ultrasensitive electrochemical biosensor for the determination of pathogenic Vibrio cholerae ( V. cholerae) DNA was developed based on polystyrene-co-acrylic acid (PSA) latex nanospheres-gold nanoparticles composite (PSA-AuNPs) DNA carrier matrix. Differential pulse voltammetry (DPV) using an electroactive anthraquninone oligonucleotide label was used for measuring the biosensor response. Loading of gold nanoparticles (AuNPs) on the DNA-latex particle electrode has significantly amplified the faradaic current of DNA hybridisation. Together with the use of a reported probe, the biosensor has demonstrated high sensitivity. The DNA biosensor yielded a reproducible and wide linear response range to target DNA from 1.0 × 10-21 to 1.0 × 10-8 M (relative standard deviation, RSD = 4.5%, n = 5) with a limit of detection (LOD) of 1.0 × 10-21 M ( R 2 = 0.99). The biosensor obtained satisfactory recovery values between 91 and 109% ( n = 3) for the detection of V. cholerae DNA in spiked samples and could be reused for six consecutive DNA assays with a repeatability RSD value of 5% ( n = 5). The electrochemical biosensor response was stable and maintainable at 95% of its original response up to 58 days of storage period.

Novel adsorbent for DNA adsorption: Fe(3+)-attached sporopollenin particles embedded composite cryogels.

PubMed

Ceylan, Şeyda; Odabaşı, Mehmet

2013-12-01

The aim of this study is to prepare supermacroporous cryogels embedded with Fe(3+)-attached sporopollenin particles (Fe(3++)-ASPs) having large surface area for high DNA adsorption capacity. Supermacroporous poly(2-hydroxyethyl methacrylate) (PHEMA)-based monolithic cryogel column embedded with Fe3+(+)-ASPs was prepared by radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N´-methylene- bis-acrylamide (MBAAm) as cross-linker directly in a plastic syringe for DNA adsorption studies. Firstly, Fe3+(+) ions were attached to the sporopollenin particles (SPs), then the supermacroporous PHEMA cryogel with embedded Fe(3++)-ASPs was produced by free radical polymerization using N,N,N´, N´-Tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as initiator/activator pair in an ice bath. Optimum conditions of adsorption experiments were performed at pH 6.0 (0.02 M Tris buffer containing 0.2 M NaCl), with flow rate of 0.5 mL/min, and at 5°C. The maximum amount of DNA adsorption from aqueous solution was very high (109 mg/g SPs) with initial concentration of 3 mg/mL. It was observed that DNA could be repeatedly adsorbed and desorbed with this composite cryogel without significant loss of adsorption capacity. As a result, higher amounts of DNA adsorbed these composite cryogels are expected to be good candidate for achieving higher removal of anti-DNA antibodies from systemic lupus erythematosus (SLE) patients plasma.

VIP Barcoding: composition vector-based software for rapid species identification based on DNA barcoding.

PubMed

Fan, Long; Hui, Jerome H L; Yu, Zu Guo; Chu, Ka Hou

2014-07-01

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time-consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user-friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two-stage algorithm. First, an alignment-free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment-based K2P distance nearest-neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods and (ii) higher scalability than alignment-based distance methods and character-based methods. These results suggest that this platform is able to deal with both large-scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/. © 2014 John Wiley & Sons Ltd.

Replication-associated mutational asymmetry in the human genome.

PubMed

Chen, Chun-Long; Duquenne, Lauranne; Audit, Benjamin; Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Huvet, Maxime; d'Aubenton-Carafa, Yves; Hyrien, Olivier; Arneodo, Alain; Thermes, Claude

2011-08-01

During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews. To date, the existence of such asymmetries associated with replication has not yet been established. Here, we compute the nucleotide substitution matrices around replication initiation zones identified as sharp peaks in replication timing profiles and associated with abrupt jumps in the compositional skew profile. We show that the substitution matrices computed in these regions fully explain the jumps in the compositional skew profile when crossing initiation zones. In intergenic regions, we observe mutational asymmetries measured as differences between complementary substitution rates; their sign changes when crossing initiation zones. These mutational asymmetries are unlikely to result from cryptic transcription but can be explained by a model based on replication errors and strand-biased repair. In transcribed regions, mutational asymmetries associated with replication superimpose on the previously described mutational asymmetries associated with transcription. We separate the substitution asymmetries associated with both mechanisms, which allows us to determine for the first time in eukaryotes, the mutational asymmetries associated with replication and to reevaluate those associated with transcription. Replication-associated mutational asymmetry may result from unequal rates of complementary base misincorporation by the DNA polymerases coupled with DNA mismatch repair (MMR) acting with different efficiencies on the leading and lagging strands. Replication, acting in germ line cells during long evolutionary times, contributed equally with transcription to produce the present abrupt jumps in the compositional skew. These results demonstrate that DNA replication is one of the major processes that shape human genome composition.

Ultrasensitive determination of DNA sequences by flow injection chemiluminescence using silver ions as labels.

PubMed

Zheng, Lichun; Liu, Xiuhui; Zhou, Min; Ma, Yongjun; Wu, Guofan; Lu, Xiaoquan

2014-10-27

We presented a new strategy for ultrasensitive detection of DNA sequences based on the novel detection probe which was labeled with Ag(+) using metallothionein (MT) as a bridge. The assay relied on a sandwich-type DNA hybridization in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on Fe3O4@Au composite magnetic nanoparticles (MNPs), and then the Ag(+)-modified detection probes were used to monitor the presence of the specific DNA targets. After being anchored on the hybrids, Ag(+) was released down through acidic treatment and sensitively determined by a coupling flow injection-chemiluminescent reaction system (Ag(+)-Mn(2+)-K2S2O8-H3PO4-luminol) (FI-CL). The experiment results showed that the CL intensities increased linearly with the concentrations of DNA targets in the range from 10 to 500 pmol L(-1) with a detection limit of 3.3 pmol L(-1). The high sensitivity in this work may be ascribed to the high molar ratio of Ag(+)-MT, the sensitive determination of Ag(+) by the coupling FI-CL reaction system and the perfect magnetic separation based on Fe3O4@Au composite MNPs. Moreover, the proposed strategy exhibited excellent selectivity against the mismatched DNA sequences and could be applied to real samples analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanical Strength and Stability of DNA-modified Gold Nanoparticle Systems

NASA Astrophysics Data System (ADS)

Lam, Letisha McLaughlin

Systems in which gold nanoparticles (AuNPs) are functionalized with DNA have the potential for a broad range of applications in gene regulation therapies, drug delivery, sensing, innovative biomaterials and material templates. The use of DNA-modified gold nanoparticle (AuNP-DNA) systems is driven by their ease of assembly with bottom-up methods as well as the tunability of the systems' mechanical, optical, and electronic properties by exploiting AuNP characteristics and behavior in a multi-particle arrangement. Periodic arrangements of AuNPs precisely distributed through ligated DNA linkers may be assembled and used on relatively large length scales, on the order of hundreds of nanometers, for use in potential nanoscale technologies and applications. However, because of the size and heterogeneous composition of AuNP-DNA systems, their stability under mechanical loading is not well understood or quantified on relevant physical scales for these applications. Hence, a large-scale specialized finite-element predictive approach with a dislocation-density based crystalline plasticity has been used to investigate the mechanical stability of AuNP-DNA-ligand systems with AuNPs within the physical dimensions required for plasmon resonance. The crystalline formulation for the AuNPs accounts for multiple crystalline slip, dislocation-density evolution, lattice rotations, and large inelastic strains. A hypoelastic formulation was used for the DNA and the ligands. The nonlinear finite-element scheme is based on accounting for finite elastic and inelastic strains. These approaches were employed to predict and understand the fundamental scale-dependent microstructural behavior, the evolving heterogeneous microstructure, and localized phenomena that can contribute to failure initiation and instability. Each system was loaded using quasi-static plane strain tension and compression to simulate application loading conditions, and the elastic and inelastic evolutions were analyzed for evidence of mechanical strengthening as well as possible failure modes. To establish a foundation for AuNP-DNA stability analysis, several different two-particle conformations were investigated, including systems with pentagonally twinned AuNPs, systems with circular AuNPs, systems with non-textured and textured cuboctahedron AuNPs with 6 nm DNA, 12 nm DNA, and 18 nm DNA. In general, the analyses indicated that the systems' stability are mainly affected by large stress gradients at AuNP-ligand interfaces, as well as large dislocation-density, normal stresses, and inelastic accumulations in the region adjacent to these interfaces between the AuNPs and the DNA. The predictions also indicate that highly faceted f.c.c. AuNPs with DNA lengths of approximately 6 nm in biaxial loading conditions were found to have the highest strength and overall stability. Furthermore, periodic AuNP-DNA superlattice composites, which mimic the crystallography of f.c.c. atomic lattices, were investigated for mechanical effectiveness as both a composite material and thin film. This investigation analyzed the stress behavior and inelastic evolution of f.c.c. AuNP-DNA superlattice systems with different Au volume fractions, matrix strengths, intrinsic nanoparticle crystallographic orientations and sizes. These analyses were also extended to superlattice f.c.c. composites on a silicon substrate. The results indicate that f.c.c. AuNP-DNA superlattices have a combination of high strength and toughness due to the ductile nature of the nanoparticles in conjunction with the physical properties of the DNA and matrix materials. The superlattice films also exhibited high strengths and toughness, with the limiting factor being the interrelated aspects of film thickness and delamination. These predictions can be used as guidelines for using these composites, superlattices, and thin films as candidates for innovative building blocks for new material systems.

Agreement Between Deoxyribonucleic Acid Base Composition and Taxometric Classification of Gram-Positive Cocci1

PubMed Central

Silvestri, L. G.; Hill, L. R.

1965-01-01

Silvestri, L. G. (Università Statale, Milan, Italy), and L. R. Hill. Agreement between deoxyribonucleic acid base composition and taxometric classification of gram-positive cocci. J. Bacteriol. 90:136–140. 1965.—It had been previously proposed, from taxometric analyses, that gram-positive, catalase-positive cocci be divided into two subgroups. Thirteen strains, representative of both subgroups, were examined for deoxyribonucleic acid (DNA) base composition, determined from melting temperatures. Per cent GC (guanine + cytosine/total bases) values fell into two groups: 30.8 to 36.5% GC and 69 to 75% GC. Strains with low per cent GC values belonged to the Staphylococcus aureus–S. saprophyticus–S. lactis taxometric subgroups, and those with high per cent GC values belonged to the S. roseus–S. afermentans subgroup. The hypothetical nature of any classification is emphasized, and, in the present work, the hypothesis derived from taxometric analyses of division into two subgroups is confirmed by the study of DNA base ratios. The two subgroups correspond, respectively, to the genera Staphylococcus and Micrococcus. PMID:16562008

Identity of the xerophilic species Aspergillus penicillioides: Integrated analysis of the genotypic and phenotypic characters.

PubMed

Tamura, Miki; Kawasaki, Hiroko; Sugiyama, Junta

1999-02-01

We examined the identity of Aspergillus penicillioides, the typical xerophilic and strictly anamorphic species, using an integrated analysis of the genotypic and phenotypic characters. Our experimental methods on two genotypic characters, i.e., DNA base composition using the HPLC method and DNA relatedness using the nitrocellulose filter hybridization technique between A. flavus, A. oryzae, and their close relations revealed a good agreement with the values by buoyant density (for DNA base composition) and spectrophotometric determination (for DNA relatedness) reported by Kurtzman et al. in 1986. On the basis of these comparisons, we examined DNA base composition and DNA relatedness of six selected strains of A. penicillioides, including IFO 8155 (originally described as A. vitricola), one strain of A. restrictus, and the respective strains from Eurotium amstelodami, E. repens, and E. rubrum. As a result, five strains within A. penicillioides, including the neotype strain NRRL 4548, had G+C contents of 46 to 49 mol%, whereas IFO 8155 had 50 mol%. A. restrictus had 52 mol%, and three Eurotium species ranged from 46 to 49 mol%. The DNA relatedness between A. penicillioides (five strains), except for IFO 8155, exhibited values greater than 70%, but the DNA complementarity between four strains and IFO 8155 in A. penicillioides revealed values of less than 40%. DNA relatedness values between three species of Eurotium were 65 to 72%. We determined 18S, 5.8S, and ITS rDNA sequences as other genotypic characters from A. penicillioides (six strains), A. restrictus, and related teleomorphic species of Eurotium. In three phylogenetic trees inferred from these sequences, five strains of A. penicillioides, including the neotype strain, were closely related to each other, whereas IFO 8155 was distantly related and grouped with other xerophilic species. Our results have suggested that A. penicillioides typified by NRRL 4548 and A. penicillioides IFO 8155 (ex holotype of A. vitricola) are not conspecific. The enzyme patterns as a genotypic character and general morphology and conidial ornamentation types as phenotypic characters supported this conclusion. Therefore the name A. vitricola Ohtsuki, typified by the holotype strain IFO 8155, should be revived. Evolutionary affinities among Aspergillus species and related teleomorphs, including the xerophilic taxa, are discussed.

A stochastic model for optimizing composite predictors based on gene expression profiles.

PubMed

Ramanathan, Murali

2003-07-01

This project was done to develop a mathematical model for optimizing composite predictors based on gene expression profiles from DNA arrays and proteomics. The problem was amenable to a formulation and solution analogous to the portfolio optimization problem in mathematical finance: it requires the optimization of a quadratic function subject to linear constraints. The performance of the approach was compared to that of neighborhood analysis using a data set containing cDNA array-derived gene expression profiles from 14 multiple sclerosis patients receiving intramuscular inteferon-beta1a. The Markowitz portfolio model predicts that the covariance between genes can be exploited to construct an efficient composite. The model predicts that a composite is not needed for maximizing the mean value of a treatment effect: only a single gene is needed, but the usefulness of the effect measure may be compromised by high variability. The model optimized the composite to yield the highest mean for a given level of variability or the least variability for a given mean level. The choices that meet this optimization criteria lie on a curve of composite mean vs. composite variability plot referred to as the "efficient frontier." When a composite is constructed using the model, it outperforms the composite constructed using the neighborhood analysis method. The Markowitz portfolio model may find potential applications in constructing composite biomarkers and in the pharmacogenomic modeling of treatment effects derived from gene expression endpoints.

Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

PubMed

Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

2018-05-09

The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

The phase behavior of cationic lipid-DNA complexes.

PubMed Central

May, S; Harries, D; Ben-Shaul, A

2000-01-01

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

Nanotechnology in plant disease management: DNA-directed silver nanoparticles on graphene oxide as an antibacterial against Xanthomonas perforans.

PubMed

Ocsoy, Ismail; Paret, Mathews L; Ocsoy, Muserref Arslan; Kunwar, Sanju; Chen, Tao; You, Mingxu; Tan, Weihong

2013-10-22

Bacterial spot caused by Xanthomonas perforans is a major disease of tomatoes, leading to reduction in production by 10-50%. While copper (Cu)-based bactericides have been used for disease management, most of the X. perforans strains isolated from tomatoes in Florida and other locations worldwide are Cu-resistant. We have developed DNA-directed silver (Ag) nanoparticles (NPs) grown on graphene oxide (GO). These Ag@dsDNA@GO composites effectively decrease X. perforans cell viability in culture and on plants. At the very low concentration of 16 ppm of Ag@dsDNA@GO, composites show excellent antibacterial capability in culture with significant advantages in improved stability, enhanced antibacterial activity, and stronger adsorption properties. Application of Ag@dsDNA@GO at 100 ppm on tomato transplants in a greenhouse experiment significantly reduced the severity of bacterial spot disease compared to untreated plants, giving results similar to those of the current grower standard treatment, with no phytotoxicity.

Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

PubMed

Gryson, Nicolas

2010-03-01

The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

Study of the controlled assembly of DNA gated PEI/Chitosan/SiO2 fluorescent sensor.

PubMed

Chang, Zheng; Mi, Yinghao; Zheng, Xingwang

2018-03-01

In this paper, polyethylenimine (PEI) and Chitosan were simultaneously one-step doped into silicon dioxide (SiO 2 ) nanoparticles to synthesize PEI/Chitosan/SiO 2 composite nanoparticles. The polymer PEI contained a large amount of amino groups, which can realize the amino functionalized SiO 2 nanoparticles. And, the good pore forming effect of Chitosan was introduced into SiO 2 nanoparticles, and the resulting composite nanoparticles also had a porous structure. In pH 7.4 phosphate buffer solution (PBS), the amino groups of PEI had positive charges, and therefore the fluorescein sodium dye molecule can be loaded into the channels of PEI/Chitosan/SiO 2 composite nanoparticles by electrostatic adsorption. Furthermore, utilizing the diversity of DNA molecular conformation, we designed a high sensitive controllable assembly of DNA gated fluorescent sensor based on PEI/Chitosan/SiO 2 composite nanoparticles as loading materials. The factors affecting the sensing performance of the sensor were investigated, and the sensing mechanism was also further studied. Copyright © 2017 John Wiley & Sons, Ltd.

A silica sands-based method for faithful analysis of microbial communities and DNA isolation from a wide range of species.

PubMed

Liu, Xia; Xu, Yongdong; Li, Zhi; Jiang, Shengwei; Yao, Shuo; Wu, Rina; An, Yingfeng

2018-04-21

A silica sands-based method has been developed to isolate high quality genomic DNAs from cells of animals, plants and microorganisms, such as Hemisalanx prognathus, Spinacia oleracea, Pichia pastoris, Bacillus licheniformis and Escherichia coli. To the best of our knowledge, no DNA isolation method has so wide application until now. In addition, this method and a commercially available kit were compared in analysis of microbial communities using high-throughput 16s rDNA sequencing. As a result, the silica sands-based method was found to be even more efficient in isolating genomic DNA from gram-positive bacteria than the kit, indicating that it would become a very valuable choice to faithfully reflect the composition of microbial communities.

Development of a DNA Sensor Based on Nanoporous Pt-Rich Electrodes

NASA Astrophysics Data System (ADS)

Van Hao, Pham; Thanh, Pham Duc; Xuan, Chu Thi; Hai, Nguyen Hoang; Tuan, Mai Anh

2017-06-01

Nanoporous Pt-rich electrodes with 72 at.% Pt composition were fabricated by sputtering a Pt-Ag alloy, followed by an electrochemical dealloying process to selectively etch away Ag atoms. The surface properties of nanoporous membranes were investigated by energy-dispersive x-ray spectroscopy (EDS), scanning electron microscopy (SEM), atomic force microscopy (AFM), a documentation system, and a gel image system (Gel Doc Imager). A single strand of probe deoxyribonucleic acid (DNA) was immobilized onto the electrode surface by physical adsorption. The DNA probe and target hybridization were measured using a lock-in amplifier and an electrochemical impedance spectroscope (EIS). The nanoporous Pt-rich electrode-based DNA sensor offers a fast response time of 3.7 s, with a limit of detection (LOD) of 4.35 × 10-10 M of DNA target.

Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

PubMed

Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

2016-12-15

Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantifying quality in DNA self-assembly

PubMed Central

Wagenbauer, Klaus F.; Wachauf, Christian H.; Dietz, Hendrik

2014-01-01

Molecular self-assembly with DNA is an attractive route for building nanoscale devices. The development of sophisticated and precise objects with this technique requires detailed experimental feedback on the structure and composition of assembled objects. Here we report a sensitive assay for the quality of assembly. The method relies on measuring the content of unpaired DNA bases in self-assembled DNA objects using a fluorescent de-Bruijn probe for three-base ‘codons’, which enables a comparison with the designed content of unpaired DNA. We use the assay to measure the quality of assembly of several multilayer DNA origami objects and illustrate the use of the assay for the rational refinement of assembly protocols. Our data suggests that large and complex objects like multilayer DNA origami can be made with high strand integration quality up to 99%. Beyond DNA nanotechnology, we speculate that the ability to discriminate unpaired from paired nucleic acids in the same macromolecule may also be useful for analysing cellular nucleic acids. PMID:24751596

Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set.

PubMed

Krueger, Andrew T; Kool, Eric T

2008-03-26

We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.

A high-sensitivity electrochemical aptasensor of carcinoembryonic antigen based on graphene quantum dots-ionic liquid-nafion nanomatrix and DNAzyme-assisted signal amplification strategy.

PubMed

Huang, Jing-Yi; Zhao, Lang; Lei, Wan; Wen, Wei; Wang, Yi-Jia; Bao, Ting; Xiong, Hua-Yu; Zhang, Xiu-Hua; Wang, Sheng-Fu

2018-01-15

In this work, we have developed an electrochemical aptasensor for high-sensitivity determination of carcinoembryonic antigen (CEA) based on lead ion (Pb 2+ )-dependent DNAzyme-assisted signal amplification and graphene quantum dot-ionic liquid-nafion (GQDs-IL-NF) composite film. We designed hairpin DNA containing CEA-specific aptamers and DNAzyme chains. In the presence of CEA, hairpin DNA recognized the target and performed a DNAzyme-assisted signal amplification reaction to yield a large number of single-stranded DNA. The GQDs-IL-NF composite film was immobilized on the glassy carbon electrode for the interaction with single-stranded DNA through noncovalent π-π stacking interaction. Therefore, the methylene blue-labeled substrate DNA (MB-substrate) was fixed on the electrode and exhibited an initial electrochemical signal. Under optimal conditions, the response current change was proportional to the concentration of CEA, demonstrating a wide linear range from 0.5fgmL -1 to 0.5ngmL -1 , with a low detection limit of 0.34fgmL -1 . Furthermore, the proposed aptasensor was successfully applied in determining CEA in serum samples, showing its superior prospects in clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of preservation method on spider monkey (Ateles geoffroyi) fecal microbiota over 8 weeks.

PubMed

Hale, Vanessa L; Tan, Chia L; Knight, Rob; Amato, Katherine R

2015-06-01

Studies of the gut microbiome have become increasingly common with recent technological advances. Gut microbes play an important role in human and animal health, and gut microbiome analysis holds great potential for evaluating health in wildlife, as microbiota can be assessed from non-invasively collected fecal samples. However, many common fecal preservation protocols (e.g. freezing at -80 °C) are not suitable for field conditions, or have not been tested for long-term (greater than 2 weeks) storage. In this study, we collected fresh fecal samples from captive spider monkeys (Ateles geoffroyi) at the Columbian Park Zoo (Lafayette, IN, USA). The samples were pooled, homogenized, and preserved for up to 8 weeks prior to DNA extraction and sequencing. Preservation methods included: freezing at -20 °C, freezing at -80 °C, immersion in 100% ethanol, application to FTA cards, and immersion in RNAlater. At 0 (fresh), 1, 2, 4, and 8 weeks from fecal collection, DNA was extracted and microbial DNA was amplified and sequenced. DNA concentration, purity, microbial diversity, and microbial composition were compared across all methods and time points. DNA concentration and purity did not correlate with microbial diversity or composition. Microbial composition of frozen and ethanol samples were most similar to fresh samples. FTA card and RNAlater-preserved samples had the least similar microbial composition and abundance compared to fresh samples. Microbial composition and diversity were relatively stable over time within each preservation method. Based on these results, if freezers are not available, we recommend preserving fecal samples in ethanol (for up to 8weeks) prior to microbial extraction and analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of polymeric-cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery.

PubMed

Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

2015-01-01

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid-polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

PubMed Central

Jain, Arvind K; Massey, Ashley; Yusuf, Helmy; McDonald, Denise M; McCarthy, Helen O; Kett, Vicky L

2015-01-01

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA. PMID:26648722

DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods

PubMed Central

Martin-Laurent, F.; Philippot, L.; Hallet, S.; Chaussod, R.; Germon, J. C.; Soulas, G.; Catroux, G.

2001-01-01

The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years. PMID:11319122

A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the Determination of Arowana Fish Gender

NASA Astrophysics Data System (ADS)

Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Chiang, Chew Poh; Rashid, Zulkafli A.; Ling, Tan Ling

2017-08-01

The present research describes a simple method for the identification of the gender of arowana fish ( Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10-18 M with a wide linear calibration range of 1.0 × 10-18 to 1.0 × 10-8 M ( R 2 = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage.

Toehold-mediated DNA displacement-based surface-enhanced Raman scattering DNA sensor utilizing an Au-Ag bimetallic nanodendrite substrate.

PubMed

Kim, Saetbyeol; Tran Ngoc, Huan; Kim, Joohoon; Yoo, So Young; Chung, Hoeil

2015-07-23

A simple and sensitive surface enhanced Raman scattering (SERS)-based DNA sensor that utilizes the toehold-mediated DNA displacement reaction as a target-capturing scheme has been demonstrated. For a SERS substrate, Au-Ag bimetallic nanodendrites were electrochemically synthesized and used as a sensor platform. The incorporation of both Ag and Au was employed to simultaneously secure high sensitivity and stability of the substrate. An optimal composition of Ag and Au that satisfied these needs was determined. A double-strand composed of 'a probe DNA (pDNA)' complementary to 'a target DNA (tDNA)' and 'an indicator DNA tagged with a Raman reporter (iDNA)' was conjugated on the substrate. The conjugation made the reporter molecule close to the surface and induced generation of the Raman signal. The tDNA released the pre-hybridized iDNA from the pDNA via toehold-mediated displacement, and the displacement of the iDNA resulted in the decrease of Raman intensity. The variation of percent intensity change was sensitive and linear in the concentration range from 200fM to 20nM, and the achieved limit of detection (LOD) was 96.3fM, superior to those reported in previous studies that adopted different signal taggings based on such as fluorescence and electrochemistry. Copyright © 2015 Elsevier B.V. All rights reserved.

Oxidative DNA damage background estimated by a system model of base excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokhansanj, B A; Wilson, III, D M

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

Sorption of DNA by diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels.

PubMed

Tozak, Kabil Özcan; Erzengin, Mahmut; Sargin, Idris; Ünlü, Nuri

2013-01-01

In this study, the DNA sorption performance of diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels were investigated for the purpose of designing a novel adsorbent that can be utilized for DNA purification, separation and immunoadsorption studies such as removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma. Poly(2-hydroxyethyl methacrylate) [p(HEMA)]-based monolithic cryogel column embedded with Zn(2+)-diatomite particles was prepared by free radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm). The polymerization reaction was initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After thawing, the monolithic composite cryogels were used for affinity sorption and then subsequent desorption of DNA molecules from aqueous solutions. Diatomite (DA) particles were characterized by XRF and BET method. The characterization of composite cryogel was done through SEM imaging. The effects of pH of the solution, initial DNA concentration, ionic strength, temperature and flow rates on adsorption were investigated to determine the optimum conditions for adsorption/desorption experiments. The particle embedding procedure was shown to yield significantly enhanced adsorption of DNA on the adsorbent. Furthermore, considering its excellent bio-compatibility, p(HEMA) cryogels are promising a candidate for further DNA sorption studies.

Sorption of DNA by diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels

PubMed Central

Tozak, Kabil Özcan; Erzengin, Mahmut; Sargin, Idris; Ünlü, Nuri

2013-01-01

In this study, the DNA sorption performance of diatomite-Zn(II) embedded supermacroporous monolithic p(HEMA) cryogels were investigated for the purpose of designing a novel adsorbent that can be utilized for DNA purification, separation and immunoadsorption studies such as removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma. Poly(2-hydroxyethyl methacrylate) [p(HEMA)]-based monolithic cryogel column embedded with Zn2+-diatomite particles was prepared by free radical cryo-copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N,N'-methylene-bis-acrylamide (MBAAm). The polymerization reaction was initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After thawing, the monolithic composite cryogels were used for affinity sorption and then subsequent desorption of DNA molecules from aqueous solutions. Diatomite (DA) particles were characterized by XRF and BET method. The characterization of composite cryogel was done through SEM imaging. The effects of pH of the solution, initial DNA concentration, ionic strength, temperature and flow rates on adsorption were investigated to determine the optimum conditions for adsorption/desorption experiments. The particle embedding procedure was shown to yield significantly enhanced adsorption of DNA on the adsorbent. Furthermore, considering its excellent bio-compatibility, p(HEMA) cryogels are promising a candidate for further DNA sorption studies. PMID:26600734

SPRi-based biosensing platforms for detection of specific DNA sequences using thiolate and dithiocarbamate assemblies

NASA Astrophysics Data System (ADS)

Drozd, Marcin; Pietrzak, Mariusz D.; Malinowska, Elżbieta

2018-05-01

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0,66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 ·1012 molecules • cm-2 (with the calculated area occupied by single nanoparticle label of 132.7 nm2), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

SPRi-Based Biosensing Platforms for Detection of Specific DNA Sequences Using Thiolate and Dithiocarbamate Assemblies.

PubMed

Drozd, Marcin; Pietrzak, Mariusz D; Malinowska, Elżbieta

2018-01-01

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0.66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 · 10 12 molecules · cm -2 (with the calculated area occupied by single nanoparticle label of ~132.7 nm 2 ), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting

PubMed Central

Eichler, Stefan; Christen, Richard; Höltje, Claudia; Westphal, Petra; Bötel, Julia; Brettar, Ingrid; Mehling, Arndt; Höfle, Manfred G.

2006-01-01

Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. RNA and DNA were extracted from the sampled water. The 16S rRNA or its genes were partially amplified by reverse transcription-PCR or PCR and analyzed by single-strand conformation polymorphism community fingerprints. The bacterial community structures of the raw water samples from the two reservoirs were very different, but no major changes of these structures occurred after flocculation and sand filtration. Chlorination of the processed raw water strongly affected bacterial community structure, as reflected by the RNA-based fingerprints. This effect was less pronounced for the DNA-based fingerprints. After chlorination, the bacterial community remained rather constant from the storage containers to the tap. Furthermore, the community structure of the tap water did not change substantially for several months. Community composition was assessed by sequencing of abundant bands and phylogenetic analysis of the sequences obtained. The taxonomic compositions of the bacterial communities from both reservoirs were very different at the species level due to their different limnologies. On the other hand, major taxonomic groups, well known to occur in freshwater, such as Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, were found in both reservoirs. Significant differences in the detection of the major groups were observed between DNA-based and RNA-based fingerprints irrespective of the reservoir. Chlorination of the drinking water seemed to promote growth of nitrifying bacteria. Detailed analysis of the community dynamics of the whole DWSS revealed a significant influence of both source waters on the overall composition of the drinking water microflora and demonstrated the relevance of the raw water microflora for the drinking water microflora provided to the end user. PMID:16517632

Imparting biomolecules to a metal-organic framework material by controlled DNA tetrahedron encapsulation

PubMed Central

Jia, Yongmei; Wei, Benmei; Duan, Ruixue; Zhang, Ying; Wang, Boya; Hakeem, Abdul; Liu, Nannan; Ou, Xiaowen; Xu, Shaofang; Chen, Zhifei; Lou, Xiaoding; Xia, Fan

2014-01-01

Recently, the incorporation of biomolecules in Metal-organic frameworks (MOFs) attracts many attentions because of controlling the functions, properties and stability of trapped molecules. Although there are few reports on protein/MOFs composites and their applications, none of DNA/MOFs composite is reported, as far as we know. Here, we report a new composite material which is self-assembled from 3D DNA (guest) and pre-synthesized MOFs (host) by electrostatic interactions and hydrophilic interactions in a well-dispersed fashion. Its biophysical characterization is well analyzed by fluorescence spectroscopy, quartz crystal microbalance (QCM) and transmission electron microscopy (TEM). This new composite material keeps 3D DNA nanostructure more stable than only 3D DNA nanostructure in DI water at room temperature, and stores amounts of genetic information. It will make DNA as a guest for MOFs and MOFs become a new platform for the development of DNA nanotechnology. PMID:25090047

High Efficiency DNA Extraction by Graphite Oxide/Cellulose/Magnetite Composites Under Na+ Free System

NASA Astrophysics Data System (ADS)

Akceoglu, Garbis Atam; Li, Oi Lun; Saito, Nagahiro

2016-04-01

DNA extraction is the key step at various research areas like biotechnology, diagnostic development, paternity determination, and forensic science . Solid support extraction is the most common method for DNA purification. In this method, Na+ ions have often been applied as binding buffers in order to obtain high extraction efficiency and high quality of DNA; however, the presence of Na+ ions might be interfering with the downstream DNA applications. In this study, we proposed graphite oxide (GO)/magnetite composite/cellulose as an innovative material for Na+-free DNA extraction. The total wt.% of GO was fixed at 4.15% in the GO/cellulose/magnetite composite . The concentration of magnetite within the composites were controlled at 0-3.98 wt.%. The extraction yield of DNA increased with increasing weight percentage of magnetite. The highest yield was achieved at 3.98 wt.% magnetite, where the extraction efficiency was reported to be 338.5 ng/µl. The absorbance ratios between 260 nm and 280 nm (A260/A280) of the DNA elution volume was demonstrated as 1.81, indicating the extracted DNA consisted of high purity. The mechanism of adsorption of DNA was provided by (1) π-π interaction between the aromatic ring in GO and nucleobases of DNA molecule, and (2) surface charge interaction between the positive charge magnetite and anions such as phosphates within the DNA molecules. The results proved that the GO/cellulose/magnetite composite provides a Na+-free method for selective DNA extraction with high extraction efficiency of pure DNA.

Functionalized gold nanoparticles as additive to form polymer/metal composite matrix for improved DNA sequencing by capillary electrophoresis.

PubMed

Zhou, Dan; Yang, Liping; Yang, Runmiao; Song, Weihua; Peng, Shuhua; Wang, Yanmei

2009-11-15

A new matrix additive, poly (N,N-dimethylacrylamide)-functionalized gold nanoparticle (GNP-PDMA), was prepared by "grafting-to" approach, and then incorporated into quasi-interpenetrating network (quasi-IPN) composed of linear polyacrylamide (LPA, 3.3 MDa) and PDMA to form novel polymer/metal composite sieving matrix (quasi-IPN/GNP-PDMA) for DNA sequencing by capillary electrophoresis. Without complete optimization, quasi-IPN/GNP-PDMA yielded a readlength of 801 bases at 98% accuracy in about 64 min by using the ABI 310 Genetic Analyzer at 50 degrees C and 150 V/cm. Compared with previous quasi-IPN/GNPs, quasi-IPN/GNP-PDMA can further improve DNA sequencing performances. This is because the presence of GNP-PDMA can improve the compatibility of GNPs with the whole sequencing system, enhance the entanglement degree of networks, and increase the GNP concentration in system, which consequently lead to higher restriction and stability, higher apparent molecular weight (MW), and smaller pore size of the total sieving networks. Furthermore, the composite matrix was also compared with quasi-IPN containing higher-MW LPA and commercial POP-6. The results indicate that the composite matrix is a promising one for DNA sequencing to achieve full automation due to the separation provided with high resolution, speediness, excellent reproducibility, and easy loading in the presence of GNP-PDMA.

Single-Molecule Electrical Random Resequencing of DNA and RNA

NASA Astrophysics Data System (ADS)

Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

2012-07-01

Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Palindromic Sequence Artifacts Generated during Next Generation Sequencing Library Preparation from Historic and Ancient DNA

PubMed Central

Star, Bastiaan; Nederbragt, Alexander J.; Hansen, Marianne H. S.; Skage, Morten; Gilfillan, Gregor D.; Bradbury, Ian R.; Pampoulie, Christophe; Stenseth, Nils Chr; Jakobsen, Kjetill S.; Jentoft, Sissel

2014-01-01

Degradation-specific processes and variation in laboratory protocols can bias the DNA sequence composition from samples of ancient or historic origin. Here, we identify a novel artifact in sequences from historic samples of Atlantic cod (Gadus morhua), which forms interrupted palindromes consisting of reverse complementary sequence at the 5′ and 3′-ends of sequencing reads. The palindromic sequences themselves have specific properties – the bases at the 5′-end align well to the reference genome, whereas extensive misalignments exists among the bases at the terminal 3′-end. The terminal 3′ bases are artificial extensions likely caused by the occurrence of hairpin loops in single stranded DNA (ssDNA), which can be ligated and amplified in particular library creation protocols. We propose that such hairpin loops allow the inclusion of erroneous nucleotides, specifically at the 3′-end of DNA strands, with the 5′-end of the same strand providing the template. We also find these palindromes in previously published ancient DNA (aDNA) datasets, albeit at varying and substantially lower frequencies. This artifact can negatively affect the yield of endogenous DNA in these types of samples and introduces sequence bias. PMID:24608104

Self-assembly of multiferroic core-shell particulate nanocomposites through DNA-DNA hybridization and magnetic field directed assembly of superstructures

NASA Astrophysics Data System (ADS)

Sreenivasulu, Gollapudi; Lochbiler, Thomas A.; Panda, Manashi; Srinivasan, Gopalan; Chavez, Ferman A.

2016-04-01

Multiferroic composites of ferromagnetic and ferroelectric phases are of importance for studies on mechanical strain mediated coupling between the magnetic and electric subsystems. This work is on DNA-assisted self-assembly of superstructures of such composites with nanometer periodicity. The synthesis involved oligomeric DNA-functionalized ferroelectric and ferromagnetic nanoparticles, 600 nm BaTiO3 (BTO) and 200 nm NiFe2O4 (NFO), respectively. Mixing BTO and NFO particles, possessing complementary DNA sequences, resulted in the formation of ordered core-shell heteronanocomposites held together by DNA hybridization. The composites were imaged by scanning electron microscopy and scanning microwave microscopy. The presence of heteroassemblies along with core-shell architecture is clearly observed. The reversible nature of the DNA hybridization allows for restructuring the composites into mm-long linear chains and 2D-arrays in the presence of a static magnetic field and ring-like structures in a rotating-magnetic field. Strong magneto-electric (ME) coupling in as-assembled composites is evident from static magnetic field H induced polarization and low-frequency magnetoelectric voltage coefficient measurements. Upon annealing the nanocomposites at high temperatures, evidence for the formation of bulk composites with excellent cross-coupling between the electric and magnetic subsystems is obtained by H-induced polarization and low-frequency ME voltage coefficient. The ME coupling strength in the self-assembled composites is measured to be much stronger than in bulk composites with randomly distributed NFO and BTO prepared by direct mixing and sintering.

Pulsatile release of biomolecules from polydimethylsiloxane (PDMS) chips with hydrolytically degradable seals.

PubMed

Intra, Janjira; Glasgow, Justin M; Mai, Hoang Q; Salem, Aliasger K

2008-05-08

We demonstrate, for the first time, a robust novel polydimethylsiloxane (PDMS) chip that can provide controlled pulsatile release of DNA based molecules, proteins and oligonucleotides without external stimuli or triggers. The PDMS chip with arrays of wells was constructed by replica molding. Poly(lactic acid-co-glycolic acid) (PLGA) polymer films of varying composition and thickness were used as seals to the wells. The composition, molecular weight and thickness of the PLGA films were all parameters used to control the degradation rate of the seals and therefore the release profiles. Degradation of the films followed the PLGA composition order of 50:50 PLGA>75:25 PLGA>85:15 PLGA at all time-points beyond week 1. Scanning electron microscopy images showed that films were initially smooth, became porous and ruptured as the osmotic pressure pushed the degrading PLGA film outwards. Pulsatile release of DNA was controlled by the composition and thickness of the PLGA used to seal the well. Transfection experiments in a model Human Embryonic Kidney 293 (HEK293) cell line showed that plasmid DNA loaded in the wells was functional after pulsatile release in comparison to control plasmid DNA at all time-points. Thicker films degraded faster than thinner films and could be used to fine-tune the release of DNA over day length periods. Finally the PDMS chip was shown to provide repeated sequential release of CpG oligonucleotides and a model antigen, Ovalbumin (OVA), indicating significant potential for this device for vaccinations or applications that require defined complex release patterns of a variety of chemicals, drugs and biomolecules.

Electrochemically-driven large amplitude pH cycling for acid-base driven DNA denaturation and renaturation.

PubMed

Wang, Yong-Chun; Lin, Cong-Bin; Su, Jian-Jia; Ru, Ying-Ming; Wu, Qiao; Chen, Zhao-Bin; Mao, Bing-Wei; Tian, Zhao-Wu

2011-06-15

In this paper, we present an electrochemically driven large amplitude pH alteration method based on a serial electrolytic cell involving a hydrogen permeable bifacial working electrode such as Pd thin foil. The method allows solution pH to be changed periodically up to ±4~5 units without additional alteration of concentration and/or composition of the system. Application to the acid-base driven cyclic denaturation and renaturation of 290 bp DNA fragments is successfully demonstrated with in situ real-time UV spectroscopic characterization. Electrophoretic analysis confirms that the denaturation and renaturation processes are reversible without degradation of the DNA. The serial electrolytic cell based electrochemical pH alteration method presented in this work would promote investigations of a wide variety of potential-dependent processes and techniques.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

PubMed

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

Nuclear DNA content and base composition in 28 taxa of Musa.

PubMed

Kamaté, K; Brown, S; Durand, P; Bureau, J M; De Nay, D; Trinh, T H

2001-08-01

The nuclear DNA content of 28 taxa of Musa was assessed by flow cytometry, using line PxPC6 of Petunia hybrida as an internal standard. The 2C DNA value of Musa balbisiana (BB genome) was 1.16 pg, whereas Musa acuminata (AA genome) had an average 2C DNA value of 1.27 pg, with a difference of 11% between its subspecies. The two haploid (IC) genomes, A and B, comprising most of the edible bananas, are therefore of similar size, 0.63 pg (610 million bp) and 0.58 pg (560 million bp), respectively. The genome of diploid Musa is thus threefold that of Arabidopsis thaliana. The genome sizes in a set of triploid Musa cultivars or clones were quite different, with 2C DNA values ranging from 1.61 to 2.23 pg. Likewise, the genome sizes of tetraploid cultivars ranged from 1.94 to 2.37 pg (2C). Apparently, tetraploids (for instance, accession I.C.2) can have a genome size that falls within the range of triploid genome sizes, and vice versa (as in the case of accession Simili Radjah). The 2C values estimated for organs such as leaf, leaf sheath, rhizome, and flower were consistent, whereas root material gave atypical results, owing to browning. The genomic base composition of these Musa taxa had a median value of 40.8% GC (SD = 0.43%).

Self-assembly of multiferroic core-shell particulate nanocomposites through DNA-DNA hybridization and magnetic field directed assembly of superstructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sreenivasulu, Gollapudi; Srinivasan, Gopalan, E-mail: srinivas@oakland.edu, E-mail: chavez@oakland.edu; Lochbiler, Thomas A.

Multiferroic composites of ferromagnetic and ferroelectric phases are of importance for studies on mechanical strain mediated coupling between the magnetic and electric subsystems. This work is on DNA-assisted self-assembly of superstructures of such composites with nanometer periodicity. The synthesis involved oligomeric DNA-functionalized ferroelectric and ferromagnetic nanoparticles, 600 nm BaTiO{sub 3} (BTO) and 200 nm NiFe{sub 2}O{sub 4} (NFO), respectively. Mixing BTO and NFO particles, possessing complementary DNA sequences, resulted in the formation of ordered core-shell heteronanocomposites held together by DNA hybridization. The composites were imaged by scanning electron microscopy and scanning microwave microscopy. The presence of heteroassemblies along with core-shellmore » architecture is clearly observed. The reversible nature of the DNA hybridization allows for restructuring the composites into mm-long linear chains and 2D-arrays in the presence of a static magnetic field and ring-like structures in a rotating-magnetic field. Strong magneto-electric (ME) coupling in as-assembled composites is evident from static magnetic field H induced polarization and low-frequency magnetoelectric voltage coefficient measurements. Upon annealing the nanocomposites at high temperatures, evidence for the formation of bulk composites with excellent cross-coupling between the electric and magnetic subsystems is obtained by H-induced polarization and low-frequency ME voltage coefficient. The ME coupling strength in the self-assembled composites is measured to be much stronger than in bulk composites with randomly distributed NFO and BTO prepared by direct mixing and sintering.« less

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

DOE PAGES

Rinke, Christian; Low, Serene; Woodcroft, Ben J.; ...

2016-09-22

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diversemore » Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rinke, Christian; Low, Serene; Woodcroft, Ben J.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diversemore » Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

PubMed Central

Low, Serene; Raina, Jean-Baptiste; Skarshewski, Adam; Le, Xuyen H.; Butler, Margaret K.; Stocker, Roman; Seymour, Justin; Tyson, Gene W.

2016-01-01

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics. PMID:27688978

Potential forensic biogeographic application of diatom colony consistency analysis employing pyrosequencing profiles of the 18S rDNA V7 region.

PubMed

Zhao, Yuancun; Chen, Xiaogang; Yang, Yiwen; Zhao, Xiaohong; Zhang, Shu; Gao, Zehua; Fang, Ting; Wang, Yufang; Zhang, Ji

2018-05-07

Diatom examination has always been used for the diagnosis of drowning in forensic practice. However, traditional examination of the microscopic features of diatom frustules is time-consuming and requires taxonomic expertise. In this study, we demonstrate a potential DNA-based method of inferring suspected drowning site using pyrosequencing (PSQ) of the V7 region of 18S ribosome DNA (18S rDNA) as a diatom DNA barcode. By employing a sparse representation-based AdvISER-M-PYRO algorithm, the original PSQ signals of diatom DNA mixtures were deciphered to determine the corresponding taxa of the composite diatoms. Additionally, we evaluated the possibility of correlating water samples to collection sites by analyzing the PSQ signal profiles of diatom mixtures contained in the water samples via multidimensional scaling. The results suggest that diatomaceous PSQ profile analysis could be used as a cost-effective method to deduce the geographical origin of an environmental bio-sample.

Core-shell magnetite-silica composite nanoparticles enhancing DNA damage induced by a photoactive platinum-diimine complex in red light.

PubMed

Zhang, Zhigang; Chai, Aiyun

2012-12-01

Lack of solubility under physiological conditions poses an additional risk for toxicity and side effects for intravenous delivery of the photodynamic therapeutic agent in vivo. Employing magnetite-silica composite nanoparticles as carriers of the photodynamic therapeutic agents may be a promising way to solve the problem. In this study, core-shell magnetite-silica composite nanoparticles were prepared by a sol-gel method, and characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy and dynamic light scattering, then they were used as carriers of a photoactive platinum diimine complex. The interactions of the photosensitizer-loaded magnetic composite nanoparticles with DNA in red light were monitored by agarose-gel electrophoresis. The results suggest that high doses of magnetite-silica composite nanoparticles might facilitate the transformation of covalently closed circular (ccc)-DNA band to open circular (oc)-DNA band though they are harmless to DNA at their low concentrations, therefore enhancing the extent of DNA damage caused by the metal complex in red light. Copyright © 2012 Elsevier Inc. All rights reserved.

Active microrheology of entangled biopolymer composites link polymer flexibility and length to molecular force response

NASA Astrophysics Data System (ADS)

Fitzpatrick, Robert; Hauer, Cole; Kyrillos, Carl; McGorty, Ryan; Robertson-Anderson, Rae

Entangled polymers have complex viscoelastic properties that are tuned by polymer lengths and flexibilities. Entangled composites of distinct polymers offer added versatility and display nonlinear mechanics, serving as a platform for multifunctional materials. To determine the role of flexibility and length in polymer composites we use optical tweezers and confocal microscopy to measure mechanical and structural properties of co-entangled actin and DNA. Actin filaments have lengths of 5-20 μm, comparable to their persistence length, while DNA of similar lengths have hundreds of persistence lengths per chain. To characterize the nonlinear mechanics of actin-DNA composites, we optically drive a microsphere through the composite and measure the induced force during and following strain. We characterize viscoelasticity and relaxation timescales; and determine the dependence of these quantities on the actin:DNA ratio (0:1-1:0) and DNA length (4-100 μm). We use confocal microscopy to image distinctly labeled co-entangled actin and DNA and characterize network homogeneity and fluctuations. Initial results show actin and DNA are well-integrated and form structurally homogenous networks that exhibit stiffness and relaxation times that increase nonlinearly with increased actin. NSF Career Award (DMR-1254340), AFOSR Young Investigator Program Award (FA95550-12-1-0315), Scialog Collaborative Innovation Award funed by Research Corp. for Scientific Advancement (24192).

A novel gene delivery composite system based on biodegradable folate-poly (ester amine) polymer and thermosensitive hydrogel for sustained gene release

PubMed Central

Yang, Yi; Zhao, Hang; Jia, YanPeng; Guo, QingFa; Qu, Ying; Su, Jing; Lu, XiaoLing; Zhao, YongXiang; Qian, ZhiYong

2016-01-01

Local anti-oncogene delivery providing high local concentration of gene, increasing antitumor effect and decreasing systemic side effects is currently attracting interest in cancer therapy. In this paper, a novel local sustained anti-oncogene delivery system, PECE thermoresponsive hydrogel containing folate-poly (ester amine) (FA-PEA) polymer/DNA (tumor suppressor) complexes, is demonstrated. First, a tumor-targeted biodegradable folate-poly (ester amine) (FA-PEA) polymer based on low-molecular-weight polyethyleneimine (PEI) was synthesized and characterized, and the application for targeted gene delivery was investigated. The polymer had slight cytotoxicity and high transfection efficiency in vitro compared with PEI 25k, which indicated that FA-PEA was a potential vector for targeted gene delivery. Meanwhile, we successfully prepared a thermoresponsive PECE hydrogel composite containing FA-PEA/DNA complexes which could contain the genes and slowly release the genes into cells. We concluded the folate-poly (ester amine) (FA-PEA) polymer would be useful for targeted gene delivery, and the novel gene delivery composite based on biodegradable folate-poly (ester amine) polymer and thermosensitive PECE hydrogel showed potential for sustained gene release. PMID:26883682

The histone variant H2A.Bbd is enriched at sites of DNA synthesis

PubMed Central

Sansoni, Viola; Casas-Delucchi, Corella S.; Rajan, Malini; Schmidt, Andreas; Bönisch, Clemens; Thomae, Andreas W.; Staege, Martin S.; Hake, Sandra B.; Cardoso, M. Cristina; Imhof, Axel

2014-01-01

Histone variants play an important role in shaping the mammalian epigenome and their aberrant expression is frequently observed in several types of cancer. However, the mechanisms that mediate their function and the composition of the variant-containing chromatin are still largely unknown. A proteomic interrogation of chromatin containing the different H2A variants macroH2A.1.2, H2A.Bbd and H2A revealed a strikingly different protein composition. Gene ontology analysis reveals a strong enrichment of splicing factors as well as components of the mammalian replisome in H2A.Bbd-containing chromatin. We find H2A.Bbd localizing transiently to sites of DNA synthesis during S-phase and during DNA repair. Cells that express H2A.Bbd have a shortened S-phase and are more susceptible to DNA damage, two phenotypes that are also observed in human Hodgkin's lymphoma cells that aberrantly express this variant. Based on our experiments we conclude that H2A.Bbd is targeted to newly synthesized DNA during replication and DNA repair. The transient incorporation of H2A.Bbd may be due to the intrinsic instability of nucleosomes carrying this variant or a faster chromatin loading. This potentially leads to a disturbance of the existing chromatin structure, which may have effects on cell cycle regulation and DNA damage sensitivity. PMID:24753410

Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles.

PubMed

Liu, Meiying; Yuan, Min; Lou, Xinhui; Mao, Hongju; Zheng, Dongmei; Zou, Ruxing; Zou, Nengli; Tang, Xiangrong; Zhao, Jianlong

2011-07-15

We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments. Copyright © 2011 Elsevier B.V. All rights reserved.

DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tarahovsky, Yury S.; Koynova, Rumiana; MacDonald, Robert C.

2010-01-18

DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugalmore » separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.« less

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking.

PubMed

Fan, Lihua; Shuai, Jiangbing; Zeng, Ruoxue; Mo, Hongfei; Wang, Suhua; Zhang, Xiaofeng; He, Yongqiang

2017-12-01

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1-38 and 3-53), a Clostridia- (gene 2-109) as well as a Bacilli-like sequence (gene 2-95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1-38 and 3-53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract.

PubMed

Burnham, Philip; Dadhania, Darshana; Heyang, Michael; Chen, Fanny; Westblade, Lars F; Suthanthiran, Manikkam; Lee, John Richard; De Vlaminck, Iwijn

2018-06-20

Urinary tract infections are one of the most common infections in humans. Here we tested the utility of urinary cell-free DNA (cfDNA) to comprehensively monitor host and pathogen dynamics in bacterial and viral urinary tract infections. We isolated cfDNA from 141 urine samples from a cohort of 82 kidney transplant recipients and performed next-generation sequencing. We found that urinary cfDNA is highly informative about bacterial and viral composition of the microbiome, antimicrobial susceptibility, bacterial growth dynamics, kidney allograft injury, and host response to infection. These different layers of information are accessible from a single assay and individually agree with corresponding clinical tests based on quantitative PCR, conventional bacterial culture, and urinalysis. In addition, cfDNA reveals the frequent occurrence of pathologies that remain undiagnosed with conventional diagnostic protocols. Our work identifies urinary cfDNA as a highly versatile analyte to monitor infections of the urinary tract.

The complete mitochondrial genome of the Asian tapirs (Tapirus indicus): the only extant Tapiridae species in the old world.

PubMed

Muangkram, Yuttamol; Wajjwalku, Worawidh; Kaolim, Nongnid; Buddhakosai, Waradee; Kamolnorranath, Sumate; Siriaroonrat, Boripat; Tipkantha, Wanlaya; Dongsaard, Khwanruean; Maikaew, Umaporn; Sanannu, Saowaphang

2016-01-01

Asian tapir (Tapirus indicus) is categorized as Endangered on the 2008 IUCN red list. The first full-length mitochondrial DNA (mtDNA) sequence of Asian tapir is 16,717 bp in length. Base composition shows 34.6% A, 27.2% T, 25.8% C and 12.3% G. Highest polymorphic site is on the control region as typical for many species.

Assessing macroinvertebrate biodiversity in freshwater ecosystems: Advances and challenges in dna-based approaches

USGS Publications Warehouse

Pfrender, M.E.; Ferrington, L.C.; Hawkins, C.P.; Hartzell, P.L.; Bagley, M.; Jackson, S.; Courtney, G.W.; Larsen, D.P.; Creutzburg, B.R.; Levesque, C.A.; Epler, J.H.; Morse, J.C.; Fend, S.; Petersen, M.J.; Ruiter, D.; Schindel, D.; Whiting, M.

2010-01-01

Assessing the biodiversity of macroinvertebrate fauna in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify water quality and measure the efficacy of remediation and restoration efforts. The accuracy and precision of biodiversity assessments based on standard morphological identifications are often limited by taxonomic resolution and sample size. Morphologically based identifications are laborious and costly, significantly constraining the sample sizes that can be processed. We suggest that the development of an assay platform based on DNA signatures will increase the precision and ease of quantifying biodiversity in freshwater ecosystems. Advances in this area will be particularly relevant for benthic and planktonic invertebrates, which are often monitored by regulatory agencies. Adopting a genetic assessment platform will alleviate some of the current limitations to biodiversity assessment strategies. We discuss the benefits and challenges associated with DNA-based assessments and the methods that are currently available. As recent advances in microarray and next-generation sequencing technologies will facilitate a transition to DNA-based assessment approaches, future research efforts should focus on methods for data collection, assay platform development, establishing linkages between DNA signatures and well-resolved taxonomies, and bioinformatics. ?? 2010 by The University of Chicago Press.

De novo DNA methylation during monkey pre-implantation embryogenesis.

PubMed

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-04-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis.

De novo DNA methylation during monkey pre-implantation embryogenesis

PubMed Central

Gao, Fei; Niu, Yuyu; Sun, Yi Eve; Lu, Hanlin; Chen, Yongchang; Li, Siguang; Kang, Yu; Luo, Yuping; Si, Chenyang; Yu, Juehua; Li, Chang; Sun, Nianqin; Si, Wei; Wang, Hong; Ji, Weizhi; Tan, Tao

2017-01-01

Critical epigenetic regulation of primate embryogenesis entails DNA methylome changes. Here we report genome-wide composition, patterning, and stage-specific dynamics of DNA methylation in pre-implantation rhesus monkey embryos as well as male and female gametes studied using an optimized tagmentation-based whole-genome bisulfite sequencing method. We show that upon fertilization, both paternal and maternal genomes undergo active DNA demethylation, and genome-wide de novo DNA methylation is also initiated in the same period. By the 8-cell stage, remethylation becomes more pronounced than demethylation, resulting in an increase in global DNA methylation. Promoters of genes associated with oxidative phosphorylation are preferentially remethylated at the 8-cell stage, suggesting that this mode of energy metabolism may not be favored. Unlike in rodents, X chromosome inactivation is not observed during monkey pre-implantation development. Our study provides the first comprehensive illustration of the 'wax and wane' phases of DNA methylation dynamics. Most importantly, our DNA methyltransferase loss-of-function analysis indicates that DNA methylation influences early monkey embryogenesis. PMID:28233770

Biomolecular damage induced by ionizing radiation: the direct and indirect effects of low-energy electrons on DNA.

PubMed

Alizadeh, Elahe; Orlando, Thomas M; Sanche, Léon

2015-04-01

Many experimental and theoretical advances have recently allowed the study of direct and indirect effects of low-energy electrons (LEEs) on DNA damage. In an effort to explain how LEEs damage the human genome, researchers have focused efforts on LEE interactions with bacterial plasmids, DNA bases, sugar analogs, phosphate groups, and longer DNA moieties. Here, we summarize the current understanding of the fundamental mechanisms involved in LEE-induced damage of DNA and complex biomolecule films. Results obtained by several laboratories on films prepared and analyzed by different methods and irradiated with different electron-beam current densities and fluencies are presented. Despite varied conditions (e.g., film thicknesses and morphologies, intrinsic water content, substrate interactions, and extrinsic atmospheric compositions), comparisons show a striking resemblance in the types of damage produced and their yield functions. The potential of controlling this damage using molecular and nanoparticle targets with high LEE yields in targeted radiation-based cancer therapies is also discussed.

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

PubMed Central

Johnson, John L.; Anderson, Robert S.; Ordal, Erling J.

1970-01-01

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA–DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA–DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid. PMID:5413826

Highly sensitive aptasensor based on synergetic catalysis activity of MoS2-Au-HE composite using cDNA-Au-GOD for signal amplification.

PubMed

Song, Hai-Yan; Kang, Tian-Fang; Lu, Li-Ping; Cheng, Shui-Yuan

2017-03-01

Single or few-layer nanosheets of MoS 2 (MoS 2 nanosheets) and a composite composed of MoS 2 nanosheets, Au nanoparticles (AuNPs) and hemin (HE) (denoted as MoS 2 -Au-HE) were prepared. The composites possessed high synergetic catalysis activity towards the electroreduction of hydrogen peroxide. Furthermore, glucose oxidase (GOD) and AuNPs were used as marker of the complementary DNA (cDNA) strand of kanamycin aptamer to prepare a conjugate (reffered as cDNA-Au-GOD) that was designed as the signal probe. Both cDNA-Au-GOD and MoS 2 -Au-HE were applied to fabricate aptasensor for kanamycin. MoS 2 -Au-HE acted as solid platform for kanamycin aptamer and signal transmitters. AuNPs were employed as the supporter of cDNA and GOD which catalyze dissolved oxygen to produce hydrogen peroxide in the presence of glucose. Then cathodic peak current of H 2 O 2 was recorded by differential pulse voltammetry (DPV). The electrochemical reduction of H 2 O 2 was catalyzed by MoS 2 -Au-HE that was modified onto the surface of a glassy carbon electrode (GCE). The cathodic peak current of H 2 O 2 was highly linearly decreased with an increase of kanamycin concentrations from 1.0ng/L to 1.0×10 5 ng/L, with a detection limit of 0.8ng/L. This aptasensor can be used to detect kanamycin in milk with high specificity, sensitivity and selectivity. Copyright © 2016 Elsevier B.V. All rights reserved.

repDNA: a Python package to generate various modes of feature vectors for DNA sequences by incorporating user-defined physicochemical properties and sequence-order effects.

PubMed

Liu, Bin; Liu, Fule; Fang, Longyun; Wang, Xiaolong; Chou, Kuo-Chen

2015-04-15

In order to develop powerful computational predictors for identifying the biological features or attributes of DNAs, one of the most challenging problems is to find a suitable approach to effectively represent the DNA sequences. To facilitate the studies of DNAs and nucleotides, we developed a Python package called representations of DNAs (repDNA) for generating the widely used features reflecting the physicochemical properties and sequence-order effects of DNAs and nucleotides. There are three feature groups composed of 15 features. The first group calculates three nucleic acid composition features describing the local sequence information by means of kmers; the second group calculates six autocorrelation features describing the level of correlation between two oligonucleotides along a DNA sequence in terms of their specific physicochemical properties; the third group calculates six pseudo nucleotide composition features, which can be used to represent a DNA sequence with a discrete model or vector yet still keep considerable sequence-order information via the physicochemical properties of its constituent oligonucleotides. In addition, these features can be easily calculated based on both the built-in and user-defined properties via using repDNA. The repDNA Python package is freely accessible to the public at http://bioinformatics.hitsz.edu.cn/repDNA/. bliu@insun.hit.edu.cn or kcchou@gordonlifescience.org Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Detecting in situ copepod diet diversity using molecular technique: development of a copepod/symbiotic ciliate-excluding eukaryote-inclusive PCR protocol.

PubMed

Hu, Simin; Guo, Zhiling; Li, Tao; Carpenter, Edward J; Liu, Sheng; Lin, Senjie

2014-01-01

Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.

Vertebrate Genome Evolution in the Light of Fish Cytogenomics and rDNAomics

PubMed Central

Howell, W. Mike

2018-01-01

To understand the cytogenomic evolution of vertebrates, we must first unravel the complex genomes of fishes, which were the first vertebrates to evolve and were ancestors to all other vertebrates. We must not forget the immense time span during which the fish genomes had to evolve. Fish cytogenomics is endowed with unique features which offer irreplaceable insights into the evolution of the vertebrate genome. Due to the general DNA base compositional homogeneity of fish genomes, fish cytogenomics is largely based on mapping DNA repeats that still represent serious obstacles in genome sequencing and assembling, even in model species. Localization of repeats on chromosomes of hundreds of fish species and populations originating from diversified environments have revealed the biological importance of this genomic fraction. Ribosomal genes (rDNA) belong to the most informative repeats and in fish, they are subject to a more relaxed regulation than in higher vertebrates. This can result in formation of a literal ‘rDNAome’ consisting of more than 20,000 copies with their high proportion employed in extra-coding functions. Because rDNA has high rates of transcription and recombination, it contributes to genome diversification and can form reproductive barrier. Our overall knowledge of fish cytogenomics grows rapidly by a continuously increasing number of fish genomes sequenced and by use of novel sequencing methods improving genome assembly. The recently revealed exceptional compositional heterogeneity in an ancient fish lineage (gars) sheds new light on the compositional genome evolution in vertebrates generally. We highlight the power of synergy of cytogenetics and genomics in fish cytogenomics, its potential to understand the complexity of genome evolution in vertebrates, which is also linked to clinical applications and the chromosomal backgrounds of speciation. We also summarize the current knowledge on fish cytogenomics and outline its main future avenues. PMID:29443947

Organizational heterogeneity of vertebrate genomes.

PubMed

Frenkel, Svetlana; Kirzhner, Valery; Korol, Abraham

2012-01-01

Genomes of higher eukaryotes are mosaics of segments with various structural, functional, and evolutionary properties. The availability of whole-genome sequences allows the investigation of their structure as "texts" using different statistical and computational methods. One such method, referred to as Compositional Spectra (CS) analysis, is based on scoring the occurrences of fixed-length oligonucleotides (k-mers) in the target DNA sequence. CS analysis allows generating species- or region-specific characteristics of the genome, regardless of their length and the presence of coding DNA. In this study, we consider the heterogeneity of vertebrate genomes as a joint effect of regional variation in sequence organization superimposed on the differences in nucleotide composition. We estimated compositional and organizational heterogeneity of genome and chromosome sequences separately and found that both heterogeneity types vary widely among genomes as well as among chromosomes in all investigated taxonomic groups. The high correspondence of heterogeneity scores obtained on three genome fractions, coding, repetitive, and the remaining part of the noncoding DNA (the genome dark matter--GDM) allows the assumption that CS-heterogeneity may have functional relevance to genome regulation. Of special interest for such interpretation is the fact that natural GDM sequences display the highest deviation from the corresponding reshuffled sequences.

Mechanisms generating long range correlation in nucleotide composition of the Borrelia Burgdorferi genome

NASA Astrophysics Data System (ADS)

Mackiewicz, P.; Gierlik, A.; Kowalczuk, M.; Szczepanik, D.; Dudek, M. R.; Cebrat, S.

1999-12-01

We have analysed protein coding and intergenic sequences in the Borrelia burgdorferi (the Lyme disease bacterium) genome using different kinds of DNA walks. Genes occupying the leading strand of DNA have significantly different nucleotide composition from genes occupying the lagging strand. Nucleotide compositional bias of the two DNA strands reflects the aminoacid composition of proteins. 96% of genes coding for ribosomal proteins lie on the leading DNA strand, which suggests that the positions of these as well as other genes are non-random. In the B. burgdorferi genome, the asymmetry in intergenic DNA sequences is lower than the asymmetry in the third positions in codons. All these characters of the B. burgdorferi genome suggest that both replication-associated mutational pressure and recombination mechanisms have established the specific structure of the genome and now any recombination leading to inversion of a gene in respect to the direction of replication is forbidden. This property of the genome allows us to assume that it is in a steady state, which enables us to fix some parameters for simulations of DNA evolution.

Composite material of DNA and cyclodextrin-immobilized poly(ethyleneimine): Accumulation of harmful compounds from multi-component solution.

PubMed

Yamada, Masanori; Hori, Minako; Tabuchi, Shinya

2010-08-01

Water-soluble beta-cyclodextrin-immobilized poly(ethyleneimine) (PEICD) was synthesized by the grafting of beta-cyclodextrin to the branched poly(ethyleneimine). In an aqueous solution, this PEICD polymer could encapsulate bisphenol A, known to be a harmful compound. Additionally, the stability constant of bisphenol A to the PEICD polymer was 1.1 x 10(4)M(-1). However, the water-solubility of PEICD has been making it difficult to utilize it as an environmental material. Therefore, we prepared the DNA-PEICD composite material by mixing the double-stranded DNA and PEICD. This DNA-PEICD composite material was extremely stable in water and possessed both properties of the intercalation into the double-stranded DNA and the encapsulation into the CD cavity. As a result, this material can accumulate various harmful compounds, such as dioxin- and polychlorobiphenyl (PCB)-derivatives and bisphenol A, from a multi-component solution. Therefore, the DNA-PEICD composite material may have the potential to be used as an environmental material. Copyright 2010 Elsevier B.V. All rights reserved.

Bioaerosol DNA Extraction Technique from Air Filters Collected from Marine and Freshwater Locations

NASA Astrophysics Data System (ADS)

Beckwith, M.; Crandall, S. G.; Barnes, A.; Paytan, A.

2015-12-01

Bioaerosols are composed of microorganisms suspended in air. Among these organisms include bacteria, fungi, virus, and protists. Microbes introduced into the atmosphere can drift, primarily by wind, into natural environments different from their point of origin. Although bioaerosols can impact atmospheric dynamics as well as the ecology and biogeochemistry of terrestrial systems, very little is known about the composition of bioaerosols collected from marine and freshwater environments. The first step to determine composition of airborne microbes is to successfully extract environmental DNA from air filters. We asked 1) can DNA be extracted from quartz (SiO2) air filters? and 2) how can we optimize the DNA yield for downstream metagenomic sequencing? Aerosol filters were collected and archived on a weekly basis from aquatic sites (USA, Bermuda, Israel) over the course of 10 years. We successfully extracted DNA from a subsample of ~ 20 filters. We modified a DNA extraction protocol (Qiagen) by adding a beadbeating step to mechanically shear cell walls in order to optimize our DNA product. We quantified our DNA yield using a spectrophotometer (Nanodrop 1000). Results indicate that DNA can indeed be extracted from quartz filters. The additional beadbeating step helped increase our yield - up to twice as much DNA product was obtained compared to when this step was omitted. Moreover, bioaerosol DNA content does vary across time. For instance, the DNA extracted from filters from Lake Tahoe, USA collected near the end of June decreased from 9.9 ng/μL in 2007 to 3.8 ng/μL in 2008. Further next-generation sequencing analysis of our extracted DNA will be performed to determine the composition of these microbes. We will also model the meteorological and chemical factors that are good predictors for microbial composition for our samples over time and space.

Amplicon-Based Sequencing of Soil Fungi from Wood Preservative Test Sites

PubMed Central

Kirker, Grant T.; Bishell, Amy B.; Jusino, Michelle A.; Palmer, Jonathan M.; Hickey, William J.; Lindner, Daniel L.

2017-01-01

Soil samples were collected from field sites in two AWPA (American Wood Protection Association) wood decay hazard zones in North America. Two field plots at each site were exposed to differing preservative chemistries via in-ground installations of treated wood stakes for approximately 50 years. The purpose of this study is to characterize soil fungal species and to determine if long term exposure to various wood preservatives impacts soil fungal community composition. Soil fungal communities were compared using amplicon-based DNA sequencing of the internal transcribed spacer 1 (ITS1) region of the rDNA array. Data show that soil fungal community composition differs significantly between the two sites and that long-term exposure to different preservative chemistries is correlated with different species composition of soil fungi. However, chemical analyses using ICP-OES found levels of select residual preservative actives (copper, chromium and arsenic) to be similar to naturally occurring levels in unexposed areas. A list of indicator species was compiled for each treatment-site combination; functional guild analyses indicate that long-term exposure to wood preservatives may have both detrimental and stimulatory effects on soil fungal species composition. Fungi with demonstrated capacity to degrade industrial pollutants were found to be highly correlated with areas that experienced long-term exposure to preservative testing. PMID:29093702

Identification of DNA-binding proteins by combining auto-cross covariance transformation and ensemble learning.

PubMed

Liu, Bin; Wang, Shanyi; Dong, Qiwen; Li, Shumin; Liu, Xuan

2016-04-20

DNA-binding proteins play a pivotal role in various intra- and extra-cellular activities ranging from DNA replication to gene expression control. With the rapid development of next generation of sequencing technique, the number of protein sequences is unprecedentedly increasing. Thus it is necessary to develop computational methods to identify the DNA-binding proteins only based on the protein sequence information. In this study, a novel method called iDNA-KACC is presented, which combines the Support Vector Machine (SVM) and the auto-cross covariance transformation. The protein sequences are first converted into profile-based protein representation, and then converted into a series of fixed-length vectors by the auto-cross covariance transformation with Kmer composition. The sequence order effect can be effectively captured by this scheme. These vectors are then fed into Support Vector Machine (SVM) to discriminate the DNA-binding proteins from the non DNA-binding ones. iDNA-KACC achieves an overall accuracy of 75.16% and Matthew correlation coefficient of 0.5 by a rigorous jackknife test. Its performance is further improved by employing an ensemble learning approach, and the improved predictor is called iDNA-KACC-EL. Experimental results on an independent dataset shows that iDNA-KACC-EL outperforms all the other state-of-the-art predictors, indicating that it would be a useful computational tool for DNA binding protein identification. .

A Well-Resolved Phylogeny of the Trees of Puerto Rico Based on DNA Barcode Sequence Data

PubMed Central

Muscarella, Robert; Uriarte, María; Erickson, David L.; Swenson, Nathan G.; Zimmerman, Jess K.; Kress, W. John

2014-01-01

Background The use of phylogenetic information in community ecology and conservation has grown in recent years. Two key issues for community phylogenetics studies, however, are (i) low terminal phylogenetic resolution and (ii) arbitrarily defined species pools. Methodology/principal findings We used three DNA barcodes (plastid DNA regions rbcL, matK, and trnH-psbA) to infer a phylogeny for 527 native and naturalized trees of Puerto Rico, representing the vast majority of the entire tree flora of the island (89%). We used a maximum likelihood (ML) approach with and without a constraint tree that enforced monophyly of recognized plant orders. Based on 50% consensus trees, the ML analyses improved phylogenetic resolution relative to a comparable phylogeny generated with Phylomatic (proportion of internal nodes resolved: constrained ML = 74%, unconstrained ML = 68%, Phylomatic = 52%). We quantified the phylogenetic composition of 15 protected forests in Puerto Rico using the constrained ML and Phylomatic phylogenies. We found some evidence that tree communities in areas of high water stress were relatively phylogenetically clustered. Reducing the scale at which the species pool was defined (from island to soil types) changed some of our results depending on which phylogeny (ML vs. Phylomatic) was used. Overall, the increased terminal resolution provided by the ML phylogeny revealed additional patterns that were not observed with a less-resolved phylogeny. Conclusions/significance With the DNA barcode phylogeny presented here (based on an island-wide species pool), we show that a more fully resolved phylogeny increases power to detect nonrandom patterns of community composition in several Puerto Rican tree communities. Especially if combined with additional information on species functional traits and geographic distributions, this phylogeny will (i) facilitate stronger inferences about the role of historical processes in governing the assembly and composition of Puerto Rican forests, (ii) provide insight into Caribbean biogeography, and (iii) aid in incorporating evolutionary history into conservation planning. PMID:25386879

A well-resolved phylogeny of the trees of Puerto Rico based on DNA barcode sequence data.

PubMed

Muscarella, Robert; Uriarte, María; Erickson, David L; Swenson, Nathan G; Zimmerman, Jess K; Kress, W John

2014-01-01

The use of phylogenetic information in community ecology and conservation has grown in recent years. Two key issues for community phylogenetics studies, however, are (i) low terminal phylogenetic resolution and (ii) arbitrarily defined species pools. We used three DNA barcodes (plastid DNA regions rbcL, matK, and trnH-psbA) to infer a phylogeny for 527 native and naturalized trees of Puerto Rico, representing the vast majority of the entire tree flora of the island (89%). We used a maximum likelihood (ML) approach with and without a constraint tree that enforced monophyly of recognized plant orders. Based on 50% consensus trees, the ML analyses improved phylogenetic resolution relative to a comparable phylogeny generated with Phylomatic (proportion of internal nodes resolved: constrained ML = 74%, unconstrained ML = 68%, Phylomatic = 52%). We quantified the phylogenetic composition of 15 protected forests in Puerto Rico using the constrained ML and Phylomatic phylogenies. We found some evidence that tree communities in areas of high water stress were relatively phylogenetically clustered. Reducing the scale at which the species pool was defined (from island to soil types) changed some of our results depending on which phylogeny (ML vs. Phylomatic) was used. Overall, the increased terminal resolution provided by the ML phylogeny revealed additional patterns that were not observed with a less-resolved phylogeny. With the DNA barcode phylogeny presented here (based on an island-wide species pool), we show that a more fully resolved phylogeny increases power to detect nonrandom patterns of community composition in several Puerto Rican tree communities. Especially if combined with additional information on species functional traits and geographic distributions, this phylogeny will (i) facilitate stronger inferences about the role of historical processes in governing the assembly and composition of Puerto Rican forests, (ii) provide insight into Caribbean biogeography, and (iii) aid in incorporating evolutionary history into conservation planning.

Human Papillomavirus-associated oropharyngeal cancer: an observational study of diagnosis, prevalence and prognosis in a UK population

PubMed Central

2013-01-01

Background The incidence of Human Papillomavirus (HPV) associated oropharyngeal cancer (OPC) is increasing. HPV-associated OPC appear to have better prognosis than HPV-negative OPC. The aim of this study was to robustly determine the prevalence of HPV-positive OPC in an unselected UK population and correlate HPV positivity with clinical outcome. Methods HPV testing by GP5+/6+ PCR, In Situ Hybridisation (ISH) and p16 immunohistochemistry (IHC) was performed on 138 OPCs diagnosed in South Wales (UK) between 2001–06. Kaplan-Meier analysis was used to correlate HPV status with clinical outcome. Results Using a composite definition of HPV positivity (HPV DNA and p16 overexpression), HPV was detected in 46/83 (55%) samples where DNA quality was assured. Five year overall survival was 75.4% (95% CI: 65.2 to 85.5) in HPV-positives vs 25.3% (95% CI: 14.2 to 36.4) in HPV negatives, corresponding to a 78% reduction in death rate (HR 0.22, p < 0.001). HPV-positives had less locoregional recurrence but second HPV-positive Head and Neck primaries occurred. Poor quality DNA in fixed pathological specimens reduced both HPV prevalence estimates and the prognostic utility of DNA-based HPV testing methods. As a single marker, p16 was least affected by sample quality and correlated well with prognosis, although was not sufficient on its own for accurate HPV prevalence reporting. Conclusions This study highlights the significant burden of OPC associated with HPV infection. HPV positive cases are clinically distinct from other OPC, and are associated with significantly better clinical outcomes. A composite definition of HPV positivity should be used for accurate prevalence reporting and up-front DNA quality assessment is recommended for any DNA-based HPV detection strategy. PMID:23634887

Submolecular Structure and Orientation of Oligonucleotide Duplexes Tethered to Gold Electrodes Probed by Infrared Reflection Absorption Spectroscopy: Effect of the Electrode Potentials.

PubMed

Kékedy-Nagy, László; Ferapontova, Elena E; Brand, Izabella

2017-02-23

Unique electronic and ligand recognition properties of the DNA double helix provide basis for DNA applications in biomolecular electronic and biosensor devices. However, the relation between the structure of DNA at electrified interfaces and its electronic properties is still not well understood. Here, potential-driven changes in the submolecular structure of DNA double helices composed of either adenine-thymine (dAdT) 25 or cytosine-guanine (dGdC) 20 base pairs tethered to the gold electrodes are for the first time analyzed by in situ polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) performed under the electrochemical control. It is shown that the conformation of the DNA duplexes tethered to gold electrodes via the C 6 alkanethiol linker strongly depends on the nucleic acid sequence composition. The tilt of purine and pyrimidine rings of the complementary base pairs (dAdT and dGdC) depends on the potential applied to the electrode. By contrast, neither the conformation nor orientation of the ionic in character phosphate-sugar backbone is affected by the electrode potentials. At potentials more positive than the potential of zero charge (pzc), a gradual tilting of the double helix is observed. In this tilted orientation, the planes of the complementary purine and pyrimidine rings lie ideally parallel to each other. These potentials do not affect the integral stability of the DNA double helix at the charged interface. At potentials more negative than the pzc, DNA helices adopt a vertical to the gold surface orientation. Tilt of the purine and pyrimidine rings depends on the composition of the double helix. In monolayers composed of (dAdT) 25 molecules the rings of the complementary base pairs lie parallel to each other. By contrast, the tilt of purine and pyrimidine rings in (dGdC) 20 helices depends on the potential applied to the electrode. Such potential-induced mobility of the complementary base pairs can destabilize the helix structure at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides.

mRNA-Based Parallel Detection of Active Methanotroph Populations by Use of a Diagnostic Microarray

PubMed Central

Bodrossy, Levente; Stralis-Pavese, Nancy; Konrad-Köszler, Marianne; Weilharter, Alexandra; Reichenauer, Thomas G.; Schöfer, David; Sessitsch, Angela

2006-01-01

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities. PMID:16461725

Analysis of conserved noncoding DNA in Drosophila reveals similar constraints in intergenic and intronic sequences.

PubMed

Bergman, C M; Kreitman, M

2001-08-01

Comparative genomic approaches to gene and cis-regulatory prediction are based on the principle that differential DNA sequence conservation reflects variation in functional constraint. Using this principle, we analyze noncoding sequence conservation in Drosophila for 40 loci with known or suspected cis-regulatory function encompassing >100 kb of DNA. We estimate the fraction of noncoding DNA conserved in both intergenic and intronic regions and describe the length distribution of ungapped conserved noncoding blocks. On average, 22%-26% of noncoding sequences surveyed are conserved in Drosophila, with median block length approximately 19 bp. We show that point substitution in conserved noncoding blocks exhibits transition bias as well as lineage effects in base composition, and occurs more than an order of magnitude more frequently than insertion/deletion (indel) substitution. Overall, patterns of noncoding DNA structure and evolution differ remarkably little between intergenic and intronic conserved blocks, suggesting that the effects of transcription per se contribute minimally to the constraints operating on these sequences. The results of this study have implications for the development of alignment and prediction algorithms specific to noncoding DNA, as well as for models of cis-regulatory DNA sequence evolution.

Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938

PubMed Central

Grande, Rossella; Celia, Christian; Mincione, Gabriella; Stringaro, Annarita; Di Marzio, Luisa; Colone, Marisa; Di Marcantonio, Maria C.; Savino, Luca; Puca, Valentina; Santoliquido, Roberto; Locatelli, Marcello; Muraro, Raffaella; Hall-Stoodley, Luanne; Stoodley, Paul

2017-01-01

Membrane vesicles (MVs) are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs), and planktonic (pMVs) phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS). Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM) analysis. eDNA (extracellular DNA) was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA). DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems. PMID:28659878

M-DNA is stabilised in G•C tracts or by incorporation of 5-fluorouracil

PubMed Central

Wood, David O.; Dinsmore, Michael J.; Bare, Grant A.; Lee, Jeremy S.

2002-01-01

M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of ∼8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)•poly(dC) and poly(dA)•poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)•(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage λ DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from Tm measurements. It is evident that the lower the pKa of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion. PMID:12000844

Diet Quality Associated with Total Sodium Intake among US Adults Aged ≥18 Years-National Health and Nutrition Examination Survey, 2009-2012.

PubMed

Mercado, Carla I; Cogswell, Mary E; Perrine, Cria G; Gillespie, Cathleen

2017-10-25

Diet quality or macronutrient composition of total daily sodium intake (dNa) <2300 mg/day in the United States (US) is unknown. Using data from 2011-2014 NHANES (National Health and Nutrition Examination Survey), we examined 24-h dietary recalls ( n = 10,142) from adults aged ≥18 years and investigated how diet composition and quality are associated with dNa. Diet quality was assessed using components of macronutrients and Healthy Eating Index 2010 (HEI-2010). Associations were tested using linear regression analysis adjusted for total energy (kcal), age, gender, and race/ethnicity. One-day dNa in the lower quartiles were more likely reported among women, older adults (≥65 years old), and lower quartiles of total energy (kcal) ( p -values ≤ 0.001). With increasing dNa, there was an increase in the mean protein, fiber, and total fat densities, while total carbohydrates densities decreased. As dNa increased, meat protein, refined grains, dairy, and total vegetables, greens and beans densities increased; while total fruit and whole fruit densities decreased. Modified HEI-2010 total score (total score without sodium component) increased as dNa increased (adjusted coefficient: 0.11, 95% confidence interval = 0.07, 0.15). Although diet quality, based on modified HEI-2010 total score, increased on days with greater dNa, there is much room for improvement with mean diet quality of about half of the optimal level.

Interlaboratory Comparison of Methods Determining the Botanical Composition of Animal Feed.

PubMed

Braglia, Luca; Morello, Laura; Gavazzi, Floriana; Gianì, Silvia; Mastromauro, Francesco; Breviario, Diego; Cardoso, Hélia Guerra; Valadas, Vera; Campos, Maria Doroteia

2018-01-01

A consortium of European enterprises and research institutions has been engaged in the Feed-Code Project with the aim of addressing the requirements stated in European Union Regulation No. 767/2009, concerning market placement and use of feed of known and ascertained botanical composition. Accordingly, an interlaboratory trial was set up to compare the performance of different assays based either on optical microscope or DNA analysis for the qualitative and quantitative identification of the composition of compound animal feeds. A tubulin-based polymorphism method, on which the Feed-Code platform was developed, provided the most accurate results. The present study highlights the need for the performance of ring trials for the determination of the botanical composition of animal feeds and raises an alarm on the actual status of analytical inaccuracy.

Two sides of the same coin: TFIIH complexes in transcription and DNA repair.

PubMed

Zhovmer, Alexander; Oksenych, Valentyn; Coin, Frédéric

2010-04-13

TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-04-01

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

Tetrodotoxin detection and species identification of pufferfish in retail roasted fish fillet by DNA barcoding in China.

PubMed

Li, Nan; Shen, Qing; Wang, Jiahui; Han, Chunhui; Ji, Rong; Li, Fengqin; Jiang, Tao

2015-01-01

This study identifies the pufferfish species and detects tetrodotoxin (TTX) in roasted fish fillet samples collected in Beijing, Qingdao and Xiamen, China. The cytochrome c oxidase I (COI) gene was used as the target gene for identification of the pufferfish species in the samples. Enzyme-linked immunosorbent assay (ELISA) screened the TTX levels in samples that had been detected as containing pufferfish by DNA barcode. A total of 125 samples were identified by DNA barcodes; 32 (26%) samples contained pufferfish composition and, among them, 26 (81%) were the highly toxic species Lagocephalus lunaris. All 32 samples containing the pufferfish composition were positive for TTX with levels ranging from 100 to 63,800 ng g(-1). Most of the 32 samples contained the highly toxic L. lunaris. Based on the results, we suggest that the monitoring of roasted fish fillet should be strengthened and the processing procedures should be standardised to minimise TTX poisoning caused by pufferfish.

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways

PubMed Central

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis

PubMed Central

Ng, Chun Kiat; Miller, Dana; Cao, Bin

2015-01-01

Introduction As bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended. Objectives This study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1) enhanced high temperature sonication during DNA extraction; 2) effect of sampling duration on DNA recoverability; and 3) an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR). Results and Findings The findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%. PMID:26619279

Exploring the Impacts of Anthropogenic Disturbance on Seawater and Sediment Microbial Communities in Korean Coastal Waters Using Metagenomics Analysis

PubMed Central

Won, Nam-Il; Kim, Ki-Hwan; Kang, Ji Hyoun; Park, Sang Rul; Lee, Hyuk Je

2017-01-01

The coastal ecosystems are considered as one of the most dynamic and vulnerable environments under various anthropogenic developments and the effects of climate change. Variations in the composition and diversity of microbial communities may be a good indicator for determining whether the marine ecosystems are affected by complex forcing stressors. DNA sequence-based metagenomics has recently emerged as a promising tool for analyzing the structure and diversity of microbial communities based on environmental DNA (eDNA). However, few studies have so far been performed using this approach to assess the impacts of human activities on the microbial communities in marine systems. In this study, using metagenomic DNA sequencing (16S ribosomal RNA gene), we analyzed and compared seawater and sediment communities between sand mining and control (natural) sites in southern coastal waters of Korea to assess whether anthropogenic activities have significantly affected the microbial communities. The sand mining sites harbored considerably lower levels of microbial diversities in the surface seawater community during spring compared with control sites. Moreover, the sand mining areas had distinct microbial taxonomic group compositions, particularly during spring season. The microbial groups detected solely in the sediment load/dredging areas (e.g., Marinobacter, Alcanivorax, Novosphingobium) are known to be involved in degradation of toxic chemicals such as hydrocarbon, oil, and aromatic compounds, and they also contain potential pathogens. This study highlights the versatility of metagenomics in monitoring and diagnosing the impacts of human disturbance on the environmental health of marine ecosystems from eDNA. PMID:28134828

Exploring the Impacts of Anthropogenic Disturbance on Seawater and Sediment Microbial Communities in Korean Coastal Waters Using Metagenomics Analysis.

PubMed

Won, Nam-Il; Kim, Ki-Hwan; Kang, Ji Hyoun; Park, Sang Rul; Lee, Hyuk Je

2017-01-27

The coastal ecosystems are considered as one of the most dynamic and vulnerable environments under various anthropogenic developments and the effects of climate change. Variations in the composition and diversity of microbial communities may be a good indicator for determining whether the marine ecosystems are affected by complex forcing stressors. DNA sequence-based metagenomics has recently emerged as a promising tool for analyzing the structure and diversity of microbial communities based on environmental DNA (eDNA). However, few studies have so far been performed using this approach to assess the impacts of human activities on the microbial communities in marine systems. In this study, using metagenomic DNA sequencing (16S ribosomal RNA gene), we analyzed and compared seawater and sediment communities between sand mining and control (natural) sites in southern coastal waters of Korea to assess whether anthropogenic activities have significantly affected the microbial communities. The sand mining sites harbored considerably lower levels of microbial diversities in the surface seawater community during spring compared with control sites. Moreover, the sand mining areas had distinct microbial taxonomic group compositions, particularly during spring season. The microbial groups detected solely in the sediment load/dredging areas (e.g., Marinobacter, Alcanivorax, Novosphingobium) are known to be involved in degradation of toxic chemicals such as hydrocarbon, oil, and aromatic compounds, and they also contain potential pathogens. This study highlights the versatility of metagenomics in monitoring and diagnosing the impacts of human disturbance on the environmental health of marine ecosystems from eDNA.

Application of differential scanning calorimetry to measure the differential binding of ions, water and protons in the unfolding of DNA molecules.

PubMed

Olsen, Chris M; Shikiya, Ronald; Ganugula, Rajkumar; Reiling-Steffensmeier, Calliste; Khutsishvili, Irine; Johnson, Sarah E; Marky, Luis A

2016-05-01

The overall stability of DNA molecules globally depends on base-pair stacking, base-pairing, polyelectrolyte effect and hydration contributions. In order to understand how they carry out their biological roles, it is essential to have a complete physical description of how the folding of nucleic acids takes place, including their ion and water binding. To investigate the role of ions, water and protons in the stability and melting behavior of DNA structures, we report here an experimental approach i.e., mainly differential scanning calorimetry (DSC), to determine linking numbers: the differential binding of ions (Δnion), water (ΔnW) and protons (ΔnH(+)) in the helix-coil transition of DNA molecules. We use DSC and temperature-dependent UV spectroscopic techniques to measure the differential binding of ions, water, and protons for the unfolding of a variety of DNA molecules: salmon testes DNA (ST-DNA), one dodecamer, one undecamer and one decamer duplexes, nine hairpin loops, and two triplexes. These methods can be applied to any conformational transition of a biomolecule. We determined complete thermodynamic profiles, including all three linking numbers, for the unfolding of each molecule. The favorable folding of a DNA helix results from a favorable enthalpy-unfavorable entropy compensation. DSC thermograms and UV melts as a function of salt, osmolyte and proton concentrations yielded releases of ions and water. Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of both counterions and water molecules. In addition, the triplex with C(+)GC base triplets yielded an uptake of protons. Furthermore, the folding of a DNA duplex is accompanied by a lower uptake of ions and a similar uptake of four water molecules as the DNA helix gets shorter. In addition, the oligomer duplexes and hairpin thermodynamic data suggest ion and water binding depends on the DNA sequence rather than DNA composition. Copyright © 2015. Published by Elsevier B.V.

Detection of Different DNA Animal Species in Commercial Candy Products.

PubMed

Muñoz-Colmenero, Marta; Martínez, Jose Luis; Roca, Agustín; Garcia-Vazquez, Eva

2016-03-01

Candy products are consumed all across the world, but there is not much information about their composition. In this study we have used a DNA-based approach for determining the animal species occurring in 40 commercial candies of different types. We extracted DNA and performed PCR amplification, cloning and sequencing for obtaining species-informative DNA sequences. Eight species were identified including fish (hake and anchovy) in 22% of the products analyzed. Bovine and porcine were the most abundant appearing in 27 samples each one. Most products contained a mixture of species. Marshmallows (7), jelly-types, and gummies (20) contained a significantly higher number of species than hard candies (9). We demonstrated the presence of DNA animal species in candy product which allow consumers to make choices and prevent allergic reaction. © 2016 Institute of Food Technologists®

Characterization and Modulation of Proteins Involved in Sulfur Mustard Vesication

DTIC Science & Technology

2000-06-01

PARP staining was present throughout the nucleus, the DBD showed a more localized punctate pattern in the region of the nucleolus and throughout the...34 oligonucleotide is synthesized that is identical in base composition to the antisense, but had a randomly generated sequence. This is an important control...reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by

Toward DNA-based facial composites: preliminary results and validation.

PubMed

Claes, Peter; Hill, Harold; Shriver, Mark D

2014-11-01

The potential of constructing useful DNA-based facial composites is forensically of great interest. Given the significant identity information coded in the human face these predictions could help investigations out of an impasse. Although, there is substantial evidence that much of the total variation in facial features is genetically mediated, the discovery of which genes and gene variants underlie normal facial variation has been hampered primarily by the multipartite nature of facial variation. Traditionally, such physical complexity is simplified by simple scalar measurements defined a priori, such as nose or mouth width or alternatively using dimensionality reduction techniques such as principal component analysis where each principal coordinate is then treated as a scalar trait. However, as shown in previous and related work, a more impartial and systematic approach to modeling facial morphology is available and can facilitate both the gene discovery steps, as we recently showed, and DNA-based facial composite construction, as we show here. We first use genomic ancestry and sex to create a base-face, which is simply an average sex and ancestry matched face. Subsequently, the effects of 24 individual SNPs that have been shown to have significant effects on facial variation are overlaid on the base-face forming the predicted-face in a process akin to a photomontage or image blending. We next evaluate the accuracy of predicted faces using cross-validation. Physical accuracy of the facial predictions either locally in particular parts of the face or in terms of overall similarity is mainly determined by sex and genomic ancestry. The SNP-effects maintain the physical accuracy while significantly increasing the distinctiveness of the facial predictions, which would be expected to reduce false positives in perceptual identification tasks. To the best of our knowledge this is the first effort at generating facial composites from DNA and the results are preliminary but certainly promising, especially considering the limited amount of genetic information about the face contained in these 24 SNPs. This approach can incorporate additional SNPs as these are discovered and their effects documented. In this context we discuss three main avenues of research: expanding our knowledge of the genetic architecture of facial morphology, improving the predictive modeling of facial morphology by exploring and incorporating alternative prediction models, and increasing the value of the results through the weighted encoding of physical measurements in terms of human perception of faces. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Plant genotyping using fluorescently tagged inter-simple sequence repeats (ISSRs): basic principles and methodology.

PubMed

Prince, Linda M

2015-01-01

Inter-simple sequence repeat PCR (ISSR-PCR) is a fast, inexpensive genotyping technique based on length variation in the regions between microsatellites. The method requires no species-specific prior knowledge of microsatellite location or composition. Very small amounts of DNA are required, making this method ideal for organisms of conservation concern, or where the quantity of DNA is extremely limited due to organism size. ISSR-PCR can be highly reproducible but requires careful attention to detail. Optimization of DNA extraction, fragment amplification, and normalization of fragment peak heights during fluorescent detection are critical steps to minimizing the downstream time spent verifying and scoring the data.

A comparison of sedimentary DNA and pollen from lake sediments in recording vegetation composition at the Siberian treeline.

PubMed

Niemeyer, Bastian; Epp, Laura S; Stoof-Leichsenring, Kathleen R; Pestryakova, Luidmila A; Herzschuh, Ulrike

2017-11-01

Reliable information on past and present vegetation is important to project future changes, especially for rapidly transitioning areas such as the boreal treeline. To study past vegetation, pollen analysis is common, while current vegetation is usually assessed by field surveys. Application of detailed sedimentary DNA (sedDNA) records has the potential to enhance our understanding of vegetation changes, but studies systematically investigating the power of this proxy are rare to date. This study compares sedDNA metabarcoding and pollen records from surface sediments of 31 lakes along a north-south gradient of increasing forest cover in northern Siberia (Taymyr peninsula) with data from field surveys in the surroundings of the lakes. sedDNA metabarcoding recorded 114 plant taxa, about half of them to species level, while pollen analyses identified 43 taxa, both exceeding the 31 taxa found by vegetation field surveys. Increasing Larix percentages from north to south were consistently recorded by all three methods and principal component analyses based on percentage data of vegetation surveys and DNA sequences separated tundra from forested sites. Comparisons of the ordinations using procrustes and protest analyses show a significant fit among all compared pairs of records. Despite similarities of sedDNA and pollen records, certain idiosyncrasies, such as high percentages of Alnus and Betula in all pollen and high percentages of Salix in all sedDNA spectra, are observable. Our results from the tundra to single-tree tundra transition zone show that sedDNA analyses perform better than pollen in recording site-specific richness (i.e., presence/absence of taxa in the vicinity of the lake) and perform as well as pollen in tracing vegetation composition. © 2017 John Wiley & Sons Ltd.

Factors That Affect Large Subunit Ribosomal DNA Amplicon Sequencing Studies of Fungal Communities: Classification Method, Primer Choice, and Error

PubMed Central

Porter, Teresita M.; Golding, G. Brian

2012-01-01

Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve Bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50–100 bp sequences. Increasing simulated sequence error reduced classification accuracy. Community shifts were detected due to sequence error and primer selection even though there was no change in the underlying community composition. Short read datasets from individual primers, as well as pooled datasets, appear to only approximate the true community composition. We hope this work informs investigators of some of the factors that affect the quality and interpretation of their environmental gene surveys. PMID:22558215

Cryptic biodiversity in streams: a comparison of macroinvertebrate communities based on morphological and DNA barcode identifications

EPA Science Inventory

Species-level identifications are difficult or impossible for many larval aquatic macroinvertebrates. We described the taxonomic composition of macroinvertebrate communities from 5 coastal streams in 3 neighboring catchments in southern California. We compared taxonomic identific...

7 CFR 91.5 - Where services are offered.

Code of Federal Regulations, 2011 CFR

2011-01-01

...) Science and Technology Programs National Science Laboratory. A variety of proximate for composition, chemical, physical, microbiological and biomolecular (DNA-based) tests and laboratory analyses performed on..., honey, meat and meat products, fiber products and processed foods are performed at the Science and...

7 CFR 91.5 - Where services are offered.

Code of Federal Regulations, 2013 CFR

2013-01-01

...) Science and Technology Programs National Science Laboratory. A variety of proximate for composition, chemical, physical, microbiological and biomolecular (DNA-based) tests and laboratory analyses performed on..., honey, meat and meat products, fiber products and processed foods are performed at the Science and...

7 CFR 91.5 - Where services are offered.

Code of Federal Regulations, 2014 CFR

2014-01-01

...) Science and Technology Programs National Science Laboratory. A variety of proximate for composition, chemical, physical, microbiological and biomolecular (DNA-based) tests and laboratory analyses performed on..., honey, meat and meat products, fiber products and processed foods are performed at the Science and...

7 CFR 91.5 - Where services are offered.

Code of Federal Regulations, 2012 CFR

2012-01-01

...) Science and Technology Programs National Science Laboratory. A variety of proximate for composition, chemical, physical, microbiological and biomolecular (DNA-based) tests and laboratory analyses performed on..., honey, meat and meat products, fiber products and processed foods are performed at the Science and...

Assessing macroinvertebrate biodiversity in freshwater ecosystems: advances and challenges in DNA-based approaches

EPA Science Inventory

Assessing the biodiversity of macroinvertebrate faunas in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify wate...

Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

PubMed Central

Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T.; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

2013-01-01

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries. PMID:24205269

Exploring the effect of asymmetric mitochondrial DNA introgression on estimating niche divergence in morphologically cryptic species.

PubMed

Wielstra, Ben; Arntzen, Jan W

2014-01-01

If potential morphologically cryptic species, identified based on differentiated mitochondrial DNA, express ecological divergence, this increases support for their treatment as distinct species. However, mitochondrial DNA introgression hampers the correct estimation of ecological divergence. We test the hypothesis that estimated niche divergence differs when considering nuclear DNA composition or mitochondrial DNA type as representing the true species range. We use empirical data of two crested newt species (Amphibia: Triturus) which possess introgressed mitochondrial DNA from a third species in part of their ranges. We analyze the data in environmental space by determining Fisher distances in a principal component analysis and in geographical space by determining geographical overlap of species distribution models. We find that under mtDNA guidance in one of the two study cases niche divergence is overestimated, whereas in the other it is underestimated. In the light of our results we discuss the role of estimated niche divergence in species delineation.

Study on a Luminol-based Electrochemiluminescent Sensor for Label-Free DNA Sensing

PubMed Central

Chu, Hai-Hong; Yan, Ji-Lin; Tu, Yi-Feng

2010-01-01

Automatic, inexpensive, simple and sensitive methods for DNA sensing and quantification are highly desirable for biomedical research. The rapid development of both the fundamentals and applications of electrochemiluminescence (ECL) over the past years has demonstrated its potential for analytical and bio-analytical chemistry. This paper reports the quenching effect of DNA on the ECL of luminol and the further development of a DNA sensing device. With the pre-functionalization by a composite of carbon nano-tubes (CNTs) and Au nanoparticles (AuNPs), the sensor provides a novel and valuable label-free approach for DNA sensing. Here the ECL intensity was remarkably decreased when more than 1.0 × 10−12 molar of DNA were adsorbed on the sensor. Linearity of the DNA amount with the reciprocal of ECL intensity was observed. A saturated sensor caused a 92.8% quenching effect. The research also proposes the mechanism for the quenching effect which could be attributed to the interaction between luminol and DNA and the elimination of reactive oxygen species (ROSs) by DNA. PMID:22163421

Identifying active foraminifera in the Sea of Japan using metatranscriptomic approach

NASA Astrophysics Data System (ADS)

Lejzerowicz, Franck; Voltsky, Ivan; Pawlowski, Jan

2013-02-01

Metagenetics represents an efficient and rapid tool to describe environmental diversity patterns of microbial eukaryotes based on ribosomal DNA sequences. However, the results of metagenetic studies are often biased by the presence of extracellular DNA molecules that are persistent in the environment, especially in deep-sea sediment. As an alternative, short-lived RNA molecules constitute a good proxy for the detection of active species. Here, we used a metatranscriptomic approach based on RNA-derived (cDNA) sequences to study the diversity of the deep-sea benthic foraminifera and compared it to the metagenetic approach. We analyzed 257 ribosomal DNA and cDNA sequences obtained from seven sediments samples collected in the Sea of Japan at depths ranging from 486 to 3665 m. The DNA and RNA-based approaches gave a similar view of the taxonomic composition of foraminiferal assemblage, but differed in some important points. First, the cDNA dataset was dominated by sequences of rotaliids and robertiniids, suggesting that these calcareous species, some of which have been observed in Rose Bengal stained samples, are the most active component of foraminiferal community. Second, the richness of monothalamous (single-chambered) foraminifera was particularly high in DNA extracts from the deepest samples, confirming that this group of foraminifera is abundant but not necessarily very active in the deep-sea sediments. Finally, the high divergence of undetermined sequences in cDNA dataset indicate the limits of our database and lack of knowledge about some active but possibly rare species. Our study demonstrates the capability of the metatranscriptomic approach to detect active foraminiferal species and prompt its use in future high-throughput sequencing-based environmental surveys.

a bare Nanocapillary for DNA Separation and Genotyping analysis in Gel-Free solutions without application of external electric field

PubMed Central

Wang, Xiayan; Wang, Shili; Veerappan, Vijaykumar; Byun, Chang Kyu; Nguyen, Han; Gendhar, Brina; Allen, Randy D.; Liu, Shaorong

2009-01-01

In this work, we demonstrate DNA separation and genotyping analysis in gel-free solutions using a nanocapillary under pressure-driven conditions without application of an external electric field. The nanocapillary is a ~50-cm-long and 500-nm-radius bare fused silica capillary. After a DNA sample is injected, the analytes are eluted out in a chromatographic separation format. The elution order of DNA molecules follows strictly with their sizes, with the longer DNA being eluted out faster than the shorter ones. High resolutions are obtained for both short (a few bases) and long (tens of thousands of base pairs) DNA fragments. Effects of key experimental parameters, such as eluent composition and elution pressure, on separation efficiency and resolution are investigated. We also apply this technique for DNA separations of real-world genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified DNA fragments are well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. Since the capillaries used to conduct the separations are uncoated, column lifetime is virtually unlimited. The only material that is consumed in these assays is the eluent, and hence the operation cost is low. PMID:18500828

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter.

PubMed

Isalan, M; Klug, A; Choo, Y

2001-07-01

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

Micro flow-through PCR in a PMMA chip fabricated by KrF excimer laser.

PubMed

Yao, Liying; Liu, Baoan; Chen, Tao; Liu, Shibing; Zuo, Tiechuan

2005-09-01

As the third PCR technology, micro flow-through PCR chip can amplify DNA specifically in an exponential fashion in vitro. Nowadays many academies in the world have successfully amplified DNA using their own-made flow-through PCR chip. In this paper, the ablation principle of PMMA at 248 nm excimer laser was studied, then a PMMA based flow-through PCR chip with 20 cycles was fabricated by excimer laser at 19 kv and 18 mm/min. The chip was bonded together with another cover chip at 105( composite function)C, 160 N and 20 minutes. In the end, it was integrated with electrical thermal thin films and Pt 100 temperature sensors. The temperature controllers was built standard PID digital temperature controller, the temperature control precision was +/- 0.2( composite function)C. The temperature grads between the three temperature zones were 16.5 and 22.2( composite function)C respectively, the gaps between the temperature zones could realize heat insulation.

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

PubMed

Adelmant, Guillaume; Calkins, Anne S; Garg, Brijesh K; Card, Joseph D; Askenazi, Manor; Miron, Alex; Sobhian, Bijan; Zhang, Yi; Nakatani, Yoshihiro; Silver, Pamela A; Iglehart, J Dirk; Marto, Jarrod A; Lazaro, Jean-Bernard

2012-08-01

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

Nanoparticle-DNA-polymer composites for hepatocellular carcinoma cell labeling, sensing, and magnetic resonance imaging.

PubMed

Leung, Ken Cham-Fai; Lee, Siu-Fung; Wong, Chi-Hin; Chak, Chun-Pong; Lai, Josie M Y; Zhu, Xiao-Ming; Wang, Yi-Xiang J; Sham, Kathy W Y; Cheng, Christopher H K

2013-12-15

This paper describes comparative studies and protocols in (1) self-assembling of ultrasmall superparamagnetic iron oxide nanoparticle (NP), circular plasmid DNA, and branched polyethylenimine (PEI) composites; (2) magnetofection; (3) gene delivery, (4) magnetic resonance imaging (MRI), and (5) cytotoxicity of the composites toward hepatocellular carcinoma HepG2 cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Heterozygote PCR product melting curve prediction.

PubMed

Dwight, Zachary L; Palais, Robert; Kent, Jana; Wittwer, Carl T

2014-03-01

Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote melting curves, 52 experimental curves were compared with brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 ± 6.7% to the composite melting curve, varying from 23%-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76%-96% of normal (depending on SNV class) and averaged 88 ± 16.4%. Based on uMelt (www.dna.utah.edu/umelt/umelt.html) with an expanded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at http://www.dna.utah.edu/hets/. © 2013 WILEY PERIODICALS, INC.

Microbial composition during Chinese soy sauce koji-making based on culture dependent and independent methods.

PubMed

Yan, Yin-zhuo; Qian, Yu-lin; Ji, Feng-di; Chen, Jing-yu; Han, Bei-zhong

2013-05-01

Koji-making is a key process for production of high quality soy sauce. The microbial composition during koji-making was investigated by culture-dependent and culture-independent methods to determine predominant bacterial and fungal populations. The culture-dependent methods used were direct culture and colony morphology observation, and PCR amplification of 16S/26S rDNA fragments followed by sequencing analysis. The culture-independent method was based on the analysis of 16S/26S rDNA clone libraries. There were differences between the results obtained by different methods. However, sufficient overlap existed between the different methods to identify potentially significant microbial groups. 16 and 20 different bacterial species were identified using culture-dependent and culture-independent methods, respectively. 7 species could be identified by both methods. The most predominant bacterial genera were Weissella and Staphylococcus. Both 6 different fungal species were identified using culture-dependent and culture-independent methods, respectively. Only 3 species could be identified by both sets of methods. The most predominant fungi were Aspergillus and Candida species. This work illustrated the importance of a comprehensive polyphasic approach in the analysis of microbial composition during soy sauce koji-making, the knowledge of which will enable further optimization of microbial composition and quality control of koji to upgrade Chinese traditional soy sauce product. Copyright © 2013 Elsevier Ltd. All rights reserved.

Community Composition and Transcriptional Activity of Ammonia-Oxidizing Prokaryotes of Seagrass Thalassia hemprichii in Coral Reef Ecosystems.

PubMed

Ling, Juan; Lin, Xiancheng; Zhang, Yanying; Zhou, Weiguo; Yang, Qingsong; Lin, Liyun; Zeng, Siquan; Zhang, Ying; Wang, Cong; Ahmad, Manzoor; Long, Lijuan; Dong, Junde

2018-01-01

Seagrasses in coral reef ecosystems play important ecological roles by enhancing coral reef resilience under ocean acidification. However, seagrass primary productivity is typically constrained by limited nitrogen availability. Ammonia oxidation is an important process conducted by ammonia-oxidizing archaea (AOA) and bacteria (AOB), yet little information is available concerning the community structure and potential activity of seagrass AOA and AOB. Therefore, this study investigated the variations in the abundance, diversity and transcriptional activity of AOA and AOB at the DNA and transcript level from four sample types: the leaf, root, rhizosphere sediment and bulk sediment of seagrass Thalassia hemprichii in three coral reef ecosystems. DNA and complementary DNA (cDNA) were used to prepare clone libraries and DNA and cDNA quantitative PCR ( q PCR) assays, targeting the ammonia monooxygenase-subunit ( amo A) genes as biomarkers. Our results indicated that the closest relatives of the obtained archaeal and bacterial amo A gene sequences recovered from DNA and cDNA libraries mainly originated from the marine environment. Moreover, all the obtained AOB sequences belong to the Nitrosomonadales cluster. Nearly all the AOA communities exhibited higher diversity than the AOB communities at the DNA level, but the q PCR data demonstrated that the abundances of AOB communities were higher than that of AOA communities based on both DNA and RNA transcripts. Collectively, most of the samples shared greater community composition similarity with samples from the same location rather than sample type. Furthermore, the abundance of archaeal amo A gene in rhizosphere sediments showed significant relationships with the ammonium concentration of sediments and the nitrogen content of plant tissue (leaf and root) at the DNA level ( P < 0.05). Conversely, no such relationships were found for the AOB communities. This work provides new insight into the nitrogen cycle, particularly nitrification of seagrass meadows in coral reef ecosystems.

Estimation of the Relative Abundance of Different Bacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

PubMed Central

Wood, Jacqueline; Scott, Karen P.; Avguštin, Gorazd; Newbold, C. James; Flint, Harry J.

1998-01-01

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples. PMID:9758785

In Vitro Cellular Gene Delivery Employing a Novel Composite Material of Single-Walled Carbon Nanotubes Associated With Designed Peptides With Pegylation.

PubMed

Ohta, Takahisa; Hashida, Yasuhiko; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

2017-03-01

Single-walled carbon nanotubes (SWCNTs) attract great interest in biomedical fields including application for drug delivery system. In this study, we developed a novel gene delivery system employing SWCNTs associated with polycationic and amphiphilic H-(-Lys-Trp-Lys-Gly-) 7 -OH [(KWKG) 7 ] peptides having pegylation. SWCNTs wrapped with (KWKG) 7 formed a complex with plasmid DNA (pDNA) in aqueous solution based on polyionic interaction but later underwent aggregation. On the other hand, a complex of pDNA and SWCNT-(KWKG) 7 modified with polyethylene glycol (PEG) chains of 12 units [SWCNT-(KWKG) 7 -(PEG) 12 ] afforded good dispersion stability for 24 h even in a cell culture medium. The in vitro cellular uptake of SWCNT-(KWKG) 7 -(PEG) 12 /pDNA complex prepared with fluorescence-labeled pDNA was evaluated with fluorescent microscopic observation and flow cytometry. The uptake by A549 human lung adenocarcinoma epithelial cells increased along with the extent of pegylation, suggesting the importance of dispersion stability in addition to the cationic charge which facilitates ionic cellular interaction. The expression of pDNA encoding the monomeric Kusabira-Orange 2 fluorescent protein in the form of the SWCNT-(KWKG) 7 -(PEG) 12 /pDNA complex demonstrated remarkable enhancement of transfection depending also on the extent of pegylation and the N/P ratio. The potential of the SWCNT composite wrapped with polycationic and amphiphilic (KWKG) 7 with pegylation as a carrier for gene delivery was demonstrated. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles.

PubMed

Souren, Nicole Y P; Lutsik, Pavlo; Gasparoni, Gilles; Tierling, Sascha; Gries, Jasmin; Riemenschneider, Matthias; Fryns, Jean-Pierre; Derom, Catherine; Zeegers, Maurice P; Walter, Jörn

2013-05-26

Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes. Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences. Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.

Enzymatic DNA molecules

NASA Technical Reports Server (NTRS)

Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

1998-01-01

The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

Comparison of Standard Culture-Based Method to Culture-Independent Method for Evaluation of Hygiene Effects on the Hand Microbiome

PubMed Central

Leff, J.; Henley, J.; Tittl, J.; De Nardo, E.; Butler, M.; Griggs, R.; Fierer, N.

2017-01-01

ABSTRACT Hands play a critical role in the transmission of microbiota on one’s own body, between individuals, and on environmental surfaces. Effectively measuring the composition of the hand microbiome is important to hand hygiene science, which has implications for human health. Hand hygiene products are evaluated using standard culture-based methods, but standard test methods for culture-independent microbiome characterization are lacking. We sampled the hands of 50 participants using swab-based and glove-based methods prior to and following four hand hygiene treatments (using a nonantimicrobial hand wash, alcohol-based hand sanitizer [ABHS], a 70% ethanol solution, or tap water). We compared results among culture plate counts, 16S rRNA gene sequencing of DNA extracted directly from hands, and sequencing of DNA extracted from culture plates. Glove-based sampling yielded higher numbers of unique operational taxonomic units (OTUs) but had less diversity in bacterial community composition than swab-based sampling. We detected treatment-induced changes in diversity only by using swab-based samples (P < 0.001); we were unable to detect changes with glove-based samples. Bacterial cell counts significantly decreased with use of the ABHS (P < 0.05) and ethanol control (P < 0.05). Skin hydration at baseline correlated with bacterial abundances, bacterial community composition, pH, and redness across subjects. The importance of the method choice was substantial. These findings are important to ensure improvement of hand hygiene industry methods and for future hand microbiome studies. On the basis of our results and previously published studies, we propose recommendations for best practices in hand microbiome research. PMID:28351915

Optimized catalytic DNA-cleaving ribozymes

NASA Technical Reports Server (NTRS)

Joyce, Gerald F. (Inventor)

1996-01-01

The present invention discloses nucleic acid enzymes capable of cleaving nucleic acid molecules, including single-stranded DNA, in a site-specific manner under physiologic conditions, as well as compositions including same. The present invention also discloses methods of making and using the disclosed enzymes and compositions.

Neighboring base damage induced by permanganate oxidation of 8-oxoguanine in DNA.

PubMed Central

Koizume, S; Inoue, H; Kamiya, H; Ohtsuka, E

1998-01-01

We found that single-stranded DNA oligomers containing a 7, 8-dihydro-8-oxoguanine (8-oxo-G) residue have high reactivity toward KMnO4; the oxidation of 8-oxo-G induces damage to the neighboring nucleotide residues. This paper describes the novel reaction in detail, including experiments that demonstrate the mechanism involved in the induction of DNA damage. The results using DNAs of various base compositions indicated that damaged G, T and C (but not A) sites caused strand scissions after hot piperidine treatment and that the damage around the 8-oxo-G occurred at G sites in both single and double strands with high frequency. The latter substrates were less sensitive to damage. Further, kinetic studies of the KMnO4reaction of single-stranded oligomers suggested that thereactivity of the DNA bases at the site 5'-adjacent to the 8-oxo-G was in the order G >A >T, C. This preference correlates with the electron donating abilities of the bases. In addition, we found that the DNA damage at the G site, which was connected with the 8-oxo-G by a long abasic chain, was inhibited in the above order by the addition of dG, dA or dC. On the other hand, the damage reactions proceeded even after the addition of scavengers for active oxygen species. This study suggests the involvement of a redox process in the unique DNA damage initiated by the oxidation of the 8-oxo-G. PMID:9671825

BECon: a tool for interpreting DNA methylation findings from blood in the context of brain.

PubMed

Edgar, R D; Jones, M J; Meaney, M J; Turecki, G; Kobor, M S

2017-08-01

Tissue differences are one of the largest contributors to variability in the human DNA methylome. Despite the tissue-specific nature of DNA methylation, the inaccessibility of human brain samples necessitates the frequent use of surrogate tissues such as blood, in studies of associations between DNA methylation and brain function and health. Results from studies of surrogate tissues in humans are difficult to interpret in this context, as the connection between blood-brain DNA methylation is tenuous and not well-documented. Here, we aimed to provide a resource to the community to aid interpretation of blood-based DNA methylation results in the context of brain tissue. We used paired samples from 16 individuals from three brain regions and whole blood, run on the Illumina 450 K Human Methylation Array to quantify the concordance of DNA methylation between tissues. From these data, we have made available metrics on: the variability of cytosine-phosphate-guanine dinucleotides (CpGs) in our blood and brain samples, the concordance of CpGs between blood and brain, and estimations of how strongly a CpG is affected by cell composition in both blood and brain through the web application BECon (Blood-Brain Epigenetic Concordance; https://redgar598.shinyapps.io/BECon/). We anticipate that BECon will enable biological interpretation of blood-based human DNA methylation results, in the context of brain.

Soil drying procedure affects the DNA quantification of Lactarius vinosus but does not change the fungal community composition.

PubMed

Castaño, Carles; Parladé, Javier; Pera, Joan; Martínez de Aragón, Juan; Alday, Josu G; Bonet, José Antonio

2016-11-01

Drying soil samples before DNA extraction is commonly used for specific fungal DNA quantification and metabarcoding studies, but the impact of different drying procedures on both the specific fungal DNA quantity and the fungal community composition has not been analyzed. We tested three different drying procedures (freeze-drying, oven-drying, and room temperature) on 12 different soil samples to determine (a) the soil mycelium biomass of the ectomycorrhizal species Lactarius vinosus using qPCR with a specifically designed TaqMan® probe and (b) the fungal community composition and diversity using the PacBio® RS II sequencing platform. Mycelium biomass of L. vinosus was significantly greater in the freeze-dried soil samples than in samples dried at oven and room temperature. However, drying procedures had no effect on fungal community composition or on fungal diversity. In addition, there were no significant differences in the proportions of fungi according to their functional roles (moulds vs. mycorrhizal species) in response to drying procedures. Only six out of 1139 operational taxonomic units (OTUs) had increased their relative proportions after soil drying at room temperature, with five of these OTUs classified as mould or yeast species. However, the magnitude of these changes was small, with an overall increase in relative abundance of these OTUs of approximately 2 %. These results suggest that DNA degradation may occur especially after drying soil samples at room temperature, but affecting equally nearly all fungi and therefore causing no significant differences in diversity and community composition. Despite the minimal effects caused by the drying procedures at the fungal community composition, freeze-drying resulted in higher concentrations of L. vinosus DNA and prevented potential colonization from opportunistic species.

Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity

PubMed Central

Thibault, Thomas; Degrouard, Jeril; Baril, Patrick; Pichon, Chantal; Midoux, Patrick

2017-01-01

Abstract Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmid-free method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide blunt-ended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 μM. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids. PMID:27899652

Electrochemical Characterization of O2 Plasma Functionalized Multi-Walled Carbon Nanotube Electrode for Legionella pneumophila DNA Sensor

NASA Astrophysics Data System (ADS)

Park, Eun Jin; Lee, Jun-Yong; Hyup Kim, Jun; Kug Kim, Sun; Lee, Cheol Jin; Min, Nam Ki

2010-08-01

An electrochemical DNA sensor for Legionella pneumophila detection was constructed using O2 plasma functionalized multi-walled carbon nanotube (MWCNT) film as a working electrode (WE). The cyclic voltammetry (CV) results revealed that the electrocatalytic activity of plasma functionalized MWCNT (pf-MWCNT) significantly changed depending on O2 plasma treatment time due to some oxygen containing functional groups on the pf-MWCNT surface. Scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra were also presented the changes of their surface morphologies and oxygen composition before and after plasma treatment. From a comparison study, it was found that the pf-MWCNT WEs had higher electrocatalytic activity and more capability of probe DNA immobilization: therefore, electrochemical signal changes by probe DNA immobilization and hybridization on pf-MWCNT WEs were larger than on Au WEs. The pf-MWCNT based DNA sensor was able to detect a concentration range of 10 pM-100 nM of target DNA to detect L. pneumophila.

Hydration Changes upon DNA Folding Studied by Osmotic Stress Experiments

PubMed Central

Nakano, Shu-ichi; Yamaguchi, Daisuke; Tateishi-Karimata, Hisae; Miyoshi, Daisuke; Sugimoto, Naoki

2012-01-01

The thermal stability of nucleic acid structures is perturbed under the conditions that mimic the intracellular environment, typically rich in inert components and under osmotic stress. We now describe the thermodynamic stability of DNA oligonucleotide structures in the presence of high background concentrations of neutral cosolutes. Small cosolutes destabilize the basepair structures, and the DNA structures consisting of the same nearest-neighbor composition show similar thermodynamic parameters in the presence of various types of cosolutes. The osmotic stress experiments reveal that water binding to flexible loops, unstable mismatches, and an abasic site upon DNA folding are almost negligible, whereas the binding to stable mismatch pairs is significant. The studies using the basepair-mimic nucleosides and the peptide nucleic acid suggest that the sugar-phosphate backbone and the integrity of the basepair conformation make important contributions to the binding of water molecules to the DNA bases and helical grooves. The study of the DNA hydration provides the basis for understanding and predicting nucleic acid structures in nonaqueous solvent systems. PMID:22735531

SiC nanoparticles-modified glassy carbon electrodes for simultaneous determination of purine and pyrimidine DNA bases.

PubMed

Ghavami, Raouf; Salimi, Abdollah; Navaee, Aso

2011-05-15

For the first time a novel and simple electrochemical method was used for simultaneous detection of DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment or separation process. Glassy carbon electrode modified with silicon carbide nanoparticles (SiCNP/GC), have been used for electrocatalytic oxidation of purine (guanine and adenine) and pyrimidine bases (thymine and cytosine) nucleotides. Field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) techniques were used to examine the structure of the SiCNP/GC modified electrode. The modified electrode shows excellent electrocatalytic activity toward guanine, adenine, thymine and cytosine. Differential pulse voltammetry (DPV) was proposed for simultaneous determination of four DNA bases. The effects of different parameters such as the thickness of SiC layer, pulse amplitude, scan rate, supporting electrolyte composition and pH were optimized to obtain the best peak potential separation and higher sensitivity. Detection limit, sensitivity and linear concentration range of the modified electrode toward proposed analytes were calculated for, guanine, adenine, thymine and cytosine, respectively. As shown this sensor can be used for nanomolar or micromolar detection of different DNA bases simultaneously or individually. This sensor also exhibits good stability, reproducibility and long lifetime. Copyright © 2011 Elsevier B.V. All rights reserved.

Detecting the borders between coding and non-coding DNA regions in prokaryotes based on recursive segmentation and nucleotide doublets statistics

PubMed Central

2012-01-01

Background Detecting the borders between coding and non-coding regions is an essential step in the genome annotation. And information entropy measures are useful for describing the signals in genome sequence. However, the accuracies of previous methods of finding borders based on entropy segmentation method still need to be improved. Methods In this study, we first applied a new recursive entropic segmentation method on DNA sequences to get preliminary significant cuts. A 22-symbol alphabet is used to capture the differential composition of nucleotide doublets and stop codon patterns along three phases in both DNA strands. This process requires no prior training datasets. Results Comparing with the previous segmentation methods, the experimental results on three bacteria genomes, Rickettsia prowazekii, Borrelia burgdorferi and E.coli, show that our approach improves the accuracy for finding the borders between coding and non-coding regions in DNA sequences. Conclusions This paper presents a new segmentation method in prokaryotes based on Jensen-Rényi divergence with a 22-symbol alphabet. For three bacteria genomes, comparing to A12_JR method, our method raised the accuracy of finding the borders between protein coding and non-coding regions in DNA sequences. PMID:23282225

Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

PubMed Central

Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J.; Waghorn, Garry C.; Janssen, Peter H.

2013-01-01

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided. PMID:24040342

Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

PubMed

Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J; Waghorn, Garry C; Janssen, Peter H

2013-01-01

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins.

PubMed

Goncearenco, Alexander; Ma, Bin-Guang; Berezovsky, Igor N

2014-03-01

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea.

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins

PubMed Central

Goncearenco, Alexander; Ma, Bin-Guang; Berezovsky, Igor N.

2014-01-01

DNA, RNA and proteins are major biological macromolecules that coevolve and adapt to environments as components of one highly interconnected system. We explore here sequence/structure determinants of mechanisms of adaptation of these molecules, links between them, and results of their mutual evolution. We complemented statistical analysis of genomic and proteomic sequences with folding simulations of RNA molecules, unraveling causal relations between compositional and sequence biases reflecting molecular adaptation on DNA, RNA and protein levels. We found many compositional peculiarities related to environmental adaptation and the life style. Specifically, thermal adaptation of protein-coding sequences in Archaea is characterized by a stronger codon bias than in Bacteria. Guanine and cytosine load in the third codon position is important for supporting the aerobic life style, and it is highly pronounced in Bacteria. The third codon position also provides a tradeoff between arginine and lysine, which are favorable for thermal adaptation and aerobicity, respectively. Dinucleotide composition provides stability of nucleic acids via strong base-stacking in ApG dinucleotides. In relation to coevolution of nucleic acids and proteins, thermostability-related demands on the amino acid composition affect the nucleotide content in the second codon position in Archaea. PMID:24371267

Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora.

PubMed

Qi, Ce; Li, Ya; Yu, Ren-Qiang; Zhou, Sheng-Li; Wang, Xing-Guo; Le, Guo-Wei; Jin, Qing-Zhe; Xiao, Hang; Sun, Jin

2017-11-28

To demonstrate that specific bacteria might release bacterial extracellular DNA (eDNA) to exert immunomodulatory functions in the mouse small intestine. Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of eDNA in the mucus layers of the small intestine and colon in healthy Male C57BL/6 mice. Composition difference of eDNA and intracellular DNA (iDNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism (T-RFLP). Stimulation of cytokine production by eDNA was studied in RAW264.7 cells in vitro . TOTO-1 iodide staining confirmed existence of eDNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the eDNA in the small intestinal mucus was significantly different from that of the iDNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the eDNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Gram-positive bacteria. Both eDNA and iDNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The eDNA induced significantly lower tumor necrosis factor-α/interleukin-10 (IL-10) and IL-6/IL-10 ratios than iDNA, suggesting the predominance for maintaining immune homeostasis of the gut. Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

FATTY ACID AND STEROL COMPOSITION OF A KARENIA BREVIS BLOOM IN THE GULF OF MEXICO

EPA Science Inventory

In the Gulf of Mexico, recurring algal blooms, caused by Karenia brevis (formerly known as Gymnodinium breve), have significant adverse health and economic impacts. K. brevis is one member of a small group of dinoflagellates, related morphologically and by DNA-based phylogenetic ...

Chemometrical characterization of four italian rice varieties based on genetic and chemical analyses.

PubMed

Brandolini, Vincenzo; Coïsson, Jean Daniel; Tedeschi, Paola; Barile, Daniela; Cereti, Elisabetta; Maietti, Annalisa; Vecchiati, Giorgio; Martelli, Aldo; Arlorio, Marco

2006-12-27

This paper describes a method for achieving qualitative identification of four rice varieties from two different Italian regions. To estimate the presence of genetic diversity among the four rice varieties, we used polymerase chain reaction-randomly amplified polymorphic DNA (PCR-RAPD) markers, and to elucidate whether a relationship exists between the ground and the specific characteristics of the product, we studied proximate composition, fatty acid composition, mineral content, and total antioxidant capacity. Using principal component analysis on genomic and compositional data, we were able to classify rice samples according to their variety and their district of production. This work also examined the discrimination ability of different parameters. It was found that genomic data give the best discrimination based on varieties, indicating that RAPD assays could be useful in discriminating among closely related species, while compositional analyses do not depend on the genetic characters only but are related to the production area.

Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

NASA Astrophysics Data System (ADS)

Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

2009-09-01

DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

PubMed

Hegedüs, Eva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

2009-09-01

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

New procedure for recovering extra- and intracellular DNA from marine sediment samples

NASA Astrophysics Data System (ADS)

Alawi, M.; Kallmeyer, J.

2012-12-01

Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Despite major methodological advances, analysis of DNA from sediment is still technically challenging, not just because of the co-elution of inhibitory substances, but also due to co-elution of extracellular DNA, which potentially leads to an overestimate of the actual diversity. Previous studies suggested that eDNA might play an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Several protocols based on the precipitation of eDNA e.g. with CTAB and ethanol have already been published. However, using these methods we did not succeed in quantifying very low amounts of eDNA (e.g. <1μg eDNA/g dry wt) in marine sediment even when using DNA carriers like glycogen. Since the recovery of eDNA by precipitation strongly depends on its concentration, these previously published procedures are not adequate for deep biosphere sediment due to the low eDNA content. We have focused on the question whether eDNA could be a source of nitrogen and phosphorus for microbes in the subseafloor biosphere. Therefore we developed a new method for the (semi)-quantitative extraction of eDNA from sediment. The new extraction procedure is based on sequential washing of the sediment to remove simultaneously eDNA and microbial cells without lysing them. After separation of the cells by centrifugation, the eDNA was extracted from the supernatant and purified by adsorption onto a solid phase, followed by removal of the solids and subsequent elution of the pure eDNA. Intracellular DNA (iDNA) was extracted and purified from the cell pellet using a commercial DNA extraction kit. Additional to a very low detection limit and reproducible quantification, this new method allows separation and purification of both extracellular and intracellular DNA to an extent that inhibitors are removed and downstream applications like PCR can be performed. To evaluate the new extraction method two sediments with rather opposing composition were analyzed. Sediment from the South Pacific Gyre, the most oligotrophic oceanic region on earth and organic-rich Baltic Sea sediment (Northern Germany) were processed. Using this new procedure high purity genomic iDNA and eDNA with a molecular size range between 20 bp and 50k bp can be simultaneously recovered even from very oligotrophic sediment with very low cell abundances. The main fraction of recovered eDNA was suitable for downstream applications like PCR and had a molecular size that indicates minimal shearing. Despite about two decades of research many questions about deep subsurface life remain unanswered. The fact that microbes can be found even in deep oligotrophic marine sediment raises the fundamental questions of the types and availability of substrates and their biogeochemical cycling. This is the first study that provides evidence that eDNA is an important potential substrate for microorganisms in the deep biosphere. Also, our results show a link between cell counts and eDNA content, indicating that the eDNA pool in the investigated sediment consist mainly of microbial DNA. Comparative sequence analysis of extracted iDNA and eDNA will provide deeper insights into the origin and turnover of eDNA and the apparent microbial community composition in the deep biosphere.

Quantitative DNA fiber mapping

DOEpatents

Gray, Joe W.; Weier, Heinz-Ulrich G.

1998-01-01

The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

[DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures].

PubMed

Antipina, M N; Gaĭnutdinov, R V; Rakhnianskaia, A A; Sergeev-Cherenkov, A N; Tolstikhina, A L; Iurova, T V; Kislov, V V; Khomutov, G B

2003-01-01

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and nanobiotechnology.

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

PubMed Central

2013-01-01

Background Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes. Results Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences. Conclusions Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies. PMID:23706164

Evaluation of DNA damage and cytotoxicity of polyurethane-based nano- and microparticles as promising biomaterials for drug delivery systems

NASA Astrophysics Data System (ADS)

Caon, Thiago; Zanetti-Ramos, Betina Giehl; Lemos-Senna, Elenara; Cloutet, Eric; Cramail, Henri; Borsali, Redouane; Soldi, Valdir; Simões, Cláudia Maria Oliveira

2010-06-01

The in vitro cytotoxicity and DNA damage evaluation of biodegradable polyurethane-based micro- and nanoparticles were carried out on animal fibroblasts. For cytotoxicity measurement and primary DNA damage evaluation, MTT and Comet assays were used, respectively. Different formulations were tested to evaluate the influence of chemical composition and physicochemical characteristics of particles on cell toxicity. No inhibition of cells growth surrounding the polyurethane particles was observed. On the other hand, a decrease of cell viability was verified when the anionic surfactant sodium dodecyl sulfate (SDS) was used as droplets stabilizer of monomeric phase. Polyurethane nanoparticles stabilized with Tween 80 and Pluronic F68 caused minor cytotoxic effects. These results indicated that the surface charge plays an important role on cytotoxicity. Particles synthesized from MDI displayed a higher cytotoxicity than those synthesized from IPDI. Size and physicochemical properties of the particles may explain the higher degree of DNA damage produced by two tested formulations. In this way, a rational choice of particles' constituents based on their cytotoxicity and genotoxicity could be very useful for conceiving biomaterials to be used as drug delivering systems.

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences

NASA Technical Reports Server (NTRS)

Ho, P. S.; Ellison, M. J.; Quigley, G. J.; Rich, A.

1986-01-01

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.

On the Sequence-Directed Nature of Human Gene Mutation: The Role of Genomic Architecture and the Local DNA Sequence Environment in Mediating Gene Mutations Underlying Human Inherited Disease

PubMed Central

Cooper, David N.; Bacolla, Albino; Férec, Claude; Vasquez, Karen M.; Kehrer-Sawatzki, Hildegard; Chen, Jian-Min

2011-01-01

Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher-order features of the genomic architecture. The human genome is now recognized to contain ‘pervasive architectural flaws’ in that certain DNA sequences are inherently mutation-prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of non-canonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair, and may serve to increase mutation frequencies in generalized fashion (i.e. both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease. PMID:21853507

An enhanced sensing platform for ultrasensitive impedimetric detection of target genes based on ordered FePt nanoparticles decorated carbon nanotubes.

PubMed

Zhang, Wei; Zong, Peisong; Zheng, Xiuwen; Wang, Libin

2013-04-15

We demonstrate a novel high-performance DNA hybridization biosensor with a carbon nanotubes (CNTs)-based nanocomposite membrane as the enhanced sensing platform. The platform was constructed by homogenously distributing ordered FePt nanoparticles (NPs) onto the CNTs matrix. The surface structure and electrochemical performance of the FePt/CNTs nanocomposite membrane were systematically investigated. Such a nanostructured composite membrane platform could combine with the advantages of FePt NPs and CNTs, greatly facilitate the electron-transfer process and the sensing behavior for DNA detection, leading to excellent sensitivity and selectivity. The complementary target genes from acute promyelocytic leukemia could be quantified in a wide range of 1.0×10⁻¹² mol/L to 1.0×10⁻⁶ mol/L using electrochemical impedance spectroscopy, and the detection limit was 2.1×10⁻¹³ mol/L under the optimal conditions. In addition, the DNA electrochemical biosensor was highly selective to discriminate single-base or double-base mismatched sequences. Copyright © 2012 Elsevier B.V. All rights reserved.

Plasmid DNA linearization in the antibacterial action of a new fluorescent Ag nanoparticle-paracetamol dimer composite

NASA Astrophysics Data System (ADS)

Sahoo, Amaresh Kumar; Sk, Md Palashuddin; Ghosh, Siddhartha Sankar; Chattopadhyay, Arun

2011-10-01

Herein, we report the generation of a composite comprised of p-hydroxyacetanilide dimer and Ag nanoparticles (NPs) by reaction of AgNO3 and p-hydroxyacetanilide. The formation of the composite was established by UV-vis, FTIR and NMR spectroscopy, transmission electron microscopy and X-ray diffraction along with substantiation by mass spectrometry. Interestingly, the composite exhibited an emission spectrum with a peak at 435 nm when excited by light of wavelength 320 nm. The composite showed superior antimicrobial activity with respect to its individual components against a wide range of Gram positive and Gram negative bacteria at relatively low concentrations of Ag NPs and at which there was no apparent cytotoxicity against mammalian cells. Our results suggest that the composite strongly interacted with the bacterial cell walls leading to cell bursting. Interestingly, enhancement in the reactive oxygen species (ROS) generation in bacteria was observed in the presence of the composite. It is proposed that the ROS generation led to oxidation of the dimer to N-acetyl-p-benzoquinone imine (NAPQI). The generated NAPQI acted as a DNA gyrase inhibitor causing cell death following linearization of DNA.Herein, we report the generation of a composite comprised of p-hydroxyacetanilide dimer and Ag nanoparticles (NPs) by reaction of AgNO3 and p-hydroxyacetanilide. The formation of the composite was established by UV-vis, FTIR and NMR spectroscopy, transmission electron microscopy and X-ray diffraction along with substantiation by mass spectrometry. Interestingly, the composite exhibited an emission spectrum with a peak at 435 nm when excited by light of wavelength 320 nm. The composite showed superior antimicrobial activity with respect to its individual components against a wide range of Gram positive and Gram negative bacteria at relatively low concentrations of Ag NPs and at which there was no apparent cytotoxicity against mammalian cells. Our results suggest that the composite strongly interacted with the bacterial cell walls leading to cell bursting. Interestingly, enhancement in the reactive oxygen species (ROS) generation in bacteria was observed in the presence of the composite. It is proposed that the ROS generation led to oxidation of the dimer to N-acetyl-p-benzoquinone imine (NAPQI). The generated NAPQI acted as a DNA gyrase inhibitor causing cell death following linearization of DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c1nr10389j

Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota.

PubMed

Horng, Katti R; Ganz, Holly H; Eisen, Jonathan A; Marks, Stanley L

2018-01-01

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and -80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer ( F -value = 6.87, DF  = 3, P  < 0.001), storage temperature ( F -value=1.77, DF  = 3, P  = 0.037), and duration of sample storage ( F -value = 3.68, DF  = 3, P  < 0.001). Changes in bacterial composition were observed in samples stored in -80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations ( DF  = 8.57, P  < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

PubMed

Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

1985-11-01

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Surface Coverage and Structure of Mixed DNA/Alkylthiol Monolayers on Gold: Characterization by XPS, NEXAFS, and Fluorescence Intensity Measurements

PubMed Central

Lee, Chi-Ying; Gong, Ping; Harbers, Gregory M.; Grainger, David W.; Castner, David G.; Gamble, Lara J.

2006-01-01

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s → π* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and 32P-radiometric assays and reported in a related study PMID:16689533

Compositions and methods for detecting single nucleotide polymorphisms

DOEpatents

Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

2016-11-22

Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

New insights into mitogenomic phylogeny and copy number in eight indigenous sheep populations based on the ATP synthase and cytochrome c oxidase genes.

PubMed

Xiao, P; Niu, L L; Zhao, Q J; Chen, X Y; Wang, L J; Li, L; Zhang, H P; Guo, J Z; Xu, H Y; Zhong, T

2017-11-16

The origins and phylogeny of different sheep breeds has been widely studied using polymorphisms within the mitochondrial hypervariable region. However, little is known about the mitochondrial DNA (mtDNA) content and phylogeny based on mtDNA protein-coding genes. In this study, we assessed the phylogeny and copy number of the mtDNA in eight indigenous (population size, n=184) and three introduced (n=66) sheep breeds in China based on five mitochondrial coding genes (COX1, COX2, ATP8, ATP6 and COX3). The mean haplotype and nucleotide diversities were 0.944 and 0.00322, respectively. We identified a correlation between the lineages distribution and the genetic distance, whereby Valley-type Tibetan sheep had a closer genetic relationship with introduced breeds (Dorper, Poll Dorset and Suffolk) than with other indigenous breeds. Similarly, the Median-joining profile of haplotypes revealed the distribution of clusters according to genetic differences. Moreover, copy number analysis based on the five mitochondrial coding genes was affected by the genetic distance combining with genetic phylogeny; we also identified obvious non-synonymous mutations in ATP6 between the different levels of copy number expressions. These results imply that differences in mitogenomic compositions resulting from geographical separation lead to differences in mitochondrial function.

Estimating population genetic parameters and comparing model goodness-of-fit using DNA sequences with error

PubMed Central

Liu, Xiaoming; Fu, Yun-Xin; Maxwell, Taylor J.; Boerwinkle, Eric

2010-01-01

It is known that sequencing error can bias estimation of evolutionary or population genetic parameters. This problem is more prominent in deep resequencing studies because of their large sample size n, and a higher probability of error at each nucleotide site. We propose a new method based on the composite likelihood of the observed SNP configurations to infer population mutation rate θ = 4Neμ, population exponential growth rate R, and error rate ɛ, simultaneously. Using simulation, we show the combined effects of the parameters, θ, n, ɛ, and R on the accuracy of parameter estimation. We compared our maximum composite likelihood estimator (MCLE) of θ with other θ estimators that take into account the error. The results show the MCLE performs well when the sample size is large or the error rate is high. Using parametric bootstrap, composite likelihood can also be used as a statistic for testing the model goodness-of-fit of the observed DNA sequences. The MCLE method is applied to sequence data on the ANGPTL4 gene in 1832 African American and 1045 European American individuals. PMID:19952140

Cell subpopulation deconvolution reveals breast cancer heterogeneity based on DNA methylation signature.

PubMed

Wen, Yanhua; Wei, Yanjun; Zhang, Shumei; Li, Song; Liu, Hongbo; Wang, Fang; Zhao, Yue; Zhang, Dongwei; Zhang, Yan

2017-05-01

Tumour heterogeneity describes the coexistence of divergent tumour cell clones within tumours, which is often caused by underlying epigenetic changes. DNA methylation is commonly regarded as a significant regulator that differs across cells and tissues. In this study, we comprehensively reviewed research progress on estimating of tumour heterogeneity. Bioinformatics-based analysis of DNA methylation has revealed the evolutionary relationships between breast cancer cell lines and tissues. Further analysis of the DNA methylation profiles in 33 breast cancer-related cell lines identified cell line-specific methylation patterns. Next, we reviewed the computational methods in inferring clonal evolution of tumours from different perspectives and then proposed a deconvolution strategy for modelling cell subclonal populations dynamics in breast cancer tissues based on DNA methylation. Further analysis of simulated cancer tissues and real cell lines revealed that this approach exhibits satisfactory performance and relative stability in estimating the composition and proportions of cellular subpopulations. The application of this strategy to breast cancer individuals of the Cancer Genome Atlas's identified different cellular subpopulations with distinct molecular phenotypes. Moreover, the current and potential future applications of this deconvolution strategy to clinical breast cancer research are discussed, and emphasis was placed on the DNA methylation-based recognition of intra-tumour heterogeneity. The wide use of these methods for estimating heterogeneity to further clinical cohorts will improve our understanding of neoplastic progression and the design of therapeutic interventions for treating breast cancer and other malignancies. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A species-specific nucleosomal signature defines a periodic distribution of amino acids in proteins.

PubMed

Quintales, Luis; Soriano, Ignacio; Vázquez, Enrique; Segurado, Mónica; Antequera, Francisco

2015-04-01

Nucleosomes are the basic structural units of chromatin. Most of the yeast genome is organized in a pattern of positioned nucleosomes that is stably maintained under a wide range of physiological conditions. In this work, we have searched for sequence determinants associated with positioned nucleosomes in four species of fission and budding yeasts. We show that mononucleosomal DNA follows a highly structured base composition pattern, which differs among species despite the high degree of histone conservation. These nucleosomal signatures are present in transcribed and non-transcribed regions across the genome. In the case of open reading frames, they correctly predict the relative distribution of codons on mononucleosomal DNA, and they also determine a periodicity in the average distribution of amino acids along the proteins. These results establish a direct and species-specific connection between the position of each codon around the histone octamer and protein composition.

The utilization of SiNWs/AuNPs-modified indium tin oxide (ITO) in fabrication of electrochemical DNA sensor.

PubMed

Rashid, Jahwarhar Izuan Abdul; Yusof, Nor Azah; Abdullah, Jaafar; Hashim, Uda; Hajian, Reza

2014-12-01

This work describes the incorporation of SiNWs/AuNPs composite as a sensing material for DNA detection on indium tin-oxide (ITO) coated glass slide. The morphology of SiNWs/AuNPs composite as the modifier layer on ITO was studied by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The morphological studies clearly showed that SiNWs were successfully decorated with 20 nm-AuNPs using self-assembly monolayer (SAM) technique. The effective surface area for SiNWs/AuNPs-modified ITO enhanced about 10 times compared with bare ITO electrode. SiNWs/AuNPs nanocomposite was further explored as a matrix for DNA probe immobilization in detection of dengue virus as a bio-sensing model to evaluate its performance in electrochemical sensors. The hybridization of complementary DNA was monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as the redox indicator. The fabricated biosensor was able to discriminate significantly complementary, non-complementary and single-base mismatch oligonucleotides. The electrochemical biosensor was sensitive to target DNA related to dengue virus in the range of 9.0-178.0 ng/ml with detection limit of 3.5 ng/ml. In addition, SiNWs/AuNPs-modified ITO, regenerated up to 8 times and its stability was up to 10 weeks at 4°C in silica gel. Copyright © 2014 Elsevier B.V. All rights reserved.

75 FR 17281 - Changes in Hourly Fee Rates for Science and Technology Laboratory Services-Fiscal Years 2010-2012

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-06

..., residue chemistry, proximate analysis for composition, and biomolecular (DNA-based) testing. A user fee... provide greater clarity of reported test analyses and laboratory determinations. DATES: Effective April 7... analyses and laboratory determinations provided by AMS laboratory services apply only to the submitted...

Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

PubMed Central

Kobayashi, H; Ngernprasirtsiri, J; Akazawa, T

1990-01-01

During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5-methylcytosine was most abundant. The presence of 5-methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run-on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts. Images Fig. 1 Fig. 2 Fig. 3. Fig. 4. Fig. 5. PMID:2303026

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adhikary, Suraj; Eichman, Brandt F.

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity,more » although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.« less

Estimation of a Killer Whale (Orcinus orca) Population’s Diet Using Sequencing Analysis of DNA from Feces

PubMed Central

Ford, Michael J.; Hempelmann, Jennifer; Hanson, M. Bradley; Ayres, Katherine L.; Baird, Robin W.; Emmons, Candice K.; Lundin, Jessica I.; Schorr, Gregory S.; Wasser, Samuel K.; Park, Linda K.

2016-01-01

Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca) in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%). Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population’s summer diet. PMID:26735849

Estimation of a Killer Whale (Orcinus orca) Population's Diet Using Sequencing Analysis of DNA from Feces.

PubMed

Ford, Michael J; Hempelmann, Jennifer; Hanson, M Bradley; Ayres, Katherine L; Baird, Robin W; Emmons, Candice K; Lundin, Jessica I; Schorr, Gregory S; Wasser, Samuel K; Park, Linda K

2016-01-01

Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca) in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%). Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population's summer diet.

Switch-on fluorescence scheme for antibiotics based on a magnetic composite probe with aptamer and hemin/G-quadruplex coimmobilized nano-Pt-luminol as signal tracer.

PubMed

Miao, Yang-Bao; Gan, Ning; Ren, Hong-Xia; Li, Tianhua; Cao, Yuting; Hu, Futao; Chen, Yinji

2016-01-15

A selective and facile fluorescence "switch-on" scheme is developed to detect antibiotics residues in food, using chloramphenicol (CAP) as model, based on a novel magnetic aptamer probe (aptamer-Pt-luminol nanocomposite labeled with hemin/G-quadruplex). Firstly, the composite probe is prepared through the immuno-reactions between the capture beads (anti-dsDNA antibody labeled on magnetic Dynabeads) and the nanotracer (nano-Pt-luminol labeled with double-strand aptamer, as ds-Apt, and hemin/G-quadruplex). When the composite probe is mixed with CAP, the aptamer preferentially reacted with CAP to decompose the double-strand aptamer to ssDNA, which cannot be recognized by the anti-dsDNA antibody on the capture probes. Thus, after magnetic separation, the nanotracer can be released into the supernatant. Because the hemin/G-quadruplex and PtNPs in nanotracer can catalyze luminol-H2O2 system to emit fluorescence. Thus a dual-amplified "switch-on" signal appeared, of which intensity is proportional to the concentration of CAP between 0.001 and 100ng mL(-1) with detection limit of 0.0005ng mL(-1) (S/N=3). Besides, our method has good selectivity and was employed for CAP detection in real milk samples. The results agree well with those from conventional gas chromatograph-mass spectrometer (GC-MS). The switch-on signal is produced by one-step substitution reaction between aptamer in nanotracer and target. When the analyte is changed, the probe can be refabricated only by changing the corresponding aptamer. Thus, all features above prove our strategy to be a facile, feasible and selective method in antibiotics screening for food safety. Copyright © 2015 Elsevier B.V. All rights reserved.

Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity.

PubMed

Thibault, Thomas; Degrouard, Jeril; Baril, Patrick; Pichon, Chantal; Midoux, Patrick; Malinge, Jean-Marc

2017-03-17

Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmid-free method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide blunt-ended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 μM. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Dispersing Zwitterions into Comb Polymers for Nonviral Transfection: Experiments and Molecular Simulation.

PubMed

Ghobadi, Ahmadreza F; Letteri, Rachel; Parelkar, Sangram S; Zhao, Yue; Chan-Seng, Delphine; Emrick, Todd; Jayaraman, Arthi

2016-02-08

Polymer-based gene delivery vehicles benefit from the presence of hydrophilic groups that mitigate the inherent toxicity of polycations and that provide tunable polymer-DNA binding strength and stable complexes (polyplexes). However, hydrophilic groups screen charge, and as such can reduce cell uptake and transfection efficiency. We report the effect of embedding zwitterionic sulfobetaine (SB) groups in cationic comb polymers, using a combination of experiments and molecular simulations. Ring-opening metathesis polymerization (ROMP) produced comb polymers with tetralysine (K4) and SB pendent groups. Dynamic light scattering, zeta potential measurements, and fluorescence-based experiments, together with coarse-grained molecular dynamics simulations, described the effect of SB groups on the size, shape, surface charge, composition, and DNA binding strength of polyplexes formed using these comb polymers. Experiments and simulations showed that increasing SB composition in the comb polymers decreased polymer-DNA binding strength, while simulations indicated that the SB groups distributed throughout the polyplex. This allows polyplexes to maintain a positive surface charge and provide high levels of gene expression in live cells. Notably, comb polymers with nearly 50 mol % SB form polyplexes that exhibit positive surface charge similarly as polyplexes formed from purely cationic comb polymers, indicating the ability to introduce an appreciable amount of SB functionality without screening surface charge. This integrated simulation-experimental study demonstrates the effectiveness of incorporating zwitterions in polyplexes, while guiding the design of new and effective gene delivery vectors.

Polymer brush coatings for DNA: fundamental polymer physics and nanofabrication applications

NASA Astrophysics Data System (ADS)

de Vries, Renko

Recombinant DNA technology allows for the production of precisely defined self-assembling protein-based polymers. So far, the major applications for such protein-based polymers have been self-assembling hydrogels and micellar structures with biomedical application. Inspired by minimal models for the self-ssembly of rod-shaped viruses such as the tobacco mosaic virus, I have developed protein-polymers that co-assemble with DNA into rod-shaped virus-like particles, and protein-polymers that provide brush coatings around single DNA molecules. In this presentation I will focus on the latter, showing that on the one hand brush coated DNA is a rich model system for exploring the physics of bottle-brush polymers, while on the other hand brush coatings of DNA can also play an important practical role in nanofabrication. A key problem in the physics of bottle-brush polymers that I will address is the scale-dependence of bottle-brush elasticity. For long-wavelength thermal deformations probed by AFM imaging I will demonstrate that there is significant stiffening due to the brush coating, while for short wavelength thermal deformations probed by force spectroscopy, we find that stiffening due to the brush coating disappears completely. DNA brush coatings can also play an important practical role in nanofabrication by acting as a compatibilizer between chemically different building blocks. I will explore the example of DNA origami in combination with gold nanoparticles: while Mg2+ ions and high concentrations of monovalent salts are crucial for the stability of DNA origami, such solution conditions are typically incompatible with the colloidal stability of gold nanoparticles.I will show how DNA brush coatings can dramatically enhance the yield of formation of isolated DNA-gold nanoparticle composite nanostructures.

Using pseudoalignment and base quality to accurately quantify microbial community composition

PubMed Central

Novembre, John

2018-01-01

Pooled DNA from multiple unknown organisms arises in a variety of contexts, for example microbial samples from ecological or human health research. Determining the composition of pooled samples can be difficult, especially at the scale of modern sequencing data and reference databases. Here we propose a novel method for taxonomic profiling in pooled DNA that combines the speed and low-memory requirements of k-mer based pseudoalignment with a likelihood framework that uses base quality information to better resolve multiply mapped reads. We apply the method to the problem of classifying 16S rRNA reads using a reference database of known organisms, a common challenge in microbiome research. Using simulations, we show the method is accurate across a variety of read lengths, with different length reference sequences, at different sample depths, and when samples contain reads originating from organisms absent from the reference. We also assess performance in real 16S data, where we reanalyze previous genetic association data to show our method discovers a larger number of quantitative trait associations than other widely used methods. We implement our method in the software Karp, for k-mer based analysis of read pools, to provide a novel combination of speed and accuracy that is uniquely suited for enhancing discoveries in microbial studies. PMID:29659582

Gemini surfactants mediate efficient mitochondrial gene delivery and expression.

PubMed

Cardoso, Ana M; Morais, Catarina M; Cruz, A Rita; Cardoso, Ana L; Silva, Sandra G; do Vale, M Luísa; Marques, Eduardo F; Pedroso de Lima, Maria C; Jurado, Amália S

2015-03-02

Gene delivery targeting mitochondria has the potential to transform the therapeutic landscape of mitochondrial genetic diseases. Taking advantage of the nonuniversal genetic code used by mitochondria, a plasmid DNA construct able to be specifically expressed in these organelles was designed by including a codon, which codes for an amino acid only if read by the mitochondrial ribosomes. In the present work, gemini surfactants were shown to successfully deliver plasmid DNA to mitochondria. Gemini surfactant-based DNA complexes were taken up by cells through a variety of routes, including endocytic pathways, and showed propensity for inducing membrane destabilization under acidic conditions, thus facilitating cytoplasmic release of DNA. Furthermore, the complexes interacted extensively with lipid membrane models mimicking the composition of the mitochondrial membrane, which predicts a favored interaction of the complexes with mitochondria in the intracellular environment. This work unravels new possibilities for gene therapy toward mitochondrial diseases.

Adansonian Analysis and Deoxyribonucleic Acid Base Composition of Serratia marcescens

PubMed Central

Colwell, R. R.; Mandel, M.

1965-01-01

Colwell, R. R. (Georgetown University, Washington, D.C.), and M. Mandel. Adansonian analysis and deoxyribonucleic acid base composition of Serratia marcescens. J. Bacteriol. 89:454–461. 1965.—A total of 33 strains of Serratia marcescens were subjected to Adansonian analysis for which more than 200 coded features for each of the organisms were included. In addition, the base composition [expressed as moles per cent guanine + cytosine (G + C)] of the deoxyribonucleic acid (DNA) prepared from each of the strains was determined. Except for four strains which were intermediate between Serratia and the Hafnia and Aerobacter group C of Edwards and Ewing, the S. marcescens species group proved to be extremely homogeneous, and the different strains showed high affinities for each other (mean similarity, ¯S = 77%). The G + C ratio of the DNA from the Serratia strains ranged from 56.2 to 58.4% G + C. Many species names have been listed for the genus, but only a single clustering of the strains was obtained at the species level, for which the species name S. marcescens was retained. S. kiliensis, S. indica, S. plymuthica, and S. marinorubra could not be distinguished from S. marcescens; it was concluded, therefore, that there is only a single species in the genus. The variety designation kiliensis does not appear to be valid, since no subspecies clustering of strains with negative Voges-Proskauer reactions could be detected. The characteristics of the species are listed, and a description of S. marcescens is presented. PMID:14255714

[Agricultural biotechnology safety assessment].

PubMed

McClain, Scott; Jones, Wendelyn; He, Xiaoyun; Ladics, Gregory; Bartholomaeus, Andrew; Raybould, Alan; Lutter, Petra; Xu, Haibin; Wang, Xue

2015-01-01

Genetically modified (GM) crops were first introduced to farmers in 1995 with the intent to provide better crop yield and meet the increasing demand for food and feed. GM crops have evolved to include a thorough safety evaluation for their use in human food and animal feed. Safety considerations begin at the level of DNA whereby the inserted GM DNA is evaluated for its content, position and stability once placed into the crop genome. The safety of the proteins coded by the inserted DNA and potential effects on the crop are considered, and the purpose is to ensure that the transgenic novel proteins are safe from a toxicity, allergy, and environmental perspective. In addition, the grain that provides the processed food or animal feed is also tested to evaluate its nutritional content and identify unintended effects to the plant composition when warranted. To provide a platform for the safety assessment, the GM crop is compared to non-GM comparators in what is typically referred to as composition equivalence testing. New technologies, such as mass spectrometry and well-designed antibody-based methods, allow better analytical measurements of crop composition, including endogenous allergens. Many of the analytical methods and their intended uses are based on regulatory guidance documents, some of which are outlined in globally recognized documents such as Codex Alimentarius. In certain cases, animal models are recommended by some regulatory agencies in specific countries, but there is typically no hypothesis or justification of their use in testing the safety of GM crops. The quality and standardization of testing methods can be supported, in some cases, by employing good laboratory practices (GLP) and is recognized in China as important to ensure quality data. Although the number of recommended, in some cases, required methods for safety testing are increasing in some regulatory agencies, it should be noted that GM crops registered to date have been shown to be comparable to their nontransgenic counterparts and safe . The crops upon which GM development are based are generally considered safe.

Extracellular DNA in single- and multiple-species unsaturated biofilms.

PubMed

Steinberger, R E; Holden, P A

2005-09-01

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.

77 FR 22333 - Prospective Grant of Exclusive License: Development of Oncolytic Viral Cancer Therapies

Federal Register 2010, 2011, 2012, 2013, 2014

2012-04-13

..., ``Recombinant Vaccinia Virus Containing a Chimeric Gene Having Foreign DNA Flanked by Vaccinia Regulatory DNA..., ``Compositions Containing Recombinant Poxviruses Having Foreign DNA Expressed under the Control of Poxvirus... entitled, ``Methods of Immunization Using Recombinant Poxviruses Having Foreign DNA Expressed under the...

A comparative study of ancient environmental DNA to pollen and macrofossils from lake sediments reveals taxonomic overlap and additional plant taxa

NASA Astrophysics Data System (ADS)

Pedersen, Mikkel Winther; Ginolhac, Aurélien; Orlando, Ludovic; Olsen, Jesper; Andersen, Kenneth; Holm, Jakob; Funder, Svend; Willerslev, Eske; Kjær, Kurt H.

2013-09-01

We use 2nd generation sequencing technology on sedimentary ancient DNA (sedaDNA) from a lake in South Greenland to reconstruct the local floristic history around a low-arctic lake and compare the results with those previously obtained from pollen and macrofossils in the same lake. Thirty-eight of thirty-nine samples from the core yielded putative DNA sequences. Using a multiple assignment strategy on the trnL g-h DNA barcode, consisting of two different phylogenetic and one sequence similarity assignment approaches, thirteen families of plants were identified, of which two (Scrophulariaceae and Asparagaceae) are absent from the pollen and macrofossil records. An age model for the sediment based on twelve radiocarbon dates establishes a chronology and shows that the lake record dates back to 10,650 cal yr BP. Our results suggest that sedaDNA analysis from lake sediments, although taxonomically less detailed than pollen and macrofossil analyses can be a complementary tool for establishing the composition of both terrestrial and aquatic local plant communities and a method for identifying additional taxa.

Exploring the Leaf Beetle Fauna (Coleoptera: Chrysomelidae) of an Ecuadorian Mountain Forest Using DNA Barcoding.

PubMed

Thormann, Birthe; Ahrens, Dirk; Marín Armijos, Diego; Peters, Marcell K; Wagner, Thomas; Wägele, Johann W

2016-01-01

Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates. Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284-289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469-481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation. Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing.

Exploring the Leaf Beetle Fauna (Coleoptera: Chrysomelidae) of an Ecuadorian Mountain Forest Using DNA Barcoding

PubMed Central

Thormann, Birthe; Ahrens, Dirk; Marín Armijos, Diego; Peters, Marcell K.; Wagner, Thomas; Wägele, Johann W.

2016-01-01

Background Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates. Methodology/Principal Findings Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284–289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469–481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation. Conclusions/Significance Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing. PMID:26849826

DOE Office of Scientific and Technical Information (OSTI.GOV)

Helfenbein, Kevin G.; Brown, Wesley M.; Boore, Jeffrey L.

We have sequenced the complete mitochondrial DNA (mtDNA) of the articulate brachiopod Terebratalia transversa. The circular genome is 14,291 bp in size, relatively small compared to other published metazoan mtDNAs. The 37 genes commonly found in animal mtDNA are present; the size decrease is due to the truncation of several tRNA, rRNA, and protein genes, to some nucleotide overlaps, and to a paucity of non-coding nucleotides. Although the gene arrangement differs radically from those reported for other metazoans, some gene junctions are shared with two other articulate brachiopods, Laqueus rubellus and Terebratulina retusa. All genes in the T. transversa mtDNA,more » unlike those in most metazoan mtDNAs reported, are encoded by the same strand. The A+T content (59.1 percent) is low for a metazoan mtDNA, and there is a high propensity for homopolymer runs and a strong base-compositional strand bias. The coding strand is quite G+T-rich, a skew that is shared by the confamilial (laqueid) specie s L. rubellus, but opposite to that found in T. retusa, a cancellothyridid. These compositional skews are strongly reflected in the codon usage patterns and the amino acid compositions of the mitochondrial proteins, with markedly different usage observed between T. retusa and the two laqueids. This observation, plus the similarity of the laqueid non-coding regions to the reverse complement of the non-coding region of the cancellothyridid, suggest that an inversion that resulted in a reversal in the direction of first-strand replication has occurred in one of the two lineages. In addition to the presence of one non-coding region in T. transversa that is comparable to those in the other brachiopod mtDNAs, there are two others with the potential to form secondary structures; one or both of these may be involved in the process of transcript cleavage.« less

The effects of food components on the digestion of DNA by pepsin.

PubMed

Zhang, Yanfang; Wang, Xingyu; Pan, Xiaoming; Liu, Yu; Wang, Hanqing; Dong, Ping; Liang, Xingguo

2016-11-01

Recently, our study found that naked nucleic acids (NAs) can be digested by pepsin. To better understand the fate of dietary DNA in the digestive tract, in this study we investigated the effects of several food compositions on its digestion. The results showed that protein inhibited the digestion of DNA when the protein:DNA ratio was higher than 80:1 (m/m). DNA found in nucleoprotein (NA), which more closely resembles the state of DNA in food, was as efficiently digested as naked DNA. When the carbohydrate:DNA ratio was 50:1-140:1 (m/m), mono-, di- and polysaccharides did not inhibit DNA digestion. NaCl exhibited an inhibitory effect at 300 mM, whereas divalent cations (Ca(2+ )and Mg(2+)) exerted a much stronger inhibitory effect even at 50 mM. The polycation compounds (e.g. chitosan and spermine) showed a significant inhibitory effect at N/P (NH3(+)/PO4(-)) = 10:1. The close relationship between food composition and DNA digestion suggests that dietary habits and food complexes are important for understanding the in vivo fate of the ingested DNA in the digestive tract.

The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia.

PubMed

Wolters, A M; Koornneef, M; Gilissen, L J

1993-09-01

This paper describes the analysis of chloroplast (cp) DNA and mitochondrial (mt) DNA in 21 somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia by means of Southern-blot hybridization. Each of these calli contained only one type of cpDNA; 14 had the N. plumbaginifolia (Np) type and seven the S. tuberosum (St) type. N. plumbaginifolia cpDNA was present in hybrids previously shown to contain predominantly N. plumbaginifolia chromosomes whereas hybrids in which S. tuberosum chromosomes predominated possessed cpDNA from potato. We have analyzed the mtDNA of these 21 somatic hybrid calli using four restriction enzyme/probe combinations. Most fusion products had only, or mostly, mtDNA fragments from the parent that predominated in the nucleus. The hybrids containing mtDNA fragments from only one parent (and new fragments) also possessed chloroplasts from the same species. The results suggest the existence of a strong nucleo-cytoplasmic incongruity which affects the genome composition of somatic hybrids between distantly related species.

The impact of base stacking on the conformations and electrostatics of single-stranded DNA.

PubMed

Plumridge, Alex; Meisburger, Steve P; Andresen, Kurt; Pollack, Lois

2017-04-20

Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

TALE-PvuII fusion proteins--novel tools for gene targeting.

PubMed

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.

Chemical and Biological Sensing Using Hybridization Chain Reaction.

PubMed

Augspurger, Erik E; Rana, Muhit; Yigit, Mehmet V

2018-05-25

Since the advent of its theoretical discovery more than 30 years ago, DNA nanotechnology has been used in a plethora of diverse applications in both the fundamental and applied sciences. The recent prominence of DNA-based technologies in the scientific community is largely due to the programmable features stored in its nucleobase composition and sequence, which allow it to assemble into highly advanced structures. DNA nanoassemblies are also highly controllable due to the precision of natural and artificial base-pairing, which can be manipulated by pH, temperature, metal ions, and solvent types. This programmability and molecular-level control have allowed scientists to create and utilize DNA nanostructures in one, two, and three dimensions (1D, 2D, and 3D). Initially, these 2D and 3D DNA lattices and shapes attracted a broad scientific audience because they are fundamentally captivating and structurally elegant; however, transforming these conceptual architectural blueprints into functional materials is essential for further advancements in the DNA nanotechnology field. Herein, the chemical and biological sensing applications of a 1D DNA self-assembly process known as hybridization chain reaction (HCR) are reviewed. HCR is a one-dimensional (1D) double stranded (ds) DNA assembly process initiated only in the presence of a specific short ssDNA (initiator) and two kinetically trapped DNA hairpin structures. HCR is considered an enzyme-free isothermal amplification process, which shows substantial promise and offers a wide range of applications for in situ chemical and biological sensing. Due to its modular nature, HCR can be programmed to activate only in the presence of highly specific biological and/or chemical stimuli. HCR can also be combined with different types of molecular reporters and detection approaches for various analytical readouts. While the long dsDNA HCR product may not be as structurally attractive as the 2D and 3D DNA networks, HCR is highly instrumental for applied biological, chemical, and environmental sciences, and has therefore been studied to foster a variety of objectives. In this review, we have focused on nucleic acid, protein, metabolite, and heavy metal ion detection using this 1D DNA nanotechnology via fluorescence, electrochemical, and nanoparticle-based methodologies.

DNA Damage among Wood Workers Assessed with the Comet Assay

PubMed Central

Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

2016-01-01

Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

A DNA Barcoding Approach to Characterize Pollen Collected by Honeybees

PubMed Central

Bruni, Ilaria; Scaccabarozzi, Daniela; Sandionigi, Anna; Barbuto, Michela; Casiraghi, Maurizio; Labra, Massimo

2014-01-01

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands. PMID:25296114

Direct Determination of Molecular Weight Distribution of Calf-Thymus DNAs and Study of Their Fragmentation under Ultrasonic and Low-Energy IR Irradiations. A Charge Detection Mass Spectrometry Investigation.

PubMed

Halim, Mohammad A; Bertorelle, Franck; Doussineau, Tristan; Antoine, Rodolphe

2018-06-09

Calf-thymus (CT-DNA) is widely used as binding agent. The commercial samples are known to be "highly polymerized DNA" samples. CT-DNA is known to be fragile in particular upon ultrasonic wave irradiation. Degradation products might have dramatic consequence on its bio-sensing activity, and an accurate determination of the molecular weight distribution and stability of commercial samples is highly demanded. We investigated the sensitivity of charge detection mass spectrometry (CDMS), a single-molecule MS method, both with single-pass and ion trap CDMS ("Benner" trap) modes to the determination of the composition and stability (under multiphoton IR irradiation) of calf-thymus DNAs. We also investigated the changes of molecular weight distributions in the course of sonication by irradiating ultrasonic wave to CT-DNA. We report for the first time, the direct molecular weight (MW) distribution of DNA sodium salt from calf-thymus revealing two populations at high (~10 MDa) and low (~3 MDa) molecular weights. We evidence a transition between the high-MW to the low-MW distribution, confirming that the low-MW distribution results from degradation of CT-DNA. Finally, we report also IRMPD experiments carried out on trapped single-stranded linear DNAs from calf-thymus allowing to extract their activation energy for unimolecular dissociation. We show that single-pass CDMS is a direct, efficient and accurate MS-based approach to determine the composition of calf-thymus DNAs. Furthermore, ion trap CDMS allows us to evaluate the stability (both under multiphoton IR irradiation and in the course of sonication by irradiating ultrasonic wave) of calf-thymus DNAs. This article is protected by copyright. All rights reserved.

Concurrent DNA Preconcentration and Separation in Bipolar Electrode-Based Microfluidic Device

PubMed Central

Song, Hongjun; Wang, Yi; Garson, Charles; Pant, Kapil

2015-01-01

This paper presents a bipolar electrode (BPE) device in a microfluidic dual-channel design for concurrent preconcentration and separation of composite DNA containing samples. The novelty of the present effort relies on the combination of BPE-induced ion concentration polarization (ICP) and end-labeled free-solution electrophoresis (ELFSE). The ion concentration polarization effect arising from the faradaic reaction on the BPE is utilized to exert opposing electrophoretic and electroosmotic forces on the DNA samples. Meanwhile, end-labeled free-solution electrophoresis alters the mass-charge ratio to enable simultaneous DNA separation in free solution. The microfluidic device was fabricated using standard and soft lithography techniques to form gold-on-glass electrode capped with a PDMS microfluidic channel. Experimental testing with various DNA samples was carried out over a range of applied electric field. Concentration ratios up to 285× within 5 minutes for a 102-mer DNA, and concurrent preconcentration and free-solution separation of binary mixture of free and bound 102-mer DNA within 6 minutes was demonstrated. The effect of applied electric field was also interrogated with respect to pertinent performance metrics of preconcentration and separation. PMID:26005497

Simulation studies of DNA at the nanoscale: Interactions with proteins, polycations, and surfaces

NASA Astrophysics Data System (ADS)

Elder, Robert M.

Understanding the nanoscale interactions of DNA, a multifunctional biopolymer with sequence-dependent properties, with other biological and synthetic substrates and molecules is essential to advancing these technologies. This doctoral thesis research is aimed at understanding the thermodynamics and molecular-level structure when DNA interacts with proteins, polycations, and functionalized surfaces. First, we investigate the ability of a DNA damage recognition protein (HMGB1a) to bind to anti-cancer drug-induced DNA damage, seeking to explain how HMGB1a differentiates between the drugs in vivo. Using atomistic molecular dynamics simulations, we show that the structure of the drug-DNA molecule exhibits drug- and base sequence-dependence that explains some of the experimentally observed differential recognition of the drugs in various sequence contexts. Then, we show how steric hindrance from the drug decreases the deformability of the drug-DNA molecule, which decreases recognition by the protein, a concept that can be applied to rational drug design. Second, we study how polycation architecture and chemistry affect polycation-DNA binding so as to design optimal polycations for high efficiency gene (DNA) delivery. Using a multiscale computational approach involving atomistic and coarse-grained simulations, we examine how rearranging polylysine from a linear to a grafted architecture, and several aspects of the grafted architecture, affect polycation-DNA binding and the structure of polycation-DNA complexes. Next, going beyond lysine we examine how oligopeptide chemistry and sequence in the grafted architecture affects polycation-DNA binding and find that strategic placement of hydrophobic peptides might be used to tailor binding strength. Third, we study the adsorption and conformations of single-stranded DNA (an amphiphilic biopolymer) on model hydrophilic and hydrophobic surfaces. Short ssDNA oligomers adsorb to both surfaces with similar strength, with the strength of adsorption to the hydrophobic surface depending on the composition of the DNA strands, i.e. purine or pyrimidine bases. Additionally, DNA-surface and DNA-water interactions near the surfaces govern the adsorption. For longer ssDNA oligomers, the effects of surface chemistry and temperature on ssDNA conformations are rather small, but either the hydrophilic surface or increased temperature favor slightly more compact conformations due to energetic and entropic effects, respectively.

Population-specific variation in haplotype composition and heterozygosity at the POLB locus.

PubMed

Yamtich, Jennifer; Speed, William C; Straka, Eva; Kidd, Judith R; Sweasy, Joann B; Kidd, Kenneth K

2009-05-01

DNA polymerase beta plays a central role in base excision repair (BER), which removes large numbers of endogenous DNA lesions from each cell on a daily basis. Little is currently known about germline polymorphisms within the POLB locus, making it difficult to study the association of variants at this locus with human diseases such as cancer. Yet, approximately thirty percent of human tumor types show variants of DNA polymerase beta. We have assessed the global frequency distributions of coding and common non-coding SNPs in and flanking the POLB gene for a total of 14 sites typed in approximately 2400 individuals from anthropologically defined human populations worldwide. We have found a marked difference between haplotype frequencies in African populations and in non-African populations.

Development of DNA-based Identification methods to track the species composition of fish larvae within nearshore areas of the Great Lakes

EPA Science Inventory

The ability to track the identity and abundance of larval fish, which are ubiquitous during spawning season, may lead to a greater understanding of fish species distributions in Great Lakes nearshore areas including early-detection of invasive fish species before they become esta...

Cytogenotoxicity of sewage sludge leachate before and after calcium oxide-based solidification in human lymphocytes.

PubMed

Gajski, Goran; Oreščanin, Višnja; Garaj-Vrhovac, Vera

2011-07-01

Present study aimed to establish the chemical composition of sewage sludge leachate before/after calcium oxide-based solidification using energy dispersive X-ray fluorescence (EDXRF). The other aim was to determine leachate effects on human lymphocyte and DNA integrity in vitro using a battery of bioassays (DNA diffusion assay, micronucleus test and comet assay) to determine effects of those complex mixtures of elements on cell and DNA integrity. EDXRF showed that nickel concentration in the leachate of untreated sludge was 18.5 times higher than the upper permissible limit for inert waste landfills. Other elements were kept below the permissible values. After sludge solidification, leachate concentrations of Cr, Mn, Fe, Ni, Cu, Zn, and Pb dropped 1.6, 2.7, 37, 5.9, 3.2, 7.8, and 2.6 times, respectively. Untreated sludge leachate was cytogenotoxic to lymphocytes, and may lead to adverse effects on the exposed human populations, but calcium oxide-based solidification reduced these effects in significant manner. Copyright © 2011 Elsevier Inc. All rights reserved.

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

PubMed

Liu, Xiao; Dang, Yunkun; Matsu-Ura, Toru; He, Yubo; He, Qun; Hong, Christian I; Liu, Yi

2017-07-20

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism. Published by Elsevier Inc.

PEG and mPEG-anthracene induce DNA condensation and particle formation.

PubMed

Froehlich, E; Mandeville, J S; Arnold, D; Kreplak, L; Tajmir-Riahi, H A

2011-08-18

In this study, we investigated the binding of DNA with poly(ethylene glycol) (PEG) of different sizes and compositions such as PEG 3350, PEG 6000, and mPEG-anthracene in aqueous solution at physiological conditions. The effects of size and composition on DNA aggregation and condensation as well as conformation were determined using Fourier transform infrared (FTIR), UV-visible, CD, fluorescence spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed moderate complex formation for PEG 3350 and PEG 6000 and weaker interaction for mPE-anthracene-DNA adducts with both hydrophilic and hydrophobic contacts. The order of ± stability of the complexes formed is K(PEG 6000) = 1.5 (±0.4) × 10(4) M(-1) > K(PEG 3350) = 7.9 (±1) × 10(3) M(-1) > K(m(PEG-anthracene))= 3.6 (±0.8) × 10(3) M(-1) with nearly 1 bound PEG molecule per DNA. No B-DNA conformational changes were observed, while DNA condensation and particle formation occurred at high PEG concentration.

A new biochromatography model based on DNA origami assembled PPARγ: construction and evaluation.

PubMed

Zhou, Jie; Meng, Lingchang; Sun, Chong; Chen, Shanshan; Sun, Fang; Luo, Pei; Zhao, Yongxing

2017-05-01

As drug targets, receptors have potential to screen drugs. Silica is an attractive support to immobilize receptors; however, the lack of biocompatibility makes it easier for receptors to lose bioactivity, which remains an obstacle to its widespread use. With the advantage of biocompatibility, DNA origami can be used as a biological carrier to improve the biocompatibility of silica and assemble receptors. In this study, a new biochromatography model based on DNA origami was constructed. A large quantity of M13ssDNA was used as a scaffold, leading to significant costs, so M13ssDNA was self-produced from the bacteriophage particles. This approach is demonstrated using the ligand binding domain of gamma isoform peroxisome proliferator-activated receptor (PPARγ-LBD) as a research object. PPARγ-LBD was assembled on DNA origami carrier and then coupled on the surface of silica. The products were packed into the column as stationary phase to construct the biochromatography with the ability to recognize drugs. Affinity and specificity of the biochromatography model were evaluated by HPLC. The final results showed that the biochromatography could recognize rosiglitazone specifically, which further proved that the model could screen chemical compositions interacted with PPARγ. It was the first time to take advantage of DNA origami to assemble PPARγ to construct biochromatography. The new biochromatography model has the advantages of being efficient, convenient, and high-throughput. This method affords a new way to rapidly and conveniently screen active ingredients from complex sample plant extracts and natural product-like libraries.

The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples.

PubMed

Maukonen, Johanna; Simões, Catarina; Saarela, Maria

2012-03-01

Recently several human health-related microbiota studies have had partly contradictory results. As some differences may be explained by methodologies applied, we evaluated how different storage conditions and commonly used DNA-extraction kits affect bacterial composition, diversity, and numbers of human fecal microbiota. According to our results, the DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after a week's storage at -20 °C, the numbers of Bacteroides spp. were 1.6-2.5 log units lower (P < 0.05). Furthermore, the numbers of predominant bacteria, Eubacterium rectale (Erec)-group, Clostridium leptum group, bifidobacteria, and Atopobium group were 0.5-4 log units higher (P < 0.05) after mechanical DNA-extraction as detected with qPCR, regardless of storage. Furthermore, the bacterial composition of Erec-group differed significantly after different DNA-extractions; after enzymatic DNA-extraction, the most prevalent genera detected were Roseburia (39% of clones) and Coprococcus (10%), whereas after mechanical DNA-extraction, the most prevalent genera were Blautia (30%), Coprococcus (13%), and Dorea (10%). According to our results, rigorous mechanical lysis enables detection of higher bacterial numbers and diversity from human fecal samples. As it was shown that the results of clostridial and actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may explain the contradictory results previously obtained. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite.

PubMed

Avelino, Karen Y P S; Frias, Isaac A M; Lucena-Silva, Norma; Gomes, Renan G; de Melo, Celso P; Oliveira, Maria D L; Andrade, César A S

2016-12-01

In the last ten years, conjugated polymers started to be used in the immobilization of nucleic acids via non-covalent interactions. In the present study, we describe the construction and use of an electrochemical DNA biosensor based on a nanostructured polyaniline-gold composite, specifically developed for the detection of the BCR/ABL chimeric oncogene. This chromosome translocation is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The working principle of the biosensor rests on measuring the conductivity resulting from the non-covalent interactions between the hybrid nanocomposite and the DNA probe. The nanostructured platform exhibits a large surface area that enhances the conductivity. Positive cases, which result from the hybridization between DNA probe and targeted gene, induce changes in the amperometric current and in the charge transfer resistance (R CT ) responses. Atomic force microscopy (AFM) images showed changes in the genosensor surface after exposure to cDNA sample of patient with leukemia, evidencing the hybridization process. This new hybrid sensing-platform displayed high specificity and selectivity, and its detection limit is estimated to be as low as 69.4 aM. The biosensor showed excellent analytical performance for the detection of the BCR/ABL oncogene in clinical samples of patients with leukemia. Hence, this electrochemical sensor appears as a simple and attractive tool for the molecular diagnosis of the BCR/ABL oncogene even in early-stage cases of leukemia and for the monitoring of minimum levels of residual disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Haplotype Phasing and Inheritance of Copy Number Variants in Nuclear Families

PubMed Central

Palta, Priit; Kaplinski, Lauris; Nagirnaja, Liina; Veidenberg, Andres; Möls, Märt; Nelis, Mari; Esko, Tõnu; Metspalu, Andres; Laan, Maris; Remm, Maido

2015-01-01

DNA copy number variants (CNVs) that alter the copy number of a particular DNA segment in the genome play an important role in human phenotypic variability and disease susceptibility. A number of CNVs overlapping with genes have been shown to confer risk to a variety of human diseases thus highlighting the relevance of addressing the variability of CNVs at a higher resolution. So far, it has not been possible to deterministically infer the allelic composition of different haplotypes present within the CNV regions. We have developed a novel computational method, called PiCNV, which enables to resolve the haplotype sequence composition within CNV regions in nuclear families based on SNP genotyping microarray data. The algorithm allows to i) phase normal and CNV-carrying haplotypes in the copy number variable regions, ii) resolve the allelic copies of rearranged DNA sequence within the haplotypes and iii) infer the heritability of identified haplotypes in trios or larger nuclear families. To our knowledge this is the first program available that can deterministically phase null, mono-, di-, tri- and tetraploid genotypes in CNV loci. We applied our method to study the composition and inheritance of haplotypes in CNV regions of 30 HapMap Yoruban trios and 34 Estonian families. For 93.6% of the CNV loci, PiCNV enabled to unambiguously phase normal and CNV-carrying haplotypes and follow their transmission in the corresponding families. Furthermore, allelic composition analysis identified the co-occurrence of alternative allelic copies within 66.7% of haplotypes carrying copy number gains. We also observed less frequent transmission of CNV-carrying haplotypes from parents to children compared to normal haplotypes and identified an emergence of several de novo deletions and duplications in the offspring. PMID:25853576

Haplotype phasing and inheritance of copy number variants in nuclear families.

PubMed

Palta, Priit; Kaplinski, Lauris; Nagirnaja, Liina; Veidenberg, Andres; Möls, Märt; Nelis, Mari; Esko, Tõnu; Metspalu, Andres; Laan, Maris; Remm, Maido

2015-01-01

DNA copy number variants (CNVs) that alter the copy number of a particular DNA segment in the genome play an important role in human phenotypic variability and disease susceptibility. A number of CNVs overlapping with genes have been shown to confer risk to a variety of human diseases thus highlighting the relevance of addressing the variability of CNVs at a higher resolution. So far, it has not been possible to deterministically infer the allelic composition of different haplotypes present within the CNV regions. We have developed a novel computational method, called PiCNV, which enables to resolve the haplotype sequence composition within CNV regions in nuclear families based on SNP genotyping microarray data. The algorithm allows to i) phase normal and CNV-carrying haplotypes in the copy number variable regions, ii) resolve the allelic copies of rearranged DNA sequence within the haplotypes and iii) infer the heritability of identified haplotypes in trios or larger nuclear families. To our knowledge this is the first program available that can deterministically phase null, mono-, di-, tri- and tetraploid genotypes in CNV loci. We applied our method to study the composition and inheritance of haplotypes in CNV regions of 30 HapMap Yoruban trios and 34 Estonian families. For 93.6% of the CNV loci, PiCNV enabled to unambiguously phase normal and CNV-carrying haplotypes and follow their transmission in the corresponding families. Furthermore, allelic composition analysis identified the co-occurrence of alternative allelic copies within 66.7% of haplotypes carrying copy number gains. We also observed less frequent transmission of CNV-carrying haplotypes from parents to children compared to normal haplotypes and identified an emergence of several de novo deletions and duplications in the offspring.

From Wolves to Dogs, and Back: Genetic Composition of the Czechoslovakian Wolfdog.

PubMed

Smetanová, Milena; Černá Bolfíková, Barbora; Randi, Ettore; Caniglia, Romolo; Fabbri, Elena; Galaverni, Marco; Kutal, Miroslav; Hulva, Pavel

2015-01-01

The Czechoslovakian Wolfdog is a unique dog breed that originated from hybridization between German Shepherds and wild Carpathian wolves in the 1950s as a military experiment. This breed was used for guarding the Czechoslovakian borders during the cold war and is currently kept by civilian breeders all round the world. The aim of our study was to characterize, for the first time, the genetic composition of this breed in relation to its known source populations. We sequenced the hypervariable part of the mtDNA control region and genotyped the Amelogenin gene, four sex-linked microsatellites and 39 autosomal microsatellites in 79 Czechoslovakian Wolfdogs, 20 German Shepherds and 28 Carpathian wolves. We performed a range of population genetic analyses based on both empirical and simulated data. Only two mtDNA and two Y-linked haplotypes were found in Czechoslovakian Wolfdogs. Both mtDNA haplotypes were of domestic origin, while only one of the Y-haplotypes was shared with German Shepherds and the other was unique to Czechoslovakian Wolfdogs. The observed inbreeding coefficient was low despite the small effective population size of the breed, possibly due to heterozygote advantages determined by introgression of wolf alleles. Moreover, Czechoslovakian Wolfdog genotypes were distinct from both parental populations, indicating the role of founder effect, drift and/or genetic hitchhiking. The results revealed the peculiar genetic composition of the Czechoslovakian Wolfdog, showing a limited introgression of wolf alleles within a higher proportion of the dog genome, consistent with the reiterated backcrossing used in the pedigree. Artificial selection aiming to keep wolf-like phenotypes but dog-like behavior resulted in a distinctive genetic composition of Czechoslovakian Wolfdogs, which provides a unique example to study the interactions between dog and wolf genomes.

Ancestral sequence reconstruction in primate mitochondrial DNA: compositional bias and effect on functional inference.

PubMed

Krishnan, Neeraja M; Seligmann, Hervé; Stewart, Caro-Beth; De Koning, A P Jason; Pollock, David D

2004-10-01

Reconstruction of ancestral DNA and amino acid sequences is an important means of inferring information about past evolutionary events. Such reconstructions suggest changes in molecular function and evolutionary processes over the course of evolution and are used to infer adaptation and convergence. Maximum likelihood (ML) is generally thought to provide relatively accurate reconstructed sequences compared to parsimony, but both methods lead to the inference of multiple directional changes in nucleotide frequencies in primate mitochondrial DNA (mtDNA). To better understand this surprising result, as well as to better understand how parsimony and ML differ, we constructed a series of computationally simple "conditional pathway" methods that differed in the number of substitutions allowed per site along each branch, and we also evaluated the entire Bayesian posterior frequency distribution of reconstructed ancestral states. We analyzed primate mitochondrial cytochrome b (Cyt-b) and cytochrome oxidase subunit I (COI) genes and found that ML reconstructs ancestral frequencies that are often more different from tip sequences than are parsimony reconstructions. In contrast, frequency reconstructions based on the posterior ensemble more closely resemble extant nucleotide frequencies. Simulations indicate that these differences in ancestral sequence inference are probably due to deterministic bias caused by high uncertainty in the optimization-based ancestral reconstruction methods (parsimony, ML, Bayesian maximum a posteriori). In contrast, ancestral nucleotide frequencies based on an average of the Bayesian set of credible ancestral sequences are much less biased. The methods involving simpler conditional pathway calculations have slightly reduced likelihood values compared to full likelihood calculations, but they can provide fairly unbiased nucleotide reconstructions and may be useful in more complex phylogenetic analyses than considered here due to their speed and flexibility. To determine whether biased reconstructions using optimization methods might affect inferences of functional properties, ancestral primate mitochondrial tRNA sequences were inferred and helix-forming propensities for conserved pairs were evaluated in silico. For ambiguously reconstructed nucleotides at sites with high base composition variability, ancestral tRNA sequences from Bayesian analyses were more compatible with canonical base pairing than were those inferred by other methods. Thus, nucleotide bias in reconstructed sequences apparently can lead to serious bias and inaccuracies in functional predictions.

A Novel Low Energy Electron Microscope for DNA Sequencing and Surface Analysis

PubMed Central

Mankos, M.; Shadman, K.; Persson, H.H.J.; N’Diaye, A.T.; Schmid, A.K.; Davis, R.W.

2014-01-01

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts. The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence. PMID:24524867

A novel low energy electron microscope for DNA sequencing and surface analysis.

PubMed

Mankos, M; Shadman, K; Persson, H H J; N'Diaye, A T; Schmid, A K; Davis, R W

2014-10-01

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts. The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel low energy electron microscope for DNA sequencing and surface analysis

DOE PAGES

Mankos, M.; Shadman, K.; Persson, H. H. J.; ...

2014-01-31

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts.more » The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Finally, experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence.« less

Rapid, sensitive, and selective fluorescent DNA detection using iron-based metal-organic framework nanorods: Synergies of the metal center and organic linker.

PubMed

Tian, Jingqi; Liu, Qian; Shi, Jinle; Hu, Jianming; Asiri, Abdullah M; Sun, Xuping; He, Yuquan

2015-09-15

Considerable recent attention has been paid to homogeneous fluorescent DNA detection with the use of nanostructures as a universal "quencher", but it still remains a great challenge to develop such nanosensor with the benefits of low cost, high speed, sensitivity, and selectivity. In this work, we report the use of iron-based metal-organic framework nanorods as a high-efficient sensing platform for fluorescent DNA detection. It only takes about 4 min to complete the whole "mix-and-detect" process with a low detection limit of 10 pM and a strong discrimination of single point mutation. Control experiments reveal the remarkable sensing behavior is a consequence of the synergies of the metal center and organic linker. This work elucidates how composition control of nanostructures can significantly impact their sensing properties, enabling new opportunities for the rational design of functional materials for analytical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

A new method for enhancer prediction based on deep belief network.

PubMed

Bu, Hongda; Gan, Yanglan; Wang, Yang; Zhou, Shuigeng; Guan, Jihong

2017-10-16

Studies have shown that enhancers are significant regulatory elements to play crucial roles in gene expression regulation. Since enhancers are unrelated to the orientation and distance to their target genes, it is a challenging mission for scholars and researchers to accurately predicting distal enhancers. In the past years, with the high-throughout ChiP-seq technologies development, several computational techniques emerge to predict enhancers using epigenetic or genomic features. Nevertheless, the inconsistency of computational models across different cell-lines and the unsatisfactory prediction performance call for further research in this area. Here, we propose a new Deep Belief Network (DBN) based computational method for enhancer prediction, which is called EnhancerDBN. This method combines diverse features, composed of DNA sequence compositional features, DNA methylation and histone modifications. Our computational results indicate that 1) EnhancerDBN outperforms 13 existing methods in prediction, and 2) GC content and DNA methylation can serve as relevant features for enhancer prediction. Deep learning is effective in boosting the performance of enhancer prediction.

Interfacing DNA nanodevices with biology: challenges, solutions and perspectives

NASA Astrophysics Data System (ADS)

Vinther, Mathias; Kjems, Jørgen

2016-08-01

The cellular machinery performs millions of complex reactions with extreme precision at nanoscale. From studying these reactions, scientists have become inspired to build artificial nanosized molecular devices with programmed functions. One of the fundamental tools in designing and creating these nanodevices is molecular self-assembly. In nature, deoxyribonucleic acid (DNA) is inarguably one of the most remarkable self-assembling molecules. Governed by the Watson-Crick base-pairing rules, DNA assembles with a structural reliability and predictability based on sequence composition unlike any other complex biological polymer. This consistency has enabled rational design of hundreds of two- and three-dimensional shapes with a molecular precision and homogeneity not preceded by any other known technology at the nanometer scale. During the last two decades, DNA nanotechnology has undergone a rapid evolution pioneered by the work of Nadrian Seeman (Kallenbach et al 1983 Nature 205 829-31). Especially the introduction of the versatile DNA Origami technique by Rothemund (2006 Nature 440 297-302) led to an efflorescence of new DNA-based self-assembled nanostructures (Andersen et al 2009 Nature 459 73-6, Douglas et al 2009 Nature 459 414-8, Dietz et al 2009 Science 325 725-30, Han et al 2011 Science 332 342-6, Iinuma et al 2014 Science 344 65-9), and variations of this technique have contributed to an increasing repertoire of DNA nanostructures (Wei et al 2012 Nature 485 623-6, Ke et al 2012 Science 338 1177-83, Benson et al 2015 Nature 523 441-4, Zhang et al 2015 Nat. Nanotechnol. 10 779-84, Scheible et al 2015 Small 11 5200-5). These advances have naturally triggered the question: What can these DNA nanostructures be used for? One of the leading proposals of use for DNA nanotechnology has been in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology.

Biophysics and protein corona analysis of Janus cyclodextrin-DNA nanocomplexes. Efficient cellular transfection on cancer cells.

PubMed

Martínez-Negro, M; Caracciolo, G; Palchetti, S; Pozzi, D; Capriotti, A L; Cavaliere, C; Laganà, A; Ortiz Mellet, C; Benito, J M; García Fernández, J M; Aicart, E; Junquera, E

2017-07-01

The self-assembling processes underlining the capabilities of facially differentiated ("Janus") polycationic amphiphilic cyclodextrins (paCDs) as non-viral gene nanocarriers have been investigated by a pluridisciplinary approach. Three representative Janus paCDs bearing a common tetradecahexanoyl multitail domain at the secondary face and differing in the topology of the cluster of amino groups at the primary side were selected for this study. All of them compact pEGFP-C3 plasmid DNA and promote transfection in HeLa and MCF-7 cells, both in absence and in presence of human serum. The electrochemical and structural characteristics of the paCD-pDNA complexes (CDplexes) have been studied by using zeta potential, DLS, SAXS, and cryo-TEM. paCDs and pDNA, when assembled in CDplexes, render effective charges that are lower than the nominal ones. The CDplexes show a self-assembling pattern corresponding to multilamellar lyotropic liquid crystal phases, characterized by a lamellar stacking of bilayers of the CD-based vectors with anionic pDNA sandwiched among them. When exposed to human serum, either in the absence or in the presence of pDNA, the surface of the cationic CD-based vector becomes coated by a protein corona (PC) whose composition has been analyzed by nanoLC-MS/MS. Some of the CDplexes herein studied showed moderate-to-high transfection levels in HeLa and MCF-7 cancer cells combined with moderate-to-high cell viabilities, as determined by FACS and MTT reduction assays. The ensemble of data provides a detail picture of the paCD-pDNA-PC association processes and a rational base to exploit the protein corona for targeted gene delivery on future in vivo applications. Copyright © 2017 Elsevier B.V. All rights reserved.

454 Pyrosequencing to Describe Microbial Eukaryotic Community Composition, Diversity and Relative Abundance: A Test for Marine Haptophytes

PubMed Central

Egge, Elianne; Bittner, Lucie; Andersen, Tom; Audic, Stéphane; de Vargas, Colomban; Edvardsen, Bente

2013-01-01

Next generation sequencing of ribosomal DNA is increasingly used to assess the diversity and structure of microbial communities. Here we test the ability of 454 pyrosequencing to detect the number of species present, and assess the relative abundance in terms of cell numbers and biomass of protists in the phylum Haptophyta. We used a mock community consisting of equal number of cells of 11 haptophyte species and compared targeting DNA and RNA/cDNA, and two different V4 SSU rDNA haptophyte-biased primer pairs. Further, we tested four different bioinformatic filtering methods to reduce errors in the resulting sequence dataset. With sequencing depth of 11000–20000 reads and targeting cDNA with Haptophyta specific primers Hap454 we detected all 11 species. A rarefaction analysis of expected number of species recovered as a function of sampling depth suggested that minimum 1400 reads were required here to recover all species in the mock community. Relative read abundance did not correlate to relative cell numbers. Although the species represented with the largest biomass was also proportionally most abundant among the reads, there was generally a weak correlation between proportional read abundance and proportional biomass of the different species, both with DNA and cDNA as template. The 454 sequencing generated considerable spurious diversity, and more with cDNA than DNA as template. With initial filtering based only on match with barcode and primer we observed 100-fold more operational taxonomic units (OTUs) at 99% similarity than the number of species present in the mock community. Filtering based on quality scores, or denoising with PyroNoise resulted in ten times more OTU99% than the number of species. Denoising with AmpliconNoise reduced the number of OTU99% to match the number of species present in the mock community. Based on our analyses, we propose a strategy to more accurately depict haptophyte diversity using 454 pyrosequencing. PMID:24069303

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

PubMed

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative Viral Community DNA Analysis Reveals the Dominance of Single-Stranded DNA Viruses in Offshore Upper Bathyal Sediment from Tohoku, Japan

PubMed Central

Yoshida, Mitsuhiro; Mochizuki, Tomohiro; Urayama, Syun-Ichi; Yoshida-Takashima, Yukari; Nishi, Shinro; Hirai, Miho; Nomaki, Hidetaka; Takaki, Yoshihiro; Nunoura, Takuro; Takai, Ken

2018-01-01

Previous studies on marine environmental virology have primarily focused on double-stranded DNA (dsDNA) viruses; however, it has recently been suggested that single-stranded DNA (ssDNA) viruses are more abundant in marine ecosystems. In this study, we performed a quantitative viral community DNA analysis to estimate the relative abundance and composition of both ssDNA and dsDNA viruses in offshore upper bathyal sediment from Tohoku, Japan (water depth = 500 m). The estimated dsDNA viral abundance ranged from 3 × 106 to 5 × 106 genome copies per cm3 sediment, showing values similar to the range of fluorescence-based direct virus counts. In contrast, the estimated ssDNA viral abundance ranged from 1 × 108 to 3 × 109 genome copies per cm3 sediment, thus providing an estimation that the ssDNA viral populations represent 96.3–99.8% of the benthic total DNA viral assemblages. In the ssDNA viral metagenome, most of the identified viral sequences were associated with ssDNA viral families such as Circoviridae and Microviridae. The principle components analysis of the ssDNA viral sequence components from the sedimentary ssDNA viral metagenomic libraries found that the different depth viral communities at the study site all exhibited similar profiles compared with deep-sea sediment ones at other reference sites. Our results suggested that deep-sea benthic ssDNA viruses have been significantly underestimated by conventional direct virus counts and that their contributions to deep-sea benthic microbial mortality and geochemical cycles should be further addressed by such a new quantitative approach. PMID:29467725

DNA Asymmetric Strand Bias Affects the Amino Acid Composition of Mitochondrial Proteins

PubMed Central

Min, Xiang Jia; Hickey, Donal A.

2007-01-01

Abstract Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins. PMID:17974594

Fungal partner shifts during the evolution of mycoheterotrophy in Neottia.

PubMed

Yagame, Takahiro; Ogura-Tsujita, Yuki; Kinoshita, Akihiko; Iwase, Koji; Yukawa, Tomohisa

2016-09-01

Few previous studies have examined how mycobionts change during the evolution from autotrophy to mycoheterotrophy based on phylogenetic hypotheses. Neottia (Orchidaceae) comprises leafy species that are autotrophic and related leafless mycoheterotrophic species, and the phylogenetic relationships among them have been clarified. Accordingly, Neottia is a suitable taxon for investigating the question above. Here we clarified the diversity of mycobionts in Neottia plants and elucidated changes in the character of symbiotic associations during the evolution of mycoheterotrophy. We sequenced the internal transcribed spacer (ITS) regions of nuclear ribosomal (nr) DNA for mycobionts of Neottia plants. Furthermore, we selected one representative DNA sample from each fungal operational taxonomic unit (OTU) and used it to amplify the large subunit (LSU) nrDNA sequences. Phylogenetic analyses of Sebacinales (basidiomycetes), the dominant mycobiont of Neottia, were conducted and sample-based rarefaction curves generated for the observed mycobiont richness on each OTU. Leafy and leafless species in Neottia were associated with Sebacinales Group B and Sebacinales Group A, respectively. The composition and specificity level of fungal partners varied among Neottia species. Fungal partner composition and specificity level changed with speciation in both leafy and leafless Neottia species. In particular, mycorrhizal associations likely shifted from Sebacinales Group B to Group A during the evolution from autotrophy to mycoheterotrophy. Partner shifts to Sebacinales Group A have also been reported in the evolution of mycoheterotrophy of other plant groups, suggesting that convergence to this fungal group occurs in association with the evolution of mycoheterotrophy. © 2016 Botanical Society of America.

DNA-binding studies of a tetraalkyl-substituted porphyrin and the mutually adaptive distortion principle.

PubMed

Ghimire, Srijana; Fanwick, Phillip E; McMillin, David R

2014-10-20

This investigation explores DNA-binding interactions of various forms of an alkyl-substituted cationic porphyrin, H2TC3 (5,10,15,20-tetra[3-(3'-methylimidazolium-1'-yl)]porphyrin). The motivating idea is that incorporating alkyl rather than aryl substituents in the meso positions will enhance the prospects for intercalative as well as external binding to DNA hosts. The ligands may also be applicable for photodynamic and/or anticancer therapy. Methods employed include absorbance, circular dichroism, and emission spectroscopies, as well as viscometry and X-ray crystallography. By comparison with the classical H2T4 system, H2TC3 exhibits a higher molar extinction coefficient but is more prone to self-association. Findings of note include that the copper(II)-containing form Cu(TC3) is adept at internalizing into single-stranded as well as B-form DNA, regardless of the base composition. Surprisingly, however, external binding of H2TC3 occurs within domains that are rich in adenine-thymine base pairs. The difference in the deformability of H2TC3 versus Cu(TC3) probably accounts for the reactivity difference. Finally, Zn(TC3) binds externally, as the metal center remains five-coordinate.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity

PubMed Central

Traverse, Charles C.

2017-01-01

ABSTRACT Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. PMID:28851848

Traceability of Plant Diet Contents in Raw Cow Milk Samples

PubMed Central

Ponzoni, Elena; Mastromauro, Francesco; Gianì, Silvia; Breviario, Diego

2009-01-01

The use of molecular marker in the dairy sector is gaining large acceptance as a reliable diagnostic approach for food authenticity and traceability. Using a PCR approach, the rbcL marker, a chloroplast-based gene, was selected to amplify plant DNA fragments in raw cow milk samples collected from stock farms or bought on the Italian market. rbcL-specific DNA fragments could be found in total milk, as well as in the skimmed and the cream fractions. When the PCR amplified fragments were sent to sequence, the nucleotide composition of the chromatogram reflected the multiple contents of the polyphytic diet. PMID:22253982

A PDDA/poly(2,6-pyridinedicarboxylic acid)-CNTs composite film DNA electrochemical sensor and its application for the detection of specific sequences related to PAT gene and NOS gene.

PubMed

Yang, Tao; Zhang, Wei; Du, Meng; Jiao, Kui

2008-05-30

2,6-Pyridinedicarboxylic acid (PDC) was electropolymerized on the glassy carbon electrode (GCE) surface combined with carboxylic group-functionalized single-walled carbon nanotubes (SWNTs) by cyclic voltammetry (CV) to form PDC-SWNTs composite film, which was rich in negatively charged carboxylic group. Then, poly(diallyldimethyl ammonium chloride) (PDDA), a linear cationic polyelectrolyte, was electrostatically adsorbed on the PDC-SWNTs/GCE surface. DNA probes with negatively charged phosphate group at the 5' end were immobilized on the PDDA/PDC-SWNTs/GCE due to the strong electrostatic attraction between PDDA and phosphate group of DNA. It has been found that modification of the electrode with PDC-SWNTs film has enhanced the effective electrode surface area and electron-transfer ability, in addition to providing negatively charged groups for the electrostatic assembly of cationic polyelectrolyte. PDDA plays a key role in the attachment of DNA probes to the PDC-SWNTs composite film and acts as a bridge to connect DNA with PDC-SWNTs film. The cathodic peak current of methylene blue (MB), an electroactive label, decreased obviously after the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA). This peak current change was used to monitor the recognition of the specific sequences related to PAT gene in the transgenic corn and the polymerase chain reaction (PCR) amplification of NOS gene from the sample of transgenic soybean with satisfactory results. Under optimal conditions, the dynamic detection range of the sensor to PAT gene target sequence was from 1.0x10(-11) to 1.0x10(-6) mol/L with the detection limit of 2.6x10(-12) mol/L.

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

PubMed Central

May, S; Ben-Shaul, A

1997-01-01

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer. PMID:9370436

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

PubMed

Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

2005-10-01

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

Simple sequence repeats in Escherichia coli: abundance, distribution, composition, and polymorphism.

PubMed

Gur-Arie, R; Cohen, C J; Eitan, Y; Shelef, L; Hallerman, E M; Kashi, Y

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.

Naked eye detection of mutagenic DNA photodimers using gold nanoparticles.

PubMed

Kim, Joong Hyun; Chung, Bong Hyun

2011-01-15

We developed a method to detect mutagenic DNA photodimers by the naked eye using gold nanoparticles. The stability of gold nanoparticles in a high ionic strength solution is maintained by straight ssDNA adsorbed physically on the AuNPs. However, we found that UV-irradiated DNA was less adsorptive onto gold nanoparticles because of a conformational change of UV-irradiated DNA. The accumulated deformation of the DNA structure by multiple-dimer formation triggered aggregation of the gold nanoparticles mixed with the UV-irradiated DNA and thus red to purple color changes of the mixture, which allowed colorimetric detection of the DNA photodimers by the naked eye. No fragmented mass and reactive oxygen species production under the UVB irradiation confirmed that the aggregation of gold nanoparticles was solely attributed to the accumulated deformation of the UV irradiated DNA. The degree of gold nanoparticles-aggregation was dependent on the UVB irradiated time and base compositions of the UV-irradiated oligonucleotides. In addition, we successfully demonstrated how to visually qualify the photosensitizing effect of chemical compounds in parallel within only 10 min by applying this new method. Since our method does not require any chemical or biochemical treatments or special instruments for purifying and qualifying the DNA photolesions, it should provide a feasible tool for the studies of the UV-induced mutagenic or carcinogenic DNA dimers and accelerate screening of a large number of drug candidates. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

PubMed Central

Yanik, Mert; Alzubi, Jamal; Lahaye, Thomas; Cathomen, Toni; Pingoud, Alfred; Wende, Wolfgang

2013-01-01

Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity. PMID:24349308

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems.

PubMed

Pawlowski, Jan; Kelly-Quinn, Mary; Altermatt, Florian; Apothéloz-Perret-Gentil, Laure; Beja, Pedro; Boggero, Angela; Borja, Angel; Bouchez, Agnès; Cordier, Tristan; Domaizon, Isabelle; Feio, Maria Joao; Filipe, Ana Filipa; Fornaroli, Riccardo; Graf, Wolfram; Herder, Jelger; van der Hoorn, Berry; Iwan Jones, J; Sagova-Mareckova, Marketa; Moritz, Christian; Barquín, Jose; Piggott, Jeremy J; Pinna, Maurizio; Rimet, Frederic; Rinkevich, Buki; Sousa-Santos, Carla; Specchia, Valeria; Trobajo, Rosa; Vasselon, Valentin; Vitecek, Simon; Zimmerman, Jonas; Weigand, Alexander; Leese, Florian; Kahlert, Maria

2018-05-15

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

PubMed

Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

2015-01-26

The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres

PubMed Central

Xiao, Hua; Wang, Feng; Wisniewski, Jan; Shaytan, Alexey K.; Ghirlando, Rodolfo; FitzGerald, Peter C.; Huang, Yingzi; Wei, Debbie; Li, Shipeng; Landsman, David; Panchenko, Anna R.; Wu, Carl

2017-01-01

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine–lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity. PMID:29074736

Adélie Penguin Population Diet Monitoring by Analysis of Food DNA in Scats

PubMed Central

Jarman, Simon N.; McInnes, Julie C.; Faux, Cassandra; Polanowski, Andrea M.; Marthick, James; Deagle, Bruce E.; Southwell, Colin; Emmerson, Louise

2013-01-01

The Adélie penguin is the most important animal currently used for ecosystem monitoring in the Southern Ocean. The diet of this species is generally studied by visual analysis of stomach contents; or ratios of isotopes of carbon and nitrogen incorporated into the penguin from its food. There are significant limitations to the information that can be gained from these methods. We evaluated population diet assessment by analysis of food DNA in scats as an alternative method for ecosystem monitoring with Adélie penguins as an indicator species. Scats were collected at four locations, three phases of the breeding cycle, and in four different years. A novel molecular diet assay and bioinformatics pipeline based on nuclear small subunit ribosomal RNA gene (SSU rDNA) sequencing was used to identify prey DNA in 389 scats. Analysis of the twelve population sample sets identified spatial and temporal dietary change in Adélie penguin population diet. Prey diversity was found to be greater than previously thought. Krill, fish, copepods and amphipods were the most important food groups, in general agreement with other Adélie penguin dietary studies based on hard part or stable isotope analysis. However, our DNA analysis estimated that a substantial portion of the diet was gelatinous groups such as jellyfish and comb jellies. A range of other prey not previously identified in the diet of this species were also discovered. The diverse prey identified by this DNA-based scat analysis confirms that the generalist feeding of Adélie penguins makes them a useful indicator species for prey community composition in the coastal zone of the Southern Ocean. Scat collection is a simple and non-invasive field sampling method that allows DNA-based estimation of prey community differences at many temporal and spatial scales and provides significant advantages over alternative diet analysis approaches. PMID:24358158

Adélie penguin population diet monitoring by analysis of food DNA in scats.

PubMed

Jarman, Simon N; McInnes, Julie C; Faux, Cassandra; Polanowski, Andrea M; Marthick, James; Deagle, Bruce E; Southwell, Colin; Emmerson, Louise

2013-01-01

The Adélie penguin is the most important animal currently used for ecosystem monitoring in the Southern Ocean. The diet of this species is generally studied by visual analysis of stomach contents; or ratios of isotopes of carbon and nitrogen incorporated into the penguin from its food. There are significant limitations to the information that can be gained from these methods. We evaluated population diet assessment by analysis of food DNA in scats as an alternative method for ecosystem monitoring with Adélie penguins as an indicator species. Scats were collected at four locations, three phases of the breeding cycle, and in four different years. A novel molecular diet assay and bioinformatics pipeline based on nuclear small subunit ribosomal RNA gene (SSU rDNA) sequencing was used to identify prey DNA in 389 scats. Analysis of the twelve population sample sets identified spatial and temporal dietary change in Adélie penguin population diet. Prey diversity was found to be greater than previously thought. Krill, fish, copepods and amphipods were the most important food groups, in general agreement with other Adélie penguin dietary studies based on hard part or stable isotope analysis. However, our DNA analysis estimated that a substantial portion of the diet was gelatinous groups such as jellyfish and comb jellies. A range of other prey not previously identified in the diet of this species were also discovered. The diverse prey identified by this DNA-based scat analysis confirms that the generalist feeding of Adélie penguins makes them a useful indicator species for prey community composition in the coastal zone of the Southern Ocean. Scat collection is a simple and non-invasive field sampling method that allows DNA-based estimation of prey community differences at many temporal and spatial scales and provides significant advantages over alternative diet analysis approaches.

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

PubMed Central

Bohannon, Kevin Patrick; Jun, Yonggun; Gross, Steven P.; Smith, Gregory Allan

2013-01-01

The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection. PMID:23569236

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture.

PubMed

Bohannon, Kevin Patrick; Jun, Yonggun; Gross, Steven P; Smith, Gregory Allan

2013-04-23

The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.

Are mutagenic non D-loop direct repeat motifs in mitochondrial DNA under a negative selection pressure?

PubMed Central

Lakshmanan, Lakshmi Narayanan; Gruber, Jan; Halliwell, Barry; Gunawan, Rudiyanto

2015-01-01

Non D-loop direct repeats (DRs) in mitochondrial DNA (mtDNA) have been commonly implicated in the mutagenesis of mtDNA deletions associated with neuromuscular disease and ageing. Further, these DRs have been hypothesized to put a constraint on the lifespan of mammals and are under a negative selection pressure. Using a compendium of 294 mammalian mtDNA, we re-examined the relationship between species lifespan and the mutagenicity of such DRs. Contradicting the prevailing hypotheses, we found no significant evidence that long-lived mammals possess fewer mutagenic DRs than short-lived mammals. By comparing DR counts in human mtDNA with those in selectively randomized sequences, we also showed that the number of DRs in human mtDNA is primarily determined by global mtDNA properties, such as the bias in synonymous codon usage (SCU) and nucleotide composition. We found that SCU bias in mtDNA positively correlates with DR counts, where repeated usage of a subset of codons leads to more frequent DR occurrences. While bias in SCU and nucleotide composition has been attributed to nucleotide mutational bias, mammalian mtDNA still exhibit higher SCU bias and DR counts than expected from such mutational bias, suggesting a lack of negative selection against non D-loop DRs. PMID:25855815

Identification of Candida lusitaniae as an opportunistic yeast in humans.

PubMed

Holzschu, D L; Presley, H L; Miranda, M; Phaff, H J

1979-08-01

Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa. Because this species has not been recognized previously as an opportunistic yeast in humans, molecular taxonomic methods were applied to confirm its identity. The nuclear deoxyribonucleic acid (DNA) base composition of two clinical isolates was shown to be 45.1 mol% guanine plus cytosine as compared to 44.7 mol% guanine plus cytosine for the type strain of this species. DNA/DNA reassociation experiments revealed more than 95% complementarity between the DNAs from the clinical isolates and that of the type strain of C. lusitaniae, thus confirming their classification by conventional taxonomy. A key is provided to differentiate C. lusitaniae from two phenotypically similar Candida species.

Bacterial communities associated with the pitcher fluids of three Nepenthes (Nepenthaceae) pitcher plant species growing in the wild.

PubMed

Chou, Lee Yiung; Clarke, Charles M; Dykes, Gary A

2014-10-01

Nepenthes pitcher plants produce modified jug-shaped leaves to attract, trap and digest insect prey. We used 16S rDNA cloning and sequencing to compare bacterial communities in pitcher fluids of each of three species, namely Nepenthes ampullaria, Nepenthes gracilis and Nepenthes mirabilis, growing in the wild. In contrast to previous greenhouse-based studies, we found that both opened and unopened pitchers harbored bacterial DNA. Pitchers of N. mirabilis had higher bacterial diversity as compared to other Nepenthes species. The composition of the bacterial communities could be different between pitcher types for N. mirabilis (ANOSIM: R = 0.340, p < 0.05). Other Nepenthes species had similar bacterial composition between pitcher types. SIMPER showed that more than 50 % of the bacterial taxa identified from the open pitchers of N. mirabilis were not found in other groups. Our study suggests that bacteria in N. mirabilis are divided into native and nonnative groups.

Compositional segmentation and complexity measurement in stock indices

NASA Astrophysics Data System (ADS)

Wang, Haifeng; Shang, Pengjian; Xia, Jianan

2016-01-01

In this paper, we introduce a complexity measure based on the entropic segmentation called sequence compositional complexity (SCC) into the analysis of financial time series. SCC was first used to deal directly with the complex heterogeneity in nonstationary DNA sequences. We already know that SCC was found to be higher in sequences with long-range correlation than those with low long-range correlation, especially in the DNA sequences. Now, we introduce this method into financial index data, subsequently, we find that the values of SCC of some mature stock indices, such as S & P 500 (simplified with S & P in the following) and HSI, are likely to be lower than the SCC value of Chinese index data (such as SSE). What is more, we find that, if we classify the indices with the method of SCC, the financial market of Hong Kong has more similarities with mature foreign markets than Chinese ones. So we believe that a good correspondence is found between the SCC of the index sequence and the complexity of the market involved.

Culturable heterotrophic bacteria from the marine sponge Dendrilla nigra: isolation and phylogenetic diversity of actinobacteria

NASA Astrophysics Data System (ADS)

Selvin, Joseph; Gandhimathi, R.; Kiran, G. Seghal; Priya, S. Shanmugha; Ravji, T. Rajeetha; Hema, T. A.

2009-09-01

Culturable heterotrophic bacterial composition of marine sponge Dendrilla nigra was analysed using different enrichments. Five media compositions including without enrichment (control), enriched with sponge extract, with growth regulator (antibiotics), with autoinducers, and complete enrichment containing sponge extract, antibiotics, and autoinducers were developed. DNA hybridization assay was performed to explore host specific bacteria and ecotypes of culturable sponge-associated bacteria. Enrichment with selective inducers (AHLs and sponge extract) and regulators (antibiotics) considerably enhanced the cultivation potential of sponge-associated bacteria. It was found that Marinobacter (MSI032), Micromonospora (MSI033), Streptomyces (MSI051), and Pseudomonas (MSI057) were sponge-associated obligate symbionts. The present findings envisaged that “ Micromonospora-Saccharomonospora-Streptomyces” group was the major culturable actinobacteria in the marine sponge D. nigra. The DNA hybridization assay was a reliable method for the analysis of culturable bacterial community in marine sponges. Based on the culturable community structure, the sponge-associated bacteria can be grouped (ecotypes) as general symbionts, specific symbionts, habitat flora, and antagonists.

Synthesis of novel Eu(III) luminescent probe based on 9- acridinecarboxylic acid skelton for sensing of ds-DNA.

PubMed

Azab, Hassan A; Hussein, Belal H M; El-Falouji, Abdullah I

2012-03-01

Eu(III)-9-acridinecarboxylate (9-ACA) complex was synthesized and characterized by elemental analysis, conductivity measurement, IR spectroscopy, thermal analysis, mass spectroscopy, (1)H-NMR, fluorescence and ultraviolet spectra. The results indicated that the composition of this complex is [Eu(III)-(9-ACA)(2)(NCS)(C(2)H(5)OH)(2)] 2.5 H(2)O and the oxygen of the carbonyl group coordinated to Eu(III). The interaction between the complex with nucleotides guanosine 5'- monophosphate (5'-GMP), adenosine 5'-diphosphates (5'-ADP), inosine (5'-IMP) and CT-DNA was studied by fluorescence spectroscopy. The fluorescence intensity of Eu(III)-9-acridinecarboxylate complex was enhanced with the addition of CT-DNA. The effect of pH values on the fluorescence intensity of Eu(III) complex was investigated. Under experimental conditions, the linear range was 9-50 ng mL(-1) for calf thymus DNA (CT- DNA) and the corresponding detection limit was 5 ng mL(-1). The results showed that Eu(III)-(9-ACA)(2) complex binds to CT-DNA with stability constant of 2.41 × 10(4) M.

Molecular analysis of microbiota along the digestive tract of juvenile Atlantic salmon (Salmo salar L.).

PubMed

Navarrete, P; Espejo, R T; Romero, J

2009-04-01

Dominant bacterial microbiota of the gut of juvenile farmed Atlantic salmon was investigated using a combination of molecular approaches. Bacterial community composition from the stomach, the pyloric caeca, and the intestine was assessed by extracting DNA directly from each gut compartment. Temporal temperature gradient gel electrophoresis (TTGE) analysis of 16S ribosomal DNA (rDNA) amplicons showed very similar bacterial compositions throughout the digestive tract. Band sequencing revealed a narrow diversity of species with a dominance of Pseudomonas in the three compartments. However, cloning revealed more diversity among the Pseudomonas sequences. To confirm these results, we analyzed the bacterial community by amplifying the variable 16S-23S rDNA intergenic spacer region (ITS). Similar ITS profiles were observed among gastrointestinal compartments of salmon, confirming the TTGE results. Moreover, the dominant ITS band at 650 bp, identified as Pseudomonas, was observed in the ITS profile from fish collected in two seasons (July 2003 and 2004). In contrast, aerobic culture analysis revealed Shewanella spp. as the most prevalent isolate. This discrepancy was resolved by evaluating 16S rDNA and ITS polymerase chain reaction amplification efficiency from both Shewanella and Pseudomonas isolates. Very similar efficiencies were observed in the two bacteria. Hence, this discrepancy may be explained by preferential cultivation of Shewanella spp. under the experimental conditions. Also, we included analyses of pelleted feed and the water influent to explore environmental influences on the bacterial composition of the gut microbiota. Overall, these results indicate a homogeneous composition of the bacterial community composition along the gastrointestinal tract of reared juvenile salmon. This community is mainly composed of Pseudomonas spp., which could be derived from water influent and may be selectively associated with salmon in this hatchery.

Changes in Liver Cell DNA Methylation Status in Diabetic Mice Affect Its FT-IR Characteristics

PubMed Central

Vidal, Benedicto de Campos; Ghiraldini, Flávia Gerelli; Mello, Maria Luiza S.

2014-01-01

Background Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. Methodology/Principal Findings The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Conclusions/Significance Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas –CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH3 groups. Other spectral differences were found at 1700–1500 cm−1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice. PMID:25019512

Electrochemical detection of aqueous Ag+ based on Ag+-assisted ligation reaction

NASA Astrophysics Data System (ADS)

Miao, Peng; Han, Kun; Wang, Bidou; Luo, Gangyin; Wang, Peng; Chen, Mingli; Tang, Yuguo

2015-03-01

In this work, a novel strategy to fabricate a highly sensitive and selective biosensor for the detection of Ag+ is proposed. Two DNA probes are designed and modified on a gold electrode surface by gold-sulfur chemistry and hybridization. In the presence of Ag+, cytosine-Ag+-cytosine composite forms and facilitates the ligation event on the electrode surface, which can block the release of electrochemical signals labeled on one of the two DNA probes during denaturation process. Ag+ can be sensitively detected with the detection limit of 0.1 nM, which is much lower than the toxicity level defined by U.S. Environmental Protection Agency. This biosensor can easily distinguish Ag+ from other interfering ions and the performances in real water samples are also satisfactory. Moreover, the two DNA probes are designed to contain the recognition sequences of a nicking endonuclease, and the ligated DNA can thus be cleaved at the original site. Therefore, the electrode can be regenerated, which allows the biosensor to be reused for additional tests.

Linking composition of extracellular polymeric substances (EPS) to the physical structure and hydraulic resistance of membrane biofilms.

PubMed

Desmond, Peter; Best, James P; Morgenroth, Eberhard; Derlon, Nicolas

2018-04-01

The effect of extracellular polymeric substances (EPS) on the meso-scale physical structure and hydraulic resistance of membrane biofilms during gravity driven membrane (GDM) filtration was investigated. Biofilms were developed on the surface of ultrafiltration membranes during dead-end filtration at ultra-low pressure (70 mbar). Biofilm EPS composition (total protein, polysaccharide and eDNA) was manipulated by growing biofilms under contrasting nutrient conditions. Nutrient conditions consisted of (i) a nutrient enriched condition with a nutrient ratio of 100:30:10 (C: N: P), (ii) a phosphorus limitation (C: N: P ratio: 100:30:0), and (iii) a nitrogen limitation (C: N: P ratio: 100:0:10). The structure of the biofilm was characterised at meso-scale using Optical Coherence Tomography (OCT). Biofilm composition was analysed with respect to total organic carbon, total cellular mass and extracellular concentrations of proteins, polysaccharides, and eDNA. 2D-confocal Raman mapping was used to characterise the functional group composition and micro-scale distribution of the biofilms EPS. Our study reveals that the composition of the EPS matrix can determine the meso-scale physical structure of membrane biofilms and in turn its hydraulic resistance. Biofilms grown under P limiting conditions were characterised by dense and homogeneous physical structures with high concentrations of polysaccharides and eDNA. Biofilm grown under nutrient enriched or N limiting conditions were characterised by heterogeneous physical structures with lower concentrations of polysaccharides and eDNA. For P limiting biofilms, 2D-confocal Raman microscopy revealed a homogeneous spatial distribution of anionic functional groups in homogeneous biofilm structures with higher polysaccharide and eDNA concentrations. This study links EPS composition, physical structure and hydraulic resistance of membrane biofilms, with practical relevance for the hydraulic performances of GDM ultrafiltration. Copyright © 2018 Elsevier Ltd. All rights reserved.

Diversity of Bacteria at Healthy Human Conjunctiva

PubMed Central

Dong, Qunfeng; Brulc, Jennifer M.; Iovieno, Alfonso; Bates, Brandon; Garoutte, Aaron; Miller, Darlene; Revanna, Kashi V.; Gao, Xiang; Antonopoulos, Dionysios A.; Slepak, Vladlen Z.

2011-01-01

Purpose. Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria. Methods. Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II). Results. Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera—Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium—were ubiquitous among the analyzed cohort and represented the putative “core” of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens. Conclusions. The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria. PMID:21571682

Facile fabrication of rice husk based silicon dioxide nanospheres loaded with silver nanoparticles as a rice antibacterial agent

PubMed Central

Cui, Jianghu; Liang, You; Yang, Desong; Liu, Yingliang

2016-01-01

Bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice, leading to reduction in production by 10–50%. In order to control this disease, various chemical bactericides have been used. Wide and prolonged application of chemical bactericides resulted in the resistant strain of Xoo that was isolated from rice. To address this problem, we were searching for an environmentally friendly alternative to the commonly used chemical bactericides. In this work, we demonstrate that silicon dioxide nanospheres loaded with silver nanoparticles (SiO2-Ag) can be prepared by using rice husk as base material precursor. The results of the antibacterial tests showed that SiO2-Ag composites displayed antibacterial activity against Xoo. At cellular level, the cell wall/membrane was damaged and intercellular contents were leaked out by slow-releasing of silver ions from SiO2-Ag composites. At molecular level, this composite induced reactive oxygen species production and inhibited DNA replication. Based on the results above, we proposed the potential antibacterial mechanism of SiO2-Ag composites. Moreover, the cytotoxicity assay indicated that the composites showed mild toxicity with rice cells. Thus, this work provided a new strategy to develop biocide derived from residual biomass. PMID:26888152

Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology

NASA Astrophysics Data System (ADS)

Samanta, Anirban; Medintz, Igor L.

2016-04-01

Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their myriad applications using examples from the literature. Overall, it is clear that this unique functional combination of nanomaterials has far more to offer than what we have seen to date and as new capabilities for each of these materials are developed, so, too, will new applications emerge.

Context based computational analysis and characterization of ARS consensus sequences (ACS) of Saccharomyces cerevisiae genome.

PubMed

Singh, Vinod Kumar; Krishnamachari, Annangarachari

2016-09-01

Genome-wide experimental studies in Saccharomyces cerevisiae reveal that autonomous replicating sequence (ARS) requires an essential consensus sequence (ACS) for replication activity. Computational studies identified thousands of ACS like patterns in the genome. However, only a few hundreds of these sites act as replicating sites and the rest are considered as dormant or evolving sites. In a bid to understand the sequence makeup of replication sites, a content and context-based analysis was performed on a set of replicating ACS sequences that binds to origin-recognition complex (ORC) denoted as ORC-ACS and non-replicating ACS sequences (nrACS), that are not bound by ORC. In this study, DNA properties such as base composition, correlation, sequence dependent thermodynamic and DNA structural profiles, and their positions have been considered for characterizing ORC-ACS and nrACS. Analysis reveals that ORC-ACS depict marked differences in nucleotide composition and context features in its vicinity compared to nrACS. Interestingly, an A-rich motif was also discovered in ORC-ACS sequences within its nucleosome-free region. Profound changes in the conformational features, such as DNA helical twist, inclination angle and stacking energy between ORC-ACS and nrACS were observed. Distribution of ACS motifs in the non-coding segments points to the locations of ORC-ACS which are found far away from the adjacent gene start position compared to nrACS thereby enabling an accessible environment for ORC-proteins. Our attempt is novel in considering the contextual view of ACS and its flanking region along with nucleosome positioning in the S. cerevisiae genome and may be useful for any computational prediction scheme.

Ability of Polyphosphate and Nucleic Acids to Trigger Blood Clotting: Some Observations and Caveats

PubMed Central

Smith, Stephanie A.; Gajsiewicz, Joshua M.; Morrissey, James H.

2018-01-01

Polyphosphate plays several roles in coagulation and inflammation, while extracellular DNA and RNA are implicated in thrombosis and as disease biomarkers. We sought to compare the procoagulant activities of polyphosphate versus DNA or RNA isolated from mammalian cells. In a recent study, we found that much of the procoagulant activity of DNA isolated from mammalian cells using Qiagen kits resisted digestion with nuclease or polyphosphatase, and even resisted boiling in acid. These kits employ spin columns packed with silica, which is highly procoagulant. Indeed, much of the apparent procoagulant activity of cellular DNA isolated with such kits was attributable to silica particles shed by the spin columns. Therefore, silica-based methods for isolating nucleic acids or polyphosphate from mammalian cells are not suitable for studying their procoagulant activities. We now report that polyphosphate readily co-purified with DNA and RNA using several popular isolation methods, including phenol/chloroform extraction. Thus, cell-derived nucleic acids are also subject to contamination with traces of cellular polyphosphate, which can be eliminated by alkaline phosphatase digestion. We further report that long-chain polyphosphate was orders of magnitude more potent than cell-derived DNA (purified via phenol/chloroform extraction) or RNA at triggering clotting. Additional experiments using RNA homopolymers found that polyG and polyI have procoagulant activity similar to polyphosphate, while polyA and polyC are not procoagulant. Thus, the procoagulant activity of RNA is rather highly dependent on base composition. PMID:29719836

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

PubMed Central

Srinivasan, Usha; Ponnaluri, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F.; Iyer, Ram K.; Misra, Dawn; Foxman, Betsy

2012-01-01

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 108 cfu/ml of Escherichia coli and 105 cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces. PMID:23071487

Gram stains: a resource for retrospective analysis of bacterial pathogens in clinical studies.

PubMed

Srinivasan, Usha; Ponnaluri, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F; Iyer, Ram K; Misra, Dawn; Foxman, Betsy

2012-01-01

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10(8) cfu/ml of Escherichia coli and 10(5) cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.

Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor.

PubMed

Kohany, Oleksiy; Gentles, Andrew J; Hankus, Lukasz; Jurka, Jerzy

2006-10-25

Repbase is a reference database of eukaryotic repetitive DNA, which includes prototypic sequences of repeats and basic information described in annotations. Updating and maintenance of the database requires specialized tools, which we have created and made available for use with Repbase, and which may be useful as a template for other curated databases. We describe the software tools RepbaseSubmitter and Censor, which are designed to facilitate updating and screening the content of Repbase. RepbaseSubmitter is a java-based interface for formatting and annotating Repbase entries. It eliminates many common formatting errors, and automates actions such as calculation of sequence lengths and composition, thus facilitating curation of Repbase sequences. In addition, it has several features for predicting protein coding regions in sequences; searching and including Pubmed references in Repbase entries; and searching the NCBI taxonomy database for correct inclusion of species information and taxonomic position. Censor is a tool to rapidly identify repetitive elements by comparison to known repeats. It uses WU-BLAST for speed and sensitivity, and can conduct DNA-DNA, DNA-protein, or translated DNA-translated DNA searches of genomic sequence. Defragmented output includes a map of repeats present in the query sequence, with the options to report masked query sequence(s), repeat sequences found in the query, and alignments. Censor and RepbaseSubmitter are available as both web-based services and downloadable versions. They can be found at http://www.girinst.org/repbase/submission.html (RepbaseSubmitter) and http://www.girinst.org/censor/index.php (Censor).

Structural features based genome-wide characterization and prediction of nucleosome organization

PubMed Central

2012-01-01

Background Nucleosome distribution along chromatin dictates genomic DNA accessibility and thus profoundly influences gene expression. However, the underlying mechanism of nucleosome formation remains elusive. Here, taking a structural perspective, we systematically explored nucleosome formation potential of genomic sequences and the effect on chromatin organization and gene expression in S. cerevisiae. Results We analyzed twelve structural features related to flexibility, curvature and energy of DNA sequences. The results showed that some structural features such as DNA denaturation, DNA-bending stiffness, Stacking energy, Z-DNA, Propeller twist and free energy, were highly correlated with in vitro and in vivo nucleosome occupancy. Specifically, they can be classified into two classes, one positively and the other negatively correlated with nucleosome occupancy. These two kinds of structural features facilitated nucleosome binding in centromere regions and repressed nucleosome formation in the promoter regions of protein-coding genes to mediate transcriptional regulation. Based on these analyses, we integrated all twelve structural features in a model to predict more accurately nucleosome occupancy in vivo than the existing methods that mainly depend on sequence compositional features. Furthermore, we developed a novel approach, named DLaNe, that located nucleosomes by detecting peaks of structural profiles, and built a meta predictor to integrate information from different structural features. As a comparison, we also constructed a hidden Markov model (HMM) to locate nucleosomes based on the profiles of these structural features. The result showed that the meta DLaNe and HMM-based method performed better than the existing methods, demonstrating the power of these structural features in predicting nucleosome positions. Conclusions Our analysis revealed that DNA structures significantly contribute to nucleosome organization and influence chromatin structure and gene expression regulation. The results indicated that our proposed methods are effective in predicting nucleosome occupancy and positions and that these structural features are highly predictive of nucleosome organization. The implementation of our DLaNe method based on structural features is available online. PMID:22449207

Biosensors for DNA sequence detection

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; Akeson, Mark

2002-01-01

DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

Nucleotide sequence composition and method for detection of neisseria gonorrhoeae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lo, A.; Yang, H.L.

1990-02-13

This patent describes a composition of matter that is specific for {ital Neisseria gonorrhoeae}. It comprises: at least one nucleotide sequence for which the ratio of the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria gonorrhoeae} to the amount of the sequence which hybridizes to chromosomal DNA of {ital Neisseria meningitidis} is greater than about five. The ratio being obtained by a method described.

Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers

NASA Technical Reports Server (NTRS)

Luu, Y. K.; Kim, K.; Hsiao, B. S.; Chu, B.; Hadjiargyrou, M.; Hadjiargyou, M. (Principal Investigator)

2003-01-01

The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.

Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation Technology.

PubMed

Schrand, Amanda M; Powell, Thomas; Robertson, Tiffany; Hussain, Saber M

2015-02-01

In this study, we examined the feasibility of extracting DNA from whole cell lysates exposed to nanoparticles using two different methodologies for evaluation of fragmentation with microfluidic electrophoretic separation. Human lung macrophages were exposed to five different carbon- and metal-based nanoparticles at two different time points (2 h, 24 h) and two different doses (5 µg/ml, 100 µg/ml). The primary difference in the banding patterns after 2 h of nanoparticle exposure is more DNA fragmentation at the higher NP concentration when examining cells exposed to nanoparticles of the same composition. However, higher doses of carbon and silver nanoparticles at both short and long dosing periods can contribute to erroneous or incomplete data with this technique. Also comparing DNA isolation methodologies, we recommend the centrifugation extraction technique, which provides more consistent banding patterns in the control samples compared to the spooling technique. Here we demonstrate that multi-walled carbon nanotubes, 15 nm silver nanoparticles and the positive control cadmium oxide cause similar DNA fragmentation at the short time point of 2 h with the centrifugation extraction technique. Therefore, the results of these studies contribute to elucidating the relationship between nanoparticle physicochemical properties and DNA fragmentation results while providing the pros and cons of altering the DNA isolation methodology. Overall, this technique provides a high throughput way to analyze subcellular alterations in DNA profiles of cells exposed to nanomaterials to aid in understanding the consequences of exposure and mechanistic effects. Future studies in microfluidic electrophoretic separation technologies should be investigated to determine the utility of protein or other assays applicable to cellular systems exposed to nanoparticles.

Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

PubMed

Tian, Yang; Li, Yan Hong

2017-01-01

To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA recovery from soils of diverse composition.

PubMed

Zhou, J; Bruns, M A; Tiedje, J M

1996-02-01

A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.

Chromatin remodelling: the industrial revolution of DNA around histones.

PubMed

Saha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R

2006-06-01

Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications regulate remodeller activity and consider a model for the exposure of nucleosomal DNA that involves the use of directional DNA translocation to pump 'DNA waves' around the nucleosome.

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

PubMed Central

Shmookler Reis, R; Timmis, J N; Ingle, J

1981-01-01

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences. Images Fig. 2. PLATE 1 Fig. 4. Fig. 10. PMID:6172117

Accelerated epigenetic aging in Werner syndrome.

PubMed

Maierhofer, Anna; Flunkert, Julia; Oshima, Junko; Martin, George M; Haaf, Thomas; Horvath, Steve

2017-04-01

Individuals suffering from Werner syndrome (WS) exhibit many clinical signs of accelerated aging. While the underlying constitutional mutation leads to accelerated rates of DNA damage, it is not yet known whether WS is also associated with an increased epigenetic age according to a DNA methylation based biomarker of aging (the "Epigenetic Clock"). Using whole blood methylation data from 18 WS cases and 18 age matched controls, we find that WS is associated with increased extrinsic epigenetic age acceleration (p=0.0072) and intrinsic epigenetic age acceleration (p=0.04), the latter of which is independent of age-related changes in the composition of peripheral blood cells. A multivariate model analysis reveals that WS is associated with an increase in DNA methylation age (on average 6.4 years, p=0.011) even after adjusting for chronological age, gender, and blood cell counts. Further, WS might be associated with a reduction in naïve CD8+ T cells (p=0.025) according to imputed measures of blood cell counts. Overall, this study shows that WS is associated with an increased epigenetic age of blood cells which is independent of changes in blood cell composition. The extent to which this alteration is a cause or effect of WS disease phenotypes remains unknown.

Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks

PubMed Central

Belotserkovskii, Boris P.; Neil, Alexander J.; Saleh, Syed Shayon; Shin, Jane Hae Soo; Mirkin, Sergei M.; Hanawalt, Philip C.

2013-01-01

The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816–12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation. PMID:23275544

DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.

PubMed

Gaget, V; Keulen, A; Lau, M; Monis, P; Brookes, J D

2017-01-01

Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing. © 2016 The Society for Applied Microbiology.

On the Origin of the Canonical Nucleobases: An Assessment of Selection Pressures across Chemical and Early Biological Evolution

PubMed Central

Rios, Andro C.

2014-01-01

The native bases of RNA and DNA are prominent examples of the narrow selection of organic molecules upon which life is based. How did nature “decide” upon these specific heterocycles? Evidence suggests that many types of heterocycles could have been present on the early Earth. It is therefore likely that the contemporary composition of nucleobases is a result of multiple selection pressures that operated during early chemical and biological evolution. The persistence of the fittest heterocycles in the prebiotic environment towards, for example, hydrolytic and photochemical assaults, may have given some nucleobases a selective advantage for incorporation into the first informational polymers. The prebiotic formation of polymeric nucleic acids employing the native bases remains, however, a challenging problem to reconcile. Hypotheses have proposed that the emerging RNA world may have included many types of nucleobases. This is supported by the extensive utilization of non-canonical nucleobases in extant RNA and the resemblance of many of the modified bases to heterocycles generated in simulated prebiotic chemistry experiments. Selection pressures in the RNA world could have therefore narrowed the composition of the nucleic acid bases. Two such selection pressures may have been related to genetic fidelity and duplex stability. Considering these possible selection criteria, the native bases along with other related heterocycles seem to exhibit a certain level of fitness. We end by discussing the strength of the N-glycosidic bond as a potential fitness parameter in the early DNA world, which may have played a part in the refinement of the alphabetic bases. PMID:25284884

Cationic lipids bearing succinic-based, acyclic and macrocyclic hydrophobic domains: Synthetic studies and in vitro gene transfer.

PubMed

Jubeli, Emile; Maginty, Amanda B; Khalique, Nada Abdul; Raju, Liji; Nicholson, David G; Larsen, Helge; Pungente, Michael D; Goldring, William P D

2017-01-05

In this communication we describe the construction of four succinic-based cationic lipids, their formulation with plasmid DNA (pDNA), and an evaluation of their in vitro gene delivery into Chinese hamster ovarian (CHO-K1) cells. The cationic lipids employed in this work possess either a dimethylamine or trimethylamine headgroup, and a macrocyclic or an acyclic hydrophobic domain composed of, or derived from two 16-atom, succinic-based acyl chains. The synthesized lipids and a co-lipid of neutral charge, either cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were formulated in an overall 3:2 cationic-to-neutral lipid molar ratio, then complexed with plasmid DNA (pDNA). The relative transfection performance was evaluated via a comparison between matched versus mismatched formulations defined by the rigidity relationship between the lipids employed. Gel electrophoresis was used to characterize the binding of the lipid formulations with plasmid DNA and the relative degree of plasmid degradation using a DNase I degradation assay. Small angle X-ray diffraction (SAXD) was employed to characterize the packing morphology of the lipid-DNA complexes. In general, the succinic unit embedded within the hydrophobic domain of the cationic lipids was found to improve lipid hydration. The transfection assays revealed a general trend in which mismatched formulations that employed a rigid lipid combined with a non-rigid (or flexible) lipid, outperformed the matched formulations. The results from this work suggest that the design of the cationic lipid structure and the composition of the lipoplex formulation play key roles in governing the transfection performance of nonviral gene delivery agents. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties

PubMed Central

Somyoonsap, Peechapack; Kitpreechavanich, Vichein

2013-01-01

A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease. PMID:25937959

The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows

PubMed Central

Vaidya, Jueeli D.; van den Bogert, Bartholomeus; Edwards, Joan E.; Boekhorst, Jos; van Gastelen, Sanne; Saccenti, Edoardo; Plugge, Caroline M.; Smidt, Hauke

2018-01-01

DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method (p < 0.001) and fraction (p < 0.001). The 260/280 ratio was not affected by extraction (p = 0.08) but was affected by fraction (p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method (p < 0.001) but not affected by fraction (p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction (p = 0.012), and that PBB (p = 0.012) and FDSS (p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota. PMID:29445366

The Effect of DNA Extraction Methods on Observed Microbial Communities from Fibrous and Liquid Rumen Fractions of Dairy Cows.

PubMed

Vaidya, Jueeli D; van den Bogert, Bartholomeus; Edwards, Joan E; Boekhorst, Jos; van Gastelen, Sanne; Saccenti, Edoardo; Plugge, Caroline M; Smidt, Hauke

2018-01-01

DNA based methods have been widely used to study the complexity of the rumen microbiota, and it is well known that the method of DNA extraction is a critical step in enabling accurate assessment of this complexity. Rumen fluid (RF) and fibrous content (FC) fractions differ substantially in terms of their physical nature and associated microorganisms. The aim of this study was therefore to assess the effect of four DNA extraction methods (RBB, PBB, FDSS, PQIAmini) differing in cell lysis and/or DNA recovery methods on the observed microbial diversity in RF and FC fractions using samples from four rumen cannulated dairy cows fed 100% grass silage (GS100), 67% GS and 33% maize silage (GS67MS33), 33% GS and 67% MS (GS33MS67), or 100% MS (MS100). An ANOVA statistical test was applied on DNA quality and yield measurements, and it was found that the DNA yield was significantly affected by extraction method ( p < 0.001) and fraction ( p < 0.001). The 260/280 ratio was not affected by extraction ( p = 0.08) but was affected by fraction ( p = 0.03). On the other hand, the 260/230 ratio was affected by extraction method ( p < 0.001) but not affected by fraction ( p = 0.8). However, all four extraction procedures yielded DNA suitable for further analysis of bacterial, archaeal and anaerobic fungal communities using quantitative PCR and pyrosequencing of relevant taxonomic markers. Redundancy analysis (RDA) of bacterial 16S rRNA gene sequence data at the family level showed that there was a significant effect of rumen fraction ( p = 0.012), and that PBB ( p = 0.012) and FDSS ( p = 0.024) also significantly contributed to explaining the observed variation in bacterial community composition. Whilst the DNA extraction method affected the apparent bacterial community composition, no single extraction method could be concluded to be ineffective. No obvious effect of DNA extraction method on the anaerobic fungi or archaea was observed, although fraction effects were evident for both. In summary, the comprehensive assessment of observed communities of bacteria, archaea and anaerobic fungi described here provides insight into a rational basis for selecting an optimal methodology to obtain a representative picture of the rumen microbiota.

Comparison of DNA preservation methods for environmental bacterial community samples

USGS Publications Warehouse

Gray, Michael A.; Pratte, Zoe A.; Kellogg, Christina A.

2013-01-01

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard™, RNAlater®, DMSO–EDTA–salt (DESS), FTA® cards, and FTA Elute® cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA® cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard™, RNAlater®, and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

iDNA-Prot: Identification of DNA Binding Proteins Using Random Forest with Grey Model

PubMed Central

Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

2011-01-01

DNA-binding proteins play crucial roles in various cellular processes. Developing high throughput tools for rapidly and effectively identifying DNA-binding proteins is one of the major challenges in the field of genome annotation. Although many efforts have been made in this regard, further effort is needed to enhance the prediction power. By incorporating the features into the general form of pseudo amino acid composition that were extracted from protein sequences via the “grey model” and by adopting the random forest operation engine, we proposed a new predictor, called iDNA-Prot, for identifying uncharacterized proteins as DNA-binding proteins or non-DNA binding proteins based on their amino acid sequences information alone. The overall success rate by iDNA-Prot was 83.96% that was obtained via jackknife tests on a newly constructed stringent benchmark dataset in which none of the proteins included has pairwise sequence identity to any other in a same subset. In addition to achieving high success rate, the computational time for iDNA-Prot is remarkably shorter in comparison with the relevant existing predictors. Hence it is anticipated that iDNA-Prot may become a useful high throughput tool for large-scale analysis of DNA-binding proteins. As a user-friendly web-server, iDNA-Prot is freely accessible to the public at the web-site on http://icpr.jci.edu.cn/bioinfo/iDNA-Prot or http://www.jci-bioinfo.cn/iDNA-Prot. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results. PMID:21935457

Compositional searching of CpG islands in the human genome

NASA Astrophysics Data System (ADS)

Luque-Escamilla, Pedro Luis; Martínez-Aroza, José; Oliver, José L.; Gómez-Lopera, Juan Francisco; Román-Roldán, Ramón

2005-06-01

We report on an entropic edge detector based on the local calculation of the Jensen-Shannon divergence with application to the search for CpG islands. CpG islands are pieces of the genome related to gene expression and cell differentiation, and thus to cancer formation. Searching for these CpG islands is a major task in genetics and bioinformatics. Some algorithms have been proposed in the literature, based on moving statistics in a sliding window, but its size may greatly influence the results. The local use of Jensen-Shannon divergence is a completely different strategy: the nucleotide composition inside the islands is different from that in their environment, so a statistical distance—the Jensen-Shannon divergence—between the composition of two adjacent windows may be used as a measure of their dissimilarity. Sliding this double window over the entire sequence allows us to segment it compositionally. The fusion of those segments into greater ones that satisfy certain identification criteria must be achieved in order to obtain the definitive results. We find that the local use of Jensen-Shannon divergence is very suitable in processing DNA sequences for searching for compositionally different structures such as CpG islands, as compared to other algorithms in literature.

Map-based cloning of a gene controlling Omega-3 fatty acid desaturation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arondel, V.; Lemieux, B.; Hwang, I.

1992-11-20

A gene from the flowering plant Arabidopsis thaliana that encodes an omega-3 desaturase was cloned on the basis of the genetic map position of a mutation affecting membrane and storage lipid fatty acid composition. Yeast artificial chromosomes covering the genetic locus were identified and used to probe a seed complementary DNA library. A complementary DNA clone for the desaturase was identified and introduced into roots of both wild-type and mutant plants by Ti plasmid-mediated transformation. Transgenic tissues of both mutant and wild-type plants had significantly increased amounts of the fatty acid produced by this desaturase. 24 refs., 2 figs., 1more » tabs.« less

Taxonomic study of extreme halophilic archaea isolated from the "Salar de Atacama", Chile.

PubMed

Lizama, C; Monteoliva-Sánchez, M; Prado, B; Ramos-Cormenzana, A; Weckesser, J; Campos, V

2001-11-01

A large number of halophilic bacteria were isolated in 1984-1992 from the Atacama Saltern (North of Chile). For this study 82 strains of extreme halophilic archaea were selected. The characterization was performed by using the phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility test. The results, together with those from reference strains, were subjected to numerical analysis, using the Simple Matching (S(SM)) coefficient and clustered by the unweighted pair group method of association (UPGMA). Fifteen phena were obtained at an 70% similarity level. The results obtained reveal a high diversity among the halophilic archaea isolated. Representative strains from the phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains. The 16S rRNA studies showed that some of these strains constitutes a new taxa of extreme halophilic archaea.

Identification of Candida lusitaniae as an opportunistic yeast in humans.

PubMed Central

Holzschu, D L; Presley, H L; Miranda, M; Phaff, H J

1979-01-01

Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa. Because this species has not been recognized previously as an opportunistic yeast in humans, molecular taxonomic methods were applied to confirm its identity. The nuclear deoxyribonucleic acid (DNA) base composition of two clinical isolates was shown to be 45.1 mol% guanine plus cytosine as compared to 44.7 mol% guanine plus cytosine for the type strain of this species. DNA/DNA reassociation experiments revealed more than 95% complementarity between the DNAs from the clinical isolates and that of the type strain of C. lusitaniae, thus confirming their classification by conventional taxonomy. A key is provided to differentiate C. lusitaniae from two phenotypically similar Candida species. PMID:292646

Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling

PubMed Central

Lu, Hsiao-Pei; Liu, Po-Yu; Wang, Yu-bin; Hsieh, Ji-Fan; Ho, Han-Chen; Huang, Shiao-Wei; Lin, Chung-Yen; Hsieh, Chih-hao; Yu, Hon-Tsen

2018-01-01

Mammalian herbivores rely on microbial activities in an expanded gut chamber to convert plant biomass into absorbable nutrients. Distinct from ruminants, small herbivores typically have a simple stomach but an enlarged cecum to harbor symbiotic microbes; however, knowledge of this specialized gut structure and characteristics of its microbial contents is limited. Here, we used leaf-eating flying squirrels as a model to explore functional characteristics of the cecal microbiota adapted to a high-fiber, toxin-rich diet. Specifically, environmental conditions across gut regions were evaluated by measuring mass, pH, feed particle size, and metabolomes. Then, parallel metagenomes and metatranscriptomes were used to detect microbial functions corresponding to the cecal environment. Based on metabolomic profiles, >600 phytochemical compounds were detected, although many were present only in the foregut and probably degraded or transformed by gut microbes in the hindgut. Based on metagenomic (DNA) and metatranscriptomic (RNA) profiles, taxonomic compositions of the cecal microbiota were dominated by bacteria of the Firmicutes taxa; they contained major gene functions related to degradation and fermentation of leaf-derived compounds. Based on functional compositions, genes related to multidrug exporters were rich in microbial genomes, whereas genes involved in nutrient importers were rich in microbial transcriptomes. In addition, genes encoding chemotaxis-associated components and glycoside hydrolases specific for plant beta-glycosidic linkages were abundant in both DNA and RNA. This exploratory study provides findings which may help to form molecular-based hypotheses regarding functional contributions of symbiotic gut microbiota in small herbivores with folivorous dietary habits. PMID:29354108

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry.

PubMed

Mariappan, S V Santhana; Cheng, Xun; van Breemen, Richard B; Silks, Louis A; Gupta, Goutam

2004-11-15

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.

Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

PubMed

Oyola, Samuel O; Otto, Thomas D; Gu, Yong; Maslen, Gareth; Manske, Magnus; Campino, Susana; Turner, Daniel J; Macinnis, Bronwyn; Kwiatkowski, Dominic P; Swerdlow, Harold P; Quail, Michael A

2012-01-03

Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Fluorescence Visual Detection of Herbal Product Substitutions at Terminal Herbal Markets by CCP-based FRET technique.

PubMed

Jiang, Chao; Yuan, Yuan; Yang, Guang; Jin, Yan; Liu, Libing; Zhao, Yuyang; Huang, Luqi

2016-10-21

Inaccurate labeling of materials used in herbal products may compromise the therapeutic efficacy and may pose a threat to medicinal safety. In this paper, a rapid (within 3 h), sensitive and visual colorimetric method for identifying substitutions in terminal market products was developed using cationic conjugated polymer-based fluorescence resonance energy transfer (CCP-based FRET). Chinese medicinal materials with similar morphology and chemical composition were clearly distinguished by the single-nucleotide polymorphism (SNP) genotyping method. Assays using CCP-based FRET technology showed a high frequency of adulterants in Lu-Rong (52.83%) and Chuan-Bei-Mu (67.8%) decoction pieces, and patented Chinese drugs (71.4%, 5/7) containing Chuan-Bei-Mu ingredients were detected in the terminal herbal market. In comparison with DNA sequencing, this protocol simplifies procedures by eliminating the cumbersome workups and sophisticated instruments, and only a trace amount of DNA is required. The CCP-based method is particularly attractive because it can detect adulterants in admixture samples with high sensitivity. Therefore, the CCP-based detection system shows great potential for routine terminal market checks and drug safety controls.

Structure, stability, and thermodynamics of lamellar DNA-lipid complexes.

PubMed Central

Harries, D; May, S; Gelbart, W M; Ben-Shaul, A

1998-01-01

We develop a statistical thermodynamic model for the phase evolution of DNA-cationic lipid complexes in aqueous solution, as a function of the ratios of charged to neutral lipid and charged lipid to DNA. The complexes consist of parallel strands of DNA intercalated in the water layers of lamellar stacks of mixed lipid bilayers, as determined by recent synchrotron x-ray measurements. Elastic deformations of the DNA and the lipid bilayers are neglected, but DNA-induced spatial inhomogeneities in the bilayer charge densities are included. The relevant nonlinear Poisson-Boltzmann equation is solved numerically, including self-consistent treatment of the boundary conditions at the polarized membrane surfaces. For a wide range of lipid compositions, the phase evolution is characterized by three regions of lipid to DNA charge ratio, rho: 1) for low rho, the complexes coexist with excess DNA, and the DNA-DNA spacing in the complex, d, is constant; 2) for intermediate rho, including the isoelectric point rho = 1, all of the lipid and DNA in solution is incorporated into the complex, whose inter-DNA distance d increases linearly with rho; and 3) for high rho, the complexes coexist with excess liposomes (whose lipid composition is different from that in the complex), and their spacing d is nearly, but not completely, independent of rho. These results can be understood in terms of a simple charging model that reflects the competition between counterion entropy and inter-DNA (rho < 1) and interbilayer (rho > 1) repulsions. Finally, our approach and conclusions are compared with theoretical work by others, and with relevant experiments. PMID:9649376

Disposable DNA biosensor with the carbon nanotubes-polyethyleneimine interface at a screen-printed carbon electrode for tests of DNA layer damage by quinazolines.

PubMed

Galandová, Júlia; Ovádeková, Renáta; Ferancová, Adriana; Labuda, Ján

2009-06-01

A screen-printed carbon working electrode within a commercially available screen-printed three-electrode assembly was modified by using a composite of multiwalled carbon nanotubes (MWCNT) dispersed in polyethylenimine (PEI) followed by covering with the calf thymus dsDNA layer. Several electrochemical methods were used to characterize the biosensor and to evaluate damage to the surface-attached DNA: square wave voltammetry of the [Ru(bpy)(3)](2+) redox indicator and mediator of the guanine moiety oxidation, cyclic voltammetry and electrochemical impedance spectroscopy in the presence of the [Fe(CN)(6)](3-/4-) indicator in solution. Due to high electroconductivity and large surface area of MWCNT and positive charge of PEI, the MWCNT-PEI composite is an advantageous platform for the DNA immobilization by the polyelectrolyte complexation and its voltammetric and impedimetric detection. In this respect, the MWCNT-PEI interface exhibited better properties than the MWCNT-chitosan one reported from our laboratory previously. A deep DNA layer damage at incubation of the biosensor in quinazoline solution was found, which depends on the quinazoline concentration and incubation time.

Characteristics and oxidative stress on rats and traffic policemen of ambient fine particulate matter from Shenyang.

PubMed

Ma, Mingyue; Li, Shuyin; Jin, Huanrong; Zhang, Yumin; Xu, Jia; Chen, Dongmei; Kuimin, Chen; Yuan, Zhou; Xiao, Chunling

2015-09-01

Fine particulate matter (PM2.5) pollution is becoming serious in China. This study aimed to investigate the impact of PM2.5 on DNA damage in Shenyang city. The concentration and composition of PM2.5 in traffic policemen's working sites including fields and indoor offices were obtained. Blood samples of field and office policemen were collected to detect DNA damage by Comet assay. Rats were used to further analyzing the oxidative DNA damage. The average concentration of PM2.5 in exposed group was significantly higher than that in control group. Composition analysis revealed that toxic heavy metal and polycyclic aromatic hydrocarbon substances were main elements of this PM2.5. DNA damage in field policemen was significantly higher than those in non-field group. Moreover, animal studies confirmed the oxidative DNA damage induced by PM2.5. Taken together, high DNA damages are found in the Shenyang traffic policemen and rats exposed to high level of airborne PM2.5. Copyright © 2015 Elsevier B.V. All rights reserved.

DNA barcoding reveals seasonal shifts in diet and consumption of deep-sea fishes in wedge-tailed shearwaters

PubMed Central

Ando, Haruko; Horikoshi, Kazuo; Suzuki, Hajime; Isagi, Yuji

2018-01-01

The foraging ecology of pelagic seabirds is difficult to characterize because of their large foraging areas. In the face of this difficulty, DNA metabarcoding may be a useful approach to analyze diet compositions and foraging behaviors. Using this approach, we investigated the diet composition and its seasonal variation of a common seabird species on the Ogasawara Islands, Japan: the wedge-tailed shearwater Ardenna pacifica. We collected fecal samples during the prebreeding (N = 73) and rearing (N = 96) periods. The diet composition of wedge-tailed shearwater was analyzed by Ion Torrent sequencing using two universal polymerase chain reaction primers for the 12S and 16S mitochondrial DNA regions that targeted vertebrates and mollusks, respectively. The results of a BLAST search of obtained sequences detected 31 and 1 vertebrate and mollusk taxa, respectively. The results of the diet composition analysis showed that wedge-tailed shearwaters frequently consumed deep-sea fishes throughout the sampling season, indicating the importance of these fishes as a stable food resource. However, there was a marked seasonal shift in diet, which may reflect seasonal changes in food resource availability and wedge-tailed shearwater foraging behavior. The collected data regarding the shearwater diet may be useful for in situ conservation efforts. Future research that combines DNA metabarcoding with other tools, such as data logging, may provide further insight into the foraging ecology of pelagic seabirds. PMID:29630670

Using faecal DNA to determine consumption by kangaroos of plants considered palatable to sheep.

PubMed

Ho, K W; Krebs, G L; McCafferty, P; van Wyngaarden, S P; Addison, J

2010-02-01

Disagreement exists within the scientific community with regards to the level of competition for feed between sheep and kangaroos in the Australian rangelands. The greatest challenge to solving this debate is finding effective means of determining the composition of the diets of these potential grazing competitors. An option is to adopt a non-invasive approach that combines faecal collection and molecular techniques that focus on faecal DNA as the primary source of dietary information. As proof-of-concept, we show that a DNA reference data bank on plant species can be established. This DNA reference data bank was then used as a library to identify plant species in kangaroo faeces collected in the southern rangelands of Western Australia. To enhance the method development and to begin the investigation of competitive grazing between sheep and kangaroos, 16 plant species known to be palatable to sheep were initially targeted for collection. To ensure that only plant sequences were studied, PCR amplification was performed using a universal primer pair previously shown to be specific to the chloroplast transfer RNA leucine (trnL) UAA gene intron. Overall, genus-specific, single and differently sized amplicons were reliably and reproducibly generated; enabling the differentiation of reference plants by PCR product length heterogeneity. However, there were a few plants that could not be clearly differentiated on the basis of size alone. This prompted the adoption of a post-PCR step that enabled further differentiation according to base sequence variation. Restriction endonucleases make sequence-specific cleavages on DNA to produce discrete and reproducible fragments having unique sizes and base compositions. Their availability, affordability and simplicity-of-use put restriction enzyme sequence (RES) profiling as a logical post-PCR step for confirming plant species identity. We demonstrate that PCR-RES profiling of plant and faecal matter is useful for the identification of plants included in the diet of kangaroos. The limitations, potential and the opportunities created for researchers interested in investigating the diet of competing herbivores in the rangelands are discussed.

A new method for locating changes in a tree reveals distinct nucleotide polymorphism vs. divergence patterns in mouse mitochondrial control region.

PubMed

Galtier, N; Boursot, P

2000-03-01

A new, model-based method was devised to locate nucleotide changes in a given phylogenetic tree. For each site, the posterior probability of any possible change in each branch of the tree is computed. This probabilistic method is a valuable alternative to the maximum parsimony method when base composition is skewed (i.e., different from 25% A, 25% C, 25% G, 25% T): computer simulations showed that parsimony misses more rare --> common than common --> rare changes, resulting in biased inferred change matrices, whereas the new method appeared unbiased. The probabilistic method was applied to the analysis of the mutation and substitution processes in the mitochondrial control region of mouse. Distinct change patterns were found at the polymorphism (within species) and divergence (between species) levels, rejecting the hypothesis of a neutral evolution of base composition in mitochondrial DNA.

A Proximity-Based Programmable DNA Nanoscale Assembly Line

PubMed Central

Gu, Hongzhou; Chao, Jie; Xiao, Shou-Jun; Seeman, Nadrian C.

2010-01-01

Our ability to synthesize nanometer-scale particles with desired shapes and compositions offers the exciting prospect of generating new functional materials and devices by combining the particles in a controlled fashion into larger structures. Self-assembly can achieve this task efficiently, but may be subject to thermodynamic and kinetic limitations: Reactants, intermediates and products may collide with each other throughout the assembly timecourse to produce non-target instead of target species. An alternative approach to nanoscale assembly uses information-containing molecules such as DNA1 to control interactions and thereby minimize unwanted crosstalk between different components. In principle, this method should allow the stepwise and programmed construction of target products by fastening individually selected nanoscale components – much as an automobile is built on an assembly line. Here, we demonstrate that a nanoscale assembly line can indeed be realized by the judicious combination of three known DNA-based modules: a DNA origami2 tile that provides a framework and track for the assembly process, cassettes containing three distinct two-state DNA machines that serve as programmable cargo-donating devices3,4 and are attached4,5 in series to the tile, and a DNA walker that can move on the track from device to device and collect cargo. As the walker traverses the pathway prescribed by the origami tile track, it encounters sequentially the three DNA devices that can be independently switched between an ‘ON’ state allowing its cargo to be transferred to the walker, and an ‘OFF’ state where no transfer occurs. We use three different types of gold nanoparticles as cargo and show that the experimental system does indeed allow the controlled fabrication of the eight different products that can be obtained with three two-state devices. PMID:20463734

Sedimentary ancient DNA and pollen reveal the composition of plant organic matter in Late Quaternary permafrost sediments of the Buor Khaya Peninsula (north-eastern Siberia)

NASA Astrophysics Data System (ADS)

Hildegard Zimmermann, Heike; Raschke, Elena; Saskia Epp, Laura; Rosmarie Stoof-Leichsenring, Kathleen; Schwamborn, Georg; Schirrmeister, Lutz; Overduin, Pier Paul; Herzschuh, Ulrike

2017-02-01

Organic matter deposited in ancient, ice-rich permafrost sediments is vulnerable to climate change and may contribute to the future release of greenhouse gases; it is thus important to get a better characterization of the plant organic matter within such sediments. From a Late Quaternary permafrost sediment core from the Buor Khaya Peninsula, we analysed plant-derived sedimentary ancient DNA (sedaDNA) to identify the taxonomic composition of plant organic matter, and undertook palynological analysis to assess the environmental conditions during deposition. Using sedaDNA, we identified 154 taxa and from pollen and non-pollen palynomorphs we identified 83 taxa. In the deposits dated between 54 and 51 kyr BP, sedaDNA records a diverse low-centred polygon plant community including recurring aquatic pond vegetation while from the pollen record we infer terrestrial open-land vegetation with relatively dry environmental conditions at a regional scale. A fluctuating dominance of either terrestrial or swamp and aquatic taxa in both proxies allowed the local hydrological development of the polygon to be traced. In deposits dated between 11.4 and 9.7 kyr BP (13.4-11.1 cal kyr BP), sedaDNA shows a taxonomic turnover to moist shrub tundra and a lower taxonomic richness compared to the older samples. Pollen also records a shrub tundra community, mostly seen as changes in relative proportions of the most dominant taxa, while a decrease in taxonomic richness was less pronounced compared to sedaDNA. Our results show the advantages of using sedaDNA in combination with palynological analyses when macrofossils are rarely preserved. The high resolution of the sedaDNA record provides a detailed picture of the taxonomic composition of plant-derived organic matter throughout the core, and palynological analyses prove valuable by allowing for inferences of regional environmental conditions.

Development and Use of Integrated Microarray-Based Genomic Technologies for Assessing Microbial Community Composition and Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, J.; Wu, L.; Gentry, T.

2006-04-05

To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approximately 2000 unique and group-specific probes with <85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50 C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appearedmore » to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ({approx}1.3{sup o} 10{sup 7} cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r{sup 2} = 0.95-0.99). Application of this microarray to naphthalene-amended enrichments and soil microcosms demonstrated that composition of the microflora varied depending on incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in enrichments, the genes involved in naphthalene degradation from Gram-negative microorganisms such as Ralstonia, Comamonas, and Burkholderia were most abundant in the soil microcosms (as well as those for polyaromatic hydrocarbon and nitrotoluene degradation). Although naphthalene degradation is widely known and studied in Pseudomonas, Pseudomonas genes were not detected in either system. Real-time PCR analysis of 4 representative genes was consistent with microarray-based quantification (r{sup 2} = 0.95). Currently, we are also applying this microarray to the study of several different microbial communities and processes at the NABIR-FRC in Oak Ridge, TN. One project involves the monitoring of the development and dynamics of the microbial community of a fluidized bed reactor (FBR) used for reducing nitrate and the other project monitors microbial community responses to stimulation of uranium reducing populations via ethanol donor additions in situ and in a model system. Additionally, we are developing novel strategies for increasing microarray hybridization sensitivity. Finally, great improvements to our methods of probe design were made by the development of a new computer program, CommOligo. CommOligo designs unique and group-specific oligo probes for whole-genomes, metagenomes, and groups of environmental sequences and uses a new global alignment algorithm to design single or multiple probes for each gene or group. We are now using this program to design a more comprehensive functional gene array for environmental studies. Overall, our results indicate that the 50mer-based microarray technology has potential as a specific and quantitative tool to reveal the composition of microbial communities and their dynamics important to processes within contaminated environments.« less

Isolation of a cDNA Encoding a Granule-Bound 152-Kilodalton Starch-Branching Enzyme in Wheat1

PubMed Central

Båga, Monica; Nair, Ramesh B.; Repellin, Anne; Scoles, Graham J.; Chibbar, Ravindra N.

2000-01-01

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5′-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80–100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum. PMID:10982440

Insight into the composition of the intercellular matrix of Streptococcus pneumoniae biofilms.

PubMed

Domenech, Mirian; García, Ernesto; Prieto, Alicia; Moscoso, Miriam

2013-02-01

Biofilm matrices consist of a mixture of extracellular polymeric substances synthesized in large part by the biofilm-producing microorganisms themselves. These matrices are responsible for the cohesion and three-dimensional architecture of biofilms. The present study demonstrates the existence of a matrix composed of extracellular DNA, proteins and polysaccharides in the biofilm formed by the human pathogen Streptococcus pneumoniae. Extracellular DNA, visualized by fluorescent labelling, was an important component of this matrix. The existence of DNA-protein complexes associated with bacterial aggregates and other polymers was hypothesized based on the unexpected DNA binding activity of lysozyme LytC, a novel moonlighting protein. Actually, a 25-amino-acid-long peptide derived from LytC (positions 408 and 432 of the mature LytC) was also capable of efficiently binding to DNA. Moreover, the presence of intercellular DNA-LytC protein complexes in pneumococcal biofilms was demonstrated by confocal laser scanning microscopy. Evidence of extracellular polysaccharide different from the capsule was obtained by staining with Calcofluor dye and four types of lectin conjugated to Alexa fluorophores, and by incubation with glycoside hydrolases. The presence of residues of Glcp(1→4) and GlcNAc(1→4) (in its deacetylated form) in the pneumococcal biofilm was confirmed by GC-MS techniques. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Two different size classes of 5S rDNA units coexisting in the same tandem array in the razor clam Ensis macha: is this region suitable for phylogeographic studies?

PubMed

Fernández-Tajes, Juan; Méndez, Josefina

2009-12-01

For a study of 5S ribosomal genes (rDNA) in the razor clam Ensis macha, the 5S rDNA region was amplified and sequenced. Two variants, so-called type I or short repeat (approximately 430 bp) and type II or long repeat (approximately 735 bp), appeared to be the main components of the 5S rDNA of this species. Their spacers differed markedly, both in length and nucleotide composition. The organization of the two variants was investigated by amplifying the genomic DNA with primers based on the sequence of the type I and type II spacers. PCR amplification products with primers EMLbF and EMSbR showed that the long and short repeats are associated within the same tandem array, suggesting an intermixed arrangement of both spacers. Nevertheless, amplifications carried out with inverse primers EMSinvF/R and EMLinvF/R revealed that some short and long repeats are contiguous in the same tandem array. This is the first report of the coexistence of two variable spacers in the same tandem array in bivalve mollusks.

Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

PubMed Central

Su, Chang; Wang, Chao; He, Lin; Yang, Chuanping; Wang, Yucheng

2014-01-01

DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch) by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees. PMID:25514241

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

PubMed Central

Desomer, Jan; Dhaese, Patrick; Montagu, Marc Van

1990-01-01

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 105/μg of DNA to 107/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination. Images PMID:16348290

Detection of Bacterial Pathogens from Broncho-Alveolar Lavage by Next-Generation Sequencing.

PubMed

Leo, Stefano; Gaïa, Nadia; Ruppé, Etienne; Emonet, Stephane; Girard, Myriam; Lazarevic, Vladimir; Schrenzel, Jacques

2017-09-20

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus , Corynebacterium jeikeium and Rothia dentocariosa , the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

On the Adsorption of DNA Origami Nanostructures in Nanohole Arrays.

PubMed

Brassat, Katharina; Ramakrishnan, Saminathan; Bürger, Julius; Hanke, Marcel; Doostdar, Mahnaz; Lindner, Jörg K N; Grundmeier, Guido; Keller, Adrian

2018-05-22

DNA origami nanostructures are versatile substrates for the controlled arrangement of molecular capture sites with nanometer precision and thus have many promising applications in single-molecule bioanalysis. Here, we investigate the adsorption of DNA origami nanostructures in nanohole arrays which represent an important class of biosensors and may benefit from the incorporation of DNA origami-based molecular probes. Nanoholes with well-defined diameter that enable the adsorption of single DNA origami triangles are fabricated in Au films on Si wafers by nanosphere lithography. The efficiency of directed DNA origami adsorption on the exposed SiO 2 areas at the bottoms of the nanoholes is evaluated in dependence of various parameters, i.e., Mg 2+ and DNA origami concentrations, buffer strength, adsorption time, and nanohole diameter. We observe that the buffer strength has a surprisingly strong effect on DNA origami adsorption in the nanoholes and that multiple DNA origami triangles with 120 nm edge length can adsorb in nanoholes as small as 120 nm in diameter. We attribute the latter observation to the low lateral mobility of once adsorbed DNA origami on the SiO 2 surface, in combination with parasitic adsorption to the Au film. Although parasitic adsorption can be suppressed by modifying the Au film with a hydrophobic self-assembled monolayer, the limited surface mobility of the adsorbed DNA origami still leads to poor localization accuracy in the nanoholes and results in many DNA origami crossing the boundary to the Au film even under optimized conditions. We discuss possible ways to minimize this effect by varying the composition of the adsorption buffer, employing different fabrication conditions, or using other substrate materials for nanohole array fabrication.

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples

PubMed Central

Arulandhu, Alfred J.; Staats, Martijn; Hagelaar, Rico; Voorhuijzen, Marleen M.; Prins, Theo W.; Scholtens, Ingrid; Costessi, Adalberto; Duijsings, Danny; Rechenmann, François; Gaspar, Frédéric B.; Barreto Crespo, Maria Teresa; Holst-Jensen, Arne; Birck, Matthew; Burns, Malcolm; Haynes, Edward; Hochegger, Rupert; Klingl, Alexander; Lundberg, Lisa; Natale, Chiara; Niekamp, Hauke; Perri, Elena; Barbante, Alessandra; Rosec, Jean-Philippe; Seyfarth, Ralf; Sovová, Tereza; Van Moorleghem, Christoff; van Ruth, Saskia; Peelen, Tamara

2017-01-01

Abstract DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance. PMID:29020743

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

PubMed

Arulandhu, Alfred J; Staats, Martijn; Hagelaar, Rico; Voorhuijzen, Marleen M; Prins, Theo W; Scholtens, Ingrid; Costessi, Adalberto; Duijsings, Danny; Rechenmann, François; Gaspar, Frédéric B; Barreto Crespo, Maria Teresa; Holst-Jensen, Arne; Birck, Matthew; Burns, Malcolm; Haynes, Edward; Hochegger, Rupert; Klingl, Alexander; Lundberg, Lisa; Natale, Chiara; Niekamp, Hauke; Perri, Elena; Barbante, Alessandra; Rosec, Jean-Philippe; Seyfarth, Ralf; Sovová, Tereza; Van Moorleghem, Christoff; van Ruth, Saskia; Peelen, Tamara; Kok, Esther

2017-10-01

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance. © The Authors 2017. Published by Oxford University Press.

Evaluation of the human hair root for DNA typing subsequent to microscopic comparison.

PubMed

Linch, C A; Smith, S L; Prahlow, J A

1998-03-01

Telogen human hairs are one of the most common useful evidence findings at crime scenes and/or on homicide victims. Occasionally, the microscopic characterization of the found telogen hair is the only physical evidence association to a victim or suspect. Recently efforts to characterize these hairs by mitochondrial DNA (mtDNA) methods have progressed. The nature of the telogen hair root morphology and ultrastructure has, however, been largely ignored. Examiners have recognized these hairs are unlikely to be typable by nuclear DNA (nuDNA) methods. Most forensic biologists have little knowledge of the complex cellular composition of anagen, catagen, and telogen hair roots or their morphogenesis. This paper reviews ex situ human hair root morphology as it relates to the likelihood of successful nuclear DNA typing. Dermatology texts of hair root morphology always demonstrate their microscopic appearance in the skin. This study investigates the use of fluorescence in situ hybridization (FISH) methods to sex type telogen head hairs, and it further investigates hair root morphology as it relates to the potential nuclear DNA content of evidence hairs. There is a need for the use of appropriate, consensus terminology for describing hair root morphology. There is also a need for standardized laboratory light microscopic methods in evaluating a hair root for DNA typing. FISH was found to be an unsuitable technique for sex determination of telogen hair clubs. It was determined that anagen/catagen hair roots without translucent sheath material are excellent candidates for nuDNA PCR-based typing and that hairs with telogen club root material only should not be submitted for nuDNA typing attempts.

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution.

PubMed

Johnson, LeeAnn K; Brown, Mary B; Carruthers, Ethan A; Ferguson, John A; Dombek, Priscilla E; Sadowsky, Michael J

2004-08-01

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.

DNA methylation-based measures of biological age: meta-analysis predicting time to death.

PubMed

Chen, Brian H; Marioni, Riccardo E; Colicino, Elena; Peters, Marjolein J; Ward-Caviness, Cavin K; Tsai, Pei-Chien; Roetker, Nicholas S; Just, Allan C; Demerath, Ellen W; Guan, Weihua; Bressler, Jan; Fornage, Myriam; Studenski, Stephanie; Vandiver, Amy R; Moore, Ann Zenobia; Tanaka, Toshiko; Kiel, Douglas P; Liang, Liming; Vokonas, Pantel; Schwartz, Joel; Lunetta, Kathryn L; Murabito, Joanne M; Bandinelli, Stefania; Hernandez, Dena G; Melzer, David; Nalls, Michael; Pilling, Luke C; Price, Timothy R; Singleton, Andrew B; Gieger, Christian; Holle, Rolf; Kretschmer, Anja; Kronenberg, Florian; Kunze, Sonja; Linseisen, Jakob; Meisinger, Christine; Rathmann, Wolfgang; Waldenberger, Melanie; Visscher, Peter M; Shah, Sonia; Wray, Naomi R; McRae, Allan F; Franco, Oscar H; Hofman, Albert; Uitterlinden, André G; Absher, Devin; Assimes, Themistocles; Levine, Morgan E; Lu, Ake T; Tsao, Philip S; Hou, Lifang; Manson, JoAnn E; Carty, Cara L; LaCroix, Andrea Z; Reiner, Alexander P; Spector, Tim D; Feinberg, Andrew P; Levy, Daniel; Baccarelli, Andrea; van Meurs, Joyce; Bell, Jordana T; Peters, Annette; Deary, Ian J; Pankow, James S; Ferrucci, Luigi; Horvath, Steve

2016-09-28

Estimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p≤8.2x10 -9 ) , independent of chronological age, even after adjusting for additional risk factors (p<5.4x10 -4 ) , and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p=7.5x10 -43 ). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality.

Drylands soil bacterial community is affected by land use change and different irrigation practices in the Mezquital Valley, Mexico.

PubMed

Lüneberg, Kathia; Schneider, Dominik; Siebe, Christina; Daniel, Rolf

2018-01-23

Dryland agriculture nourishes one third of global population, although crop irrigation is often mandatory. As freshwater sources are scarce, treated and untreated wastewater is increasingly used for irrigation. Here, we investigated how the transformation of semiarid shrubland into rainfed farming or irrigated agriculture with freshwater, dam-stored or untreated wastewater affects the total (DNA-based) and active (RNA-based) soil bacterial community composition, diversity, and functionality. To do this we collected soil samples during the dry and rainy seasons and isolated DNA and RNA. Soil moisture, sodium content and pH were the strongest drivers of the bacterial community composition. We found lineage-specific adaptations to drought and sodium content in specific land use systems. Predicted functionality profiles revealed gene abundances involved in nitrogen, carbon and phosphorous cycles differed among land use systems and season. Freshwater irrigated bacterial community is taxonomically and functionally susceptible to seasonal environmental changes, while wastewater irrigated ones are taxonomically susceptible but functionally resistant to them. Additionally, we identified potentially harmful human and phytopathogens. The analyses of 16 S rRNA genes, its transcripts and deduced functional profiles provided extensive understanding of the short-term and long-term responses of bacterial communities associated to land use, seasonality, and water quality used for irrigation in drylands.

Selenium nanoparticles synthesized in aqueous extract of Allium sativum perturbs the structural integrity of Calf thymus DNA through intercalation and groove binding.

PubMed

Ezhuthupurakkal, Preedia Babu; Polaki, Lokeswara Rao; Suyavaran, Arumugam; Subastri, Ariraman; Sujatha, Venugopal; Thirunavukkarasu, Chinnasamy

2017-05-01

Biomedical application of selenium nanoparticles (SeNPs) demands the eco-friendly composite for synthesis of SeNPs. The present study reports an aqueous extract of Allium sativum (AqEAS) plug-up the current need. Modern spectroscopic, microscopic and gravimetric techniques were employed to characterize the synthesized nanoparticles. Characterization studies revealed the formation of crystalline spherical shaped SeNPs. FTIR spectrum brings out the presence of different functional groups in AqEAS, which influence the SeNPs formation and stabilization. Furthermore the different aspects of the interaction between SeNPs and CT-DNA were scrutinized by various spectroscopic and cyclic voltametric studies. The results reveals the intercalation and groove binding mode of interaction of SeNPs with stacked base pair of CT-DNA. The Stern-Volmer quenching constant (K SV ) were found to be 7.02×10 6 M- 1 (ethidium bromide), 4.22×10 6 M- 1 (acridine orange) and 7.6×10 6 M- 1 (Hoechst) indicating strong binding of SeNPs with CT-DNA. The SeNPs - CT-DNA interactions were directly visualized by atomic force microscopy. The present study unveils the cost effective, innocuous, highly stable SeNPs intricate mechanism of DNA interaction, which will be a milestone in DNA targeted chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

A complete mitochondrial genome sequence of Asian black bear Sichuan subspecies (Ursus thibetanus mupinensis)

PubMed Central

Hou, Wan-ru; Chen, Yu; Wu, Xia; Hu, Jin-chu; Peng, Zheng-song; Yang, Jung; Tang, Zong-xiang; Zhou, Cai-Quan; Li, Yu-ming; Yang, Shi-kui; Du, Yu-jie; Kong, Ling-lu; Ren, Zheng-long; Zhang, Huai-yu; Shuai, Su-rong

2007-01-01

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16 868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%). PMID:17205108

Simple and universal model for electron-impact ionization of complex biomolecules

NASA Astrophysics Data System (ADS)

Tan, Hong Qi; Mi, Zhaohong; Bettiol, Andrew A.

2018-03-01

We present a simple and universal approach to calculate the total ionization cross section (TICS) for electron impact ionization in DNA bases and other biomaterials in the condensed phase. Evaluating the electron impact TICS plays a vital role in ion-beam radiobiology simulation at the cellular level, as secondary electrons are the main cause of DNA damage in particle cancer therapy. Our method is based on extending the dielectric formalism. The calculated results agree well with experimental data and show a good comparison with other theoretical calculations. This method only requires information of the chemical composition and density and an estimate of the mean binding energy to produce reasonably accurate TICS of complex biomolecules. Because of its simplicity and great predictive effectiveness, this method could be helpful in situations where the experimental TICS data are absent or scarce, such as in particle cancer therapy.

DNA origami based Au-Ag-core-shell nanoparticle dimers with single-molecule SERS sensitivity

NASA Astrophysics Data System (ADS)

Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.; Bald, I.

2016-03-01

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled. Electronic supplementary information (ESI) available: Additional information about materials and methods, designs of DNA origami templates, height profiles, additional SERS spectra, assignment of DNA bands, SEM images, additional AFM images, FDTD simulations, additional reference spectra for Cy3 and detailed description of EF estimation, simulated absorption and scattering spectra. See DOI: 10.1039/c5nr08674d

Development and evaluation of a 16S ribosomal DNA array-based approach for describing complex microbial communities in ready-to-eat vegetable salads packed in a modified atmosphere.

PubMed

Rudi, Knut; Flateland, Signe L; Hanssen, Jon Fredrik; Bengtsson, Gunnar; Nissen, Hilde

2002-03-01

There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets 16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4 degrees C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10 degrees C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10 degrees C. In that batch, the Enterobacteriaceae also were abundant after storage at 4 degrees C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions.

Development and Evaluation of a 16S Ribosomal DNA Array-Based Approach for Describing Complex Microbial Communities in Ready-To-Eat Vegetable Salads Packed in a Modified Atmosphere

PubMed Central

Rudi, Knut; Flateland, Signe L.; Hanssen, Jon Fredrik; Bengtsson, Gunnar; Nissen, Hilde

2002-01-01

There is a clear need for new approaches in the field of microbial community analyses, since the methods used can be severely biased. We have developed a DNA array-based method that targets16S ribosomal DNA (rDNA), enabling the direct detection and quantification of microorganisms from complex communities without cultivation. The approach is based on the construction of specific probes from the 16S rDNA sequence data retrieved directly from the communities. The specificity of the assay is obtained through a combination of DNA array hybridization and enzymatic labeling of the constructed probes. Cultivation-dependent assays (enrichment and plating) and cultivation-independent assays (direct fluorescence microscopy and scanning electron microscopy) were used as reference methods in the development and evaluation of the method. The description of microbial communities in ready-to-eat vegetable salads in a modified atmosphere was used as the experimental model. Comparisons were made with respect to the effect of storage at different temperatures for up to 12 days and with respect to the geographic origin of the crisphead lettuce (Spanish or Norwegian), the main salad component. The conclusion drawn from the method comparison was that the DNA array-based method gave an accurate description of the microbial communities. Pseudomonas spp. dominated both of the salad batches, containing either Norwegian or Spanish lettuce, before storage and after storage at 4°C. The Pseudomonas population also dominated the batch containing Norwegian lettuce after storage at 10°C. On the contrary, Enterobacteriaceae and lactic acid bacteria dominated the microbial community of the batch containing Spanish lettuce after storage at 10°C. In that batch, the Enterobacteriaceae also were abundant after storage at 4°C as well as before storage. The practical implications of these results are that microbial communities in ready-to-eat vegetable salads can be diverse and that microbial composition is dependent both on the origin of the raw material and on the storage conditions. PMID:11872462

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study.

PubMed

Valentini, Elisabetta; Zampieri, Michele; Malavolta, Marco; Bacalini, Maria Giulia; Calabrese, Roberta; Guastafierro, Tiziana; Reale, Anna; Franceschi, Claudio; Hervonen, Antti; Koller, Bernhard; Bernhardt, Jürgen; Slagboom, P Eline; Toussaint, Olivier; Sikora, Ewa; Gonos, Efstathios S; Breusing, Nicolle; Grune, Tilman; Jansen, Eugène; Dollé, Martijn E T; Moreno-Villanueva, María; Sindlinger, Thilo; Bürkle, Alexander; Ciccarone, Fabio; Caiafa, Paola

2016-08-29

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project 'MARK-AGE'. The results provide evidence for an age-related decline of TET1 , TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly.

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study

PubMed Central

Valentini, Elisabetta; Zampieri, Michele; Malavolta, Marco; Bacalini, Maria Giulia; Calabrese, Roberta; Guastafierro, Tiziana; Reale, Anna; Franceschi, Claudio; Hervonen, Antti; Koller, Bernhard; Bernhardt, Jürgen; Slagboom, P. Eline; Toussaint, Olivier; Sikora, Ewa; Gonos, Efstathios S.; Breusing, Nicolle; Grune, Tilman; Jansen, Eugène; Dollé, Martijn E.T.; Moreno-Villanueva, María; Sindlinger, Thilo; Bürkle, Alexander; Ciccarone, Fabio; Caiafa, Paola

2016-01-01

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly. PMID:27587280

Evidence of sexually dimorphic introgression in Pinaleno Mountain Apache trout

USGS Publications Warehouse

Porath, M.T.; Nielsen, J.L.

2003-01-01

The high-elevation headwater streams of the Pinaleno Mountains support small populations of threatened Apache trout Oncorhynchus apache that were stocked following the chemical removal of nonnative salmonids in the 1960s. A fisheries survey to assess population composition, growth, and size structure confirmed angler reports of infrequent occurrences of Oncorhynchus spp. exhibiting the external morphological characteristics of both Apache trout and rainbow trout O. mykiss. Nonlethal tissue samples were collected from 50 individuals in the headwaters of each stream. Mitochondrial DNA (mtDNA) sequencing and amplification of nuclear microsatellite loci were used to determine the levels of genetic introgression by rainbow trout in Apache trout populations at these locations. Sexually dimorphic introgression from the spawning of male rainbow trout with female Apache trout was detected using mtDNA and microsatellites. Estimates of the degree of hybridization based on three microsatellite loci were 10-88%. The use of nonlethal DNA genetic analyses can supplement information obtained from standard survey methods and be useful in assessing the relative importance of small and sensitive populations with a history of nonnative introductions.

On the efficacy of malaria DNA vaccination with magnetic gene vectors.

PubMed

Nawwab Al-Deen, Fatin; Ma, Charles; Xiang, Sue D; Selomulya, Cordelia; Plebanski, Magdalena; Coppel, Ross L

2013-05-28

We investigated the efficacy and types of immune responses from plasmid malaria DNA vaccine encoding VR1020-PyMSP119 condensed on the surface of polyethyleneimine (PEI)-coated SPIONs. In vivo mouse studies were done firstly to determine the optimum magnetic vector composition, and then to observe immune responses elicited when magnetic vectors were introduced via different administration routes. Higher serum antibody titers against PyMSP119 were observed with intraperitoneal and intramuscular injections than subcutaneous and intradermal injections. Robust IgG2a and IgG1 responses were observed for intraperitoneal administration, which could be due to the physiology of peritoneum as a major reservoir of macrophages and dendritic cells. Heterologous DNA prime followed by single protein boost vaccination regime also enhanced IgG2a, IgG1, and IgG2b responses, indicating the induction of appropriate memory immunity that can be elicited by protein on recall. These outcomes support the possibility to design superparamagnetic nanoparticle-based DNA vaccines to optimally evoke desired antibody responses, useful for a variety of diseases including malaria. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA Data Visualization (DDV): Software for Generating Web-Based Interfaces Supporting Navigation and Analysis of DNA Sequence Data of Entire Genomes.

PubMed

Neugebauer, Tomasz; Bordeleau, Eric; Burrus, Vincent; Brzezinski, Ryszard

2015-01-01

Data visualization methods are necessary during the exploration and analysis activities of an increasingly data-intensive scientific process. There are few existing visualization methods for raw nucleotide sequences of a whole genome or chromosome. Software for data visualization should allow the researchers to create accessible data visualization interfaces that can be exported and shared with others on the web. Herein, novel software developed for generating DNA data visualization interfaces is described. The software converts DNA data sets into images that are further processed as multi-scale images to be accessed through a web-based interface that supports zooming, panning and sequence fragment selection. Nucleotide composition frequencies and GC skew of a selected sequence segment can be obtained through the interface. The software was used to generate DNA data visualization of human and bacterial chromosomes. Examples of visually detectable features such as short and long direct repeats, long terminal repeats, mobile genetic elements, heterochromatic segments in microbial and human chromosomes, are presented. The software and its source code are available for download and further development. The visualization interfaces generated with the software allow for the immediate identification and observation of several types of sequence patterns in genomes of various sizes and origins. The visualization interfaces generated with the software are readily accessible through a web browser. This software is a useful research and teaching tool for genetics and structural genomics.

Whole-proteome phylogeny of large dsDNA viruses and parvoviruses through a composition vector method related to dynamical language model

PubMed Central

2010-01-01

Background The vast sequence divergence among different virus groups has presented a great challenge to alignment-based analysis of virus phylogeny. Due to the problems caused by the uncertainty in alignment, existing tools for phylogenetic analysis based on multiple alignment could not be directly applied to the whole-genome comparison and phylogenomic studies of viruses. There has been a growing interest in alignment-free methods for phylogenetic analysis using complete genome data. Among the alignment-free methods, a dynamical language (DL) method proposed by our group has successfully been applied to the phylogenetic analysis of bacteria and chloroplast genomes. Results In this paper, the DL method is used to analyze the whole-proteome phylogeny of 124 large dsDNA viruses and 30 parvoviruses, two data sets with large difference in genome size. The trees from our analyses are in good agreement to the latest classification of large dsDNA viruses and parvoviruses by the International Committee on Taxonomy of Viruses (ICTV). Conclusions The present method provides a new way for recovering the phylogeny of large dsDNA viruses and parvoviruses, and also some insights on the affiliation of a number of unclassified viruses. In comparison, some alignment-free methods such as the CV Tree method can be used for recovering the phylogeny of large dsDNA viruses, but they are not suitable for resolving the phylogeny of parvoviruses with a much smaller genome size. PMID:20565983

Genetic origin and composition of a natural hybrid poplar Populus × jrtyschensis from two distantly related species.

PubMed

Jiang, Dechun; Feng, Jianju; Dong, Miao; Wu, Guili; Mao, Kangshan; Liu, Jianquan

2016-04-18

The factors that contribute to and maintain hybrid zones between distinct species are highly variable, depending on hybrid origins, frequencies and fitness. In this study, we aimed to examine genetic origins, compositions and possible maintenance of Populus × jrtyschensis, an assumed natural hybrid between two distantly related species. This hybrid poplar occurs mainly on the floodplains along the river valleys between the overlapping distributions of the two putative parents. We collected 566 individuals from 45 typical populations of P. × jrtyschensis, P. nigra and P. laurifolia. We genotyped them based on the sequence variations of one maternally inherited chloroplast DNA (cpDNA) fragment and genetic polymorphisms at 20 SSR loci. We further sequenced eight nuclear genes for 168 individuals from 31 populations. Two groups of cpDNA haplotypes characteristic of P. nigra and P. laurifolia respectively were both recovered for P. × jrtyschensis. Genetic structures and coalescent tests of two sets of nuclear population genetic data suggested that P. × jrtyschensis originated from hybridizations between the two assumed parental species. All examined populations of P. × jrtyschensis comprise mainly F1 hybrids from interspecific hybridizations between P. nigra and P. laurifolia. In the habitats of P. × jrtyschensis, there are lower concentrations of soil nitrogen than in the habitats occupied by the other two species. Our extensive examination of the genetic composition of P. × jrtyschensis suggested that it is typical of F1-dominated hybrid zones. This finding plus the low concentration of soil nitrogen in the floodplain soils support the F1-dominated bounded hybrid superiority hypothesis of hybrid zone maintenance for this particular hybrid poplar.

Polymeric vs hydroxyapatite-based scaffolds on dental pulp stem cell proliferation and differentiation

PubMed Central

Khojasteh, Arash; Motamedian, Saeed Reza; Rad, Maryam Rezai; Shahriari, Mehrnoosh Hasan; Nadjmi, Nasser

2015-01-01

AIM: To evaluate adhesion, proliferation and differentiation of human dental pulp stem cells (hDPSCs) on four commercially available scaffold biomaterials. METHODS: hDPSCs were isolated from human dental pulp tissues of extracted wisdom teeth and established in stem cell growth medium. hDPSCs at passage 3-5 were seeded on four commercially available scaffold biomaterials, SureOss (Allograft), Cerabone (Xenograft), PLLA (Synthetic), and OSTEON II Collagen (Composite), for 7 and 14 d in osteogenic medium. Cell adhesion and morphology to the scaffolds were evaluated by scanning electron microscopy (SEM). Cell proliferation and differentiation into osteogenic lineage were evaluated using DNA counting and alkaline phosphatase (ALP) activity assay, respectively. RESULTS: All scaffold biomaterials except SureOss (Allograft) supported hDPSC adhesion, proliferation and differentiation. hDPSCs seeded on PLLA (Synthetic) scaffold showed the highest cell proliferation and attachment as indicated with both SEM and DNA counting assay. Evaluating the osteogenic differentiation capability of hDPSCs on different scaffold biomaterials with ALP activity assay showed high level of ALP activity on cells cultured on PLLA (Synthetic) and OSTEON II Collagen (Composite) scaffolds. SEM micrographs also showed that in the presence of Cerabone (Xenograft) and OSTEON II Collagen (Composite) scaffolds, the hDPSCs demonstrated the fibroblastic phenotype with several cytoplasmic extension, while the cells on PLLA scaffold showed the osteoblastic-like morphology, round-like shape. CONCLUSION: PLLA scaffold supports adhesion, proliferation and osteogenic differentiation of hDPSCs. Hence, it may be useful in combination with hDPSCs for cell-based reconstructive therapy. PMID:26640621

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae

PubMed Central

Carvalho, Natalia D. M.; Carmo, Edson; Neves, Rogerio O.; Schneider, Carlos Henrique; Gross, Maria Claudia

2016-01-01

Abstract Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by Cot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by Cot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using Cot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, Cot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of Cot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position. PMID:27551343

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae.

PubMed

Carvalho, Natalia D M; Carmo, Edson; Neves, Rogerio O; Schneider, Carlos Henrique; Gross, Maria Claudia

2016-01-01

Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by C ot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by C ot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using C ot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, C ot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of C ot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position.

Controllable g5p-Protein-Directed Aggregation of ssDNA-Gold Nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, S.; Maye, M; Zhang, Y

We assembled single-stranded DNA (ssDNA) conjugated nanoparticles using the phage M13 gene 5 protein (g5p) as the molecular glue to bind two antiparallel noncomplementary ssDNA strands. The entire process was controlled tightly by the concentration of the g5p protein and the presence of double-stranded DNA. The g5p-ssDNA aggregate was disintegrated by hybridization with complementary ssDNA (C-ssDNA) that triggers the dissociation of the complex. Polyhistidine-tagged g5p was bound to nickel nitrilotriacetic acid (Ni2+-NTA) conjugated nanoparticles and subsequently used to coassemble the ssDNA-conjugated nanoparticles into multiparticle-type aggregates. Our approach offers great promise for designing biologically functional, controllable protein/nanoparticle composites.

Chromatin organization in sperm may be the major functional consequence of base composition variation in the human genome.

PubMed

Vavouri, Tanya; Lehner, Ben

2011-04-01

Chromatin in sperm is different from that in other cells, with most of the genome packaged by protamines not nucleosomes. Nucleosomes are, however, retained at some genomic sites, where they have the potential to transmit paternal epigenetic information. It is not understood how this retention is specified. Here we show that base composition is the major determinant of nucleosome retention in human sperm, predicting retention very well in both genic and non-genic regions of the genome. The retention of nucleosomes at GC-rich sequences with high intrinsic nucleosome affinity accounts for the previously reported retention at transcription start sites and at genes that regulate development. It also means that nucleosomes are retained at the start sites of most housekeeping genes. We also report a striking link between the retention of nucleosomes in sperm and the establishment of DNA methylation-free regions in the early embryo. Taken together, this suggests that paternal nucleosome transmission may facilitate robust gene regulation in the early embryo. We propose that chromatin organization in the male germline, rather than in somatic cells, is the major functional consequence of fine-scale base composition variation in the human genome. The selective pressure driving base composition evolution in mammals could, therefore, be the need to transmit paternal epigenetic information to the zygote.

Auto-assembly of nanometer thick, water soluble layers of plasmid DNA complexed with diamines and basic amino acids on graphite: Greatest DNA protection is obtained with arginine.

PubMed

Khalil, T T; Boulanouar, O; Heintz, O; Fromm, M

2017-02-01

We have investigated the ability of diamines as well as basic amino acids to condense DNA onto highly ordered pyrolytic graphite with minimum damage after re-dissolution in water. Based on a bibliographic survey we briefly summarize DNA binding properties with diamines as compared to basic amino acids. Thus, solutions of DNA complexed with these linkers were drop-cast in order to deposit ultra-thin layers on the surface of HOPG in the absence or presence of Tris buffer. Atomic Force Microscopy analyses showed that, at a fixed ligand-DNA mixing ratio of 16, the mean thickness of the layers can be statistically predicted to lie in the range 0-50nm with a maximum standard deviation ±6nm, using a simple linear law depending on the DNA concentration. The morphology of the layers appears to be ligand-dependent. While the layers containing diamines present holes, those formed in the presence of basic amino acids, except for lysine, are much more compact and dense. X-ray Photoelectron Spectroscopy measurements provide compositional information indicating that, compared to the maximum number of DNA sites to which the ligands may bind, the basic amino acids Arg and His are present in large excess. Conservation of the supercoiled topology of the DNA plasmids was studied after recovery of the complex layers in water. Remarkably, arginine has the best protection capabilities whether Tris was present or not in the initial solution. Copyright © 2016 Elsevier B.V. All rights reserved.

B-DNA Structure and Stability as Function of Nucleic Acid Composition: Dispersion-Corrected DFT Study of Dinucleoside Monophosphate Single and Double Strands

PubMed Central

Barone, Giampaolo; Fonseca Guerra, Célia; Bickelhaupt, F Matthias

2013-01-01

We have computationally investigated the structure and stability of all 16 combinations of two out of the four natural DNA bases A, T, G and C in a di-2′-deoxyribonucleoside-monophosphate model DNA strand as well as in 10 double-strand model complexes thereof, using dispersion-corrected density functional theory (DFT-D). Optimized geometries with B-DNA conformation were obtained through the inclusion of implicit water solvent and, in the DNA models, of sodium counterions, to neutralize the negative charge of the phosphate groups. The results obtained allowed us to compare the relative stability of isomeric single and double strands. Moreover, the energy of the Watson–Crick pairing of complementary single strands to form double-helical structures was calculated. The latter furnished the following increasing stability trend of the double-helix formation energy: d(TpA)2

Improving accuracy of DNA diet estimates using food tissue control materials and an evaluation of proxies for digestion bias.

PubMed

Thomas, Austen C; Jarman, Simon N; Haman, Katherine H; Trites, Andrew W; Deagle, Bruce E

2014-08-01

Ecologists are increasingly interested in quantifying consumer diets based on food DNA in dietary samples and high-throughput sequencing of marker genes. It is tempting to assume that food DNA sequence proportions recovered from diet samples are representative of consumer's diet proportions, despite the fact that captive feeding studies do not support that assumption. Here, we examine the idea of sequencing control materials of known composition along with dietary samples in order to correct for technical biases introduced during amplicon sequencing and biological biases such as variable gene copy number. Using the Ion Torrent PGM(©) , we sequenced prey DNA amplified from scats of captive harbour seals (Phoca vitulina) fed a constant diet including three fish species in known proportions. Alongside, we sequenced a prey tissue mix matching the seals' diet to generate tissue correction factors (TCFs). TCFs improved the diet estimates (based on sequence proportions) for all species and reduced the average estimate error from 28 ± 15% (uncorrected) to 14 ± 9% (TCF-corrected). The experimental design also allowed us to infer the magnitude of prey-specific digestion biases and calculate digestion correction factors (DCFs). The DCFs were compared with possible proxies for differential digestion (e.g. fish protein%, fish lipid%) revealing a strong relationship between the DCFs and percent lipid of the fish prey, suggesting prey-specific corrections based on lipid content would produce accurate diet estimates in this study system. These findings demonstrate the value of parallel sequencing of food tissue mixtures in diet studies and offer new directions for future research in quantitative DNA diet analysis. © 2013 John Wiley & Sons Ltd.

Comparison of DNA preservation methods for environmental bacterial community samples.

PubMed

Gray, Michael A; Pratte, Zoe A; Kellogg, Christina A

2013-02-01

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard(™), RNAlater(®), DMSO-EDTA-salt (DESS), FTA(®) cards, and FTA Elute(®) cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA(®) cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard(™), RNAlater(®), and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae).

PubMed

Alvarenga, Elenice M; Mondin, Mateus; Rodrigues, Vera L C C; Andrade, Larissa M; Vidal, Benedicto de Campos; Mello, Maria Luiza S

2012-11-01

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus. Copyright © 2011 Elsevier GmbH. All rights reserved.

In Silico Design and Characterization of DNA Nanomaterials

NASA Astrophysics Data System (ADS)

Nash, Jessica A.

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) function biologically as carriers of genetic information. However, due to their ability to self-assemble via base pairing, nucleic acid molecules have become widely used in nanotechnology. In this dissertation, in silico techniques are used to probe the structure-property relationships of nucleic acid based nanomaterials. In Part 1, computational methods are employed to formulate nanoparticle design rules for applications in nucleic acid packaging and delivery. Nanoparticles (NPs) play increasingly important roles in nanomedicine, where the surface chemistry allows for control over interactions with biomolecules. Understanding how DNA and RNA compaction occurs is relevant to biological systems and systems in nanotechnology, and critical for the development of more efficient and effective nanoparticle carriers. Computational modeling allows for the description of bio-nano systems and processes with unprecedented detail, and can provide insights and guidelines for the creation of new nanomaterials. Using all-atom molecular dynamics simulations, the effect of nanoparticle surface chemistry, size, and solvent ionic strength on interactions with DNA and RNA are reported. In Chapter 2, a systematic study of the effect of nanoparticle charge on ability to bend and wrap short sequences of DNA and RNA is presented. To cause bending of DNA, a nanoparticle charge of at least +30 is required. Higher nanoparticle charges cause a greater degree of compaction. For RNA, however, charged ligand end-groups bind internally and prevent RNA bending. Nanoparticles were designed to test the influence of NP ligand shell shape and length on RNA binding using these results. In Chapter 3, all-atom simulation of NPs with long double stranded RNA are reported. Simulations show that by shortening NP ligand length, double stranded RNA can be wrapped. In Chapter 4, we consider compaction of long DNA by nanoparticles. NPs with +120 charge can fully compact DNA, but the wrapping is unordered on the surface. Chapter 5 reports the influence of NPs on the structure of single stranded DNA and RNA, showing that NPs have a greater influence on poly-pyrimidine strands than poly-purine strands, and can interrupt hydrogen bonds and pi-pi stacking. In Part II of this dissertation, computational techniques are applied to study DNA tiles and origami. Due to base-pairing DNA can be used to place objects with nanoscale precision, with applications in nanoscience and nanomedicine. Chapter 6 presents the development of anticoagulants using DNA weave tiles and aptamers. More effective anticoagulants can be created by varying the DNA aptamer used, and increasing local concentration by attaching aptamers to a DNA tile. Molecular dynamics simulations show that increasing the number of helices on a DNA weave tile increases tile flexibility. Chapter 7 introduces a tool developed for visualization of DNA origami design. We develop circle map visualizations for DNA origami and maps of the base composition, allowing for visualizations of DNA origami that were not previously available. This tool is currently available online via nanohub (open source) for users around the world. The results reported here provide a fundamental understanding of the behavior of DNA systems in nanotechnology. Results are expected to aid in the development of more effective NP compaction agents, DNA delivery vehicles, and DNA origami design.

Comparison of American Fisheries Society (AFS) standard fish sampling techniques and environmental DNA for characterizing fish communities in a large reservoir

USGS Publications Warehouse

Perez, Christina R.; Bonar, Scott A.; Amberg, Jon J.; Ladell, Bridget; Rees, Christopher B.; Stewart, William T.; Gill, Curtis J.; Cantrell, Chris; Robinson, Anthony

2017-01-01

Recently, methods involving examination of environmental DNA (eDNA) have shown promise for characterizing fish species presence and distribution in waterbodies. We evaluated the use of eDNA for standard fish monitoring surveys in a large reservoir. Specifically, we compared the presence, relative abundance, biomass, and relative percent composition of Largemouth Bass Micropterus salmoides and Gizzard Shad Dorosoma cepedianum measured through eDNA methods and established American Fisheries Society standard sampling methods for Theodore Roosevelt Lake, Arizona. Catches at electrofishing and gillnetting sites were compared with eDNA water samples at sites, within spatial strata, and over the entire reservoir. Gizzard Shad were detected at a higher percentage of sites with eDNA methods than with boat electrofishing in both spring and fall. In contrast, spring and fall gillnetting detected Gizzard Shad at more sites than eDNA. Boat electrofishing and gillnetting detected Largemouth Bass at more sites than eDNA; the exception was fall gillnetting, for which the number of sites of Largemouth Bass detection was equal to that for eDNA. We observed no relationship between relative abundance and biomass of Largemouth Bass and Gizzard Shad measured by established methods and eDNA copies at individual sites or lake sections. Reservoirwide catch composition for Largemouth Bass and Gizzard Shad (numbers and total weight [g] of fish) as determined through a combination of gear types (boat electrofishing plus gillnetting) was similar to the proportion of total eDNA copies from each species in spring and fall field sampling. However, no similarity existed between proportions of fish caught via spring and fall boat electrofishing and the proportion of total eDNA copies from each species. Our study suggests that eDNA field sampling protocols, filtration, DNA extraction, primer design, and DNA sequencing methods need further refinement and testing before incorporation into standard fish sampling surveys.

New insights into chromatin folding and dynamics from multi-scale modeling

NASA Astrophysics Data System (ADS)

Olson, Wilma

The dynamic organization of chromatin plays an essential role in the regulation of gene expression and in other fundamental cellular processes. The underlying physical basis of these activities lies in the sequential positioning, chemical composition, and intermolecular interactions of the nucleosomes-the familiar assemblies of roughly 150 DNA base pairs and eight histone proteins-found on chromatin fibers. We have developed a mesoscale model of short nucleosomal arrays and a computational framework that make it possible to incorporate detailed structural features of DNA and histones in simulations of short chromatin constructs with 3-25 evenly spaced nucleosomes. The correspondence between the predicted and observed effects of nucleosome composition, spacing, and numbers on long-range communication between regulatory proteins bound to the ends of designed nucleosome arrays lends credence to the model and to the molecular insights gleaned from the simulated structures. We have extracted effective nucleosome-nucleosome potentials from the mesoscale simulations and introduced the potentials in a larger scale computational treatment of regularly repeating chromatin fibers. Our results reveal a remarkable influence of nucleosome spacing on chromatin flexibility. Small changes in the length of the DNA fragments linking successive nucleosomes introduce marked changes in the local interactions of the nucleosomes and in the spatial configurations of the fiber as a whole. The changes in nucleosome positioning influence the statistical properties of longer chromatin constructs with 100-10,000 nucleosomes. We are investigating the extent to which the `local' interactions of regularly spaced nucleosomes contribute to the corresponding interactions in chains with mixed spacings as a step toward the treatment of fibers with nucleosomes positioned at the sites mapped at base-pair resolution on genomic sequences. Support of the work by USPHS R01 GM 34809 is gratefully acknowledged.

Fluorescent aptasensor for antibiotic detection using magnetic bead composites coated with gold nanoparticles and a nicking enzyme.

PubMed

Luo, Zewei; Wang, Yimin; Lu, Xiaoyong; Chen, Junman; Wei, Fujing; Huang, Zhijun; Zhou, Chen; Duan, Yixiang

2017-09-01

Antibiotic abuse has been bringing serious pollution in water, which is closely related to human health. It is desirable to develop a new strategy for antibiotic detection. To address this problem, a sensitive fluorescent aptasensor for antibiotic detection was developed by utilizing gold nanoparticles modified magnetic bead composites (AuNPs/MBs) and nicking enzyme. AuNPs/MBs were synthesized with the help of polyethylenimine (PEI). The prepared AuNPs/MBs acted as dual-functional scaffolds that owned excellent magnetic separation capacity and strong covalent bio-conjugation. The non-specifically absorbed aptamers in AuNPs/MBs were less than that in MBs. Hence, the fluorescent aptasensor based on AuNPs/MBs show a better signal to background ratio than that based on carboxyl modified magnetic beads (MBs). In this work, ampicillin was employed as a model analyte. In the presence of ampicillin, the specific binding between ampicillin and aptamer induced structure-switching that led to the release of partial complementary DNA (cDNA) of aptamer. Then, the released cDNA initiated the cycle of nicking enzyme assisted signal amplification (NEASA). Therefore, a large amount of taqman probes were cleaved and fluorescence signal was amplified. The prepared fluorescent aptasensor bring sensitive detection in range of 0.1-100 ng mL -1 with the limit of detection of 0.07 ng mL -1 . Furthermore, this aptasensor was also successfully applied in real sample detection with acceptable accuracy. The fluorescent aptasensor provides a promising method for efficient, rapid and sensitive antibiotic detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Binding of the Biogenic Polyamines to Deoxyribonucleic Acids of Varying Base Composition: Base Specificity and the Associated Energetics of the Interaction

PubMed Central

Kabir, Ayesha; Suresh Kumar, Gopinatha

2013-01-01

Background The thermodynamics of the base pair specificity of the binding of the polyamines spermine, spermidine, putrescine, and cadaverine with three genomic DNAs Clostridium perfringens, 27% GC, Escherichia coli, 50% GC and Micrococcus lysodeikticus, 72% GC have been studied using titration calorimetry and the data supplemented with melting studies, ethidium displacement and circular dichroism spectroscopy results. Methodology/Principal Findings Isothermal titration calorimetry, differential scanning calorimetry, optical melting studies, ethidium displacement, circular dichroism spectroscopy are the various techniques employed to characterize the interaction of four polyamines, spermine, spermidine, putersine and cadaverine with the DNAs. Polyamines bound stronger with AT rich DNA compared to the GC rich DNA and the binding varied depending on the charge on the polyamine as spermine>spermidine >putrescine>cadaverine. Thermodynamics of the interaction revealed that the binding was entropy driven with small enthalpy contribution. The binding was influenced by salt concentration suggesting the contribution from electrostatic forces to the Gibbs energy of binding to be the dominant contributor. Each system studied exhibited enthalpy-entropy compensation. The negative heat capacity changes suggested a role for hydrophobic interactions which may arise due to the non polar interactions between DNA and polyamines. Conclusion/Significance From a thermodynamic analysis, the AT base specificity of polyamines to DNAs has been elucidated for the first time and supplemented by structural studies. PMID:23894663

Towards robust and repeatable sampling methods in eDNA based studies.

PubMed

Dickie, Ian A; Boyer, Stephane; Buckley, Hannah; Duncan, Richard P; Gardner, Paul; Hogg, Ian D; Holdaway, Robert J; Lear, Gavin; Makiola, Andreas; Morales, Sergio E; Powell, Jeff R; Weaver, Louise

2018-05-26

DNA based techniques are increasingly used for measuring the biodiversity (species presence, identity, abundance and community composition) of terrestrial and aquatic ecosystems. While there are numerous reviews of molecular methods and bioinformatic steps, there has been little consideration of the methods used to collect samples upon which these later steps are based. This represents a critical knowledge gap, as methodologically sound field sampling is the foundation for subsequent analyses. We reviewed field sampling methods used for metabarcoding studies of both terrestrial and freshwater ecosystem biodiversity over a nearly three-year period (n = 75). We found that 95% (n = 71) of these studies used subjective sampling methods, inappropriate field methods, and/or failed to provide critical methodological information. It would be possible for researchers to replicate only 5% of the metabarcoding studies in our sample, a poorer level of reproducibility than for ecological studies in general. Our findings suggest greater attention to field sampling methods and reporting is necessary in eDNA-based studies of biodiversity to ensure robust outcomes and future reproducibility. Methods must be fully and accurately reported, and protocols developed that minimise subjectivity. Standardisation of sampling protocols would be one way to help to improve reproducibility, and have additional benefits in allowing compilation and comparison of data from across studies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

CRISPR-Based Technologies and the Future of Food Science.

PubMed

Selle, Kurt; Barrangou, Rodolphe

2015-11-01

The on-going CRISPR craze is focused on the use of Cas9-based technologies for genome editing applications in eukaryotes, with high potential for translational medicine and next-generation gene therapy. Nevertheless, CRISPR-Cas systems actually provide adaptive immunity in bacteria, and have much promise for various applications in food bacteria that include high-resolution typing of pathogens, vaccination of starter cultures against phages, and the genesis of programmable and specific antibiotics that can selectively modulate bacterial population composition. Indeed, the molecular machinery from these DNA-encoded, RNA-mediated, DNA-targeting systems can be harnessed in native hosts, or repurposed in engineered systems for a plethora of applications that can be implemented in all organisms relevant to the food chain, including agricultural crops trait-enhancement, livestock breeding, and fermentation-based manufacturing, and for the genesis of next-generation food products with enhanced quality and health-promoting functionalities. CRISPR-based applications are now poised to revolutionize many fields within food science, from farm to fork. In this review, we describe CRISPR-Cas systems and highlight their potential for the development of enhanced foods. © 2015 Institute of Food Technologists®

Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis (Carr 1958) comb. nov.

PubMed

Lisdiyanti, Puspita; Kawasaki, Hiroko; Seki, Tatsuji; Yamada, Yuzo; Uchimura, Tai; Komagata, Kazuo

2000-06-01

Thirty-one Acetobacter strains obtained from culture collections and 45 Acetobacter strains isolated from Indonesian sources were investigated for their phenotypic characteristics, ubiquinone systems, DNA base compositions, and levels of DNA-DNA relatedness. Of 31 reference strains, six showed the presence of ubiquinone 10 (Q-10). These strains were eliminated from the genus Acetobacter. The other 25 reference strains and 45 Indonesian isolates were subjected to a systematic study and separated into 8 distinct groups on the basis of DNA-DNA relatedness. The known species, Acetobacter aceti, A. pasteurianus, and A. peroxydans are retained for three of these groups. New combinations, A. orleanensis (Henneberg 1906) comb. nov., A. lovaniensis (Frateur 1950) comb. nov., and A. estunensis (Carr 1958) comb. nov. are proposed for three other groups. Two new species, A. indonesiensis sp. nov. and A. tropicalis sp. nov. are proposed for the remaining two. No Indonesian isolates were identified as A. aceti, A. estunensis, and A. peroxydans. Phylogenetic analysis on the basis of 16S rDNA sequences was carried out for representative strains from each of the groups. This supported that the eight species belonged to the genus Acetobacter. Several strains previously assigned to the species of A. aceti and A. pasteurianus were scattered over the different species. It is evident that the value of DNA-DNA relatedness between strains comprising a new species should be determined for the establishment of the species. Thus current bacterial species without data of DNA-DNA relatedness should be reexamined for the stability of bacterial nomenclature.

Effect of DNA Extraction Methods on the Apparent Structure of Yak Rumen Microbial Communities as Revealed by 16S rDNA Sequencing.

PubMed

Chen, Ya-Bing; Lan, Dao-Liang; Tang, Cheng; Yang, Xiao-Nong; Li, Jian

2015-01-01

To more efficiently identify the microbial community of the yak rumen, the standardization of DNA extraction is key to ensure fidelity while studying environmental microbial communities. In this study, we systematically compared the efficiency of several extraction methods based on DNA yield, purity, and 16S rDNA sequencing to determine the optimal DNA extraction methods whose DNA products reflect complete bacterial communities. The results indicate that method 6 (hexadecyltrimethylammomium bromide-lysozyme-physical lysis by bead beating) is recommended for the DNA isolation of the rumen microbial community due to its high yield, operational taxonomic unit, bacterial diversity, and excellent cell-breaking capability. The results also indicate that the bead-beating step is necessary to effectively break down the cell walls of all of the microbes, especially Gram-positive bacteria. Another aim of this study was to preliminarily analyze the bacterial community via 16S rDNA sequencing. The microbial community spanned approximately 21 phyla, 35 classes, 75 families, and 112 genera. A comparative analysis showed some variations in the microbial community between yaks and cattle that may be attributed to diet and environmental differences. Interestingly, numerous uncultured or unclassified bacteria were found in yak rumen, suggesting that further research is required to determine the specific functional and ecological roles of these bacteria in yak rumen. In summary, the investigation of the optimal DNA extraction methods and the preliminary evaluation of the bacterial community composition of yak rumen support further identification of the specificity of the rumen microbial community in yak and the discovery of distinct gene resources.

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

PubMed

Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

2011-01-31

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

Unravelling the active microbial community in a thermophilic anaerobic digester-microbial electrolysis cell coupled system under different conditions.

PubMed

Cerrillo, Míriam; Viñas, Marc; Bonmatí, August

2017-03-01

Thermophilic anaerobic digestion (AD) of pig slurry coupled to a microbial electrolysis cell (MEC) with a recirculation loop was studied at lab-scale as a strategy to increase AD stability when submitted to organic and nitrogen overloads. The system performance was studied, with the recirculation loop both connected and disconnected, in terms of AD methane production, chemical oxygen demand removal (COD) and volatile fatty acid (VFA) concentrations. Furthermore, the microbial population was quantitatively and qualitatively assessed through DNA and RNA-based qPCR and high throughput sequencing (MiSeq), respectively to identify the RNA-based active microbial populations from the total DNA-based microbial community composition both in the AD and MEC reactors under different operational conditions. Suppression of the recirculation loop reduced the AD COD removal efficiency (from 40% to 22%) and the methane production (from 0.32 to 0.03 m 3  m -3  d -1 ). Restoring the recirculation loop led to a methane production of 0.55 m 3  m -3  d -1 concomitant with maximum MEC COD and ammonium removal efficiencies of 29% and 34%, respectively. Regarding microbial analysis, the composition of the AD and MEC anode populations differed from really active microorganisms. Desulfuromonadaceae was revealed as the most active family in the MEC (18%-19% of the RNA relative abundance), while hydrogenotrophic methanogens (Methanobacteriaceae) dominated the AD biomass. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation of Fe 3O 4/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride) by emulsifier-free emulsion polymerization and its interaction with DNA

NASA Astrophysics Data System (ADS)

Li, Xiaolong; Liu, Guoqiang; Yan, Wei; Chu, Paul K.; Yeung, Kelvin W. K.; Wu, Shuilin; Yi, Changfeng; Xu, Zushun

2012-04-01

Cationic magnetic polymer particles Fe3O4/poly(styrene-butyl acrylate-[2-(methacryloxy)ethyl]trimethylammonium chloride), a type of potential gene carrier, were prepared by emulsifier-free emulsion polymerization with oleic acid modified magnetite Fe3O4, styrene, butyl acrylate and [2-(methacryloxy)ethyl]trimethylammonium chloride) (METAC). The morphology of the particles was characterized by transmission electron microscopy and the composites of particles were characterized by FT-IR spectroscopy, X-ray diffraction. These results showed that magnetic particles were well dispersed in polymers with the content of about 15%(wt/wt). The composites exhibited superparamagnetism and possessed a certain level of magnetic response. The interactions between the particles with calf-thymus DNA (ct DNA) were confirmed by zeta potential measurement, UV-vis spectroscopy and fluorescence spectroscopy. The DNA-binding capacity determined by the agarose gel electrophoresis showed good binding capacity of the emulsion to DNA. These results suggested the potential of the cationic magnetic polymer emulsion as gene target delivery carrier.

STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER.

PubMed

MAGGIO, R; SIEKEVITZ, P; PALADE, G E

1963-08-01

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl(2) followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl(2). Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that approximately 88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that approximately 90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of approximately 22.3 per cent DNA, approximately 4.7 per cent RNA, and approximately 73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.

Capillary electrophoretic separation-based approach to determine the labeling kinetics of oligodeoxynucleotides

PubMed Central

Kanavarioti, Anastassia; Greenman, Kevin L.; Hamalainen, Mark; Jain, Aakriti; Johns, Adam M.; Melville, Chris R.; Kemmish, Kent; Andregg, William

2014-01-01

With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined capillary electrophoresis (CE) protocol to assess the label stability, reactivity, and selectivity. We report our protocol using osmium tetroxide 2,2′-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in single-stranded (ss) DNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM. PMID:23147698

Nitrogen-fixing and cellulose-producing Gluconacetobacter kombuchae sp. nov., isolated from Kombucha tea.

PubMed

Dutta, Debasree; Gachhui, Ratan

2007-02-01

A few members of the family Acetobacteraceae are cellulose-producers, while only six members fix nitrogen. Bacterial strain RG3T, isolated from Kombucha tea, displays both of these characteristics. A high bootstrap value in the 16S rRNA gene sequence-based phylogenetic analysis supported the position of this strain within the genus Gluconacetobacter, with Gluconacetobacter hansenii LMG 1527T as its nearest neighbour (99.1 % sequence similarity). It could utilize ethanol, fructose, arabinose, glycerol, sorbitol and mannitol, but not galactose or xylose, as sole sources of carbon. Single amino acids such as L-alanine, L-cysteine and L-threonine served as carbon and nitrogen sources for growth of strain RG3T. Strain RG3T produced cellulose in both nitrogen-free broth and enriched medium. The ubiquinone present was Q-10 and the DNA base composition was 55.8 mol% G+C. It exhibited low values of 5.2-27.77 % DNA-DNA relatedness to the type strains of related gluconacetobacters, which placed it within a separate taxon, for which the name Gluconacetobacter kombuchae sp. nov. is proposed, with the type strain RG3T (=LMG 23726T=MTCC 6913T).

Methylation-Sensitive High Resolution Melting (MS-HRM).

PubMed

Hussmann, Dianna; Hansen, Lise Lotte

2018-01-01

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization.

PubMed

Cavalli, Roberta; Bisazza, Agnese; Trotta, Michele; Argenziano, Monica; Civra, Andrea; Donalisio, Manuela; Lembo, David

2012-01-01

The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery.

New chitosan nanobubbles for ultrasound-mediated gene delivery: preparation and in vitro characterization

PubMed Central

Cavalli, Roberta; Bisazza, Agnese; Trotta, Michele; Argenziano, Monica; Civra, Andrea; Donalisio, Manuela; Lembo, David

2012-01-01

Background The development of nonviral gene delivery systems is one of the most intriguing topics in nanomedicine. However, despite the advances made in recent years, several key issues remain unsettled. One of the main problems relates to the difficulty in designing nanodevices for targeted delivery of genes and other drugs to specific anatomic sites. In this study, we describe the development of a novel chitosan nanobubble-based gene delivery system for ultrasound-triggered release. Methods and results Chitosan was selected for the nanobubble shell because of its low toxicity, low immunogenicity, and excellent biocompatibility, while the core consisted of perfluoropentane. DNA-loaded chitosan nanobubbles were formed with a mean diameter of less than 300 nm and a positive surface charge. Transmission electron microscopic analysis confirmed composition of the core-shell structure. The ability of the chitosan nanobubbles to complex with and protect DNA was confirmed by agarose gel assay. Chitosan nanobubbles were found to be stable following insonation (2.5 MHz) for up to 3 minutes at 37°C. DNA release was evaluated in vitro in both the presence and absence of ultrasound. The release of chitosan nanobubble-bound plasmid DNA occurred after just one minute of insonation. In vitro transfection experiments were performed by exposing adherent COS7 cells to ultrasound in the presence of different concentrations of plasmid DNA-loaded nanobubbles. In the absence of ultrasound, nanobubbles failed to trigger transfection at all concentrations tested. In contrast, 30 seconds of ultrasound promoted a moderate degree of transfection. Cell viability experiments demonstrated that neither ultrasound nor the nanobubbles affected cell viability under these experimental conditions. Conclusion Based on these results, chitosan nanobubbles have the potential to be promising tools for ultrasound-mediated DNA delivery. PMID:22802689

Voltammetric determination of the Escherichia coli DNA using a screen-printed carbon electrode modified with polyaniline and gold nanoparticles.

PubMed

Shoaie, Nahid; Forouzandeh, Mehdi; Omidfar, Kobra

2018-03-12

The authors describe an electrochemical assay for fast detection of Escherichia coli (E. coli). It is based on a dual signal amplification strategy and the use of a screen-printed carbon electrode (SPCE) whose surface was modified with a polyaniline (PANI) film and gold nanoparticles (AuNPs) via cyclic voltammetry (CV). In the next step, avidin was covalently immobilized on the PANI/AuNP composite on the SPCE surface. Subsequently, the biotinylated DNA capture probe was immobilized onto the PANI/AuNP/avidin-modified SPCE by biotin-avidin interaction. Then, DNA of E.coli, digoxigenin-labeled DNA detector probe and anti-digoxigenin-labeled horseradish peroxidase (HRP) were placed on the electrode. 3,3',5,5'-Tetramethylbenzidine (TMB) and H 2 O 2 solution were added and the CV electrochemical signal was generated at a potential of -0.1 V (vs. Ag/AgCl) and a scan rate 50 mV.s -1 . The assay can detect 4 × 10 6 to 4 CFU of E. coli without DNA amplification. The biosensor is highly specific over other pathogens including Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Staphylococcus haemolyticus and Pseudomonas aeruginosa. It can be concluded that this genosensor has an excellent potential for rapid and accurate diagnosis of E.coli inflicted infections. Graphical Abstract Schematic of an electrochemical E. coli genosensor based on sandwich assay on a polyaniline/gold nanoparticle-modified screen printed carbon electrode (SPCE). The biosensor can detect 4 × 10 6 to 4 CFU of E. coli without DNA amplification.

New cobalt(II) and nickel(II) complexes of benzyl carbazate Schiff bases: Syntheses, crystal structures, in vitro DNA and HSA binding studies.

PubMed

Nithya, Palanivelu; Helena, Sannasi; Simpson, Jim; Ilanchelian, Malaichamy; Muthusankar, Aathi; Govindarajan, Subbiah

2016-12-01

In the present study, new Schiff base complexes with the composition [M(NCS) 2 (L1) 2 ]·nH 2 O, where M=Co (n=0) (1) and Ni (n=2) (2); [M(NCS) 2 (L2) 2 ], M=Co (3) and Ni (4) as well as [M(NCS) 2 (L3) 2 ], M=Co (5) and Ni (6); (L1=benzyl 2-(propan-2-ylidene)hydrazinecarboxylate, L2=benzyl 2-(butan-2-ylidene)hydrazinecarboxylate and L3=benzyl 2-(pentan-3-ylidene)hydrazinecarboxylate) have been synthesized by a template method. The complexes were characterised by analytical methods, spectroscopic studies, thermal and X-ray diffraction techniques. The structures of all the complexes explore that the metal(II) cation has a trans-planar coordination environment, the monomeric units containing a six-coordinated metal center in octahedral geometry with N-bound isothiocyanate anions coordinated as terminal ligands. Furthermore, the binding of the two Schiff base ligands to the metal centers involves the azomethine nitrogen and the carbonyl oxygen in mutually trans configuration. The binding interactions of all the complexes with Calf thymus-deoxyribonucleic acid (CT-DNA) and human serum albumin (HSA) have been investigated using absorption and emission spectral techniques. The CT-DNA binding properties of these complexes reveal that they bind to CT-DNA through a partial intercalation mode and the binding constant values were calculated using the absorption and emission spectral data. The binding constant values (~10×10 6 moldm -3 ) indicate strong binding of metal complexes with CT-DNA. HSA binding interaction studies showed that the cobalt and nickel complexes can quench the intrinsic fluorescence of HSA through static quenching process. Also, molecular docking studies were supported out to apprehend the binding interactions of these complexes with DNA and HSA which offer new understandings into the experimental model observations. Copyright © 2016 Elsevier B.V. All rights reserved.

[Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I].

PubMed

Shematorova, E K; Shpakovskiĭ, G V

2002-01-01

In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

Understanding DNA replication by the bacteriophage T4 replisome.

PubMed

Benkovic, Stephen J; Spiering, Michelle M

2017-11-10

The T4 replisome has provided a unique opportunity to investigate the intricacies of DNA replication. We present a comprehensive review of this system focusing on the following: its 8-protein composition, their individual and synergistic activities, and assembly in vitro and in vivo into a replisome capable of coordinated leading/lagging strand DNA synthesis. We conclude with a brief comparison with other replisomes with emphasis on how coordinated DNA replication is achieved. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

PubMed

de la Bastide, Paul Y; Leung, Wai Lam; Hintz, William E

2015-01-01

The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Estimating Diversity of Florida Keys Zooplankton Using New Environmental DNA Methods

NASA Astrophysics Data System (ADS)

Djurhuus, A.; Goldsmith, D. B.; Sawaya, N. A.; Breitbart, M.

2016-02-01

Zooplankton are of great importance in marine food webs, where they serve to link the phytoplankton and bacteria with higher trophic levels. Zooplankton are a diverse group containing molluscs, crustaceans, fish larvae and many other taxa. The sheer number of species and often minor morphological distinctions between species makes it challenging and exceptionally time consuming to identify the species composition of marine zooplankton samples. As a part of the Marine Biodiversity Observation Network (MBON) project, we have developed and groundtruthed an alternative, relatively time-efficient method for zooplankton identification using environmental DNA (eDNA). Samples were collected from Molasses reef, Looe Key, and Western Sambo along the Florida Keys from five bi-monthly cruises on board the RV Walton Smith. Samples were collected for environmental DNA (eDNA) by filtering 1 L of water on to a 0.22 µm filter and zooplankton samples were collected using nets with three mesh sizes (64μm, 200μm, and 500μm) to catch different size fractions. Half of zooplankton samples were fixed in 70% ethanol and half in 10% formalin, for DNA extraction and morphological identification, respectively. Individuals representing visually abundant taxa were picked into individual wells for PCR with universal 18S rRNA gene primers and subsequent sequencing to build a reference barcode database for zooplankton species commonly found in the study region. PCR and Illumina MiSeq next generation sequencing was applied to the eDNA extracted from the 0.22 μm filters and sequences were be compared to our local custom database as well as publicly available databases to determine zooplankton community composition. Finally, composition and diversity analyses were performed to compare results obtained with the new eDNA approach to standard morphological classification of zooplankton communities. Results show that the eDNA approach can enable the determination of zooplankton diversity through collection of a single water sample, which, when combined with bacterial and archaeal diversity analyses, will help us understand the coupling between different trophic levels and the drivers of plankton dynamics in the sub-tropical Florida Keys.

Classification of thermophilic actinobacteria isolated from arid desert soils, including the description of Amycolatopsis deserti sp. nov.

PubMed

Busarakam, Kanungnid; Brown, Ros; Bull, Alan T; Tan, Geok Yuan Annie; Zucchi, Tiago D; da Silva, Leonardo José; de Souza, Wallace Rafael; Goodfellow, Michael

2016-02-01

The taxonomic position of 26 filamentous actinobacteria isolated from a hyper-arid Atacama Desert soil and 2 from an arid Australian composite soil was established using a polyphasic approach. All of the isolates gave the diagnostic amplification product using 16S rRNA oligonucleotide primers specific for the genus Amycolatopsis. Representative isolates had chemotaxonomic and morphological properties typical of members of the genus Amycolatopsis. 16S rRNA gene analyses showed that all of the isolates belong to the Amycolatopsis methanolica 16S rRNA gene clade. The Atacama Desert isolates were assigned to one or other of two recognised species, namely Amycolatopsis ruanii and Amycolatopsis thermalba, based on 16S rRNA gene sequence, DNA:DNA relatedness and phenotypic data; emended descriptions are given for these species. In contrast, the two strains from the arid Australian composite soil, isolates GY024(T) and GY142, formed a distinct branch at the periphery of the A. methanolica 16S rRNA phyletic line, a taxon that was supported by all of the tree-making algorithms and by a 100 % bootstrap value. These strains shared a high degree of DNA:DNA relatedness and have many phenotypic properties in common, some of which distinguished them from all of the constituent species classified in the A. methanolica 16S rRNA clade. Isolates GY024(T) and GY142 merit recognition as a new species within the A. methanolica group of thermophilic strains. The name proposed for the new species is Amycolatopsis deserti sp. nov.; the type strain is GY024(T) (=NCIMB 14972(T) = NRRL B-65266(T)).

How Gene Marker and PCR Primer Choice Influence DNA Metabarcoding of Aquatic Communities

EPA Science Inventory

Molecular genetic tools like DNA barcoding/metabarcoding hold promise for improving our ability to characterize biotic composition, with relevance to detecting invasive species, monitoring species of concern, or describing diversity for bioassessments. To be most effective, these...

Does DNA barcoding improve performance of traditional stream bioassessment metrics?

EPA Science Inventory

Benthic macroinvertebrate community composition is used to assess wetland and stream condition and to help differentiate the effects of stressors among sites. Deoxyribonucleic acid (DNA) barcoding has been promoted as a way to increase taxonomic resolution and, thereby, to increa...

Archaeogenetics of Late Iron Age Çemialo Sırtı, Batman: Investigating maternal genetic continuity in north Mesopotamia since the Neolithic.

PubMed

Yaka, Reyhan; Birand, Ayşegül; Yılmaz, Yasemin; Caner, Ceren; Açan, Sinan Can; Gündüzalp, Sidar; Parvizi, Poorya; Erim Özdoğan, Aslı; Togan, İnci; Somel, Mehmet

2018-05-01

North Mesopotamia has witnessed dramatic social change during the Holocene, but the impact of these events on its demographic history is poorly understood. Here, we study this question by analysing genetic data from the recently excavated Late Iron Age settlement of Çemialo Sırtı in Batman, southeast Turkey. Archaeological and radiocarbon evidence indicate that the site was inhabited during the second and first millennia BCE. Çemialo Sırtı reveals nomadic items of the Early Iron Age, as well as items associated with the Late Achaemenid and subsequent Hellenistic Periods. We compare Çemialo Sırtı mitochondrial DNA profiles with earlier and later populations from west Eurasia to describe genetic continuity patterns in the region. A total of 16 Çemialo Sırtı individuals' remains were studied. PCR and Sanger sequencing were used to obtain mitochondrial DNA HVRI-HVRII sequences. We studied haplotype diversity and pairwise genetic distances using F ST , comparing the Çemialo Sırtı population with ancient and modern-day populations from west Eurasia. Coalescent simulations were carried out to test continuity for specific population comparisons. Mitochondrial DNA (mtDNA) haplotypes from 12 Çemialo Sırtı individuals reveal high haplotype diversity in this population, conspicuously higher than early Holocene west Eurasian populations, which supports the notion of increasing population admixture in west Eurasia through the Holocene. In its mtDNA composition, Çemialo Sırtı shows highest affinity to Neolithic north Syria and Neolithic Anatolia among ancient populations studied, and to modern-day southwest Asian populations. Based on population genetic simulations we cannot reject continuity between Neolithic and Iron Age, or between Iron Age and present-day populations of the region. Despite the region's complex sociopolitical history and indication for increased genetic diversity over time, we find no evidence for sharp shifts in north Mesopotamian maternal genetic composition within the last 10,000 years. © 2018 Wiley Periodicals, Inc.

TU-F-CAMPUS-T-04: Using Gold Nanoparticles to Target Mitochondria in Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

McNamara, A; McMahon, S; Lin, Y

2015-06-15

Purpose: The mitochondrion, like the cell nucleus, contains genetic material and plays several critical roles that determine the cell viability, including neutralization of free radicals within the cell. Studies have shown that irradiated cells with impaired mitochondria will incur more damage to the cell nucleus. This study investigates the potential use of GNPs to enhance radiation-induced damage to the organelle. Methods: The compositions of the organelles of a JURKAT cell were determined experimentally. Using Monte Carlo simulations, we investigate the significance of dose enhancement in a monoenergetic (10–50 keV and 6 MeV) x-ray irradiated cell cytoplasm, consisting of the experimentallymore » determined composition. We also investigate the track structure of secondary electrons in the mitochondria using Geant4-DNA in the presence and absence of GNPs for incident protons and photons. The biological effect was determined using an approach based on the local effect model, assuming the mitochondrial DNA (mtDNA) was the primary target. Results: Adding 0.01% of gold to the cell cytoplasm material can cause substantial dose enhancement, dependent on the incident x-ray energy. Track structure Monte Carlo (MC) simulations show an increased number of ionization events within the mitochondrion structure. The close proximity of GNPs to the mtDNA storing nucleoid may cause the mtDNA to receive doses above ∼100 Gy for keV x-rays, leading to mitochondrial dysfunction. Conclusion: A substantial increase in ionization events can occur in the mitochondria in the presence of GNPs. If GNPs can be delivered to tumors and attached to a sufficient number of mitochondria inside the tumor cells, mitochondrial induced cell death could be a prevalent cause of cell death. The biological structures developed here will be included in the biological MC toolkit, TOPAS-nBio.« less

A large-scale dataset of single and mixed-source short tandem repeat profiles to inform human identification strategies: PROVEDIt.

PubMed

Alfonse, Lauren E; Garrett, Amanda D; Lun, Desmond S; Duffy, Ken R; Grgicak, Catherine M

2018-01-01

DNA-based human identity testing is conducted by comparison of PCR-amplified polymorphic Short Tandem Repeat (STR) motifs from a known source with the STR profiles obtained from uncertain sources. Samples such as those found at crime scenes often result in signal that is a composite of incomplete STR profiles from an unknown number of unknown contributors, making interpretation an arduous task. To facilitate advancement in STR interpretation challenges we provide over 25,000 multiplex STR profiles produced from one to five known individuals at target levels ranging from one to 160 copies of DNA. The data, generated under 144 laboratory conditions, are classified by total copy number and contributor proportions. For the 70% of samples that were synthetically compromised, we report the level of DNA damage using quantitative and end-point PCR. In addition, we characterize the complexity of the signal by exploring the number of detected alleles in each profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemical and genetic discrimination of Cistanches Herba based on UPLC-QTOF/MS and DNA barcoding.

PubMed

Zheng, Sihao; Jiang, Xue; Wu, Labin; Wang, Zenghui; Huang, Linfang

2014-01-01

Cistanches Herba (Rou Cong Rong), known as "Ginseng of the desert", has a striking curative effect on strength and nourishment, especially in kidney reinforcement to strengthen yang. However, the two plant origins of Cistanches Herba, Cistanche deserticola and Cistanche tubulosa, vary in terms of pharmacological action and chemical components. To discriminate the plant origin of Cistanches Herba, a combined method system of chemical and genetic--UPLC-QTOF/MS technology and DNA barcoding--were firstly employed in this study. The results indicated that three potential marker compounds (isomer of campneoside II, cistanoside C, and cistanoside A) were obtained to discriminate the two origins by PCA and OPLS-DA analyses. DNA barcoding enabled to differentiate two origins accurately. NJ tree showed that two origins clustered into two clades. Our findings demonstrate that the two origins of Cistanches Herba possess different chemical compositions and genetic variation. This is the first reported evaluation of two origins of Cistanches Herba, and the finding will facilitate quality control and its clinical application.

Effective Design of Multifunctional Peptides by Combining Compatible Functions

PubMed Central

Diener, Christian; Garza Ramos Martínez, Georgina; Moreno Blas, Daniel; Castillo González, David A.; Corzo, Gerardo; Castro-Obregon, Susana; Del Rio, Gabriel

2016-01-01

Multifunctionality is a common trait of many natural proteins and peptides, yet the rules to generate such multifunctionality remain unclear. We propose that the rules defining some protein/peptide functions are compatible. To explore this hypothesis, we trained a computational method to predict cell-penetrating peptides at the sequence level and learned that antimicrobial peptides and DNA-binding proteins are compatible with the rules of our predictor. Based on this finding, we expected that designing peptides for CPP activity may render AMP and DNA-binding activities. To test this prediction, we designed peptides that embedded two independent functional domains (nuclear localization and yeast pheromone activity), linked by optimizing their composition to fit the rules characterizing cell-penetrating peptides. These peptides presented effective cell penetration, DNA-binding, pheromone and antimicrobial activities, thus confirming the effectiveness of our computational approach to design multifunctional peptides with potential therapeutic uses. Our computational implementation is available at http://bis.ifc.unam.mx/en/software/dcf. PMID:27096600

DNA origami based Au–Ag-core–shell nanoparticle dimers with single-molecule SERS sensitivity† †Electronic supplementary information (ESI) available: Additional information about materials and methods, designs of DNA origami templates, height profiles, additional SERS spectra, assignment of DNA bands, SEM images, additional AFM images, FDTD simulations, additional reference spectra for Cy3 and detailed description of EF estimation, simulated absorption and scattering spectra. See DOI: 10.1039/c5nr08674d Click here for additional data file.

PubMed Central

Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.

2016-01-01

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled. PMID:26892770

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase.

PubMed

Samai, Poulami; Shuman, Stewart

2011-06-24

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.

Linking Specific Heterotrophic Bacterial Populations to Bioreduction of Uranium and Nitrate in Contaminated Subsurface Sediments by Using Stable Isotope Probing▿†

PubMed Central

Akob, Denise M.; Kerkhof, Lee; Küsel, Kirsten; Watson, David B.; Palumbo, Anthony V.; Kostka, Joel E.

2011-01-01

Shifts in terminal electron-accepting processes during biostimulation of uranium-contaminated sediments were linked to the composition of stimulated microbial populations using DNA-based stable isotope probing. Nitrate reduction preceded U(VI) and Fe(III) reduction in [13C]ethanol-amended microcosms. The predominant, active denitrifying microbial groups were identified as members of the Betaproteobacteria, whereas Actinobacteria dominated under metal-reducing conditions. PMID:21948831

Synthesis and studies of polypeptide materials: Self-assembled block copolypeptide amphiphiles, DNA-condensing block copolypeptides and membrane-interactive random copolypeptides

NASA Astrophysics Data System (ADS)

Wyrsta, Michael Dmytro

A new class of transition metal initiators for the controlled polymerization of alpha-aminoacid-N-carboxyanhydrides (alpha-NCAs), has been developed by Deming et al. This discovery has allowed for the synthesis of well-defined "protein-like" polymers. Using this chemistry we have made distinct block/random copolypeptides for biomedical applications. Drug delivery, gene delivery, and antimicrobial polymers were the focus of our research efforts. The motivation for the synthesis and study of synthetic polypeptide based materials comes from proteins. Natural proteins are able to adopt a staggeringly large amount of uniquely well-defined folded structures. These structures account for the diversity in properties of proteins. As catalysts (enzymes) natural proteins perform some of the most difficult chemistry with ease and precision at ambient pressures and temperatures. They also exhibit incredible structural properties that directly result from formation of complex hierarchical assemblies. Self-assembling block copolymers were synthesized with various compositions and architectures. In general, di- and tri-block amphiphiles were studied for their self-assembling properties. Both spherical and tubular vesicles were found to assemble from di- and tri-block amphiphiles, respectively. In addition to self-assembly, pH responsiveness was engineered into these amphiphiles by the incorporation of basic residues (lysine) into the hydrophobic block. Another form of self-assembly studied was the condensation of DNA using cationic block copolymers. It was found that cationic block copolymers could condense DNA into compact, ordered, water-soluble aggregates on the nanoscale. These aggregates sufficiently protected DNA from nucleases and yet were susceptible to proteases. These studies form the basis of a gene delivery platform. The ease with which NCAs are polymerized renders them completely amenable to parallel synthetic methods. We have employed this technique to discover new antimicrobial polypeptides. The polymers studied were themselves the antimicrobial agent, not a self-assembled aggregate that contained antibiotics. It was found that powerful antibacterial polymers could be readily prepared with simple binary compositions. Antibacterial activity was sensitive to copolymer composition, bacterial cell-wall type, and insensitive to chain length (within reason).

Degenerative minimalism in the genome of a psyllid endosymbiont.

PubMed

Clark, M A; Baumann, L; Thao, M L; Moran, N A; Baumann, P

2001-03-01

Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection. We have sequenced 37 kb of DNA of the genome of Carsonella ruddii, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont Buchnera aphidicola. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA. These features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base composition has a large effect on proteins: in polypeptides of C. ruddii, half of the residues consist of five amino acids with codons low in guanine plus cytosine. Furthermore, the proteins of C. ruddii are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria. These observations suggest that the C. ruddii genome is not subject to constraints that limit the evolution of other known eubacteria.

DNA methylation-based measures of biological age: meta-analysis predicting time to death

PubMed Central

Chen, Brian H.; Marioni, Riccardo E.; Colicino, Elena; Peters, Marjolein J.; Ward-Caviness, Cavin K.; Tsai, Pei-Chien; Roetker, Nicholas S.; Just, Allan C.; Demerath, Ellen W.; Guan, Weihua; Bressler, Jan; Fornage, Myriam; Studenski, Stephanie; Vandiver, Amy R.; Moore, Ann Zenobia; Tanaka, Toshiko; Kiel, Douglas P.; Liang, Liming; Vokonas, Pantel; Schwartz, Joel; Lunetta, Kathryn L.; Murabito, Joanne M.; Bandinelli, Stefania; Hernandez, Dena G.; Melzer, David; Nalls, Michael; Pilling, Luke C.; Price, Timothy R.; Singleton, Andrew B.; Gieger, Christian; Holle, Rolf; Kretschmer, Anja; Kronenberg, Florian; Kunze, Sonja; Linseisen, Jakob; Meisinger, Christine; Rathmann, Wolfgang; Waldenberger, Melanie; Visscher, Peter M.; Shah, Sonia; Wray, Naomi R.; McRae, Allan F.; Franco, Oscar H.; Hofman, Albert; Uitterlinden, André G.; Absher, Devin; Assimes, Themistocles; Levine, Morgan E.; Lu, Ake T.; Tsao, Philip S.; Hou, Lifang; Manson, JoAnn E.; Carty, Cara L.; LaCroix, Andrea Z.; Reiner, Alexander P.; Spector, Tim D.; Feinberg, Andrew P.; Levy, Daniel; Baccarelli, Andrea; van Meurs, Joyce; Bell, Jordana T.; Peters, Annette; Deary, Ian J.; Pankow, James S.; Ferrucci, Luigi; Horvath, Steve

2016-01-01

Estimates of biological age based on DNA methylation patterns, often referred to as “epigenetic age”, “DNAm age”, have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cause mortality in four human cohorts. Here, we expanded our original observation to 13 different cohorts for a total sample size of 13,089 individuals, including three racial/ethnic groups. In addition, we examined whether incorporating information on blood cell composition into the epigenetic age metrics improves their predictive power for mortality. All considered measures of epigenetic age acceleration were predictive of mortality (p≤8.2×10−9), independent of chronological age, even after adjusting for additional risk factors (p<5.4×10−4), and within the racial/ethnic groups that we examined (non-Hispanic whites, Hispanics, African Americans). Epigenetic age estimates that incorporated information on blood cell composition led to the smallest p-values for time to death (p=7.5×10−43). Overall, this study a) strengthens the evidence that epigenetic age predicts all-cause mortality above and beyond chronological age and traditional risk factors, and b) demonstrates that epigenetic age estimates that incorporate information on blood cell counts lead to highly significant associations with all-cause mortality. PMID:27690265

Simple Sequence Repeats in Escherichia coli: Abundance, Distribution, Composition, and Polymorphism

PubMed Central

Gur-Arie, Riva; Cohen, Cyril J.; Eitan, Yuval; Shelef, Leora; Hallerman, Eric M.; Kashi, Yechezkel

2000-01-01

Computer-based genome-wide screening of the DNA sequence of Escherichia coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1 to 6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in noncoding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in noncoding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the noncoding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in noncoding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AF209020–209030 and AF209508–209518.] PMID:10645951

Persistence of immersed blood and hair DNA: A preliminary study based on casework.

PubMed

Frippiat, Christophe; Gastaldi, Agathe; Van Grunderbeeck, Séverine

2017-10-01

In some cases, evidence is collected from rivers, canals, lakes or sink pipes. To determine the utility of analyzing these samples and for cases in which DNA was recovered from submerged bulletproof vest parts, we evaluated the time necessary to degrade the blood and, subsequently, DNA on bulletproof vests. In a second experiment, also based on cases, blood was diluted in water from a kitchen sink pipe and incubated at room temperature for different times. Subsequently, DNA quality was assessed. In a parallel experiment, hair roots were incubated in spring water for different time periods. This study demonstrates that after one week of immersion of the bulletproof vest parts in a canal only one sample from more than 100 samples gave a partial genetic profile. No genetic profile were obtained for the 99 other samples. After one month immersion and despite the finding that blood remained detectable on bulletproof vest parts, no genetic profile was obtained for all samples using the classical STR approach. For longer immersion times, no genetic profiles were obtained. In sink pipe water, an incubation time of 72 h (h) was necessary before significant blood degradation occurred. Nevertheless, high inter-sample variability was observed. This high variability may be explained by the variability of water composition coming from nine different sink pipes. For hair root cells incubated in water, we observed that more than 90% of the DNA was degraded after 72 h. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Photophysical Characterization of Enhanced 6-Methylisoxanthopterin Fluorescence in Duplex DNA.

PubMed

Moreno, Andrew; Knee, J L; Mukerji, Ishita

2016-12-08

The structure and dynamic motions of bases in DNA duplexes and other constructs are important for understanding mechanisms of selectivity and recognition of DNA-binding proteins. The fluorescent guanine analogue, 6-methylisoxanthopterin 6-MI, is well suited to this purpose as it exhibits an unexpected 3- to 4-fold increase in relative quantum yield upon duplex formation when incorporated into the following sequences: ATFAA, AAFTA, or ATFTA (where F represents 6-MI). To better understand some of the factors leading to the 6-MI fluorescence increase upon duplex formation, we characterized the effect of local sequence and structural perturbations on 6-MI photophysics through temperature melts, quantum yield measurements, fluorescence quenching assays, and fluorescence lifetime measurements. By examining 21 sequences we have determined that the duplex-enhanced fluorescence (DEF) depends on the composition of bases adjacent to 6-MI and the presence of adenines at locations n ± 2 from the probe. Investigation of duplex stability and local solvent accessibility measurements support a model in which the DEF arises from a constrained geometry of 6-MI in the duplex, which remains H-bonded to cytosine, stacked with adjacent bases and inaccessible to quenchers. Perturbation of DNA structure through the introduction of an unpaired base 3' to 6-MI or a mismatched basepair increases 6-MI dynamic motion leading to fluorescence quenching and a reduction in quantum yield. Molecular dynamics simulations suggest the enhanced fluorescence results from a greater degree of twist at the X-F step relative to the quenched duplexes examined. These results point to a model where adenine residues located at n ± 2 from 6-MI induce a structural geometry with greater twist in the duplex that hinders local motion reducing dynamic quenching and producing an increase in 6-MI fluorescence.

Magnetic wire trap arrays for biomarker-based molecular detection

NASA Astrophysics Data System (ADS)

Vieira, Gregory; Mahajan, Kalpesh; Ruan, Gang; Winter, Jessica; Sooryakumar, R.

2012-02-01

Submicrometer-scale magnetic devices built on chip-based platforms have recently been shown to present opportunities for new particle trapping and manipulation technologies. Meanwhile, advances in nanoparticle fabrication allow for the building of custom-made particles with precise control of their size, composition, and other properties such as magnetism, fluorescence, and surface biomarker characteristics. In particular, carefully tailored surface biomarkers facilitate precise binding to targeted molecules, self-actuated construction of hybrid structures, and fluorescence-based detection schemes. Based on these progresses, we present an on-chip detection mechanism for molecules with known surface markers. Hybrid nanostructures consisting of micelle nanoparticles, fluorescent quantum dots, and superparamagnetic iron oxide nanoparticles are used to detect proteins or DNA molecules. The target is detected by the magnetic and fluorescent functionalities of the composite nanostructure, whereas in the absence of the target these signals are not present. Underlying this approach is the simultaneous manipulation via ferromagnetic zigzag nanowire arrays and imaging via quantum dot excitation. This chip-based detection technique could provide a powerful, low cost tool for ultrasensitive molecule detection with ramifications in healthcare diagnostics and small-scale chemical synthesis.

The best of both worlds: A combined approach for analyzing microalgal diversity via metabarcoding and morphology-based methods

PubMed Central

Kahlert, Maria; Fink, Patrick

2017-01-01

An increasing number of studies use next generation sequencing (NGS) to analyze complex communities, but is the method sensitive enough when it comes to identification and quantification of species? We compared NGS with morphology-based identification methods in an analysis of microalgal (periphyton) communities. We conducted a mesocosm experiment in which we allowed two benthic grazer species to feed upon benthic biofilms, which resulted in altered periphyton communities. Morphology-based identification and 454 (Roche) pyrosequencing of the V4 region in the small ribosomal unit (18S) rDNA gene were used to investigate the community change caused by grazing. Both the NGS-based data and the morphology-based method detected a marked shift in the biofilm composition, though the two methods varied strongly in their abilities to detect and quantify specific taxa, and neither method was able to detect all species in the biofilms. For quantitative analysis, we therefore recommend using both metabarcoding and microscopic identification when assessing the community composition of eukaryotic microorganisms. PMID:28234997

Patterns of cell division, DNA base compositions, and fine structures of some radiation-resistant vegetative bacteria found in food

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanders, S.W.; Maxcy, R.B.

1979-01-01

Representative highly radiation-resistant Moraxella-Acinetobacter (M-A), Pseudomonas radiora, Micrococcus radiodurans, and Micrococcus radiophilus exhibited a wide variety of division systems and cell wall characteristics. However, the more resistant M-A possessed unusually thick cell walls, indicating a possible role of the cell wall in radiation resistance in the M-A. Thick septation was present in most of the bacteria studied, but was absent in P. radiora, thus excluding this as a necessity for high resistance. Reliable determination of the number of division planes of the M-A for use as a taxonomic criterion was achieved by the direct observation of dividing cells. The highlymore » resistant M-A were found to divide in multiple planes and had base compositions of 54.0 to 57.5%, unlike typical Moraxella and/or Acinetobacter species. The taxonomic position of most highly resistant bacteria remains unclear.« less

Accurate phylogenetic classification of DNA fragments based onsequence composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

McHardy, Alice C.; Garcia Martin, Hector; Tsirigos, Aristotelis

2006-05-01

Metagenome studies have retrieved vast amounts of sequenceout of a variety of environments, leading to novel discoveries and greatinsights into the uncultured microbial world. Except for very simplecommunities, diversity makes sequence assembly and analysis a verychallenging problem. To understand the structure a 5 nd function ofmicrobial communities, a taxonomic characterization of the obtainedsequence fragments is highly desirable, yet currently limited mostly tothose sequences that contain phylogenetic marker genes. We show that forclades at the rank of domain down to genus, sequence composition allowsthe very accurate phylogenetic 10 characterization of genomic sequence.We developed a composition-based classifier, PhyloPythia, for de novophylogenetic sequencemore » characterization and have trained it on adata setof 340 genomes. By extensive evaluation experiments we show that themethodis accurate across all taxonomic ranks considered, even forsequences that originate fromnovel organisms and are as short as 1kb.Application to two metagenome datasets 15 obtained from samples ofphosphorus-removing sludge showed that the method allows the accurateclassification at genus level of most sequence fragments from thedominant populations, while at the same time correctly characterizingeven larger parts of the samples at higher taxonomic levels.« less

The relationship between genetic and chemotypic diversity in American ginseng (Panax quinquefolius L.).

PubMed

Schlag, Erin M; McIntosh, Marla S

2013-09-01

Ginseng is one of the world's most important herbals used as an adaptogen and a cure for an impressively large range of ailments. Differences in the medicinal properties of ginseng roots have been attributed to variation in ginsenoside composition. In this study, the association between genetic and chemotypic profiles of wild and cultivated American ginseng (Panax quinquefolius L.) roots grown in Maryland was investigated. Ginseng roots were classified into chemotypes based on their relative composition of Re and Rg1. Genetic profiles of these roots were determined from the analysis of 38 polymorphic RAPD markers and used for a cluster analysis of genetic similarities. The close correspondence between chemotype and genetic cluster provides the first DNA-based evidence for the genetic basis of ginsenoside composition. Results of this research are significant for plant breeding and conservation, phytochemical research, and clinical and pharmacological studies. Also, the correlation between RAPD markers and chemotype indicates the potential to use RAPD markers as a reliable and practical method for identification and certification of ginseng roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

Classification of Sharks in the Egyptian Mediterranean Waters Using Morphological and DNA Barcoding Approaches

PubMed Central

Moftah, Marie; Abdel Aziz, Sayeda H.; Elramah, Sara; Favereaux, Alexandre

2011-01-01

The identification of species constitutes the first basic step in phylogenetic studies, biodiversity monitoring and conservation. DNA barcoding, i.e. the sequencing of a short standardized region of DNA, has been proposed as a new tool for animal species identification. The present study provides an update on the composition of shark in the Egyptian Mediterranean waters off Alexandria, since the latest study to date was performed 30 years ago, DNA barcoding was used in addition to classical taxonomical methodologies. Thus, 51 specimen were DNA barcoded for a 667 bp region of the mitochondrial COI gene. Although DNA barcoding aims at developing species identification systems, some phylogenetic signals were apparent in the data. In the neighbor-joining tree, 8 major clusters were apparent, each of them containing individuals belonging to the same species, and most with 100% bootstrap value. This study is the first to our knowledge to use DNA barcoding of the mitochondrial COI gene in order to confirm the presence of species Squalus acanthias, Oxynotus centrina, Squatina squatina, Scyliorhinus canicula, Scyliorhinus stellaris, Mustelus mustelus, Mustelus punctulatus and Carcharhinus altimus in the Egyptian Mediterranean waters. Finally, our study is the starting point of a new barcoding database concerning shark composition in the Egyptian Mediterranean waters (Barcoding of Egyptian Mediterranean Sharks [BEMS], http://www.boldsystems.org/views/projectlist.php?&#Barcoding%20Fish%20%28FishBOL%29). PMID:22087242

Association of dietary type with fecal microbiota in vegetarians and omnivores in Slovenia.

PubMed

Matijašić, Bojana Bogovič; Obermajer, Tanja; Lipoglavšek, Luka; Grabnar, Iztok; Avguštin, Gorazd; Rogelj, Irena

2014-06-01

The purpose of this study was to discover differences in the human fecal microbiota composition driven by long-term omnivore versus vegan/lacto-vegetarian dietary pattern. In addition, the possible association of demographic characteristics and dietary habits such as consumption of particular foods with the fecal microbiota was examined. This study was conducted on a Slovenian population comprising 31 vegetarian participants (11 lacto-vegetarians and 20 vegans) and 29 omnivore participants. Bacterial DNA was extracted from the frozen fecal samples by Maxwell 16 Tissue DNA Purification Kit (Promega). Relative quantification of selected bacterial groups was performed by real-time PCR. Differences in fecal microbiota composition were evaluated by PCR-DGGE fingerprinting of the V3 16S rRNA region. Participants' demographic characteristics, dietary habits and health status information were collected through a questionnaire. Vegetarian diet was associated with higher ratio (% of group-specific DNA in relation to all bacterial DNA) of Bacteroides-Prevotella, Bacteroides thetaiotaomicron, Clostridium clostridioforme and Faecalibacterium prausnitzii, but with lower ratio (%) of Clostridium cluster XIVa. Real-time PCR also showed a higher concentration and ratio of Enterobacteriaceae (16S rDNA copies/g and %) in female participants (p < 0.05 and p < 0.01) and decrease in Bifidobacterium with age (p < 0.01). DGGE analysis of the 16S rRNA V3 region showed that relative quantity of DGGE bands from certain bacterial groups was lower (Bifidobacterium, Streptococus, Collinsella and Lachnospiraceae) or higher (Subdoligranulum) among vegetarians, indicating the association of dietary type with bacterial community composition. Sequencing of selected DGGE bands revealed the presence of common representatives of fecal microbiota: Bacteroides, Eubacterium, Faecalibacterium, Ruminococcaceae, Bifidobacterium and Lachnospiraceae. Up to 4 % of variance in microbial community analyzed by DGGE could be explained by the vegetarian type of diet. Long-term vegetarian diet contributed to quantity and associated bacterial community shifts in fecal microbiota composition. Consumption of foods of animal origin (eggs, red meat, white meat, milk, yoghurt, other dairy products, fish and seafood) and vegetarian type of diet explained the largest share of variance in microbial community structure. Fecal microbiota composition was also associated with participants' age, gender and body mass.

Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

PubMed

Vera-Rodriguez, M; Diez-Juan, A; Jimenez-Almazan, J; Martinez, S; Navarro, R; Peinado, V; Mercader, A; Meseguer, M; Blesa, D; Moreno, I; Valbuena, D; Rubio, C; Simon, C

2018-04-01

What is the origin and composition of cell-free DNA in human embryo spent culture media? Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. This study was limited by the sample size and the number of cells analyzed by FISH. This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. This work was funded by Igenomix S.L. There are no competing interests.

Carbon stable isotope composition of DNA isolated from an incipient paleosol

NASA Astrophysics Data System (ADS)

Jahren, A. Hope; Kelm, Kellie; Wendland, Beverly; Petersen, Gitte; Seberg, Ole

2006-05-01

We determined the carbon isotope (δ13C) value of double-stranded DNA (dsDNA) isolated from the organic horizons of a Delaware soil that is actively being covered by an encroaching sand dune. The soil belongs to a Nymphaea odorata Ait. (water lily) wetland, and we regard its active acquisition of a thick (˜24 cm) surface mantle to embody the process of paleopedogenesis; therefore, we have termed it an “incipient paleosol.” In this study, we compared the δ13C value of paleosol dsDNA to the bulk δ13C value of N. odorata, as well as to the δ13C value of plants that had colonized the surface mantle. The isotopic offset between paleosol δ13CdsDNA and N. odorata δ13Ctissue was identical to the relationship between δ13CdsDNA and δ13Ctissue for tracheophytes, which we had previously determined. In contrast, the isotopic offset between paleosol δ13CdsDNA and the δ13Ctissue of plants colonizing the surface mantle differed from this relationship by as much as 4‰. Similarly, the δ13C value of bulk paleosol organic matter was extremely heterogeneous and varied across 6‰. All paleosol DNA polymerase chain reaction (PCR) products produced clear, sharp, 350 base-pair (bp) fragments of rbcL, a gene shared by all photosynthetic organisms. These results open the exciting possibility that stable isotope analysis of dsDNA isolated from paleosol organic matter can be used to infer the δ13C value of the plant that dominated the nucleic acid contribution.

Label-free probing of genes by time-domain terahertz sensing.

PubMed

Haring Bolivar, P; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Büttner, R

2002-11-07

A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a freespace analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

Casting inorganic structures with DNA molds

PubMed Central

Sun, Wei; Boulais, Etienne; Hakobyan, Yera; Wang, Wei Li; Guan, Amy; Bathe, Mark; Yin, Peng

2014-01-01

We report a general strategy for designing and synthesizing inorganic nanostructures with arbitrarily prescribed three-dimensional shapes. Computationally designed DNA strands self-assemble into a stiff “nano-mold” that contains a user-specified three-dimensional cavity and encloses a nucleating gold “seed”. Under mild conditions, this seed grows into a larger cast structure that fills and thus replicates the cavity. We synthesized a variety of nanoparticles with three nanometer resolution: three distinct silver cuboids with three independently tunable dimensions, silver and gold nanoparticles with diverse cross sections, and composite structures with homo-/heterogeneous components. The designer equilateral silver triangular and spherical nanoparticles exhibited plasmonic properties consistent with electromagnetism-based simulations. Our framework is generalizable to more complex geometries and diverse inorganic materials, offering a range of applications in biosensing, photonics, and nanoelectronics. PMID:25301973

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

PubMed

Guza, Rebecca; Kotandeniya, Delshanee; Murphy, Kristopher; Dissanayake, Thakshila; Lin, Chen; Giambasu, George Madalin; Lad, Rahul R; Wojciechowski, Filip; Amin, Shantu; Sturla, Shana J; Hudson, Robert H E; York, Darrin M; Jankowiak, Ryszard; Jones, Roger; Tretyakova, Natalia Y

2011-05-01

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine. © The Author(s) 2011. Published by Oxford University Press.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA

PubMed Central

Guza, Rebecca; Kotandeniya, Delshanee; Murphy, Kristopher; Dissanayake, Thakshila; Lin, Chen; Giambasu, George Madalin; Lad, Rahul R.; Wojciechowski, Filip; Amin, Shantu; Sturla, Shana J.; Hudson, Robert H.E.; York, Darrin M.; Jankowiak, Ryszard; Jones, Roger; Tretyakova, Natalia Y.

2011-01-01

Endogenous 5-methylcytosine (MeC) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational ‘hotspots’ for smoking induced lung cancer. MeC enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5′-CCCGGCACCC GC[15N3,13C1-G]TCCGCG-3′, + strand) were prepared containing [15N3, 13C1]-guanine opposite unsubstituted cytosine, MeC, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2′-deoxynucleosides, N2-BPDE-dG adducts formed at the [15N3, 13C1]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N2-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE–DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N2 position of guanine. PMID:21245046

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis.

PubMed

Danhorn, Thomas; Young, Curtis R; DeLong, Edward F

2012-11-01

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary 'next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Comparison of Standard Culture-Based Method to Culture-Independent Method for Evaluation of Hygiene Effects on the Hand Microbiome.

PubMed

Zapka, C; Leff, J; Henley, J; Tittl, J; De Nardo, E; Butler, M; Griggs, R; Fierer, N; Edmonds-Wilson, S

2017-03-28

Hands play a critical role in the transmission of microbiota on one's own body, between individuals, and on environmental surfaces. Effectively measuring the composition of the hand microbiome is important to hand hygiene science, which has implications for human health. Hand hygiene products are evaluated using standard culture-based methods, but standard test methods for culture-independent microbiome characterization are lacking. We sampled the hands of 50 participants using swab-based and glove-based methods prior to and following four hand hygiene treatments (using a nonantimicrobial hand wash, alcohol-based hand sanitizer [ABHS], a 70% ethanol solution, or tap water). We compared results among culture plate counts, 16S rRNA gene sequencing of DNA extracted directly from hands, and sequencing of DNA extracted from culture plates. Glove-based sampling yielded higher numbers of unique operational taxonomic units (OTUs) but had less diversity in bacterial community composition than swab-based sampling. We detected treatment-induced changes in diversity only by using swab-based samples ( P < 0.001); we were unable to detect changes with glove-based samples. Bacterial cell counts significantly decreased with use of the ABHS ( P < 0.05) and ethanol control ( P < 0.05). Skin hydration at baseline correlated with bacterial abundances, bacterial community composition, pH, and redness across subjects. The importance of the method choice was substantial. These findings are important to ensure improvement of hand hygiene industry methods and for future hand microbiome studies. On the basis of our results and previously published studies, we propose recommendations for best practices in hand microbiome research. IMPORTANCE The hand microbiome is a critical area of research for diverse fields, such as public health and forensics. The suitability of culture-independent methods for assessing effects of hygiene products on microbiota has not been demonstrated. This is the first controlled laboratory clinical hand study to have compared traditional hand hygiene test methods with newer culture-independent characterization methods typically used by skin microbiologists. This study resulted in recommendations for hand hygiene product testing, development of methods, and future hand skin microbiome research. It also demonstrated the importance of inclusion of skin physiological metadata in skin microbiome research, which is atypical for skin microbiome studies. Copyright © 2017 Zapka et al.

Physical disturbance to ecological niches created by soil structure alters community composition of methanotrophs.

PubMed

Kumaresan, Deepak; Stralis-Pavese, Nancy; Abell, Guy C J; Bodrossy, Levente; Murrell, J Colin

2011-10-01

Aggregates of different sizes and stability in soil create a composite of ecological niches differing in terms of physico-chemical and structural characteristics. The aim of this study was to identify, using DNA-SIP and mRNA-based microarray analysis, whether shifts in activity and community composition of methanotrophs occur when ecological niches created by soil structure are physically perturbed. Landfill cover soil was subject to three treatments termed: 'control' (minimal structural disruption), 'sieved' (sieved soil using 2 mm mesh) and 'ground' (grinding using mortar and pestle). 'Sieved' and 'ground' soil treatments exhibited higher methane oxidation potentials compared with the 'control' soil treatment. Analysis of the active community composition revealed an effect of physical disruption on active methanotrophs. Type I methanotrophs were the most active methanotrophs in 'sieved' and 'ground' soil treatments, whereas both Type I and Type II methanotrophs were active in the 'control' soil treatment. The result emphasize that changes to a particular ecological niche may not result in an immediate change to the active bacterial composition and change in composition will depend on the ability of the bacterial communities to respond to the perturbation. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

The complete mitochondrial genome of the three-spot seahorse, Hippocampus trimaculatus (Teleostei, Syngnathidae).

PubMed

Chang, Chia-Hao; Shao, Kwang-Tsao; Lin, Yeong-Shin; Liao, Yun-Chih

2013-12-01

The complete mitochondrial genome of the three-spot seahorse was sequenced using a polymerase chain reaction-based method. The total length of mitochondrial DNA is 16,535 bp and includes 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. The mitochondrial gene order of the three-spot seahorse also conforms to the distinctive vertebrate mitochondrial gene order. The base composition of the genome is A (32.7%), T (29.3%), C (23.4%), and G (14.6%) with an A + T-rich hallmark as that of other vertebrate mitochondrial genomes.

The complete mitochondrial genome of the tiger tail seahorse, Hippocampus comes (Teleostei, Syngnathidae).

PubMed

Chang, Chia-Hao; Lin, Han-Yang; Jang-Liaw, Nian-Hong; Shao, Kwang-Tsao; Lin, Yeong-Shin; Ho, Hsuan-Ching

2013-06-01

The complete mitochondrial genome of the tiger tail seahorse was sequenced using a polymerase chain reaction-based method. The total length of mitochondrial DNA is 16,525 bp and includes 13 protein-coding genes, 2 ribosomal RNA, 22 transfer RNA genes, and a control region. The mitochondrial gene arrangement of the tiger tail seahorse is also matching the one observed in the most vertebrate creatures. Base composition of the genome is A (32.8%), T (29.8%), C (23.0%), and G (14.4%) with an A+T-rich hallmark as that of other vertebrate mitochondrial genomes.

Markov models of genome segmentation

NASA Astrophysics Data System (ADS)

Thakur, Vivek; Azad, Rajeev K.; Ramaswamy, Ram

2007-01-01

We introduce Markov models for segmentation of symbolic sequences, extending a segmentation procedure based on the Jensen-Shannon divergence that has been introduced earlier. Higher-order Markov models are more sensitive to the details of local patterns and in application to genome analysis, this makes it possible to segment a sequence at positions that are biologically meaningful. We show the advantage of higher-order Markov-model-based segmentation procedures in detecting compositional inhomogeneity in chimeric DNA sequences constructed from genomes of diverse species, and in application to the E. coli K12 genome, boundaries of genomic islands, cryptic prophages, and horizontally acquired regions are accurately identified.

Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil

PubMed Central

Nüsslein, Klaus; Tiedje, James M.

1999-01-01

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium–Agrobacterium assemblages in the pasture soil. PMID:10427058

Comparing Ecological and Genetic Diversity Within the Marine Diatom Genus Pseudo-nitzschia: A Multiregional Synthesis

NASA Astrophysics Data System (ADS)

Hubbard, K.; Bruzek, S.

2016-02-01

The globally distributed marine diatom genus Pseudo-nitzschia consists of approximately 40 species, more than half of which occur in US coastal waters. Here, sensitive genetic tools targeting a variable portion of the internal transcribed spacer 1 (ITS1) region of the rRNA gene were used to assess Pseudo-nitzschia spp. diversity in more than 600 environmental DNA samples collected from US Atlantic, Pacific, and Gulf of Mexico waters. Community-based approaches employed genus-specific primers for environmental DNA fingerprinting and targeted sequencing. For the Gulf of Mexico samples especially, a nested PCR approach (with or without degenerate primers) improved resolution of species diversity. To date, more than 40 unique ITS1 amplicon sizes have been repeatedly observed in ITS1 fingerprints. Targeted sequencing of environmental DNA as well as single chains isolated from live samples indicate that many of these represent novel and known inter- and intra-specific Pseudo-nitzschia diversity. A few species (e.g., P. pungens, P. cuspidata) occur across all three regions, whereas other species and intraspecific variants occurred at local to regional spatial scales only. Generally, species frequently co-occur in complex assemblages, and transitions in Pseudo-nitzschia community composition occur seasonally, prior to bloom initiation, and across (cross-shelf, latitudinal, and vertical) environmental gradients. These observations highlight the dynamic nature of diatom community composition in the marine environment and the importance of classifying diversity at relevant ecological and/or taxonomic scales.

Molecular scatology as a tool to study diet: analysis of prey DNA in scats from captive Steller sea lions.

PubMed

Deagle, B E; Tollit, D J; Jarman, S N; Hindell, M A; Trites, A W; Gales, N J

2005-05-01

The DNA of prey present in animal scats may provide a valuable source of information for dietary studies. We conducted a captive feeding trial to test whether prey DNA could be reliably detected in scat samples from Steller sea lions (Eumetopias jubatus). Two sea lions were fed a diet of fish (five species) and squid (one species), and DNA was extracted from the soft component of collected scats. Most of the DNA obtained came from the predator, but prey DNA could be amplified using prey-specific primers. The four prey species fed in consistent daily proportions throughout the trial were detected in more than 90% of the scat DNA extractions. Squid and sockeye salmon, which were fed as a relatively small percentage of the daily diet, were detected as reliably as the more abundant diet items. Prey detection was erratic in scats collected when the daily diet was fed in two meals that differed in prey composition, suggesting that prey DNA is passed in meal specific pulses. Prey items that were removed from the diet following one day of feeding were only detected in scats collected within 48 h of ingestion. Proportions of fish DNA present in eight scat samples (evaluated through the screening of clone libraries) were roughly proportional to the mass of prey items consumed, raising the possibility that DNA quantification methods could provide semi-quantitative diet composition data. This study should be of broad interest to researchers studying diet since it highlights an approach that can accurately identify prey species and is not dependent on prey hard parts surviving digestion.

DNA-PK assay

DOEpatents

Anderson, Carl W.; Connelly, Margery A.

2004-10-12

The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

The chloroplast and mitochondrial genome sequences of the charophyte Chaetosphaeridium globosum: Insights into the timing of the events that restructured organelle DNAs within the green algal lineage that led to land plants

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2002-01-01

The land plants and their immediate green algal ancestors, the charophytes, form the Streptophyta. There is evidence that both the chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) underwent substantial changes in their architecture (intron insertions, gene losses, scrambling in gene order, and genome expansion in the case of mtDNA) during the evolution of streptophytes; however, because no charophyte organelle DNAs have been sequenced completely thus far, the suite of events that shaped streptophyte organelle genomes remains largely unknown. Here, we have determined the complete cpDNA (131,183 bp) and mtDNA (56,574 bp) sequences of the charophyte Chaetosphaeridium globosum (Coleochaetales). At the levels of gene content (124 genes), intron composition (18 introns), and gene order, Chaetosphaeridium cpDNA is remarkably similar to land-plant cpDNAs, implying that most of the features characteristic of land-plant lineages were gained during the evolution of charophytes. Although the gene content of Chaetosphaeridium mtDNA (67 genes) closely resembles that of the bryophyte Marchantia polymorpha (69 genes), this charophyte mtDNA differs substantially from its land-plant relatives at the levels of size, intron composition (11 introns), and gene order. Our finding that it shares only one intron with its land-plant counterparts supports the idea that the vast majority of mitochondrial introns in land plants appeared after the emergence of these organisms. Our results also suggest that the events accounting for the spacious intergenic spacers found in land-plant mtDNAs took place late during the evolution of charophytes or coincided with the transition from charophytes to land plants. PMID:12161560

Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants.

PubMed

Hill, Cian J; Brown, Jillian R M; Lynch, Denise B; Jeffery, Ian B; Ryan, C Anthony; Ross, R Paul; Stanton, Catherine; O'Toole, Paul W

2016-05-10

Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p < 0.001). When the microbiota profiles were analysed at the operational taxonomic unit (OTU) level, three infant datasets in the study did not cluster together, while only one elderly dataset did not. The lower microbiota diversity of the infant gut microbiota compared to the elderly gut microbiota (p < 0.001) means that any alteration in the infant datasets has a proportionally larger effect. The commercial storage vials appear to be suitable for high diversity microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is unavoidable, a consistent storage regime should be used. We would recommend extraction ideally within the first week of storage.

Engineering the Structure and Properties of DNA-Nanoparticle Superstructures Using Polyvalent Counterions.

PubMed

Chou, Leo Y T; Song, Fayi; Chan, Warren C W

2016-04-06

DNA assembly of nanoparticles is a powerful approach to control their properties and prototype new materials. However, the structure and properties of DNA-assembled nanoparticles are labile and sensitive to interactions with counterions, which vary with processing and application environment. Here we show that substituting polyamines in place of elemental counterions significantly enhanced the structural rigidity and plasmonic properties of DNA-assembled metal nanoparticles. These effects arose from the ability of polyamines to condense DNA and cross-link DNA-coated nanoparticles. We further used polyamine wrapped DNA nanostructures as structural templates to seed the growth of polymer multilayers via layer-by-layer assembly, and controlled the degree of DNA condensation, plasmon coupling efficiency, and material responsiveness to environmental stimuli by varying polyelectrolyte composition. These results highlight counterion engineering as a versatile strategy to tailor the properties of DNA-nanoparticle assemblies for various applications, and should be applicable to other classes of DNA nanostructures.

GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules

PubMed Central

Sarrion-Perdigones, Alejandro; Falconi, Erica Elvira; Zandalinas, Sara I.; Juárez, Paloma; Fernández-del-Carmen, Asun; Granell, Antonio; Orzaez, Diego

2011-01-01

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described. PMID:21750718

GoldenBraid: an iterative cloning system for standardized assembly of reusable genetic modules.

PubMed

Sarrion-Perdigones, Alejandro; Falconi, Erica Elvira; Zandalinas, Sara I; Juárez, Paloma; Fernández-del-Carmen, Asun; Granell, Antonio; Orzaez, Diego

2011-01-01

Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop ("braid") topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.

PEGylated Polyaniline Nanofibers: Antifouling and Conducting Biomaterial for Electrochemical DNA Sensing.

PubMed

Hui, Ni; Sun, Xiaotian; Niu, Shuyan; Luo, Xiliang

2017-01-25

Biofouling arising from nonspecific adsorption is a substantial outstanding challenge in diagnostics and disease monitoring, and antifouling sensing interfaces capable of reducing the nonspecific adsorption of proteins from biological complex samples are highly desirable. We present herein the preparation of novel composite nanofibers through the grafting of polyethylene glycol (PEG) polymer onto polyaniline (PANI) nanofibers and their application in the development of antifouling electrochemical biosensors. The PEGylated PANI (PANI/PEG) nanofibers possessed large surface area and remained conductive and at the same time demonstrated excellent antifouling performances in single protein solutions as well as complex human serum samples. Sensitive and low fouling electrochemical biosensors for the breast cancer susceptibility gene (BRCA1) can be easily fabricated through the attachment of DNA probes to the PANI/PEG nanofibers. The biosensor showed a very high sensitivity to target BRCA1 with a linear range from 0.01 pM to 1 nM and was also efficient enough to detect DNA mismatches with satisfactory selectivity. Moreover, the DNA biosensor based on the PEGylated PANI nanofibers supported the quantification of BRCA1 in complex human serum, indicating great potential of this novel biomaterial for application in biosensors and bioelectronics.

Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds.

PubMed

Kang, Yun Gyeong; Wei, Jie; Shin, Ji Won; Wu, Yan Ru; Su, Jiacan; Park, Young Shik; Shin, Jung-Woog

2018-01-01

Successful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity. In this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w). Evaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group. These results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration.

Institutional Biosafety Committees and the Inadequacies of Risk Regulation.

ERIC Educational Resources Information Center

Bereano, Philip L.

1984-01-01

Discusses institutional biosafety committees (IBC) which provide quasi-independent reviews of recombinant DNA work done at an institution. Considers the nature of IBC operation, the National Institutes of Health "Guidelines for Recombinant DNA Research," the composition of IBCs, the intellectual content of IBC deliberations, and issues…

The influence of selenium status on body composition, oxidative DNA damage and total antioxidant capacity in newly diagnosed type 2 diabetes mellitus: A case-control study.

PubMed

Othman, Fatimah Binti; Mohamed, Hamid Jan Bin Jan; Sirajudeen, K N S; Noh, Mohd Fairulnizal B Md; Rajab, Nor Fadilah

2017-09-01

Selenium is involved in the complex system of defense against oxidative stress in diabetes through its biological function of selenoproteins and the antioxidant enzyme. A case-control study was carried out to determine the association of plasma selenium with oxidative stress and body composition status presented in Type 2 Diabetes Mellitus (T2DM) patient and healthy control. This study involved 82 newly diagnosed T2DM patients and 82 healthy controls. Plasma selenium status was determined with Graphite Furnace Atomic Absorption Spectrometry. Body Mass Index, total body fat and visceral fat was assessed for body composition using Body Composition Analyzer (TANITA). Oxidative DNA damage and total antioxidant capacity were determined for oxidative stress biomarker status. In age, gender and BMI adjustment, no significant difference of plasma selenium level between T2DM and healthy controls was observed. There was as a significant difference of Oxidative DNA damage and total antioxidant capacity between T2DM patients and healthy controls with tail DNA% 20.62 [95% CI: 19.71,21.49] (T2DM), 17.67 [95% CI: 16.87,18.56] (control); log tail moment 0.41[95% CI: 0.30,0.52] (T2DM), 0.41[95% CI: 0.30,0.52] (control); total antioxidant capacity 0.56 [95% CI: 0.54,0.58] (T2DM), 0.60 [95% CI: 0.57,0.62] (control). Waist circumference, BMI, visceral fat, body fat and oxidative DNA damage in the T2DM group were significantly lower in the first plasma selenium tertile (38.65-80.90μg/L) compared to the second (80.91-98.20μg/L) and the third selenium tertiles (98.21-158.20μg/L). A similar trend, but not statistically significant, was observed in the control group. Copyright © 2016 Elsevier GmbH. All rights reserved.

Analysis of Structure and Composition of Bacterial Core Communities in Mature Drinking Water Biofilms and Bulk Water of a Citywide Network in Germany

PubMed Central

Henne, Karsten; Kahlisch, Leila; Brettar, Ingrid

2012-01-01

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity. PMID:22389373

Analysis of structure and composition of bacterial core communities in mature drinking water biofilms and bulk water of a citywide network in Germany.

PubMed

Henne, Karsten; Kahlisch, Leila; Brettar, Ingrid; Höfle, Manfred G

2012-05-01

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity.

A Review of Conventional PCR Assays for the Detection of Selected Phytopathogens of Wheat.

PubMed

Kuzdraliński, Adam; Kot, Anna; Szczerba, Hubert; Nowak, Michał; Muszyńska, Marta

2017-01-01

Infection of phyllosphere (stems, leaves, husks, and grains) by pathogenic fungi reduces the wheat yield and grain quality. Detection of the main wheat pathogenic fungi provides information about species composition and allows effective and targeted plant treatment. Since conventional procedures for the detection of these organisms are unreliable and time consuming, diagnostic DNA-based methods are required. Nucleic acid amplification technologies are independent of the morphological and biochemical characteristics of fungi. Microorganisms do not need to be cultured. Therefore, a number of PCR-based methodologies have been developed for the identification of key pathogenic fungi, such as Fusarium spp., Puccinia spp., Zymoseptoria tritici, Parastagonospora nodorum, Blumeria graminis f. sp. tritici, and Pyrenophora tritici-repentis. This article reviews frequently used DNA regions for fungus identification and discusses already known PCR assays for detection of the aforementioned wheat pathogens. We demonstrate that PCR-based wheat pathogen identification assays require further research. In particular, the number of diagnostic tests for Fusarium graminearum, Puccinia spp., and P. tritici-repentis are insufficient. © 2017 S. Karger AG, Basel.

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

PubMed

Scala, Giovanni; Affinito, Ornella; Palumbo, Domenico; Florio, Ermanno; Monticelli, Antonella; Miele, Gennaro; Chiariotti, Lorenzo; Cocozza, Sergio

2016-11-25

CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

Predicting DNA binding proteins using support vector machine with hybrid fractal features.

PubMed

Niu, Xiao-Hui; Hu, Xue-Hai; Shi, Feng; Xia, Jing-Bo

2014-02-21

DNA-binding proteins play a vitally important role in many biological processes. Prediction of DNA-binding proteins from amino acid sequence is a significant but not fairly resolved scientific problem. Chaos game representation (CGR) investigates the patterns hidden in protein sequences, and visually reveals previously unknown structure. Fractal dimensions (FD) are good tools to measure sizes of complex, highly irregular geometric objects. In order to extract the intrinsic correlation with DNA-binding property from protein sequences, CGR algorithm, fractal dimension and amino acid composition are applied to formulate the numerical features of protein samples in this paper. Seven groups of features are extracted, which can be computed directly from the primary sequence, and each group is evaluated by the 10-fold cross-validation test and Jackknife test. Comparing the results of numerical experiments, the group of amino acid composition and fractal dimension (21-dimension vector) gets the best result, the average accuracy is 81.82% and average Matthew's correlation coefficient (MCC) is 0.6017. This resulting predictor is also compared with existing method DNA-Prot and shows better performances. © 2013 The Authors. Published by Elsevier Ltd All rights reserved.

Unlocking biodiversity and conservation studies in high diversity environments using environmental DNA (eDNA): a test with Guianese freshwater fishes.

PubMed

Cilleros, Kévin; Valentini, Alice; Allard, Luc; Dejean, Tony; Etienne, Roselyne; Grenouillet, Gaël; Iribar, Amaia; Taberlet, Pierre; Vigouroux, Régis; Brosse, Sébastien

2018-05-16

Determining the species compositions of local assemblages is a prerequisite to understanding how anthropogenic disturbances affect biodiversity. However, biodiversity measurements often remain incomplete due to the limited efficiency of sampling methods. This is particularly true in freshwater tropical environments that host rich fish assemblages, for which assessments are uncertain and often rely on destructive methods. Developing an efficient and non-destructive method to assess biodiversity in tropical freshwaters is highly important. In this study, we tested the efficiency of environmental DNA (eDNA) metabarcoding to assess the fish diversity of 39 Guianese sites. We compared the diversity and composition of assemblages obtained using traditional and metabarcoding methods. More than 7,000 individual fish belonging to 203 Guianese fish species were collected by traditional sampling methods, and ~17 million reads were produced by metabarcoding, among which ~8 million reads were assigned to 148 fish taxonomic units, including 132 fish species. The two methods detected a similar number of species at each site, but the species identities partially matched. The assemblage compositions from the different drainage basins were better discriminated using metabarcoding, revealing that while traditional methods provide a more complete but spatially limited inventory of fish assemblages, metabarcoding provides a more partial but spatially extensive inventory. eDNA metabarcoding can therefore be used for rapid and large-scale biodiversity assessments, while at a local scale, the two approaches are complementary and enable an understanding of realistic fish biodiversity. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Annual time-series analysis of aqueous eDNA reveals ecologically relevant dynamics of lake ecosystem biodiversity

PubMed Central

Bista, Iliana; Carvalho, Gary R.; Walsh, Kerry; Seymour, Mathew; Hajibabaei, Mehrdad; Lallias, Delphine; Christmas, Martin; Creer, Simon

2017-01-01

The use of environmental DNA (eDNA) in biodiversity assessments offers a step-change in sensitivity, throughput and simultaneous measures of ecosystem diversity and function. There remains, however, a need to examine eDNA persistence in the wild through simultaneous temporal measures of eDNA and biota. Here, we use metabarcoding of two markers of different lengths, derived from an annual time series of aqueous lake eDNA to examine temporal shifts in ecosystem biodiversity and in an ecologically important group of macroinvertebrates (Diptera: Chironomidae). The analyses allow different levels of detection and validation of taxon richness and community composition (β-diversity) through time, with shorter eDNA fragments dominating the eDNA community. Comparisons between eDNA, community DNA, taxonomy and UK species abundance data further show significant relationships between diversity estimates derived across the disparate methodologies. Our results reveal the temporal dynamics of eDNA and validate the utility of eDNA metabarcoding for tracking seasonal diversity at the ecosystem scale. PMID:28098255

Annual time-series analysis of aqueous eDNA reveals ecologically relevant dynamics of lake ecosystem biodiversity

NASA Astrophysics Data System (ADS)

Bista, Iliana; Carvalho, Gary R.; Walsh, Kerry; Seymour, Mathew; Hajibabaei, Mehrdad; Lallias, Delphine; Christmas, Martin; Creer, Simon

2017-01-01

The use of environmental DNA (eDNA) in biodiversity assessments offers a step-change in sensitivity, throughput and simultaneous measures of ecosystem diversity and function. There remains, however, a need to examine eDNA persistence in the wild through simultaneous temporal measures of eDNA and biota. Here, we use metabarcoding of two markers of different lengths, derived from an annual time series of aqueous lake eDNA to examine temporal shifts in ecosystem biodiversity and in an ecologically important group of macroinvertebrates (Diptera: Chironomidae). The analyses allow different levels of detection and validation of taxon richness and community composition (β-diversity) through time, with shorter eDNA fragments dominating the eDNA community. Comparisons between eDNA, community DNA, taxonomy and UK species abundance data further show significant relationships between diversity estimates derived across the disparate methodologies. Our results reveal the temporal dynamics of eDNA and validate the utility of eDNA metabarcoding for tracking seasonal diversity at the ecosystem scale.

Effect of potentially interfering substances on the measurement of HIV-1 viral load by the bDNA assay.

PubMed

Alonso, R; García de Viedma, D; Rodríguez-Creixems, M; Bouza, E

1999-03-01

As high heterogeneity of plasma composition may be responsible for interference with HIV-1 viral load determination by the bDNA assay, the potential interference caused by a number of plasma components was examined. Among the biochemical substances assayed, cholesterol, bilirubin, and triglycerides did not affect viral load quantification. Hemoglobin did not interfere with the assay at concentrations lower than or equal to 14 g/dl. Above this concentration, measurements decreased by up to 0.78 log, but these hemoglobin levels do not usually occur in the clinical setting. None of the antiretroviral drugs assayed (AZT, dDC, d4T, 3TC and Indinavir) interfered with the measurement. HIV bDNA is a robust assay even in those frequent circumstances in which plasma composition differs notably from normal.

Membrane-containing virus particles exhibit the mechanics of a composite material for genome protection.

PubMed

Azinas, S; Bano, F; Torca, I; Bamford, D H; Schwartz, G A; Esnaola, J; Oksanen, H M; Richter, R P; Abrescia, N G

2018-04-26

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

PubMed

Pavco, P A; Van Tuyle, G C

1985-01-01

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.

Genomes of the T4-related bacteriophages as windows on microbial genome evolution.

PubMed

Petrov, Vasiliy M; Ratnayaka, Swarnamala; Nolan, James M; Miller, Eric S; Karam, Jim D

2010-10-28

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature.

Genomes of the T4-related bacteriophages as windows on microbial genome evolution

PubMed Central

2010-01-01

The T4-related bacteriophages are a group of bacterial viruses that share morphological similarities and genetic homologies with the well-studied Escherichia coli phage T4, but that diverge from T4 and each other by a number of genetically determined characteristics including the bacterial hosts they infect, the sizes of their linear double-stranded (ds) DNA genomes and the predicted compositions of their proteomes. The genomes of about 40 of these phages have been sequenced and annotated over the last several years and are compared here in the context of the factors that have determined their diversity and the diversity of other microbial genomes in evolution. The genomes of the T4 relatives analyzed so far range in size between ~160,000 and ~250,000 base pairs (bp) and are mosaics of one another, consisting of clusters of homology between them that are interspersed with segments that vary considerably in genetic composition between the different phage lineages. Based on the known biological and biochemical properties of phage T4 and the proteins encoded by the T4 genome, the T4 relatives reviewed here are predicted to share a genetic core, or "Core Genome" that determines the structural design of their dsDNA chromosomes, their distinctive morphology and the process of their assembly into infectious agents (phage morphogenesis). The Core Genome appears to be the most ancient genetic component of this phage group and constitutes a mere 12-15% of the total protein encoding potential of the typical T4-related phage genome. The high degree of genetic heterogeneity that exists outside of this shared core suggests that horizontal DNA transfer involving many genetic sources has played a major role in diversification of the T4-related phages and their spread to a wide spectrum of bacterial species domains in evolution. We discuss some of the factors and pathways that might have shaped the evolution of these phages and point out several parallels between their diversity and the diversity generally observed within all groups of interrelated dsDNA microbial genomes in nature. PMID:21029436

A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

PubMed

Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

2016-12-15

The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. Copyright © 2016 Elsevier B.V. All rights reserved.

Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

Treesearch

Fei Cheng; Lin Hou; Keith Woeste; Zhengchun Shang; Xiaobang Peng; Peng Zhao; Shuoxin Zhang

2016-01-01

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high...

Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

PubMed

Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

1999-02-05

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

Eukaryotic DNA Replication Fork.

PubMed

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

PubMed Central

Baumeister, Mark A.; Zhang, Nan; Beas, Hilda; Brooks, Jesse R.; Canchola, Jesse A.; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R.

2012-01-01

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements. PMID:22479381

A sensitive branched DNA HIV-1 signal amplification viral load assay with single day turnaround.

PubMed

Baumeister, Mark A; Zhang, Nan; Beas, Hilda; Brooks, Jesse R; Canchola, Jesse A; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R

2012-01-01

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) ("Versant Assay") currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16-18 h to 2.5 h, composition of only the "Lysis Diluent" solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements.

Sandwiching spherical 1,2-dioleoyltrimethylammoniumpropane liposome in gold nanoparticle on solid transducer for electrochemical ultrasensitive DNA detection and transfection.

PubMed

Shankara Narayanan, Jeyaraman; Bhuvana, Mohanlal; Dharuman, Venkataraman

2014-08-15

Cationic N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium propane (DOTAP) liposome is spherically sandwiched in gold nanoparticle (abbreviated as sDOTAP-AuNP) onto a gold electrode surface. The sDOTAP-AuNP is applied for electrochemical label free DNA sensing and Escherichia coli cell transfection for the first time. Complementary target (named as hybridized), non-complementary target (un-hybridized) and single base mismatch target (named as SMM) hybridized surfaces are discriminated sensitively and selectively in presence of [Fe(CN)6](3-/4-). Double strand specific intercalator methylene blue in combination with [Fe(CN)6](3-) is used to enhance target detection limit down to femtomolar concentration. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV) techniques are used for characterizing DNA sensing. High Resolution Transmission Electron Microscopy (HRTEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Dynamic Light Scattering (DLS) techniques are used to confirm the spherical nature of the sDOTAP-AuNP-DNA composite in solution and on the solid surface. DNA on the sDOTAP-ssDNA is transferred by potential stripping method (+0.2V (Ag/AgCl)) into buffer solution containing E. coli cells. The transfection is confirmed by the contrast images for the transfected and non-transfected cell from Confocal Laser Scanning Microscopy (CLSM). The results demonstrate effectiveness of the electrochemical DNA transfection method developed and could be applied for other cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

PubMed

Yu, Zhongtang; Yu, Marie; Morrison, Mark

2006-04-01

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

Ribosomal DNA Organization Before and After Magnification in Drosophila melanogaster

PubMed Central

Bianciardi, Alessio; Boschi, Manuela; Swanson, Ellen E.; Belloni, Massimo; Robbins, Leonard G.

2012-01-01

In all eukaryotes, the ribosomal RNA genes are stably inherited redundant elements. In Drosophila melanogaster, the presence of a Ybb− chromosome in males, or the maternal presence of the Ribosomal exchange (Rex) element, induces magnification: a heritable increase of rDNA copy number. To date, several alternative classes of mechanisms have been proposed for magnification: in situ replication or extra-chromosomal replication, either of which might act on short or extended strings of rDNA units, or unequal sister chromatid exchange. To eliminate some of these hypotheses, none of which has been clearly proven, we examined molecular-variant composition and compared genetic maps of the rDNA in the bb2 mutant and in some magnified bb+ alleles. The genetic markers used are molecular-length variants of IGS sequences and of R1 and R2 mobile elements present in many 28S sequences. Direct comparison of PCR products does not reveal any particularly intensified electrophoretic bands in magnified alleles compared to the nonmagnified bb2 allele. Hence, the increase of rDNA copy number is diluted among multiple variants. We can therefore reject mechanisms of magnification based on multiple rounds of replication of short strings. Moreover, we find no changes of marker order when pre- and postmagnification maps are compared. Thus, we can further restrict the possible mechanisms to two: replication in situ of an extended string of rDNA units or unequal exchange between sister chromatids. PMID:22505623

Diversity of Bacterial Communities in Container Habitats of Mosquitoes

PubMed Central

Ponnusamy, Loganathan; Xu, Ning; Stav, Gil; Wesson, Dawn M.; Schal, Coby

2010-01-01

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n=12), cemetery urns (n=23), and miscellaneous containers that included two tree holes (n=19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. PMID:18373113

A methodology to annotate systems biology markup language models with the synthetic biology open language.

PubMed

Roehner, Nicholas; Myers, Chris J

2014-02-21

Recently, we have begun to witness the potential of synthetic biology, noted here in the form of bacteria and yeast that have been genetically engineered to produce biofuels, manufacture drug precursors, and even invade tumor cells. The success of these projects, however, has often failed in translation and application to new projects, a problem exacerbated by a lack of engineering standards that combine descriptions of the structure and function of DNA. To address this need, this paper describes a methodology to connect the systems biology markup language (SBML) to the synthetic biology open language (SBOL), existing standards that describe biochemical models and DNA components, respectively. Our methodology involves first annotating SBML model elements such as species and reactions with SBOL DNA components. A graph is then constructed from the model, with vertices corresponding to elements within the model and edges corresponding to the cause-and-effect relationships between these elements. Lastly, the graph is traversed to assemble the annotating DNA components into a composite DNA component, which is used to annotate the model itself and can be referenced by other composite models and DNA components. In this way, our methodology can be used to build up a hierarchical library of models annotated with DNA components. Such a library is a useful input to any future genetic technology mapping algorithm that would automate the process of composing DNA components to satisfy a behavioral specification. Our methodology for SBML-to-SBOL annotation is implemented in the latest version of our genetic design automation (GDA) software tool, iBioSim.

Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase*

PubMed Central

Samai, Poulami; Shuman, Stewart

2011-01-01

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5′-phosphate nucleotide and the 3′-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps. PMID:21527793

Impedimetric genosensor for ultratrace detection of hepatitis B virus DNA in patient samples assisted by zeolites and MWCNT nano-composites.

PubMed

Narang, Jagriti; Singhal, Chaitali; Malhotra, Nitesh; Narang, Sumit; Pn, Anoop Krishna; Gupta, Riya; Kansal, Ruby; Pundir, C S

2016-12-15

Nanocrystals of zeolites (Nanocrys Zeo) and Multi-walled carbon nanotubes (MWCNT) based diagnostic genosensor was employed for detection of polymerase chain (PCR) amplified HBVDNA in blood of hepatitis B patients. The ssDNA-nanocomposite modified electrode was characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The hybridization between ss DNA probe and target ss DNA was detected by reduction in current, generated by interaction of methylene blue (MB) with free guanine (3'G) of ssDNA. Nanocrys zeo were deposited on the Fluorine doped tin oxide glass electrode (FTO) by drop-casting method for better immobilization of ss DNA while MWCNTs are incorporated into the zeolite-assembly to enhance the electro-conductivity of the present genosensor. The ssDNA-nanocomposite modified FTO electrode exhibited optimum current within 5s, at pH 5.6, and incubation temperature of 45°C. The value of charge transfer resistance (Rct) was linear with the number of copies of target DNA between 150 and 10(6) copies/ml. The limit of detection (LOD) of the sensor was 50 copies/ml. Within and between batches coefficients of variation (CV) were 2.5% and 3.2% respectively. Results obtained with our genosensor were also correlated with those by RT-PCR and r(2) value found with good accuracy of 97%. The electrode was reused by dipping it into 0.1M NaOH for 3min and lost 50% of its initial activity in 4 weeks. Furthermore the technique employed for detection of HBV is EIS, which is convenient and required less analysis time. Copyright © 2016 Elsevier B.V. All rights reserved.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

PubMed

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped. Copyright © 2017 Traverse and Ochman.

Quantification of DNA in simple eukaryotic cells using Fourier transform infrared spectroscopy.

PubMed

Whelan, Donna R; Bambery, Keith R; Puskar, Ljiljana; McNaughton, Don; Wood, Bayden R

2013-10-01

A technique capable of detecting and monitoring nucleic acid concentration offers potential in diagnosing cancer and further developing an understanding of the biochemistry of disease. The application of Fourier transform infrared (FTIR) spectroscopy has previously been hindered by the supposed non-Beer-Lambert absorption behavior of DNA in intact cells making elucidation of the DNA bands difficult. We use known composition DNA/hemoglobin standards to successfully estimate the DNA content in avian erythrocyte nuclei (44.2%) and intact erythrocytes (12.8%). Furthermore we demonstrate that the absorption of cellular DNA does follow the Beer-Lambert Law and highlights the role of conformation and hydration in FTIR spectroscopy of biological samples. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Environmental filtering and phylogenetic clustering correlate with the distribution patterns of cryptic protist species.

PubMed

Singer, David; Kosakyan, Anush; Seppey, Christophe V W; Pillonel, Amandine; Fernández, Leonardo D; Fontaneto, Diego; Mitchell, Edward A D; Lara, Enrique

2018-04-01

The community composition of any group of organisms should theoretically be determined by a combination of assembly processes including resource partitioning, competition, environmental filtering, and phylogenetic legacy. Environmental DNA studies have revealed a huge diversity of protists in all environments, raising questions about the ecological significance of such diversity and the degree to which they obey to the same rules as macroscopic organisms. The fast-growing cultivable protist species on which hypotheses are usually experimentally tested represent only a minority of the protist diversity. Addressing these questions for the lesser known majority can only be inferred through observational studies. We conducted an environmental DNA survey of the genus Nebela, a group of closely related testate (shelled) amoeba species, in different habitats within Sphagnum-dominated peatlands. Identification based on the mitochondrial cytochrome c oxidase 1 gene, allowed species-level resolution as well as phylogenetic reconstruction. Community composition varied strongly across habitats and associated environmental gradients. Species showed little overlap in their realized niche, suggesting resource partitioning, and a strong influence of environmental filtering driving community composition. Furthermore, phylogenetic clustering was observed in the most nitrogen-poor samples, supporting phylogenetic inheritance of adaptations in the group of N. guttata. This study showed that the studied free-living unicellular eukaryotes follow to community assembly rules similar to those known to determine plant and animal communities; the same may be true for much of the huge functional and taxonomic diversity of protists. © 2018 by the Ecological Society of America.

Introducing a model of pairing based on base pair specific interactions between identical DNA sequences

NASA Astrophysics Data System (ADS)

(O' Lee, Dominic J.

2018-02-01

At present, there have been suggested two types of physical mechanism that may facilitate preferential pairing between DNA molecules, with identical or similar base pair texts, without separation of base pairs. One mechanism solely relies on base pair specific patterns of helix distortion being the same on the two molecules, discussed extensively in the past. The other mechanism proposes that there are preferential interactions between base pairs of the same composition. We introduce a model, built on this second mechanism, where both thermal stretching and twisting fluctuations are included, as well as the base pair specific helix distortions. Firstly, we consider an approximation for weak pairing interactions, or short molecules. This yields a dependence of the energy on the square root of the molecular length, which could explain recent experimental data. However, analysis suggests that this approximation is no longer valid at large DNA lengths. In a second approximation, for long molecules, we define two adaptation lengths for twisting and stretching, over which the pairing interaction can limit the accumulation of helix disorder. When the pairing interaction is sufficiently strong, both adaptation lengths are finite; however, as we reduce pairing strength, the stretching adaptation length remains finite but the torsional one becomes infinite. This second state persists to arbitrarily weak values of the pairing strength; suggesting that, if the molecules are long enough, the pairing energy scales as length. To probe differences between the two pairing mechanisms, we also construct a model of similar form. However, now, pairing between identical sequences solely relies on the intrinsic helix distortion patterns. Between the two models, we see interesting qualitative differences. We discuss our findings, and suggest new work to distinguish between the two mechanisms.

Micromonospora halotolerans sp. nov., isolated from the rhizosphere of a Pisum sativum plant.

PubMed

Carro, Lorena; Pukall, Rüdiger; Spröer, Cathrin; Kroppenstedt, Reiner M; Trujillo, Martha E

2013-06-01

A filamentous actinomycete strain designated CR18(T) was isolated on humic acid agar from the rhizosphere of a Pisum sativum plant collected in Spain. This isolate was observed to grow optimally at 28 °C, pH 7.0 and in the presence of 5 % NaCl. Phylogenetic analyses based on the 16S rRNA gene sequence indicated a close relationship with the type strains of Micromonospora chersina and Micromonospora endolithica. A further analysis based on a concatenated DNA sequence stretch of 4,523 bp that included partial sequences of the atpD, gyrB, recA, rpoB and 16S rRNA genes clearly differentiated the new strain from recognized Micromonospora species compared. DNA-DNA hybridization studies further supported the taxonomic position of strain CR18(T) as a novel genomic species. Chemotaxonomic analyses which included whole cell sugars, polar lipids, fatty acid profiles and menaquinone composition confirmed the affiliation of the new strain to the genus Micromonospora and also highlighted differences at the species level. These studies were finally complemented with an array of physiological tests to help differentiate between the new strain and its phylogenetic neighbours. Consequently, strain CR18(T) (= CECT 7890(T) = DSM 45598(T)) is proposed as the type strain of a novel species, Micromonospora halotolerans sp. nov.

Molecular characterization of total and metabolically active bacterial communities of "white colonizations" in the Altamira Cave, Spain.

PubMed

Portillo, M Carmen; Saiz-Jimenez, Cesareo; Gonzalez, Juan M

2009-01-01

Caves with paleolithic paintings are influenced by bacterial development. Altamira Cave (Spain) contains some of the most famous paintings from the Paleolithic era. An assessment of the composition of bacterial communities that have colonized this cave represents a first step in understanding and potentially controlling their proliferation. In this study, areas showing colonization with uncolored microorganisms, referred to as "white colonizations", were analyzed. Microorganisms present in these colonizations were studied using DNA analysis, and those showing significant metabolic activity were detected in RNA-based RNA analysis. Bacterial community fingerprints were obtained both from DNA and RNA analyses, indicating differences between the microorganisms present and metabolically active in these white colonizations. Metabolically active microorganisms represented only a fraction of the total bacterial community present in the colonizations. 16S rRNA gene libraries were used to identify the major representative members of the studied communities. Proteobacteria constituted the most frequently found division both among metabolically active microorganisms (from RNA-based analysis) and those present in the community (from DNA analysis). Results suggest the existence of a huge variety of taxa in white colonizations of the Altamira Cave which represent a potential risk for the conservation of the cave and its paintings.

Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring

PubMed Central

Vierheilig, J.; Savio, D.; Ley, R. E.; Mach, R. L.; Farnleitner, A. H.

2016-01-01

The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multicompartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

Protein adsorption and biomimetic mineralization behaviors of PLL-DNA multilayered films assembled onto titanium

NASA Astrophysics Data System (ADS)

Gao, Wenli; Feng, Bo; Ni, Yuxiang; Yang, Yongli; Lu, Xiong; Weng, Jie

2010-11-01

Titanium and its alloys are frequently used as surgical implants in load bearing situations, such as hip prostheses and dental implants, owing to their biocompatibility, mechanical and physical properties. In this paper, a layer-by-layer (LBL) self-assembly technique, based on the polyelectrolyte-mediated electrostatic adsorption of poly-L-lysine (PLL) and DNA, was used to the formation of multilayer on titanium surfaces. Then bovine serum albumin (BSA) adsorption and biomimetic mineralization of modified surfaces were studied. The chemical composition and wettability of assembled substrates were investigated by X-ray photoelectron spectroscopy (XPS), fluorescence microscopy and water contact angle measurement, respectively. The XPS analysis indicated that the layers were assembled successfully through electrostatic attractions. The measurement with ultraviolet (UV) spectrophotometer revealed that the LBL films enhanced ability of BSA adsorption onto titanium. The adsorption quantity of BSA on the surface terminated with PLL was higher than that of the surface terminated with DNA, and the samples of TiOH/P/D/P absorbed BSA most. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) showed that samples of assembled PLL or/and DNA had better bioactivity in inducing HA formation. Thus the assembling of PLL and DNA onto the surface of titanium in turn via a layer-by-layer self-assembly technology can improve the bioactivity of titanium.

A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN-γ

NASA Astrophysics Data System (ADS)

Miao, Xiangmin; Ko, Chung-Nga; Vellaisamy, Kasipandi; Li, Zongbing; Yang, Guanjun; Leung, Chung-Hang; Ma, Dik-Lung

2017-02-01

A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples.

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

PubMed

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2012-01-01

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

A Support Vector Machine based method to distinguish proteobacterial proteins from eukaryotic plant proteins

PubMed Central

2012-01-01

Background Members of the phylum Proteobacteria are most prominent among bacteria causing plant diseases that result in a diminution of the quantity and quality of food produced by agriculture. To ameliorate these losses, there is a need to identify infections in early stages. Recent developments in next generation nucleic acid sequencing and mass spectrometry open the door to screening plants by the sequences of their macromolecules. Such an approach requires the ability to recognize the organismal origin of unknown DNA or peptide fragments. There are many ways to approach this problem but none have emerged as the best protocol. Here we attempt a systematic way to determine organismal origins of peptides by using a machine learning algorithm. The algorithm that we implement is a Support Vector Machine (SVM). Result The amino acid compositions of proteobacterial proteins were found to be different from those of plant proteins. We developed an SVM model based on amino acid and dipeptide compositions to distinguish between a proteobacterial protein and a plant protein. The amino acid composition (AAC) based SVM model had an accuracy of 92.44% with 0.85 Matthews correlation coefficient (MCC) while the dipeptide composition (DC) based SVM model had a maximum accuracy of 94.67% and 0.89 MCC. We also developed SVM models based on a hybrid approach (AAC and DC), which gave a maximum accuracy 94.86% and a 0.90 MCC. The models were tested on unseen or untrained datasets to assess their validity. Conclusion The results indicate that the SVM based on the AAC and DC hybrid approach can be used to distinguish proteobacterial from plant protein sequences. PMID:23046503

Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.

PubMed

Eustermann, Sebastian; Schall, Kevin; Kostrewa, Dirk; Lakomek, Kristina; Strauss, Mike; Moldt, Manuela; Hopfner, Karl-Peter

2018-04-01

In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers 1-3 such as the 15-subunit INO80 complex 4 . INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and -1 nucleosomes at promoter DNA 5-8 . The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA + ATPase heterohexamer is an assembly scaffold for the complex and acts as a 'stator' for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location -6, unwraps approximately 15 base pairs, disrupts the H2A-DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations -2 and -3 to act as a counter grip for the motor, on the other side of the H2A-H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A-H2B near the 'acidic patch'. Our structure together with biochemical data 8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A-H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A-H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange.

Different Amounts of DNA in Newborn Cells of Escherichia coli Preclude a Role for the Chromosome in Size Control According to the "Adder" Model.

PubMed

Huls, Peter G; Vischer, Norbert O E; Woldringh, Conrad L

2018-01-01

According to the recently-revived adder model for cell size control, newborn cells of Escherichia coli will grow and divide after having added a constant size or length, ΔL , irrespective of their size at birth. Assuming exponential elongation, this implies that large newborns will divide earlier than small ones. The molecular basis for the constant size increment is still unknown. As DNA replication and cell growth are coordinated, the constant ΔL could be based on duplication of an equal amount of DNA, ΔG , present in newborn cells. To test this idea, we measured amounts of DNA and lengths of nucleoids in DAPI-stained cells growing in batch culture at slow and fast rates. Deeply-constricted cells were divided in two subpopulations of longer and shorter lengths than average; these were considered to represent large and small prospective daughter cells, respectively. While at slow growth, large and small prospective daughter cells contained similar amounts of DNA, fast growing cells with multiforked replicating chromosomes, showed a significantly higher amount of DNA (20%) in the larger cells. This observation precludes the hypothesis that Δ L is based on the synthesis of a constant ΔG . Growth curves were constructed for siblings generated by asymmetric division and growing according to the adder model. Under the assumption that all cells at the same growth rate exhibit the same time between initiation of DNA replication and cell division (i.e., constant C+D -period), the constructions predict that initiation occurs at different sizes ( Li ) and that, at fast growth, large newborn cells transiently contain more DNA than small newborns, in accordance with the observations. Because the state of segregation, measured as the distance between separated nucleoids, was found to be more advanced in larger deeply-constricted cells, we propose that in larger newborns nucleoid separation occurs faster and at a shorter length, allowing them to divide earlier. We propose a composite model in which both differential initiation and segregation leads to an adder-like behavior of large and small newborn cells.

Casting inorganic structures with DNA molds

DOE PAGES

Sun, Wei; Boulais, Etienne; Hakobyan, Yera; ...

2014-10-09

Here we report a general strategy for designing and synthesizing inorganic nanostructures with arbitrarily prescribed three-dimensional shapes. Computationally designed DNA strands self-assemble into a stiff “nano-mold” that contains a user-specified three-dimensional cavity and encloses a nucleating gold “seed”. Under mild conditions, this seed grows into a larger cast structure that fills and thus replicates the cavity. We synthesized a variety of nanoparticles with three nanometer resolution: three distinct silver cuboids with three independently tunable dimensions, silver and gold nanoparticles with diverse cross sections, and composite structures with homo-/heterogeneous components. The designer equilateral silver triangular and spherical nanoparticles exhibited plasmonic propertiesmore » consistent with electromagnetism-based simulations. Our framework is generalizable to more complex geometries and diverse inorganic materials, offering a range of applications in biosensing, photonics, and nanoelectronics.« less

A convenient and adaptable package of DNA sequence analysis programs for microcomputers.

PubMed Central

Pustell, J; Kafatos, F C

1982-01-01

We describe a package of DNA data handling and analysis programs designed for microcomputers. The package is convenient for immediate use by persons with little or no computer experience, and has been optimized by trial in our group for a year. By typing a single command, the user enters a system which asks questions or gives instructions in English. The system will enter, alter, and manage sequence files or a restriction enzyme library. It generates the reverse complement, translates, calculates codon usage, finds restriction sites, finds homologies with various degrees of mismatch, and graphs amino acid composition or base frequencies. A number of options for data handling and printing can be used to produce figures for publication. The package will be available in ANSI Standard FORTRAN for use with virtually any FORTRAN compiler. PMID:6278412

Casting inorganic structures with DNA molds.

PubMed

Sun, Wei; Boulais, Etienne; Hakobyan, Yera; Wang, Wei Li; Guan, Amy; Bathe, Mark; Yin, Peng

2014-11-07

We report a general strategy for designing and synthesizing inorganic nanostructures with arbitrarily prescribed three-dimensional shapes. Computationally designed DNA strands self-assemble into a stiff "nanomold" that contains a user-specified three-dimensional cavity and encloses a nucleating gold "seed." Under mild conditions, this seed grows into a larger cast structure that fills and thus replicates the cavity. We synthesized a variety of nanoparticles with 3-nanometer resolution: three distinct silver cuboids with three independently tunable dimensions, silver and gold nanoparticles with diverse cross sections, and composite structures with homo- and heterogeneous components. The designer equilateral silver triangular and spherical nanoparticles exhibited plasmonic properties consistent with electromagnetism-based simulations. Our framework is generalizable to more complex geometries and diverse inorganic materials, offering a range of applications in biosensing, photonics, and nanoelectronics. Copyright © 2014, American Association for the Advancement of Science.

DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

DOE PAGES

None

2014-12-01

The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore » 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

Population diversity of ammonium oxidizers investigated by specific PCR amplification

USGS Publications Warehouse

Ward, B.B.; Voytek, M.A.; Witzel, K.-P.

1997-01-01

The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

Maternal Genetic Composition of a Medieval Population from a Hungarian-Slavic Contact Zone in Central Europe.

PubMed

Csákyová, Veronika; Szécsényi-Nagy, Anna; Csősz, Aranka; Nagy, Melinda; Fusek, Gabriel; Langó, Péter; Bauer, Miroslav; Mende, Balázs Gusztáv; Makovický, Pavol; Bauerová, Mária

2016-01-01

The genetic composition of the medieval populations of Central Europe has been poorly investigated to date. In particular, the region of modern-day Slovakia is a blank spot in archaeogenetic research. This paper reports the study of mitochondrial DNA (mtDNA) in ancient samples from the 9th-12th centuries originating from the cemeteries discovered in Nitra-Šindolka and Čakajovce, located in western Slovakia (Central Europe). This geographical region is interesting to study because its medieval multi-ethnic population lived in the so-called contact zone of the territory of the Great Moravian and later Hungarian state formations. We described 16 different mtDNA haplotypes in 19 individuals, which belong to the most widespread European mtDNA haplogroups: H, J, T, U and R0. Using comparative statistical and population genetic analyses, we showed the differentiation of the European gene pool in the medieval period. We also demonstrated the heterogeneous genetic characteristics of the investigated population and its affinity to the populations of modern Europe.

Maternal Genetic Composition of a Medieval Population from a Hungarian-Slavic Contact Zone in Central Europe

PubMed Central

Csákyová, Veronika; Szécsényi-Nagy, Anna; Csősz, Aranka; Nagy, Melinda; Fusek, Gabriel; Langó, Péter; Bauer, Miroslav; Mende, Balázs Gusztáv; Makovický, Pavol; Bauerová, Mária

2016-01-01

The genetic composition of the medieval populations of Central Europe has been poorly investigated to date. In particular, the region of modern-day Slovakia is a blank spot in archaeogenetic research. This paper reports the study of mitochondrial DNA (mtDNA) in ancient samples from the 9th–12th centuries originating from the cemeteries discovered in Nitra-Šindolka and Čakajovce, located in western Slovakia (Central Europe). This geographical region is interesting to study because its medieval multi-ethnic population lived in the so-called contact zone of the territory of the Great Moravian and later Hungarian state formations. We described 16 different mtDNA haplotypes in 19 individuals, which belong to the most widespread European mtDNA haplogroups: H, J, T, U and R0. Using comparative statistical and population genetic analyses, we showed the differentiation of the European gene pool in the medieval period. We also demonstrated the heterogeneous genetic characteristics of the investigated population and its affinity to the populations of modern Europe. PMID:26963389

Planktonic foraminifera-derived environmental DNA extracted from abyssal sediments preserves patterns of plankton macroecology

NASA Astrophysics Data System (ADS)

Morard, Raphaël; Lejzerowicz, Franck; Darling, Kate F.; Lecroq-Bennet, Béatrice; Winther Pedersen, Mikkel; Orlando, Ludovic; Pawlowski, Jan; Mulitza, Stefan; de Vargas, Colomban; Kucera, Michal

2017-06-01

Deep-sea sediments constitute a unique archive of ocean change, fueled by a permanent rain of mineral and organic remains from the surface ocean. Until now, paleo-ecological analyses of this archive have been mostly based on information from taxa leaving fossils. In theory, environmental DNA (eDNA) in the sediment has the potential to provide information on non-fossilized taxa, allowing more comprehensive interpretations of the fossil record. Yet, the process controlling the transport and deposition of eDNA onto the sediment and the extent to which it preserves the features of past oceanic biota remains unknown. Planktonic foraminifera are the ideal taxa to allow an assessment of the eDNA signal modification during deposition because their fossils are well preserved in the sediment and their morphological taxonomy is documented by DNA barcodes. Specifically, we re-analyze foraminiferal-specific metabarcodes from 31 deep-sea sediment samples, which were shown to contain a small fraction of sequences from planktonic foraminifera. We confirm that the largest portion of the metabarcode originates from benthic bottom-dwelling foraminifera, representing the in situ community, but a small portion (< 10 %) of the metabarcodes can be unambiguously assigned to planktonic taxa. These organisms live exclusively in the surface ocean and the recovered barcodes thus represent an allochthonous component deposited with the rain of organic remains from the surface ocean. We take advantage of the planktonic foraminifera portion of the metabarcodes to establish to what extent the structure of the surface ocean biota is preserved in sedimentary eDNA. We show that planktonic foraminifera DNA is preserved in a range of marine sediment types, the composition of the recovered eDNA metabarcode is replicable and that both the similarity structure and the diversity pattern are preserved. Our results suggest that sedimentary eDNA could preserve the ecological structure of the entire pelagic community, including non-fossilized taxa, thus opening new avenues for paleoceanographic and paleoecological studies.

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

PubMed Central

Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

2011-01-01

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

An epigenome-wide association meta-analysis of prenatal maternal stress in neonates: A model approach for replication

PubMed Central

Rijlaarsdam, Jolien; Pappa, Irene; Walton, Esther; Bakermans-Kranenburg, Marian J.; Mileva-Seitz, Viara R.; Rippe, Ralph C.A.; Roza, Sabine J.; Jaddoe, Vincent W.V.; Verhulst, Frank C.; Felix, Janine F.; Cecil, Charlotte A.M.; Relton, Caroline L.; Gaunt, Tom R.; McArdle, Wendy; Mill, Jonathan; Barker, Edward D.; Tiemeier, Henning; van IJzendoorn, Marinus H.

2016-01-01

ABSTRACT Prenatal maternal stress exposure has been associated with neonatal differential DNA methylation. However, the available evidence in humans is largely based on candidate gene methylation studies, where only a few CpG sites were evaluated. The aim of this study was to examine the association between prenatal exposure to maternal stress and offspring genome-wide cord blood methylation using different methods. First, we conducted a meta-analysis and follow-up pathway analyses. Second, we used novel region discovery methods [i.e., differentially methylated regions (DMRs) analyses]. To this end, we used data from two independent population-based studies, the Generation R Study (n = 912) and the Avon Longitudinal Study of Parents and Children (ALSPAC, n = 828), to (i) measure genome-wide DNA methylation in cord blood and (ii) extract a prenatal maternal stress composite. The meta-analysis (ntotal = 1,740) revealed no epigenome-wide (meta P <1.00e-07) associations of prenatal maternal stress exposure with neonatal differential DNA methylation. Follow-up analyses of the top hits derived from our epigenome-wide meta-analysis (meta P <1.00e-04) indicated an over-representation of the methyltransferase activity pathway. We identified no Bonferroni-corrected (P <1.00e-06) DMRs associated with prenatal maternal stress exposure. Combining data from two independent population-based samples in an epigenome-wide meta-analysis, the current study indicates that there are no large effects of prenatal maternal stress exposure on neonatal DNA methylation. Such replication efforts are essential in the search for robust associations, whether derived from candidate gene methylation or epigenome-wide studies. PMID:26889969

Vibrio japonicus sp. nov., a novel member of the Nereis clade in the genus Vibrio isolated from the coast of Japan

PubMed Central

2017-01-01

A novel Vibrio strain, JCM 31412T, was isolated from seawater collected from the Inland Sea (Setonaikai), Japan, and characterized as a Gram-negative, oxidase-positive, catalase-negative, facultatively anaerobic, motile, ovoid-shaped bacterium with one polar flagellum. Based on 16S rDNA gene identity, strain JCM 31412T showed a close relationship with type strains of Vibrio brasiliensis (LMG 20546T, 98.2% identity), V. harveyi (NBRC 15634T, 98.2%), V. caribbeanicus (ATCC BAA-2122T, 97.8%) and V. proteolyticus (NBRC 13287T, 97.8%). The G+C content of strain JCM 31412T DNA was 46.8%. Multi-locus sequence analysis (MLSA) of eight loci (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA; 5535bp) further clustered strain JCM 31412T in the Nereis clade, genus Vibrio. Phenotypically, strain JCM 31412T differed from the closest related Vibrio species in its utilization of melibiose and raffinose, and its lack of casein and gelatin hydrolysis. It was further differentiated based on its fatty acid composition, specifically properties of C12:03OH and summed features, which were significantly different from those of V. brasiliensis, V. nigripulchritudo and V. caribbeanicus type strains. Overall, the results of DNA-DNA hybridization, and physiological and biochemical analysis differentiated strain JCM 31412T from other described species of the genus Vibrio. Based on these polyphasic taxonomic findings, it was therefore concluded that JCM 31412T was a novel Vibrio species, for which the name Vibrio japonicus sp. nov. was proposed, with JCM 31412T (= LMG 29636T = ATCC TSD-62T) as the type strain. PMID:28231272

Polymer multilayer tattooing for enhanced DNA vaccination

NASA Astrophysics Data System (ADS)

Demuth, Peter C.; Min, Younjin; Huang, Bonnie; Kramer, Joshua A.; Miller, Andrew D.; Barouch, Dan H.; Hammond, Paula T.; Irvine, Darrell J.

2013-04-01

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These ‘multilayer tattoo’ DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

Chamber Bioaerosol Study: Outdoor Air and Human Occupants as Sources of Indoor Airborne Microbes

PubMed Central

Adams, Rachel I.; Bhangar, Seema; Pasut, Wilmer; Arens, Edward A.; Taylor, John W.; Lindow, Steven E.; Nazaroff, William W.; Bruns, Thomas D.

2015-01-01

Human occupants are an important source of microbes in indoor environments. In this study, we used DNA sequencing of filter samples to assess the fungal and bacterial composition of air in an environmental chamber under different levels of occupancy, activity, and exposed or covered carpeting. In this office-like, mechanically ventilated environment, results showed a strong influence of outdoor-derived particles, with the indoor microbial composition tracking that of outdoor air for the 2-hour sampling periods. The number of occupants and their activity played a significant but smaller role influencing the composition of indoor bioaerosols. Human-associated taxa were observed but were not particularly abundant, except in the case of one fungus that appeared to be transported into the chamber on the clothing of a study participant. Overall, this study revealed a smaller signature of human body-associated taxa than had been expected based on recent studies of indoor microbiomes, suggesting that occupants may not exert a strong influence on bioaerosol microbial composition in a space that, like many offices, is well ventilated with air that is moderately filtered and moderately occupied. PMID:26024222

Comparative Analysis of the Base Compositions of the Pre-mRNA 3′ Cleaved-Off Region and the mRNA 3′ Untranslated Region Relative to the Genomic Base Composition in Animals and Plants

PubMed Central

Li, Xiu-Qing

2014-01-01

The precursor messenger RNA (pre-mRNA) three-prime cleaved-off region (3′COR) and the mRNA three-prime untranslated region (3′UTR) play critical roles in regulating gene expression. The differences in base composition between these regions and the corresponding genomes are still largely uncharacterized in animals and plants. In this study, the base compositions of non-redundant 3′CORs and 3′UTRs were compared with the corresponding whole genomes of eleven animals, four dicotyledonous plants, and three monocotyledonous (cereal) plants. Among the four bases (A, C, G, and U for adenine, cytosine, guanine, and uracil, respectively), U (which corresponds to T, for thymine, in DNA) was the most frequent, A the second most frequent, G the third most frequent, and C the least frequent in most of the species in both the 3′COR and 3′UTR regions. In comparison with the whole genomes, in both regions the U content was usually the most overrepresented (particularly in the monocotyledonous plants), and the C content was the most underrepresented. The order obtained for the species groups, when ranked from high to low according to the U contents in the 3′COR and 3′UTR was as follows: dicotyledonous plants, monocotyledonous plants, non-mammal animals, and mammals. In contrast, the genomic T content was highest in dicotyledonous plants, lowest in monocotyledonous plants, and intermediate in animals. These results suggest the following: 1) there is a mechanism operating in both animals and plants which is biased toward U and against C in the 3′COR and 3′UTR; 2) the 3′UTR and 3′COR, as functional units, minimized the difference between dicotyledonous and monocotyledonous plants, while the dicotyledonous and monocotyledonous genomes evolved into two extreme groups in terms of base composition. PMID:24941005

Degenerative Minimalism in the Genome of a Psyllid Endosymbiont

PubMed Central

Clark, Marta A.; Baumann, Linda; Thao, MyLo Ly; Moran, Nancy A.; Baumann, Paul

2001-01-01

Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection. We have sequenced 37 kb of DNA of the genome of Carsonella ruddii, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont Buchnera aphidicola. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA. These features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base composition has a large effect on proteins: in polypeptides of C. ruddii, half of the residues consist of five amino acids with codons low in guanine plus cytosine. Furthermore, the proteins of C. ruddii are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria. These observations suggest that the C. ruddii genome is not subject to constraints that limit the evolution of other known eubacteria. PMID:11222582

Subspecies composition and founder contribution of the captive U.S. chimpanzee (Pan troglodytes) population.

PubMed

Ely, John J; Dye, Brent; Frels, William I; Fritz, Jo; Gagneux, Pascal; Khun, Henry H; Switzer, William M; Lee, D Rick

2005-10-01

Chimpanzees are presently classified into three subspecies: Pan troglodytes verus from west Africa, P.t. troglodytes from central Africa, and P.t. schweinfurthii from east Africa. A fourth subspecies (P.t. vellerosus), from Cameroon and northern Nigeria, has been proposed. These taxonomic designations are based on geographical origins and are reflected in sequence variation in the first hypervariable region (HVR-I) of the mtDNA D-loop. Although advances have been made in our understanding of chimpanzee phylogenetics, little has been known regarding the subspecies composition of captive chimpanzees. We sequenced part of the mtDNA HVR-I region in 218 African-born population founders and performed a phylogenetic analysis with previously characterized African sequences of known provenance to infer subspecies affiliations. Most founders were P.t. verus (95.0%), distantly followed by the troglodytes schweinfurthii clade (4.6%), and a single P.t. vellerosus (0.4%). Pedigree-based estimates of genomic representation in the descendant population revealed that troglodytes schweinfurthii founder representation was reduced in captivity, vellerosus representation increased due to prolific breeding by a single male, and reproductive variance resulted in uneven representation among male P.t.verus founders. No increase in mortality was evident from between-subspecies interbreeding, indicating a lack of outbreeding depression. Knowledge of subspecies and their genomic representation can form the basis for phylogenetically informed genetic management of extant chimpanzees to preserve rare genetic variation for research, conservation, or possible future breeding. Copyright 2005 Wiley-Liss, Inc.

Barcode DNA length polymorphisms vs fatty acid profiling for adulteration detection in olive oil.

PubMed

Uncu, Ali Tevfik; Uncu, Ayse Ozgur; Frary, Anne; Doganlar, Sami

2017-04-15

The aim of this study was to compare the performance of a DNA-barcode assay with fatty acid profile analysis to authenticate the botanical origin of olive oil. To achieve this aim, we performed a PCR-capillary electrophoresis (PCR-CE) approach on olive oil: seed oil blends using the plastid trnL (UAA) intron barcode. In parallel to genomic analysis, we subjected the samples to gas chromatography analysis of fatty acid composition. While the PCR-CE assay proved equally efficient as gas chromatography analysis in detecting adulteration with soybean, palm, rapeseed, sunflower, sesame, cottonseed and peanut oils, it was superior to the widely utilized analytical chemistry approach in revealing the adulterant species and detecting small quantities of corn and safflower oils in olive oil. Moreover, the DNA-based test correctly identified all tested olive oil: hazelnut oil blends whereas it was not feasible to detect hazelnut oil adulteration through fatty acid profile analysis. Thus, the present research has shown the feasibility of a PCR-CE barcode assay to detect adulteration in olive oil. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chemical and Genetic Discrimination of Cistanches Herba Based on UPLC-QTOF/MS and DNA Barcoding

PubMed Central

Zheng, Sihao; Jiang, Xue; Wu, Labin; Wang, Zenghui; Huang, Linfang

2014-01-01

Cistanches Herba (Rou Cong Rong), known as “Ginseng of the desert”, has a striking curative effect on strength and nourishment, especially in kidney reinforcement to strengthen yang. However, the two plant origins of Cistanches Herba, Cistanche deserticola and Cistanche tubulosa, vary in terms of pharmacological action and chemical components. To discriminate the plant origin of Cistanches Herba, a combined method system of chemical and genetic –UPLC-QTOF/MS technology and DNA barcoding–were firstly employed in this study. The results indicated that three potential marker compounds (isomer of campneoside II, cistanoside C, and cistanoside A) were obtained to discriminate the two origins by PCA and OPLS-DA analyses. DNA barcoding enabled to differentiate two origins accurately. NJ tree showed that two origins clustered into two clades. Our findings demonstrate that the two origins of Cistanches Herba possess different chemical compositions and genetic variation. This is the first reported evaluation of two origins of Cistanches Herba, and the finding will facilitate quality control and its clinical application. PMID:24854031

Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2017-06-01

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

An improved model for whole genome phylogenetic analysis by Fourier transform.

PubMed

Yin, Changchuan; Yau, Stephen S-T

2015-10-07

DNA sequence similarity comparison is one of the major steps in computational phylogenetic studies. The sequence comparison of closely related DNA sequences and genomes is usually performed by multiple sequence alignments (MSA). While the MSA method is accurate for some types of sequences, it may produce incorrect results when DNA sequences undergone rearrangements as in many bacterial and viral genomes. It is also limited by its computational complexity for comparing large volumes of data. Previously, we proposed an alignment-free method that exploits the full information contents of DNA sequences by Discrete Fourier Transform (DFT), but still with some limitations. Here, we present a significantly improved method for the similarity comparison of DNA sequences by DFT. In this method, we map DNA sequences into 2-dimensional (2D) numerical sequences and then apply DFT to transform the 2D numerical sequences into frequency domain. In the 2D mapping, the nucleotide composition of a DNA sequence is a determinant factor and the 2D mapping reduces the nucleotide composition bias in distance measure, and thus improving the similarity measure of DNA sequences. To compare the DFT power spectra of DNA sequences with different lengths, we propose an improved even scaling algorithm to extend shorter DFT power spectra to the longest length of the underlying sequences. After the DFT power spectra are evenly scaled, the spectra are in the same dimensionality of the Fourier frequency space, then the Euclidean distances of full Fourier power spectra of the DNA sequences are used as the dissimilarity metrics. The improved DFT method, with increased computational performance by 2D numerical representation, can be applicable to any DNA sequences of different length ranges. We assess the accuracy of the improved DFT similarity measure in hierarchical clustering of different DNA sequences including simulated and real datasets. The method yields accurate and reliable phylogenetic trees and demonstrates that the improved DFT dissimilarity measure is an efficient and effective similarity measure of DNA sequences. Due to its high efficiency and accuracy, the proposed DFT similarity measure is successfully applied on phylogenetic analysis for individual genes and large whole bacterial genomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

PubMed

Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

2014-01-01

Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.

The Use of Lead Isotope and Rare Earth Element Geochemistry for Forensic Geographic Provenancing

NASA Astrophysics Data System (ADS)

Carey, A.; Darrah, T.; Harrold, Z.; Prutsman-Pfeiffer, J.; Poreda, R.

2008-12-01

Lead isotope and rare earth element composition of modern human bones are analyzed to explore their utility for geographical provenancing. DNA analysis is the standard for identification of individuals. DNA analysis requires a DNA match for comparison. Thus, DNA analysis is of limited use in cases involving unknown remains. Trace elements are incorporated into bones and teeth during biomineralization, recording the characteristics of an individual's geochemical environment. Teeth form during adolescence, recording the geochemical environment of an individual's youth. Bones remodel throughout an individual's lifetime. Bones consist of two types of bone tissue (cortical and trabecular) that remodel at different rates, recording the geochemical environment at the time of biomineralization. Cortical bone tissue, forming the outer surface of bones, is dense, hard tissue that remodels in 25-30 yrs. Conversely, trabecular bone tissue, the inner cavity of bones, is low density, porous and remodels in 2-5 years. Thus, analyzing teeth and both bone tissues allows for the development of a geographical time line capable of tracking immigration patterns through time instead of only an individual's youth. Geochemical isotopic techniques (Sr, O, C, N) have been used for geographical provenancing in physical anthropology. The isotopic values of Sr, C, O, N are predominantly a function of soil compositions in areas where food is grown or water is consumed. Application of these provenancing techniques has become difficult as an individual's diet may reflect the isotopic composition of foods obtained at the local grocer as opposed to local soil compositions. Thus, we explore the use of REEs and Pb isotopes for geographical provenancing. Pb and REEs are likely more reliable indicators of modern geographical location as their composition are high in bio-available sources such as local soils, atmospheric aerosols, and dust as opposed to Sr, C, O, N that are controlled by food and drinking water. Lead isotope and REE analysis of trabecular and cortical bone tissue of 60 femoral heads resected during hip replacement surgery at the Univ. of Roch. Medical Center were analyzed by a combination of TIMS and ICP-MS. Results show that Pb compositions are consistent with local soil with variable inputs from known environmental sources. Several samples demonstrate inputs from known environmental sources (e.g. Mississippi Valley ore) that was used in paint, solder, and US gasoline. Additionally, results suggest bioincorporation of Pb with isotopic composition consistent with that observed for Canadian gasoline aerosols. Immigrants included in the study show Pb compositions distinctly different than local residents.

Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds

PubMed Central

Kang, Yun Gyeong; Wei, Jie; Shin, Ji Won; Wu, Yan Ru; Su, Jiacan; Park, Young Shik; Shin, Jung-Woog

2018-01-01

Background Successful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity. Methods In this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w). Results Evaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group. Conclusion These results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration. PMID:29520139

Disturbance of DNA conformation by the binding of testosterone-based platinum drugs via groove-face and intercalative interactions: a molecular dynamics simulation study

PubMed Central

2013-01-01

Background To explore novel platinum-based anticancer agents that are distinct from the structure and interaction mode of the traditional cisplatin by forming the bifunctional intrastrand 1,2 GpG adduct, the monofunctional platinum + DNA adducts with extensive non-covalent interactions had been studied. It was reported that the monofunctional testosterone-based platinum(II) agents present the high anticancer activity. Moreover, it was also found that the testosterone-based platinum agents could cause the DNA helix to undergo significant unwinding and bending over the non-testosterone-based platinum agents. However, the interaction mechanisms of these platinum agents with DNA at the atomic level are not yet clear so far. Results In the present work, we used molecular dynamics (MD) simulations and DNA conformational dynamics calculations to study the DNA distortion properties of the testosterone-based platinum + DNA, the improved testosterone-based platinum + DNA and the non-testosterone-based platinum + DNA adducts. The results show that the intercalative interaction of the improved flexible testosterone-based platinum agent with DNA molecule could cause larger DNA conformational distortion than the groove-face interaction of the rigid testosterone-based platinum agent with DNA molecule. Further investigations for the non-testosterone-based platinum agent reveal the occurrence of insignificant change of DNA conformation due to the absence of testosterone ligand in such agent. Based on the DNA dynamics analysis, the DNA base motions relating to DNA groove parameter changes and hydrogen bond destruction of DNA base pairs were also discussed in this work. Conclusions The flexible linker in the improved testosterone-based platinum agent causes an intercalative interaction with DNA in the improved testosterone-based platinum + DNA adduct, which is different from the groove-face interaction caused by a rigid linker in the testosterone-based platinum agent. The present investigations provide useful information of DNA conformation affected by a testosterone-based platinum complex at the atomic level. PMID:23517640

Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents

PubMed Central

Dezi, Manuela; Di Cicco, Aurelie; Bassereau, Patricia; Lévy, Daniel

2013-01-01

Giant unilamellar vesicles (GUVs) are convenient biomimetic systems of the same size as cells that are increasingly used to quantitatively address biophysical and biochemical processes related to cell functions. However, current approaches to incorporate transmembrane proteins in the membrane of GUVs are limited by the amphiphilic nature or proteins. Here, we report a method to incorporate transmembrane proteins in GUVs, based on concepts developed for detergent-mediated reconstitution in large unilamellar vesicles. Reconstitution is performed either by direct incorporation from proteins purified in detergent micelles or by fusion of purified native vesicles or proteoliposomes in preformed GUVs. Lipid compositions of the membrane and the ionic, protein, or DNA compositions in the internal and external volumes of GUVs can be controlled. Using confocal microscopy and functional assays, we show that proteins are unidirectionally incorporated in the GUVs and keep their functionality. We have successfully tested our method with three types of transmembrane proteins. GUVs containing bacteriorhodopsin, a photoactivable proton pump, can generate large transmembrane pH and potential gradients that are light-switchable and stable for hours. GUVs with FhuA, a bacterial porin, were used to follow the DNA injection by T5 phage upon binding to its transmembrane receptor. GUVs incorporating BmrC/BmrD, a bacterial heterodimeric ATP-binding cassette efflux transporter, were used to demonstrate the protein-dependent translocation of drugs and their interactions with encapsulated DNA. Our method should thus apply to a wide variety of membrane or peripheral proteins for producing more complex biomimetic GUVs. PMID:23589883

Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks.

PubMed

Heinz, Kathrin S; Casas-Delucchi, Corella S; Török, Timea; Cmarko, Dusan; Rapp, Alexander; Raska, Ivan; Cardoso, M Cristina

2018-05-10

The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance.

The Making of the African mtDNA Landscape

PubMed Central

Salas, Antonio; Richards, Martin; De la Fe, Tomás; Lareu, María-Victoria; Sobrino, Beatriz; Sánchez-Diz, Paula; Macaulay, Vincent; Carracedo, Ángel

2002-01-01

Africa presents the most complex genetic picture of any continent, with a time depth for mitochondrial DNA (mtDNA) lineages >100,000 years. The most recent widespread demographic shift within the continent was most probably the Bantu dispersals, which archaeological and linguistic evidence suggest originated in West Africa 3,000–4,000 years ago, spreading both east and south. Here, we have carried out a thorough phylogeographic analysis of mtDNA variation in a total of 2,847 samples from throughout the continent, including 307 new sequences from southeast African Bantu speakers. The results suggest that the southeast Bantu speakers have a composite origin on the maternal line of descent, with ∼44% of lineages deriving from West Africa, ∼21% from either West or Central Africa, ∼30% from East Africa, and ∼5% from southern African Khoisan-speaking groups. The ages of the major founder types of both West and East African origin are consistent with the likely timing of Bantu dispersals, with those from the west somewhat predating those from the east. Despite this composite picture, the southeastern African Bantu groups are indistinguishable from each other with respect to their mtDNA, suggesting that they either had a common origin at the point of entry into southeastern Africa or have undergone very extensive gene flow since. PMID:12395296

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

Exploring the Impact of Food on the Gut Ecosystem Based on the Combination of Machine Learning and Network Visualization.

PubMed

Shima, Hideaki; Masuda, Shizuka; Date, Yasuhiro; Shino, Amiu; Tsuboi, Yuuri; Kajikawa, Mizuho; Inoue, Yoshihiro; Kanamoto, Taisei; Kikuchi, Jun

2017-12-01

Prebiotics and probiotics strongly impact the gut ecosystem by changing the composition and/or metabolism of the microbiota to improve the health of the host. However, the composition of the microbiota constantly changes due to the intake of daily diet. This shift in the microbiota composition has a considerable impact; however, non-pre/probiotic foods that have a low impact are ignored because of the lack of a highly sensitive evaluation method. We performed comprehensive acquisition of data using existing measurements (nuclear magnetic resonance, next-generation DNA sequencing, and inductively coupled plasma-optical emission spectroscopy) and analyses based on a combination of machine learning and network visualization, which extracted important factors by the Random Forest approach, and applied these factors to a network module. We used two pteridophytes, Pteridium aquilinum and Matteuccia struthiopteris , for the representative daily diet. This novel analytical method could detect the impact of a small but significant shift associated with Matteuccia struthiopteris but not Pteridium aquilinum intake, using the functional network module. In this study, we proposed a novel method that is useful to explore a new valuable food to improve the health of the host as pre/probiotics.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

PubMed Central

Milrot, Elad; Mutsafi, Yael; Ben-Dor, Shifra; Levin, Yishai; Savidor, Alon; Kartvelishvily, Elena

2016-01-01

ABSTRACT The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed “viral factories,” where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as “production lines” of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed “viral factories,” are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles. PMID:27581975

Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii.

PubMed

Zheng, Li; Pang, Wei; Qi, Zanmei; Luo, Enjie; Cui, Liwang; Cao, Yaming

2016-08-08

Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Evolution of EF-hand calcium-modulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins

NASA Technical Reports Server (NTRS)

Kretsinger, R. H.; Nakayama, S.

1993-01-01

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

Biological nanopore MspA for DNA sequencing

NASA Astrophysics Data System (ADS)

Manrao, Elizabeth A.

Unlocking the information hidden in the human genome provides insight into the inner workings of complex biological systems and can be used to greatly improve health-care. In order to allow for widespread sequencing, new technologies are required that provide fast and inexpensive readings of DNA. Nanopore sequencing is a third generation DNA sequencing technology that is currently being developed to fulfill this need. In nanopore sequencing, a voltage is applied across a small pore in an electrolyte solution and the resulting ionic current is recorded. When DNA passes through the channel, the ionic current is partially blocked. If the DNA bases uniquely modulate the ionic current flowing through the channel, the time trace of the current can be related to the sequence of DNA passing through the pore. There are two main challenges to realizing nanopore sequencing: identifying a pore with sensitivity to single nucleotides and controlling the translocation of DNA through the pore so that the small single nucleotide current signatures are distinguishable from background noise. In this dissertation, I explore the use of Mycobacterium smegmatis porin A (MspA) for nanopore sequencing. In order to determine MspA's sensitivity to single nucleotides, DNA strands of various compositions are held in the pore as the resulting ionic current is measured. DNA is immobilized in MspA by attaching it to a large molecule which acts as an anchor. This technique confirms the single nucleotide resolution of the pore and additionally shows that MspA is sensitive to epigenetic modifications and single nucleotide polymorphisms. The forces from the electric field within MspA, the effective charge of nucleotides, and elasticity of DNA are estimated using a Freely Jointed Chain model of single stranded DNA. These results offer insight into the interactions of DNA within the pore. With the nucleotide sensitivity of MspA confirmed, a method is introduced to controllably pass DNA through the pore. Using a DNA polymerase, DNA strands are stepped through MspA one nucleotide at a time. The steps are observable as distinct levels on the ionic-current time-trace and are related to the DNA sequence. These experiments overcome the two fundamental challenges to realizing MspA nanopore sequencing and pave the way to the development of a commercial technology.

Tracing the phylogeography of human populations in Britain based on 4th-11th century mtDNA genotypes.

PubMed

Töpf, A L; Gilbert, M T P; Dumbacher, J P; Hoelzel, A R

2006-01-01

Some of the transitional periods of Britain during the first millennium A.D. are traditionally associated with the movement of people from continental Europe, composed largely of invading armies (e.g., the Roman, Saxon, and Viking invasions). However, the extent to which these were migrations (as opposed to cultural exchange) remains controversial. We investigated the history of migration by women by amplifying mitochondrial DNA (mtDNA) from ancient Britons who lived between approximately A.D. 300-1,000 and compared these with 3,549 modern mtDNA database genotypes from England, Europe, and the Middle East. The objective was to assess the dynamics of the historical population composition by comparing genotypes in a temporal context. Towards this objective we test and calibrate the use of rho statistics to identify relationships between founder and source populations. We find evidence for shared ancestry between the earliest sites (predating Viking invasions) with modern populations across the north of Europe from Norway to Estonia, possibly reflecting common ancestors dating back to the last glacial epoch. This is in contrast with a late Saxon site in Norwich, where the genetic signature is consistent with more recent immigrations from the south, possibly as part of the Saxon invasions.

A compositional segmentation of the human mitochondrial genome is related to heterogeneities in the guanine mutation rate

PubMed Central

Samuels, David C.; Boys, Richard J.; Henderson, Daniel A.; Chinnery, Patrick F.

2003-01-01

We applied a hidden Markov model segmentation method to the human mitochondrial genome to identify patterns in the sequence, to compare these patterns to the gene structure of mtDNA and to see whether these patterns reveal additional characteristics important for our understanding of genome evolution, structure and function. Our analysis identified three segmentation categories based upon the sequence transition probabilities. Category 2 segments corresponded to the tRNA and rRNA genes, with a greater strand-symmetry in these segments. Category 1 and 3 segments covered the protein- coding genes and almost all of the non-coding D-loop. Compared to category 1, the mtDNA segments assigned to category 3 had much lower guanine abundance. A comparison to two independent databases of mitochondrial mutations and polymorphisms showed that the high substitution rate of guanine in human mtDNA is largest in the category 3 segments. Analysis of synonymous mutations showed the same pattern. This suggests that this heterogeneity in the mutation rate is partly independent of respiratory chain function and is a direct property of the genome sequence itself. This has important implications for our understanding of mtDNA evolution and its use as a ‘molecular clock’ to determine the rate of population and species divergence. PMID:14530452

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro.

PubMed

Larson, Erik D; Nickens, David; Drummond, James T

2002-02-01

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G.T mismatch was achieved by placing a nick 10-40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.

Characterization of a fused protein specified by the adenovirus type 2-simian virus 40 hybrid Ad2+ND1 dp2.

PubMed Central

Fey, G; Lewis, J B; Grodzicker, T; Bothwell, A

1979-01-01

The adenovirus type 2-simian virus 40 (SV40) hybrid virus Ad2+ND1 dp2 (E. Lukanidin, manuscript in preparation) specified two proteins (molecular weights, 24,000 and 23,000) that are, in part, products of an insertion of SV40 early DNA sequences. This was demonstrated by translation in vitro from viral mRNA that had been selected by hybridization to SV40 DNA. These two phosphorylated, nonvirion proteins were produced late in infection in amounts similar to adenovirus 2 structural proteins and were closely related to each other in tryptic peptide composition. The portion of SV40 DNA (map units 0.17 to 0.22 on the SV40 genome) coding for these proteins was joined to sequences coding for the amino-terminal part of the adenovirus type 2 structural protein IV (fiber). The Ad2+ND1 dp2 23,000- and 24,000-molecular-weight proteins were hybrid polypeptides, with about two-thirds of their tryptic peptides contributed by the fiber protein and the remainder contributed by SV40 T-antigen. They shared with T-antigen (molecular weight, 96,000) a carboxy-terminal proline-rich tryptic peptide. Together, the tryptic peptide composition of these proteins and the known SV40 DNA sequences suggested the reading frame for the translation of T-antigen. The carboxy terminus for T-anigen would then be located on the SV40 genome map next to the TAA terminator triplet at position 0.175, 910 bases away from the cleavage site of the restriction endonuclease EcoRI. Seven host range mutants from Ad2+ND1 dp2 were isolated that had lost the capacity to propagate on monkey cells. They did not induce detectable levels of the hybrid proteins. Three of these mutants had lost the SV40 DNA insertion that codes in part for these proteins. Thus, in analogy to the Ad2+ND1 30,000-molecular-weight protein, the presence of these proteins correlates with the presence of the helper function for adenovirus replication on monkey cells. Images PMID:225516

Composition and carbon dynamics of forests in northeastern North America in a future, warmer world

Treesearch

Jacqueline E. Mohan; Roger M. Cox; Louis R. Iverson

2009-01-01

Increasing temperatures, precipitation extremes, and other anthropogenic influences (pollutant deposition, increasing carbon dioxide) will influence future forest composition and productivity in the northeastern United States and eastern Canada. This synthesis of empirical and modeling studies includes tree DNA evidence suggesting tree...

SIMILARITY OF PARTICLE-ASSOCIATED AND FREE-LIVING BACTERIAL COMMUNITIES IN NORTHERN SAN FRANCISCO BAY, CALIFORNIA

EPA Science Inventory

We used denaturing gradient gel electrophoresis (DGGE) of 16S rDNA PCR amplicons to analyze the composition of Bacteria communities in samples collected during the summer, low flow season from northern San Francisco Bay, California. There were clear compositional differences in ...