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Sample records for acid dna base

  1. Semisynthetic DNA-protein conjugates for fabrication of nucleic acid based nanostructures

    NASA Astrophysics Data System (ADS)

    Rabe, Kersten S.; Feldkamp, Udo; Niemeyer, Christof M.

    2008-10-01

    We here report on the developments of semisynthetic DNA-protein conjugates and their assembly into multi-component nanostructures. We describe the improvement of the DNA sequences embedded in such nanostructures by computational and analytical methods. Moreover, we report on the exploration of novel DNA conjugates of streptavidin or redox proteins with improved properties for the assembly of nucleic acid based nanostructures.

  2. DNA and RNA "traffic lights": synthetic wavelength-shifting fluorescent probes based on nucleic acid base substitutes for molecular imaging.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2013-08-01

    The DNA base substitute approach by the (S)-3-amino-1,2-propanediol linker allows placing two fluorophores in a precise way inside a given DNA framework. The double helical architecture around the fluorophores, especially the DNA-induced twist, is crucial for the desired photophysical interactions. Excitonic, excimer, and energy transfer interactions yield fluorescent DNA and RNA probes with dual emission color readout. Especially, our DNA and RNA "traffic light" that combines the green emission of TO with the red emission of TR represents an important tool for molecular imaging and can be applied as aptasensors and as probes to monitor the siRNA delivery into cells. The concept can be extended to the synthetically easier to access postsynthetic 2'-modifications and the NIR range. Thereby, the pool of tailor-made fluorescent nucleic acid conjugates can be extended. PMID:23796243

  3. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.; Selvaganapathy, M.; Mahalakshmi, R.

    2012-10-01

    Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2rad -) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

  4. Label-free nucleic acids detection based on DNA templated silver nanoclusters fluorescent probe.

    PubMed

    Zhao, Haiyan; Wang, Lei; Zhu, Jing; Wei, Haiping; Jiang, Wei

    2015-06-01

    Based on DNA templated Ag NCs (DNA/Ag NCs) fluorescent probe, a label-free fluorescent method was developed for the detection of clinical significant DNA fragments from human immunodeficiency virus type 1 (HIV-1) DNA. Firstly, a hairpin probe, containing target DNA recognition sequence and guanine-rich sequence, was designed to hybridize with the target DNA and form a blunt 3'-terminus DNA duplex. Then, exonuclease III (Exo III) was employed to stepwise hydrolyze the mononucleotides from formed blunt 3'-terminus DNA duplex, releasing the target DNA and guanine-rich sequence. Finally, DNA/Ag NCs fluorescent probe was introduced to hybridize with the guanine-rich sequence, leading to an enhanced fluorescence signal for detection. The proposed method could detect as low as 2.9×10(-10) mol L(-1) HIV-1 DNA and exhibited excellent selectivity against mismatched target DNA. Furthermore, the method possessed perfect recoveries in cells lysate and human serum, showing potential to be used in biological samples. PMID:25863386

  5. "Nano-oddities": unusual nucleic acid assemblies for DNA-based nanostructures and nanodevices.

    PubMed

    Yatsunyk, Liliya A; Mendoza, Oscar; Mergny, Jean-Louis

    2014-06-17

    CONSPECTUS: DNA is an attractive polymer building material for nanodevices and nanostructures due to its ability for self-recognition and self-assembly. Assembly relies on the formation of base-specific interactions that allow strands to adopt structures in a controllable fashion. Most DNA-based higher order structures such as DNA cages, 2D and 3D DNA crystals, or origamis are based on DNA double helices stabilized by Watson-Crick complementarity. A number of nonclassical pairing patterns are possible between or among DNA strands; these interactions result in formation of unusual structures that include, but are not limited to, G-quadruplexes, i-motifs, triplexes, and parallel-stranded duplexes. These structures create greater diversity of DNA-based building blocks for nanomaterials and have certain advantages over conventional duplex DNA, such as enhanced thermal stability and sensitivity to chemical stimuli. In this Account, we briefly introduce these alternative DNA structures and describe in detail their utilization in a variety of nanomaterials and nanomachines. The field of DNA "nano-oddities" emerged in the late 1990s when for the first time a DNA nanomachine was designed based on equilibrium between B-DNA and noncanonical, left-handed Z-DNA. Soon after, "proof-of-principle" DNA nanomachines based on several DNA "oddities" were reported. These machines were set in motion by the addition of complementary strands (a principle used by many B-DNA-based nanodevices), by the addition of selected cations, small molecules, or proteins, or by a change in pH or temperature. Today, we have fair understanding of the mechanism of action of these devices, excellent control over their performance, and knowledge of basic principles of their design. pH sensors and pH-controlled devices occupy a central niche in the field. They are usually based on i-motifs or triplex DNA, are amazingly simple, robust, and reversible, and create no waste apart from salt and water. G

  6. Development of artificial nucleic acid that recognizes a CG base pair in triplex DNA formation.

    PubMed

    Hari, Yoshiyuki

    2013-01-01

    An oligonucleotide that can form a triplex with double-stranded DNA is called a triplex-forming oligonucleotide (TFO). TFOs have gained considerable attention because of their potential as gene targeting tools. However, triplex DNA formation involves inherent problems for practical use. The most important problem is that natural nucleotides in TFO do not have sufficient affinity and base pair-selectivity to pyrimidine-purine base pair, like a CG or TA base pair, within dsDNA. This suggests that dsDNA region including a CG or TA base pair cannot be targeted. Therefore, artificial nucleotides, especially with non-natural nucleobases, capable of direct recognition of a CG or TA base pair via hydrogen bond formation have been developed; however, nucleotides with better selectivity and stronger affinity are necessary for implementing this dsDNA-targeting technology using TFOs. Under such a background, we considered that facile and efficient synthesis of various nucleobase derivatives in TFOs would be useful for finding an ideal nucleobase for recognition of a CG or TA base pair because detailed and rational exploration of nucleobase structures is facilitated. Recently, to develop a nucleobase recognizing a CG base pair, we have used post-elongation modification, i.e., modification after oligonucleotide synthesis, for the facile synthesis of nucleobase derivatives. This review mainly summarizes our recent findings on the development of artificial nucleobases and nucleotides for recognition of a CG base pair in triplexes formed between dsDNA and TFOs. PMID:24189561

  7. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications. PMID:24647836

  8. Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: A comparative approach

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sakthivel, A.; Pravin, N.

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 102 to 105 indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical.

  9. Exploring DNA binding and nucleolytic activity of few 4-aminoantipyrine based amino acid Schiff base complexes: a comparative approach.

    PubMed

    Raman, N; Sakthivel, A; Pravin, N

    2014-05-01

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes were synthesized from Schiff base(s), obtained by the condensation of 4-aminoantipyrine with furfural and amino acid (glycine(L1)/alanine(L2)/valine(L3)) and respective metal(II) chloride. Their structural features and other properties were explored from the analytical and spectral methods. The binding behaviors of the complexes to calf thymus DNA were investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The intrinsic binding constants for the above synthesized complexes are found to be in the order of 10(2) to 10(5) indicating that most of the synthesized complexes are good intercalators. The binding constant values (Kb) clearly indicate that valine Schiff-base complexes have more intercalating ability than alanine and glycine Schiff-base complexes. The results indicate that the complexes bind to DNA through intercalation and act as efficient cleaving agents. The in vitro antibacterial and antifungal assay indicates that these complexes are good antimicrobial agents against various pathogens. The IC50 values of [Ni(L1)2] and [Zn(L1)2] complexes imply that these complexes have preferable ability to scavenge hydroxyl radical. PMID:24566120

  10. DNA interaction with octahedral and square planar Ni(II) complexes of aspartic-acid Schiff-bases

    NASA Astrophysics Data System (ADS)

    Sallam, S. A.; Orabi, A. S.; Abbas, A. M.

    2011-12-01

    Ni(II) complexes of (S,E)-2-(2-OHbenzilydene)aspartic acid; (S,E)-2-(2,3-diOHbenzilydene)aspartic acid-; (S,E)-2-(2,4-diOH-benzilydene)aspartic acid; (S,E)-2-(2,5-diOHbenzilydene)aspartic acid and (S,E)-2-((2-OHnaphthalene-1-yl)methylene)aspartic acid Schiff-bases have been synthesized by template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and 1H nmr spectra as well as thermal analysis (TG, DTG, DTA). The Schiff-bases are dibasic tridentate or tetradentate donors and the complexes have square planar and octahedral structures. The complexes decompose in two or three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy.

  11. Synthesis, physicochemical studies, embryos toxicity and DNA interaction of some new Iron(II) Schiff base amino acid complexes

    NASA Astrophysics Data System (ADS)

    Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.

    2013-05-01

    New Fe(II) Schiff base amino acid complexes derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized by elemental analysis, IR, electronic spectra, and conductance measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. The investigated Schiff bases exhibited tridentate coordination mode with the general formulae [Fe(HL)2]·nH2O for all amino acids except L-histidine. But in case of L-histidine, the ligand acts as tetradentate ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their toxicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. The interaction between CT-DNA and the investigated complexes were followed by spectrophotometry and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA cleavage activity with the sequence: nhi > nari > nali > nasi > nphali. The thermodynamic Profile of the binding of nphali complex and CT-DNA was constructed by analyzing the experimental data of absorption titration and UV melting studies with the McGhee equation, van't Hoff's equation, and the Gibbs-Helmholtz equation.

  12. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    PubMed Central

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-01-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/. PMID:26482832

  13. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  14. Gold-mercaptopropionic acid-polyethylenimine composite based DNA sensor for early detection of rheumatic heart disease.

    PubMed

    Singh, Swati; Kaushal, Ankur; Khare, Shashi; Kumar, Pradeep; Kumar, Ashok

    2014-07-21

    The first gold-mercaptopropionic acid-polyethylenimine composite based electrochemical DNA biosensor was fabricated for the early detection of Streptococcus pyogenes infection in humans causing rheumatic heart disease (heart valve damage). No biosensor is available for the detection of rheumatic heart disease (RHD). Therefore, the mga gene based sensor was developed by the covalent immobilization of a 5'-carboxyl modified single stranded DNA probe onto the gold composite electrode. The immobilized probe was hybridized with the genomic DNA (G-DNA) of S. pyogenes from throat swabs and the electrochemical response was measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance (EI). Covalent immobilization of the probe onto the gold composite and its hybridization with G-DNA was characterized by FTIR and SEM. The sensitivity of the sensor was 110.25 μA cm(-2) ng(-1) with DPV and the lower limit of detection was 10 pg per 6 μL. The sensor was validated with patient throat swab samples and results were compared with available methods. The sensor is highly specific to S. pyogenes and can prevent damage to heart valves by the early detection of the infection in only 30 min. PMID:24875529

  15. DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Brown, W. Ted; Junaid, Mohammed A.

    2016-01-01

    It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases. PMID:27199632

  16. Label-free DNA biosensor based on a peptide nucleic acid-functionalized microstructured optical fiber-Bragg grating

    NASA Astrophysics Data System (ADS)

    Candiani, Alessandro; Bertucci, Alessandro; Giannetti, Sara; Konstantaki, Maria; Manicardi, Alex; Pissadakis, Stavros; Cucinotta, Annamaria; Corradini, Roberto; Selleri, Stefano

    2013-05-01

    We describe a novel sensing approach based on a functionalized microstructured optical fiber-Bragg grating for specific DNA target sequences detection. The inner surface of a microstructured fiber, where a Bragg grating was previously inscribed, has been functionalized by covalent linking of a peptide nucleic acid probe targeting a DNA sequence bearing a single point mutation implicated in cystic fibrosis (CF) disease. A solution of an oligonucleotide (ON) corresponding to a tract of the CF gene containing the mutated DNA has been infiltrated inside the fiber capillaries and allowed to hybridize to the fiber surface according to the Watson-Crick pairing. In order to achieve signal amplification, ON-functionalized gold nanoparticles were then infiltrated and used in a sandwich-like assay. Experimental measurements show a clear shift of the reflected high order mode of a Bragg grating for a 100 nM DNA solution, and fluorescence measurements have confirmed the successful hybridization. Several experiments have been carried out on the same fiber using the identical concentration, showing the same modulation trend, suggesting the possibility of the reuse of the sensor. Measurements have also been made using a 100 nM mismatched DNA solution, containing a single nucleotide mutation and corresponding to the wild-type gene, and the results demonstrate the high selectivity of the sensor.

  17. A Spherical Nucleic Acids Platform Based on Self-Assembled DNA Biopolymer for High Performance Cancer Therapy

    PubMed Central

    Zheng, Jing; Zhu, Guizhi; Li, Yinhui; Li, Chunmei; You, Mingxu; Chen, Tao; Song, Erqun

    2013-01-01

    Based on their enhanced cellular uptake, stability, biocompatibility, and versatile surface functionalization, spherical nucleic acids (SNAs) have become a potentially useful platform in biological application. It still remains important to expand the SNAs “toolbox”, especially given the current interest in multimodal or theranostic nanomaterials, that is, composites capable of multiple simultaneous applications such as imaging, sensing, and drug delivery. In this paper, we have engineered a nanoparticle-conjugated initiator that triggers a cascade of hybridization reaction resulting in the formation of a long DNA polymer as the nanoparticle shell. By employing different DNA fragments, self-assembled multifunctional SNAs can be constructed. Therefore, using one capped ligand, these SNAs can combine imaging fluorescent tags, target recognition element, and targeted delivery molecules together. Since these SNAs possess high drug loading capacity and high specificity by the incorporation of an aptamer, our approach might find potential applications in new drug development, existing drug improvement, and drug delivery for cancer therapy. PMID:23841478

  18. Hydrogen-bonding studies of amino acid side-chains with DNA base pairs

    NASA Astrophysics Data System (ADS)

    Deepa, P.; Kolandaivel, P.; Senthilkumar, K.

    2011-08-01

    The interactions of the amino acid side-chains arginine (ARG), aspartic acid (ASP), asparagine (ASN), lysine (LYS) and serine (SER) with nucleic acid base pairs have been investigated using theoretical methods. The interaction energy of the short intermolecular N-H ... N, N-H ... O, O-H ... O, O-H ... N, C-H ... O and C-H ... N hydrogen bonds present in both isolated base pairs and complexes and its role in providing stability to the complexes have been explored. The homonuclear interactions are found to be stronger than the heteronuclear interactions. An improper hydrogen bond has been observed for some of the N-H ... O and N-H ... N hydrogen-bond interactions with the contraction of the N-H bond varying from 0.001 to 0.0260 Å and the corresponding blue shift of the stretching frequency by 4-291 cm-1. Localized molecular orbital energy decomposition analysis (LMOEDA) reveals that the major contributions to the energetics are from the long-range polarization (PL) interaction, and the short-range attractive (ES, EX) and repulsive (REP) interactions. The Bader's atoms in molecules (AIM) theory shows good correlation for the electron density and its Laplacian at the bond critical points (BCP) with the N-H ... N and N-H ... O hydrogen-bond lengths in the complexes, and gives a proper explanation for the stability of the structure. The charge-transfer from the proton acceptor to the antibonding orbital of the X-H bond in the complexes was studied using natural bond orbital (NBO) analysis.

  19. Genomic DNA Methylation Changes in Response to Folic Acid Supplementation in a Population-Based Intervention Study among Women of Reproductive Age

    PubMed Central

    Berry, Robert J.; Hao, Ling; Li, Zhu; Maneval, David; Yang, Thomas P.; Rasmussen, Sonja A.; Yang, Quanhe; Zhu, Jiang-Hui; Hu, Dale J.; Bailey, Lynn B.

    2011-01-01

    Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (−14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation. PMID:22163281

  20. Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform.

    PubMed

    Lin, Meihua; Song, Ping; Zhou, Guobao; Zuo, Xiaolei; Aldalbahi, Ali; Lou, Xiaoding; Shi, Jiye; Fan, Chunhai

    2016-07-01

    The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h. PMID:27310264

  1. Ternary copper(II) complexes with amino acid chains and heterocyclic bases: DNA binding, cytotoxic and cell apoptosis induction properties.

    PubMed

    Ma, Tieliang; Xu, Jun; Wang, Yuan; Yu, Hao; Yang, Yong; Liu, Yang; Ding, Weiliang; Zhu, Wenjiao; Chen, Ruhua; Ge, Zhijun; Tan, Yongfei; Jia, Lei; Zhu, Taofeng

    2015-03-01

    Nowadays, chemotherapy is a common means of oncology. However, it is difficult to find excellent chemotherapy drugs. Here we reported three new ternary copper(II) complexes which have potential chemotherapy characteristics with reduced Schiff base ligand and heterocyclic bases (TBHP), [Cu(phen)(TBHP)]H2O (1), [Cu(dpz)(TBHP)]H2O (2) and [Cu(dppz)(TBHP)]H2O (3) (phen=1,10-phenanthroline, dpz=dipyrido [3,2:2',3'-f]quinoxaline, dppz=dipyrido [3,2-a:2',3'-c]phenazine, H2TBHP=2-(3,5-di-tert-butyl-2-hydroxybenzylamino)-2-benzyl-acetic acid). The DNA-binding properties of the complexes were investigated by spectrometric titrations, ethidium bromide displacement experiments and viscosity measurements. The results indicated that the three complexes, especially the complex 13, can strongly bind to calf-thymus DNA (CT-DNA). The intrinsic binding constants Kb of the ternary copper(II) complexes with CT-DNA were 1.37×10(5), 1.81×10(5) and 3.21×10(5) for 1, 2 and 3 respectively. Comparative cytotoxic activities of the copper(II) complexes were also determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that the ternary copper(II) complexes had significant cytotoxic activity against the human lung cancer (A549), human esophageal cancer (Eca109) and human gastric cancer (SGC7901) cell lines. Cell apoptosis were detected by AnnexinV/PI flow cytometry and by Western blotting with the protein expression of p53, Bax and Bcl-2. All the three copper complexes can effectively induce apoptosis of the three human tumor cells. PMID:25555321

  2. Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods.

    PubMed

    Jara, C; Mateo, E; Guillamón, J M; Torija, M J; Mas, A

    2008-12-10

    Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery. PMID:18950887

  3. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  4. Development of PCR-Based DNA markers flanking three low phytic acid mutant loci in barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytic acid (PA) is the most abundant form of phosphorus (P) in cereal grains. PA chelates mineral cations to form an indigestible salt, and is thus regarded as an antinutritional agent and a contributor to water pollution. Grain with low phytic acid (lpa) genotypes could aid in mitigating this prob...

  5. Indole-3-acetic acid biosensor based on G-rich DNA labeled AuNPs as chemiluminescence probe coupling the DNA signal amplification

    NASA Astrophysics Data System (ADS)

    Hun, Xu; Mei, Zhenghua; Wang, Zhouping; He, Yunhua

    2012-09-01

    A highly sensitive chemiluminescence (CL) method for detection of phytohormone indole-3-acetic acid (IAA) was developed by using G-rich DNA labeled gold nanoparticles (AuNPs) as CL probe coupling the DNA signal amplification technology. The IAA antibody was immobilized on carboxyl terminated magnetic beads (MBs). In the presence of IAA, antibody labeled AuNPs were captured by antibody functionalized MBs. The DNA on AuNPs is released by a ligand exchange process induced by the addition of DTT. The released DNA is then acted as the linker and hybridized with the capture DNA on MBs and probe DNA on AuNPs CL probe. The CL signal is obtained via the instantaneous derivatization reaction between a specific CL reagent, 3,4,5-trimethoxyl-phenylglyoxal (TMPG), and the G-rich DNA on AuNPs CL probe. IAA can be detected in the concentration range from 0.02 ng/mL to 30 ng/mL, and the limit of detection is 0.01 ng/mL.

  6. A label-free fluorescent probe based on DNA-templated silver nanoclusters and exonuclease III-assisted recycling amplification detection of nucleic acid.

    PubMed

    Yang, Wen; Tian, Jianniao; Ma, Yefei; Wang, Lijun; Zhao, Yanchun; Zhao, Shulin

    2015-11-01

    A number of specific nucleic acids are closely related with many serious diseases, in the current research, a platform taking advantage of exonuclease III (Exo III) to realize double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters (DNA-AgNCs) for detecting of nucleic acid had been developed. In this method, a molecular beacon (MB) with 3'-protruding termini and a single-stranded cytosine-rich (C-rich) probe were designed that coexist stably with Exo III. Once the target DNA appeared, portion of the MB could hybridize with target DNA and was digested by Exo III, which allowed the release of target DNA and a residual sequence. Subsequently, the residual sequence could trigger the Exo III to digest C-rich probe, and the DNA-AgNCs was not able to be synthesized because of the C-rich probe was destroyed; finally the fluorescent of solution was quenched. This assay enables to monitor human hemochromatosis gene (as a model) with high sensitivity, the detection limit is as low as 120 pM compared with other fluorescence DNA-AgNCs methods, this assay also exhibits superior specificity even against single base mismatch. The strategy is applied to detect human hemochromatosis gene in real human serum samples successfully. PMID:26572843

  7. Crosslink Mapping at Amino Acid-Base Resolution Reveals the Path of Scrunched DNA in Initial Transcribing Complexes.

    PubMed

    Winkelman, Jared T; Winkelman, Bradford T; Boyce, Julian; Maloney, Michael F; Chen, Albert Y; Ross, Wilma; Gourse, Richard L

    2015-09-01

    RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RPO) is the same as in high-resolution X-ray structures, we define the path following substrate addition in "scrunched" complexes (RPITC). The DNA bulges that form within the transcription bubble in RPITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on interactions between the β flap, β' clamp, and σ3.2. We propose that this stress contributes to σ3.2 displacement from the RNA exit channel, facilitating promoter escape. PMID:26257284

  8. Structural analysis of DNA interaction with retinol and retinoic acid.

    PubMed

    Mandeville, J S; N'soukpoé-Kossi, C N; Neault, J F; Tajmir-Riahi, H A

    2010-06-01

    Dietary constituents of fresh fruits and vegetables may play a relevant role in DNA adduct formation by inhibiting enzymatic activities. Studies have shown the important role of antioxidant vitamins A, C, and E in the protection against cancer and cardiovascular diseases. The antioxidant activity of vitamin A and beta-carotene may consist of scavenging oxygen radicals and preventing DNA damage. This study was designed to examine the interaction of calf-thymus DNA with retinol and retinoic acid in aqueous solution at physiological conditions using a constant DNA concentration and various retinoid contents. Fourier transform infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopic methods were used to determine retinoid binding mode, the binding constant, and the effects of retinol and retinoic acid complexation on DNA conformation and aggregation. Structural analysis showed that retinol and retinoic acid bind DNA via G-C and A-T base pairs and the backbone phosphate groups with overall binding constants of Kret = 3.0 (+/-0.50) x 10(3) (mol.L(-1))(-1) and Kretac = 1.0 (+/-0.20) x 10(4) (mol.L(-1))(-1). The number of bound retinoids per DNA were 0.84 for retinol and 1.3 for retinoic acid. Hydrophobic interactions were also observed at high retinol and retinoic acid contents. At a high retinoid concentration, major DNA aggregation occurred, while DNA remained in the B-family structure. PMID:20555389

  9. Metal based pharmacologically active agents: Synthesis, structural characterization, molecular modeling, CT-DNA binding studies and in vitro antimicrobial screening of iron(II) bromosalicylidene amino acid chelates

    NASA Astrophysics Data System (ADS)

    Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.; Ismael, Mohamed; Seleem, Amin Abdou

    2014-01-01

    In recent years, great interest has been focused on Fe(II) Schiff base amino acid complexes as cytotoxic and antitumor drugs. Thus a series of new iron(II) complexes based on Schiff bases amino acids ligands have been designed and synthesized from condensation of 5-bromosalicylaldehyde (bs) and α-amino acids (L-alanine (ala), L-phenylalanine (phala), L-aspartic acid (aspa), L-histidine (his) and L-arginine (arg)). The structure of the investigated iron(II) complexes was elucidated using elemental analyses, infrared, ultraviolet-visible, thermogravimetric analysis, as well as conductivity and magnetic susceptibility measurements. Moreover, the stoichiometry and the stability constants of the prepared complexes have been determined spectrophotometrically. The results suggest that 5-bromosalicylaldehyde amino acid Schiff bases (bs:aa) behave as dibasic tridentate ONO ligands and coordinate to Fe(II) in octahedral geometry according to the general formula [Fe(bs:aa)2]ṡnH2O. The conductivity values between 37 and 64 ohm-1 mol-1 cm2 in ethanol imply the presence of nonelectrolyte species. The structure of the complexes was validated using quantum mechanics calculations based on accurate DFT methods. Geometry optimization of the Fe-Schiff base amino acid complexes showed that all complexes had octahedral coordination. In addition, the interaction of these complexes with (CT-DNA) was investigated at pH = 7.2, by using UV-vis absorption, viscosity and agarose gel electrophoresis measurements. Results indicated that the investigated complexes strongly bind to calf thymus DNA via intercalative mode and showed a different DNA binding according to the sequence: bsari > bshi > bsali > bsasi > bsphali. Moreover, the prepared compounds are screened for their in vitro antibacterial and antifungal activity against three types of bacteria, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus and three types of anti fungal cultures, Penicillium purpurogenium, Aspergillus

  10. The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex

    NASA Astrophysics Data System (ADS)

    Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

    2013-07-01

    A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy = 2,2-bipyridine, Hbcpip = 2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5 ± 1.4) × 105 M-1 in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived.

  11. Synthesis, spectral characterization and DNA binding of Schiff-base metal complexes derived from 2-amino-3-hydroxyprobanoic acid and acetylacetone

    NASA Astrophysics Data System (ADS)

    Hosny, Nasser Mohammed; Hussien, Mostafa A.; Radwan, Fatima M.; Nawar, Nagwa

    2014-11-01

    Four new metal complexes derived from the reaction of Cu(II), Co(II), Ni(II) and Zn(II) acetates with the Schiff-base ligand (H3L) resulted from the condensation of the amino acid 2-amino-3-hydroxyprobanoic acid (serine) and acetylacetone have been synthesized and characterized by, elemental analyses, ES-MS, IR, UV-Vis., 1H NMR, 13C NMR, ESR, thermal analyses (TGA and DTG) and magnetic measurements. The results showed that the Schiff-base ligand acts as bi-negative tridentate through the azomethine nitrogen, the deprotonated carboxylate oxygen and the enolic carbonyl oxygen. The optical band gaps measurements indicated the semi-conducting nature of these complexes. Molecular docking was used to predict the binding between the Schiff base ligand with the receptor of prostate cancer mutant H874Y. The interactions between the Cu(II) complex and calf thymus DNA (CT-DNA) have been studied by UV spectra. The results confirm that the Cu(II) complex binds to CT-DNA in an intercalative mode.

  12. DNA binding proteins that alter nucleic acid flexibility

    NASA Astrophysics Data System (ADS)

    McCauley, Micah; Hardwidge, Philip R.; Maher, L. J., III; Williams, Mark C.

    2007-09-01

    Dual - beam optical tweezers experiments subject single molecules of DNA to high forces (~ 300 pN) with 0.1 pN accuracy, probing the energy and specificity of nucleic acid - ligand structures. Stretching phage λ-DNA reveals an increase in the applied force up to a critical force known as the overstretching transition. In this region, base pairing and stacking are disrupted as double stranded DNA (dsDNA) is melted. Proteins that bind to the double strand will tend to stabilize dsDNA, and melting will occur at higher forces. Proteins that bind to single stranded DNA (ssDNA) destabilize melting, provided that the rate of association is comparable to the pulling rate of the experiment. Many proteins, however, exhibit some affinity for both dsDNA and ssDNA. We describe experiments upon DNA + HMGB2 (box A), a nuclear protein that is believed to facilitate transcription. By characterizing changes in the structure of dsDNA with a polymer model of elasticity, we have determined the equilibrium association constant for HMGB2 to be K ds = 0.15 +/- 0.7 10 9 M -1 for dsDNA binding. Analysis of the melting transition reveals an equilibrium association constant for HMGB2 to ssDNA to be K ss = 0.039 +/- 0.019 10 9 M -1 for ssDNA binding.

  13. DNA cleavage by new oxovanadium(IV) complexes of N-salicylidene alpha-amino acids and phenanthroline bases in the photodynamic therapy window.

    PubMed

    Sasmal, Pijus K; Patra, Ashis K; Nethaji, Munirathinam; Chakravarty, Akhil R

    2007-12-24

    Oxovanadium(IV) complexes [VO(salmet)(B)] (1-3) and [VO(saltrp)(B)] (4-6), where salmet and saltrp are N-salicylidene-l-methionate and N-salicylidene-l-tryptophanate, respectively, and B is a N,N-donor heterocyclic base (viz. 1,10-phenanthroline (phen, 1, 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2, 5), and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3, 6)) are prepared and characterized and their DNA binding and photoinduced DNA cleavage activity studied. Complexes 1, 2, and 4 are structurally characterized by single-crystal X-ray crystallography. The molecular structure shows the presence of a vanadyl group in the VO3N3 coordination geometry. The dianionic alpha-amino acid Schiff base acts as a tridentate O,N,O-donor ligand in a meridional binding mode. The N,N-donor heterocyclic base displays a chelating mode of bonding with a N-donor site trans to the oxo group. The complexes show a d-d band in the range of 680-710 nm in DMF with a shoulder near 840 nm. They exhibit an irreversible oxidative cyclic voltammetric response near 0.8 V assignable to the V(V)/V(IV) couple and a quasi-reversible V(IV)/V(III) redox couple near -1.1 V vs SCE in DMF-0.1 M TBAP. The complexes show good binding propensity to calf thymus DNA giving binding constant values in the range from 5.2 x 10(4) to 7.2 x 10(5) M(-1). The binding site size, thermal melting, and viscosity data suggest DNA surface and/or groove binding nature of the complexes. The complexes show poor "chemical nuclease" activity in the dark in the presence of 3-mercaptopropionic acid or hydrogen peroxide. The dpq and dppz complexes show efficient DNA cleavage activity on irradiation with UV-A light of 365 nm via a mechanistic pathway involving formation of singlet oxygen as the reactive species. They also show significant DNA cleavage activity on photoexcitation in red light (>750 nm) by (1)O2 species. Observation of red-light-induced cleavage of DNA is unprecedented in the vanadium chemistry. The DNA cleavage activity is

  14. Electrochemical DNA biosensor based on poly(2,6-pyridinedicarboxylic acid) modified glassy carbon electrode for the determination of anticancer drug gemcitabine.

    PubMed

    Tığ, Gözde Aydoğdu; Zeybek, Bülent; Pekyardımcı, Şule

    2016-07-01

    In this study, a simple methodology was used to develop a new electrochemical DNA biosensor based on poly(2,6-pyridinedicarboxylic acid) (P(PDCA)) modified glassy carbon electrode (GCE). This modified electrode was used to monitor for the electrochemical interaction between the dsDNA and gemcitabine (GEM) for the first time. A decrease in oxidation signals of guanine after the interaction of the dsDNA with the GEM was used as an indicator for the selective determination of the GEM via differential pulse voltammetry (DPV). The guanine oxidation peak currents were linearly proportional to the concentrations of the GEM in the range of 1-30mgL(‒1). Limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.276mgL(‒1) and 0.922mgL(‒1), respectively. The reproducibility, repeatability, and applicability of the analysis to pharmaceutical dosage forms and human serum samples were also examined. In addition to DPV method, UV-vis and viscosity measurements were utilized to propose the interaction mechanism between the GEM and the dsDNA. The novel DNA biosensor could serve for sensitive, accurate and rapid determination of the GEM. PMID:27154680

  15. Nucleic acid based molecular devices.

    PubMed

    Krishnan, Yamuna; Simmel, Friedrich C

    2011-03-28

    In biology, nucleic acids are carriers of molecular information: DNA's base sequence stores and imparts genetic instructions, while RNA's sequence plays the role of a messenger and a regulator of gene expression. As biopolymers, nucleic acids also have exciting physicochemical properties, which can be rationally influenced by the base sequence in myriad ways. Consequently, in recent years nucleic acids have also become important building blocks for bottom-up nanotechnology: as molecules for the self-assembly of molecular nanostructures and also as a material for building machinelike nanodevices. In this Review we will cover the most important developments in this growing field of nucleic acid nanodevices. We also provide an overview of the biochemical and biophysical background of this field and the major "historical" influences that shaped its development. Particular emphasis is laid on DNA molecular motors, molecular robotics, molecular information processing, and applications of nucleic acid nanodevices in biology. PMID:21432950

  16. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.

    PubMed

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui

    2016-08-31

    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. PMID:27506345

  17. Design, characterization, teratogenicity testing, antibacterial, antifungal and DNA interaction of few high spin Fe(II) Schiff base amino acid complexes

    NASA Astrophysics Data System (ADS)

    Abdel-Rahman, Laila H.; El-Khatib, Rafat M.; Nassr, Lobna A. E.; Abu-Dief, Ahmed M.; Lashin, Fakhr El-Din

    2013-07-01

    In this study, new Fe(II) Schiff base amino acid chelates derived from the condensation of o-hydroxynaphthaldehyde with L-alanine, L-phenylalanine, L-aspartic acid, L-histidine and L-arginine were synthesized and characterized via elemental, thermogravimetric analysis, molar conductance, IR, electronic, mass spectra and magnetic moment measurements. The stoichiometry and the stability constants of the complexes were determined spectrophotometrically. Correlation of all spectroscopic data suggested that Schiff bases ligands exhibited tridentate with ONO sites coordinating to the metal ions via protonated phenolic-OH, azomethine-N and carboxylate-O with the general formulae [Fe(HL)2]·nH2O. But in case of L-histidine, the ligand acts as tetradentate via deprotonated phenolic-OH, azomethine-N, carboxylate-O and N-imidazole ring ([FeL(H2O)2]·2H2O), where HL = mono anion and L = dianion of the ligand. The structure of the prepared complexes is suggested to be octahedral. The prepared complexes were tested for their teratogenicity on chick embryos and found to be safe until a concentration of 100 μg/egg with full embryos formation. Moreover, the interaction between CT-DNA and the investigated complexes were followed by spectrophotometric and viscosity measurements. It was found that, the prepared complexes bind to DNA via classical intercalative mode and showed a different DNA activity with the sequence: nhi > nari > nali > nasi > nphali. Furthermore, the free ligands and their complexes are screened for their in vitro antibacterial and antifungal activity against three types of bacteria, Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus and three types of anti fungal cultures, Penicillium purpurogenium, Aspergillus flavus and Trichotheium rosium in order to assess their antimicrobial potential. The results show that the metal complexes are more reactive with respect to their corresponding Schiff base amino acid ligands.

  18. [Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence].

    PubMed

    Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

    2016-01-01

    An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen. PMID:27239860

  19. A facile, sensitive, and highly specific trinitrophenol assay based on target-induced synergetic effects of acid induction and electron transfer towards DNA-templated copper nanoclusters.

    PubMed

    Li, Haiyin; Chang, Jiafu; Hou, Ting; Ge, Lei; Li, Feng

    2016-11-01

    Reliable, selective and sensitive approaches for trinitrophenol (TNP) detection are highly desirable with respect to national security and environmental protection. Herein, a simple and novel fluorescent strategy for highly sensitive and specific TNP assay has been successfully developed, which is based on the quenching of the fluorescent poly(thymine)-templated copper nanoclusters (DNA-CuNCs), through the synergetic effects of acid induction and electron transfer. Upon the addition of TNP, donor-acceptor complexes between the electron-deficient nitro-groups in TNP and the electron-donating DNA templates are formed, resulting in the close proximity between TNP and CuNCs. Moreover, the acidity of TNP contributes to the pH decrease of the system. These factors combine to dramatically quench the fluorescence of DNA-CuNCs, providing a "signal-off" strategy for TNP sensing. The as-proposed strategy demonstrates high sensitivity for TNP assay, and a detection limit of 0.03μM is obtained, which is lower than those reported by using organic fluorescent materials. More significantly, this approach shows outstanding selectivity over a number of TNP analogues, such as 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitrophenol (DNP), 3-nitrophenol (NP), nitrobenzene (NB), phenol (BP), and toluene (BT). Compared with previous studies, this method does not need complex DNA sequence design, fluorescent dye labeling, or sophisticated organic reactions, rendering the strategy with additional advantages of simplicity and cost-effectiveness. In addition, the as-proposed strategy has been adopted for the detection of TNP in natural water samples, indicating its great potential to be applied in the fields of public safety and environmental monitoring. PMID:27591641

  20. DNA-Based Applications in Nanobiotechnology

    PubMed Central

    Abu-Salah, Khalid M.; Ansari, Anees A.; Alrokayan, Salman A.

    2010-01-01

    Biological molecules such as deoxyribonucleic acid (DNA) have shown great potential in fabrication and construction of nanostructures and devices. The very properties that make DNA so effective as genetic material also make it a very suitable molecule for programmed self-assembly. The use of DNA to assemble metals or semiconducting particles has been extended to construct metallic nanowires and functionalized nanotubes. This paper highlights some important aspects of conjugating the unique physical properties of dots or wires with the remarkable recognition capabilities of DNA which could lead to miniaturizing biological electronics and optical devices, including biosensors and probes. Attempts to use DNA-based nanocarriers for gene delivery are discussed. In addition, the ecological advantages and risks of nanotechnology including DNA-based nanobiotechnology are evaluated. PMID:20652049

  1. Spectral characterization, optical band gap calculations and DNA binding of some binuclear Schiff-base metal complexes derived from 2-amino-ethanoic acid and acetylacetone

    NASA Astrophysics Data System (ADS)

    Hussien, Mostafa A.; Nawar, Nagwa; Radwan, Fatima M.; Hosny, Nasser Mohammed

    2015-01-01

    Bi-nuclear metal complexes derived from the reaction of Cu(II), Co(II), Ni(II) and Zn(II) acetates with the Schiff-base ligand (H2L) resulted from the condensation of 2-amino-ethanoic acid (glycine) and acetylacetone have been synthesized and characterized by elemental analyses, Raman spectra, FT-IR, ES-MS, UV-Vis., 1H NMR, ESR, thermal analyses (TG, DTG and DTA) and magnetic measurements. The results showed that, the Schiff base ligand can bind two metal ions in the same time. It coordinates to the first metal ion as mono-negative bi-dentate through azomethine nitrogen and enolic carbonyl after deprotonation. At the same time, it binds to the second metal ion via carboxylate oxygen after deprotonation. The thermodynamic parameters E∗, ΔH∗, ΔG∗ and ΔS∗ have been calculated by Coats-Redfern (CR) and Horowitz-Metzger (HM) methods. The optical band gaps of the isolated complexes have been calculated from absorption spectra and the results indicated semi-conducting nature of the investigated complexes. The interactions between the copper (II) complex and calf thymus DNA (CT-DNA) have been studied by UV spectra. The results confirm that the Cu(II) complex binds to CT-DNA.

  2. Targeting DNA G-Quadruplex Structures with Peptide Nucleic Acids

    PubMed Central

    Panyutin, Igor G.; Onyshchenko, Mykola I.; Englund, Ethan A.; Appella, Daniel H.; Neumann, Ronald D.

    2012-01-01

    Regulation of genetic functions based on targeting DNA or RNA sequences with complementary oligonucleotides is especially attractive in the post-genome era. Oligonucleotides can be rationally designed to bind their targets based on simple nucleic acid base pairing rules. However, the use of natural DNA and RNA oligonucleotides as targeting probes can cause numerous off-target effects. In addition, natural nucleic acids are prone to degradation in vivo by various nucleases. To address these problems, nucleic acid mimics such as peptide nucleic acids (PNA) have been developed. They are more stable, show less off-target effects, and, in general, have better binding affinity to their targets. However, their high affinity to DNA can reduce their sequence-specificity. The formation of alternative DNA secondary structures, such as the G-quadruplex, provides an extra level of specificity as targets for PNA oligomers. PNA probes can target the loops of G-quadruplex, invade the core by forming PNA-DNA guanine-tetrads, or bind to the open bases on the complementary cytosine-rich strand. Not only could the development of such G-quadruplex-specific probes allow regulation of gene expression, but it will also provide a means to clarify the biological roles G-quadruplex structures may possess. PMID:22376112

  3. Target-induced quenching for highly sensitive detection of nucleic acids based on label-free luminescent supersandwich DNA/silver nanoclusters.

    PubMed

    Wang, Guangfeng; Zhu, Yanhong; Chen, Ling; Wang, Lun; Zhang, Xiaojun

    2014-01-01

    Luminescent silver nanoclusters (AgNCs) were anchored by designed oligonucleotides, acting as fluorescent labels. They hybridized with specific nucleic acid targets to form a supersandwich structure resulting in the fluorescence intensity of the DNA/AgNCs decreasing linearly with respect to the concentration of the target DNA. PMID:24244937

  4. Complexing of amino acids to DNA by chromate in intact cells.

    PubMed Central

    Voitkun, V; Zhitkovich, A; Costa, M

    1994-01-01

    Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7843108

  5. DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery.

    PubMed

    Rosa, Mónica; Penacho, Nuno; Simöes, Sérgio; Lima, Maria C P; Lindman, Björn; Miguel, Maria G

    2008-01-01

    A study related to the development and characterization of a new gene delivery system was performed. The approach consists in both the pre-condensation of plasmid DNA with an arginine-based cationic surfactant, arginine-N-lauroyl amide dihydrochloride (ALA), which was found not to be toxic, and the incorporation of the blood protein transferrin (Tf) into the formulations. Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP:Chol) and the other a pH sensitive formulation constituted of DOTAP, Chol, Dioleoyl phosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS). Particles with different ALA/DNA and cationic lipid/DNA charge ratios were produced and a physicochemical characterization of the systems developed was performed. DNA conformational changes in the presence of ALA were studied by Circular Dichroism (CD) and the ALA binding to DNA was followed by surface tension measurements. Insight into the structure and morphology of the various ALA-complexes (complexes composed of ALA, DNA, Tf and liposomes) was obtained by cryogenic-Transmission Electron Microscopy (cryo-TEM) and the sizes of the ALA-complexes were determined through Photon Correlation Spectroscopy (PCS). We found that the transfection capacity of these systems is directly related with the presence of ALA and the lipidic composition. Complexes based on the pH sensitive liposome formulation present better transfection profiles. The correlation between the inner structure, density and size of the ALA-complexes and their biological activity is discussed. Overall, we demonstrate that the presence of ALA improves the transfection efficiency when conjugated with cationic liposome systems. PMID:18097953

  6. QPSO-Based Adaptive DNA Computing Algorithm

    PubMed Central

    Karakose, Mehmet; Cigdem, Ugur

    2013-01-01

    DNA (deoxyribonucleic acid) computing that is a new computation model based on DNA molecules for information storage has been increasingly used for optimization and data analysis in recent years. However, DNA computing algorithm has some limitations in terms of convergence speed, adaptability, and effectiveness. In this paper, a new approach for improvement of DNA computing is proposed. This new approach aims to perform DNA computing algorithm with adaptive parameters towards the desired goal using quantum-behaved particle swarm optimization (QPSO). Some contributions provided by the proposed QPSO based on adaptive DNA computing algorithm are as follows: (1) parameters of population size, crossover rate, maximum number of operations, enzyme and virus mutation rate, and fitness function of DNA computing algorithm are simultaneously tuned for adaptive process, (2) adaptive algorithm is performed using QPSO algorithm for goal-driven progress, faster operation, and flexibility in data, and (3) numerical realization of DNA computing algorithm with proposed approach is implemented in system identification. Two experiments with different systems were carried out to evaluate the performance of the proposed approach with comparative results. Experimental results obtained with Matlab and FPGA demonstrate ability to provide effective optimization, considerable convergence speed, and high accuracy according to DNA computing algorithm. PMID:23935409

  7. Cinnamate-based DNA photolithography

    PubMed Central

    Romulus, Joy; Li, Minfeng; Sha, Ruojie; Royer, John; Wu, Kun-Ta; Xu, Qin

    2013-01-01

    As demonstrated by means of DNA nanoconstructs[1], as well as DNA functionalization of nanoparticles[2-4] and micrometre-scale colloids[5-8], complex self-assembly processes require components to associate with particular partners in a programmable fashion. In many cases the reversibility of the interactions between complementary DNA sequences is an advantage[9]. However, permanently bonding some or all of the complementary pairs may allow for flexibility in design and construction[10]. Here, we show that the substitution of a pair of complementary bases by a cinnamate group provides an efficient, addressable, UV light-based method to covalently bond complementary DNA. To show the potential of this approach, we wrote micrometre-scale patterns on a surface via UV light and demonstrate the reversible attachment of conjugated DNA and DNA-coated colloids. Our strategy enables both functional DNA photolithography and multi-step, specific binding in self-assembly processes. PMID:23685865

  8. DNA-LCEB: a high-capacity and mutation-resistant DNA data-hiding approach by employing encryption, error correcting codes, and hybrid twofold and fourfold codon-based strategy for synonymous substitution in amino acids.

    PubMed

    Hafeez, Ibbad; Khan, Asifullah; Qadir, Abdul

    2014-11-01

    Data-hiding in deoxyribonucleic acid (DNA) sequences can be used to develop an organic memory and to track parent genes in an offspring as well as in genetically modified organism. However, the main concerns regarding data-hiding in DNA sequences are the survival of organism and successful extraction of watermark from DNA. This implies that the organism should live and reproduce without any functional disorder even in the presence of the embedded data. Consequently, performing synonymous substitution in amino acids for watermarking becomes a primary option. In this regard, a hybrid watermark embedding strategy that employs synonymous substitution in both twofold and fourfold codons of amino acids is proposed. This work thus presents a high-capacity and mutation-resistant watermarking technique, DNA-LCEB, for hiding secret information in DNA of living organisms. By employing the different types of synonymous codons of amino acids, the data storage capacity has been significantly increased. It is further observed that the proposed DNA-LCEB employing a combination of synonymous substitution, lossless compression, encryption, and Bose-Chaudary-Hocquenghem coding is secure and performs better in terms of both capacity and robustness compared to existing DNA data-hiding schemes. The proposed DNA-LCEB is tested against different mutations, including silent, miss-sense, and non-sense mutations, and provides substantial improvement in terms of mutation detection/correction rate and bits per nucleotide. A web application for DNA-LCEB is available at http://111.68.99.218/DNA-LCEB. PMID:25195035

  9. Carbohydrate-interactive pDNA and siRNA gene vectors based on boronic acid functionalized poly(amido amine)s.

    PubMed

    Piest, Martin; Ankoné, Marc; Engbersen, Johan F J

    2013-08-10

    In order to evaluate the influence of incorporation of boronic acid groups on the properties of poly(amido amine)s as gene vectors, a novel poly(amido amine) copolymer p(CBA-ABOL/2AMPBA) containing ortho-aminomethylphenylboronic acid (2AMPBA) moieties was prepared by Michael-type polyaddition of a mixture of 1,4-aminobutanol (ABOL) and 2-((4-aminobutylamino)methyl)phenyl boronic acid to N,N'-cystamine bisacrylamide (CBA). It appeared that the presence of the boronic acid moieties as side groups along the polymer chain strongly enhances the stability of the self-assembled nanoparticles and nanosized polyplexes formed from this polymer; no aggregation was observed after storage for 6days at 37°C. This strong stabilization can be attributed to intermolecular Lewis acid-base interactions between the 2AMPBA groups and the alcohol and amine groups present in the polymer, leading to dynamical (reversible) crosslinking in the nanoparticles. Moreover, since the boronic acids can reversibly form boronic esters with vicinal diol groups, the presence of the 2AMPBA groups add carbohydrate-interactive properties to these polymers that strongly influence their behavior as gene delivery vectors. DNA transfection with p(CBA-ABOL/2AMPBA) polyplexes gave transfection efficiencies that were approximately similar to commercial PEI in different cell lines (COS-7, HUH-6 and H1299-Fluc), but lower than those obtained with reference polyplexes from p(CBA-ABOL). It is hypothesized that the uptake of the boronated polyplexes is suppressed by binding to the glycocalyx of the cells. This is supported by the observation that addition of sorbitol or dextran to the transfection medium significantly enhances the transfection efficiency, which can be attributed to increased cellular uptake of the polyplexes due to boronic ester formation with these agents. AFM, SEM and confocal microscopy showed that polyplexes of p(CBA-ABOL/2AMPBA) become decorated with a dextran layer in the presence of 0.9% (w

  10. Amino Acid Racemization and the Preservation of Ancient DNA

    NASA Technical Reports Server (NTRS)

    Poinar, Hendrik N.; Hoss, Matthias

    1996-01-01

    The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.

  11. Acid-Base Homeostasis

    PubMed Central

    Nakhoul, Nazih; Hering-Smith, Kathleen S.

    2015-01-01

    Acid-base homeostasis and pH regulation are critical for both normal physiology and cell metabolism and function. The importance of this regulation is evidenced by a variety of physiologic derangements that occur when plasma pH is either high or low. The kidneys have the predominant role in regulating the systemic bicarbonate concentration and hence, the metabolic component of acid-base balance. This function of the kidneys has two components: reabsorption of virtually all of the filtered HCO3− and production of new bicarbonate to replace that consumed by normal or pathologic acids. This production or generation of new HCO3− is done by net acid excretion. Under normal conditions, approximately one-third to one-half of net acid excretion by the kidneys is in the form of titratable acid. The other one-half to two-thirds is the excretion of ammonium. The capacity to excrete ammonium under conditions of acid loads is quantitatively much greater than the capacity to increase titratable acid. Multiple, often redundant pathways and processes exist to regulate these renal functions. Derangements in acid-base homeostasis, however, are common in clinical medicine and can often be related to the systems involved in acid-base transport in the kidneys. PMID:26597304

  12. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  13. Acid-base chemistry

    SciTech Connect

    Hand, C.W.; Blewit, H.L.

    1985-01-01

    The book is not a research compendium and there are no references to the literature. It is a teaching text covering the entire range of undergraduate subject matter dealing with acid-base chemistry (some of it remotely) as taught in inorganic, analytical, and organic chemistry courses. The excellent chapters VII through IX deal in detail with the quantitative aspects of aqueous acid-base equilibria (salt hydrolysis and buffer, titrations, polyprotic and amphoteric substances).

  14. Dark States in Single DNA Bases and DNA Base Polymers

    NASA Astrophysics Data System (ADS)

    Kohler, Bern; Hare, Patrick M.; Middleton, Chris T.

    2009-06-01

    DNA is vulnerable to photochemical modification by UV light. The excited electronic states that initiate DNA damage have been difficult to characterize due to their ultrashort lifetimes, and most excitations in single DNA bases decay to the electronic ground state in hundreds of femtoseconds. Although many workers have now located conical intersections between various electronic states of the nucleobases, there is still confusion over the precise dynamics that lead to deactivation. This is especially true for the pyrimidine bases where the initial Franck-Condon population bifurcates with some molecules decaying to the ground state and others relaxing to a relatively long-lived ^1nπ* state. Results from UV/UV and UV/mid-IR transient absorption experiments will be presented that illustrate these dual decay pathways. Evidence suggests that the ^1nπ* state mediates intersystem crossing to the triplet state. Finally, current understanding of how these single-base decay pathways are modified by interactions in DNA polymers will be discussed.

  15. DNA based arithmetic function: a half adder based on DNA strand displacement

    NASA Astrophysics Data System (ADS)

    Li, Wei; Zhang, Fei; Yan, Hao; Liu, Yan

    2016-02-01

    Biomolecular programming utilizes the reactions and information stored in biological molecules, such as proteins and nucleic acids, for computational purposes. DNA has proven itself an excellent candidate for building logic operating systems due to its highly predictable molecular behavior. In this work we designed and realized an XOR logic gate and an AND logic gate based on DNA strand displacement reactions. These logic gates utilize ssDNA as input and output signals. The XOR gate and the AND gate were used as building blocks for constructing a half adder logic circuit, which is a primary step in constructing a full adder, a basic arithmetic unit in computing. This work provides the field of DNA molecular programming with a potential universal arithmetic tool.Biomolecular programming utilizes the reactions and information stored in biological molecules, such as proteins and nucleic acids, for computational purposes. DNA has proven itself an excellent candidate for building logic operating systems due to its highly predictable molecular behavior. In this work we designed and realized an XOR logic gate and an AND logic gate based on DNA strand displacement reactions. These logic gates utilize ssDNA as input and output signals. The XOR gate and the AND gate were used as building blocks for constructing a half adder logic circuit, which is a primary step in constructing a full adder, a basic arithmetic unit in computing. This work provides the field of DNA molecular programming with a potential universal arithmetic tool. Electronic supplementary information (ESI) available: Detailed descriptions of DNA logic gate design, materials and methods, and additional data analysis. See DOI: 10.1039/c5nr08497k

  16. DNA-based control of protein activity

    PubMed Central

    Engelen, W.; Janssen, B. M. G.

    2016-01-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  17. DNA-based control of protein activity.

    PubMed

    Engelen, W; Janssen, B M G; Merkx, M

    2016-03-01

    DNA has emerged as a highly versatile construction material for nanometer-sized structures and sophisticated molecular machines and circuits. The successful application of nucleic acid based systems greatly relies on their ability to autonomously sense and act on their environment. In this feature article, the development of DNA-based strategies to dynamically control protein activity via oligonucleotide triggers is discussed. Depending on the desired application, protein activity can be controlled by directly conjugating them to an oligonucleotide handle, or expressing them as a fusion protein with DNA binding motifs. To control proteins without modifying them chemically or genetically, multivalent ligands and aptamers that reversibly inhibit their function provide valuable tools to regulate proteins in a noncovalent manner. The goal of this feature article is to give an overview of strategies developed to control protein activity via oligonucleotide-based triggers, as well as hurdles yet to be taken to obtain fully autonomous systems that interrogate, process and act on their environments by means of DNA-based protein control. PMID:26812623

  18. DNA based computing for understanding complex shapes.

    PubMed

    Ullah, A M M Sharif; D'Addona, Doriana; Arai, Nobuyuki

    2014-03-01

    This study deals with a computing method called DNA based computing (DBC) that takes inspiration from the Central Dogma of Molecular Biology. The proposed DBC uses a set of user-defined rules to create a DNA-like sequence from a given piece of problem-relevant information (e.g., image data) in a dry-media (i.e., in an ordinary computer). It then uses another set of user-defined rules to create an mRNA-like sequence from the DNA. Finally, it uses the genetic code to translate the mRNA (or directly the DNA) to a protein-like sequence (a sequence of amino acids). The informational characteristics of the protein (entropy, absence, presence, abundance of some selected amino acids, and relationships among their likelihoods) can be used to solve problems (e.g., to understand complex shapes from their image data). Two case studies ((1) fractal geometry generated shape of a fern-leaf and (2) machining experiment generated shape of the worn-zones of a cutting tool) are presented elucidating the shape understanding ability of the proposed DBC in the presence of a great deal of variability in the image data of the respective shapes. The implication of the proposed DBC from the context of Internet-aided manufacturing system is also described. Further study can be carried out in solving other complex computational problems by using the proposed DBC and its derivatives. PMID:24447435

  19. [Forced Oscillations of DNA Bases].

    PubMed

    Yakushevich, L V; Krasnobaeva, L A

    2016-01-01

    This paper presents the results of the studying of forced angular oscillations of the DNA bases with the help of the mathematical model consisting of two coupled nonlinear differential equations that take into account the effects of dissipation and the influence of an external periodic field. The calculation results are illustrated for sequence of gene encoding interferon alpha 17 (IFNA 17). PMID:27192830

  20. Accurate and easy-to-use assessment of contiguous DNA methylation sites based on proportion competitive quantitative-PCR and lateral flow nucleic acid biosensor.

    PubMed

    Xu, Wentao; Cheng, Nan; Huang, Kunlun; Lin, Yuehe; Wang, Chenguang; Xu, Yuancong; Zhu, Longjiao; Du, Dan; Luo, Yunbo

    2016-06-15

    Many types of diagnostic technologies have been reported for DNA methylation, but they require a standard curve for quantification or only show moderate accuracy. Moreover, most technologies have difficulty providing information on the level of methylation at specific contiguous multi-sites, not to mention easy-to-use detection to eliminate labor-intensive procedures. We have addressed these limitations and report here a cascade strategy that combines proportion competitive quantitative PCR (PCQ-PCR) and lateral flow nucleic acid biosensor (LFNAB), resulting in accurate and easy-to-use assessment. The P16 gene with specific multi-methylated sites, a well-studied tumor suppressor gene, was used as the target DNA sequence model. First, PCQ-PCR provided amplification products with an accurate proportion of multi-methylated sites following the principle of proportionality, and double-labeled duplex DNA was synthesized. Then, a LFNAB strategy was further employed for amplified signal detection via immune affinity recognition, and the exact level of site-specific methylation could be determined by the relative intensity of the test line and internal reference line. This combination resulted in all recoveries being greater than 94%, which are pretty satisfactory recoveries in DNA methylation assessment. Moreover, the developed cascades show significantly high usability as a simple, sensitive, and low-cost tool. Therefore, as a universal platform for sensing systems for the detection of contiguous multi-sites of DNA methylation without external standards and expensive instrumentation, this PCQ-PCR-LFNAB cascade method shows great promise for the point-of-care diagnosis of cancer risk and therapeutics. PMID:26914373

  1. DNA based arithmetic function: a half adder based on DNA strand displacement.

    PubMed

    Li, Wei; Zhang, Fei; Yan, Hao; Liu, Yan

    2016-02-14

    Biomolecular programming utilizes the reactions and information stored in biological molecules, such as proteins and nucleic acids, for computational purposes. DNA has proven itself an excellent candidate for building logic operating systems due to its highly predictable molecular behavior. In this work we designed and realized an XOR logic gate and an AND logic gate based on DNA strand displacement reactions. These logic gates utilize ssDNA as input and output signals. The XOR gate and the AND gate were used as building blocks for constructing a half adder logic circuit, which is a primary step in constructing a full adder, a basic arithmetic unit in computing. This work provides the field of DNA molecular programming with a potential universal arithmetic tool. PMID:26814628

  2. Morphine Induces Redox-Based Changes in Global DNA Methylation and Retrotransposon Transcription by Inhibition of Excitatory Amino Acid Transporter Type 3–Mediated Cysteine Uptake

    PubMed Central

    Trivedi, Malav; Shah, Jayni; Hodgson, Nathaniel; Byun, Hyang-Min

    2014-01-01

    Canonically, opioids influence cells by binding to a G protein–coupled opioid receptor, initiating intracellular signaling cascades, such as protein kinase, phosphatidylinositol 3-kinase, and extracellular receptor kinase pathways. This results in several downstream effects, including decreased levels of the reduced form of glutathione (GSH) and elevated oxidative stress, as well as epigenetic changes, especially in retrotransposons and heterochromatin, although the mechanism and consequences of these actions are unclear. We characterized the acute and long-term influence of morphine on redox and methylation status (including DNA methylation levels) in cultured neuronal SH-SY5Y cells. Acting via μ-opioid receptors, morphine inhibits excitatory amino acid transporter type 3–mediated cysteine uptake via multiple signaling pathways, involving different G proteins and protein kinases in a temporal manner. Decreased cysteine uptake was associated with decreases in both the redox and methylation status of neuronal cells, as defined by the ratios of GSH to oxidized forms of glutathione and S-adenosylmethionine to S-adenosylhomocysteine levels, respectively. Further, morphine induced global DNA methylation changes, including CpG sites in long interspersed nuclear elements (LINE-1) retrotransposons, resulting in increased LINE-1 mRNA. Together, these findings illuminate the mechanism by which morphine, and potentially other opioids, can influence neuronal-cell redox and methylation status including DNA methylation. Since epigenetic changes are implicated in drug addiction and tolerance phenomenon, this study could potentially extrapolate to elucidate a novel mechanism of action for other drugs of abuse. PMID:24569088

  3. Folic acid binds DNA and RNA at different locations.

    PubMed

    Bourassa, P; Tajmir-Riahi, H A

    2015-03-01

    We located multiple binding sites for folic acid on DNA and tRNA at physiological conditions, using FTIR, CD, fluorescence spectroscopic methods and molecular modeling. Structural analysis revealed that folic acid binds DNA and tRNA at multiple sites via hydrophilic, hydrophobic and H-bonding contacts with overall binding constants of Kfolic acid-DNA=1.1 (±0.3)×10(4) M(-1) and Kfolic acid-tRNA=6.4 (±0.5)×10(3) M(-1). Molecular modeling showed the participation of several nucleobases in folic acid complexes with DNA and tRNA, stabilized by H-bonding network. Two types of complexes were located for folic acid-tRNA adducts, one at the major groove and the other with TΨC loop, while acid binding occurs at major and minor grooves of DNA duplex. Folic acid complexation induced more alterations of DNA structure than tRNA. PMID:25555838

  4. Single and double stranded DNA detection using locked nucleic acid (LNA) functionalized nanoparticles

    NASA Astrophysics Data System (ADS)

    McKenzie, Fiona; Stokes, Robert; Faulds, Karen; Graham, Duncan

    2008-08-01

    Gold and silver nanoparticles functionalized with oligonucleotides can be used for the detection of specific sequences of DNA. We show that gold nanoparticles modified with locked nucleic acid (LNA) form stronger duplexes with a single stranded DNA target and offer better discrimination against single base pair mismatches than analogous DNA probes. Our LNA nanoparticle probes have also been used to detect double stranded DNA through triplex formation, whilst still maintaining selectivity for only complementary targets. Nanoparticle conjugates embedded with suitable surface enhanced resonance Raman scattering (SERRS) labels have been synthesized enabling simultaneous detection and identification of multiple DNA targets.

  5. Synthesis of DNA oligonucleotides containing C5-ethynylbenzenesulfonamide-modified nucleotides (EBNA) by polymerases towards the construction of base functionalized nucleic acids.

    PubMed

    Goubet, Astrid; Chardon, Antoine; Kumar, Pawan; Sharma, Pawan K; Veedu, Rakesh N

    2013-02-01

    C5-Ethynylbenzenesulfonamide-modified nucleotide (EBNA) was investigated as substrate of various DNA polymerases. The experiments revealed that KOD, Phusion and Klenow DNA polymerases successfully accepted EBNA-T nucleotide as a substrate and yielded the fully extended DNA. KOD DNA polymerase was found to be the most efficient enzyme to furnish EBNA-T containing DNA in good yields. Phusion DNA polymerase efficiently amplified the template containing EBNA-T nucleotides by PCR. PMID:23265899

  6. Antioxidant and DNA damage protection potentials of selected phenolic acids.

    PubMed

    Sevgi, Kemal; Tepe, Bektas; Sarikurkcu, Cengiz

    2015-03-01

    In this study, ten different phenolic acids (caffeic, chlorogenic, cinnamic, ferulic, gallic, p-hydroxybenzoic, protocatechuic, rosmarinic, syringic, and vanillic acids) were evaluated for their antioxidant and DNA damage protection potentials. Antioxidant activity was evaluated by using four different test systems named as β-carotene bleaching, DPPH free radical scavenging, reducing power and chelating effect. In all test systems, rosmarinic acid showed the maximum activity potential, while protocatechuic acid was determined as the weakest antioxidant in β-carotene bleaching, DPPH free radical scavenging, and chelating effect assays. Phenolic acids were also screened for their protective effects on pBR322 plasmid DNA against the mutagenic and toxic effects of UV and H2O2. Ferulic acid was found as the most active phytochemical among the others. Even at the lowest concentration value (0.002 mg/ml), ferulic acid protected all of the bands in the presence of H2O2 and UV. It is followed by caffeic, rosmarinic, and vanillic acids. On the other hand, cinnamic acid (at 0.002 mg/ml), gallic acid (at 0.002 mg/ml), p-hydroxybenzoic acid (at 0.002 and 0.004 mg/ml), and protocatechuic acid (at 0.002 and 0.004 mg/ml) could not protect plasmid DNA. PMID:25542528

  7. DNA-based watermarks using the DNA-Crypt algorithm

    PubMed Central

    Heider, Dominik; Barnekow, Angelika

    2007-01-01

    Background The aim of this paper is to demonstrate the application of watermarks based on DNA sequences to identify the unauthorized use of genetically modified organisms (GMOs) protected by patents. Predicted mutations in the genome can be corrected by the DNA-Crypt program leaving the encrypted information intact. Existing DNA cryptographic and steganographic algorithms use synthetic DNA sequences to store binary information however, although these sequences can be used for authentication, they may change the target DNA sequence when introduced into living organisms. Results The DNA-Crypt algorithm and image steganography are based on the same watermark-hiding principle, namely using the least significant base in case of DNA-Crypt and the least significant bit in case of the image steganography. It can be combined with binary encryption algorithms like AES, RSA or Blowfish. DNA-Crypt is able to correct mutations in the target DNA with several mutation correction codes such as the Hamming-code or the WDH-code. Mutations which can occur infrequently may destroy the encrypted information, however an integrated fuzzy controller decides on a set of heuristics based on three input dimensions, and recommends whether or not to use a correction code. These three input dimensions are the length of the sequence, the individual mutation rate and the stability over time, which is represented by the number of generations. In silico experiments using the Ypt7 in Saccharomyces cerevisiae shows that the DNA watermarks produced by DNA-Crypt do not alter the translation of mRNA into protein. Conclusion The program is able to store watermarks in living organisms and can maintain the original information by correcting mutations itself. Pairwise or multiple sequence alignments show that DNA-Crypt produces few mismatches between the sequences similar to all steganographic algorithms. PMID:17535434

  8. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications. PMID:26614075

  9. Method for sequencing DNA base pairs

    DOEpatents

    Sessler, Andrew M.; Dawson, John

    1993-01-01

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source.

  10. Thermodynamically based DNA strand design

    PubMed Central

    Tulpan, Dan; Andronescu, Mirela; Chang, Seo Bong; Shortreed, Michael R.; Condon, Anne; Hoos, Holger H.; Smith, Lloyd M.

    2005-01-01

    We describe a new algorithm for design of strand sets, for use in DNA computations or universal microarrays. Our algorithm can design sets that satisfy any of several thermodynamic and combinatorial constraints, which aim to maximize desired hybridizations between strands and their complements, while minimizing undesired cross-hybridizations. To heuristically search for good strand sets, our algorithm uses a conflict-driven stochastic local search approach, which is known to be effective in solving comparable search problems. The PairFold program of Andronescu et al. [M. Andronescu, Z. C. Zhang and A. Condon (2005) J. Mol. Biol., 345, 987–1001; M. Andronescu, R. Aguirre-Hernandez, A. Condon, and H. Hoos (2003) Nucleic Acids Res., 31, 3416–3422.] is used to calculate the minimum free energy of hybridization between two mismatched strands. We describe new thermodynamic measures of the quality of strand sets. With respect to these measures of quality, our algorithm consistently finds, within reasonable time, sets that are significantly better than previously published sets in the literature. PMID:16145053

  11. Evaluation of cellular uptake and intracellular trafficking as determining factors of gene expression for amino acid-substituted gemini surfactant-based DNA nanoparticles

    PubMed Central

    2012-01-01

    Background Gene transfer using non-viral vectors offers a non-immunogenic and safe method of gene delivery. Cellular uptake and intracellular trafficking of the nanoparticles can impact on the transfection efficiency of these vectors. Therefore, understanding the physicochemical properties that may influence the cellular uptake and the intracellular trafficking can aid the design of more efficient non-viral gene delivery systems. Recently, we developed novel amino acid-substituted gemini surfactants that showed higher transfection efficiency than their parent compound. In this study, we evaluated the mechanism of cellular uptake of the plasmid/gemini surfactant/helper lipid nanoparticles and their effect on the transfection efficiency. Results Nanoparticles were incubated with Sf 1 Ep cells in the presence of different endocytic inhibitors and gene expression (interferon-γ) was measured using ELISA. Clathrin-mediated and caveolae-mediated uptake were found to be equally contributing to cellular internalization of both P/12-7NH-12/L (parent gemini surfactant) and P/12-7NGK-12/L (amino acid-substituted gemini surfactant) nanoparticles. The plasmid and the helper lipid were fluorescently tagged to track the nanoparticles inside the cells, using confocal laser scanning microscopy. Transmission electron microscopy images showed that the P/12-7NGK-12/L particles were cylindrical while the P/12-7NH-12/L particles were spherical which may influence the cellular uptake behaviour of these particles. Dye exclusion assay and pH-titration of the nanoparticles suggested that high buffering capacity, pH-dependent increase in particle size and balanced DNA binding properties may be contributing to a more efficient endosomal escape of P/12-7NGK-12/L compared to the P/12-7NH-12/L nanoparticles, leading to higher gene expression. Conclusion Amino-acid substitution in the spacer of gemini surfactant did not alter the cellular uptake pathway, showing similar pattern to the

  12. Cationic Lipid-Based Nucleic Acid Vectors.

    PubMed

    Jubeli, Emile; Goldring, William P D; Pungente, Michael D

    2016-01-01

    The delivery of nucleic acids into cells remains an important laboratory cell culture technique and potential clinical therapy, based upon the initial cellular uptake, then translation into protein (in the case of DNA), or gene deletion by RNA interference (RNAi). Although viral delivery vectors are more efficient, the high production costs, limited cargo capacity, and the potential for clinical adverse events make nonviral strategies attractive. Cationic lipids are the most widely applied and studied nonviral vectors; however, much remains to be solved to overcome limitations of these systems. Advances in the field of cationic lipid-based nucleic acid (lipoplex) delivery rely upon the development of robust and reproducible lipoplex formulations, together with the use of cell culture assays. This chapter provides detailed protocols towards the formulation, delivery, and assessment of in vitro cationic lipid-based delivery of DNA. PMID:27436310

  13. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells

    PubMed Central

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  14. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells.

    PubMed

    Song, Eun Ah; Kim, Hyeyoung

    2016-01-01

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells' molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies. PMID:27527148

  15. Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

    PubMed Central

    Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

    2003-01-01

    The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) ‘clamps’ for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by ‘hybrid’ phosphorothioate–LNA ODNs induces tumour necrosis factor-α production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination. PMID:14530430

  16. NMR analysis of base-pair opening kinetics in DNA

    PubMed Central

    Szulik, Marta W.; Voehler, Markus; Stone, Michael P.

    2014-01-01

    Base pairing in nucleic acids plays a crucial role in their structure and function. Differences in the base pair opening and closing kinetics of individual double stranded DNA sequences or between chemically modified base pairs provide insight into the recognition of these base pairs by DNA processing enzymes. This unit describes how to quantify the kinetics for localized base pairs by observing changes in the imino proton signals by nuclear magnetic resonance spectroscopy. The determination of all relevant parameters using state of the art techniques and NMR instrumentation, including cryoprobes, is discussed. PMID:25501592

  17. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers.

    PubMed

    Jolly, Pawan; Estrela, Pedro; Ladomery, Michael

    2016-06-30

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  18. Unscheduled deoxyribonucleic acid (DNA) synthesis assays for toxicological studies. May 1977-March 1990 (A Bibliography from the NTIS data base). Report for May 1977-March 1990

    SciTech Connect

    Not Available

    1990-04-01

    This bibliography contains citations concerning the unscheduled DNA synthesis (UDS) assay for toxicological studies. UDS assays provide very sensitive measures of damage to DNA by detecting induction of DNA synthesis in non-S-phase cells. UDS toxicological studies analyzing gamma radiation, drugs, pesticides, nerve gas, jet engine fuels, ultraviolet light, chlorated organic compounds, and aromatic compounds are discussed. UDS studies using both human and animal tissue cultures are described. (Contains 57 citations fully indexed and including a title list.)

  19. Gibberellic Acid Enhancement of DNA Turnover in Barley Aleurone Cells 1

    PubMed Central

    Taiz, Lincoln; Starks, Jayum E.

    1977-01-01

    When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [3H]thymidine-labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse-chase studies demonstrated that the newly synthesized DNA is metabolically labile. The buoyant density on CsCl density gradients of hormone-treated aleurone DNA is identical with that of DNA extracted from whole seedlings. After density-labeling halfseed DNA with 5-bromodeoxyuridine, a bimodal absorption profile is obtained in neutral CsCl. The light band (1.70 g/ml) corresponds to unsubstituted DNA, while the heavy band (1.725-1.74 g/ml) corresponds to a hybrid density-labeled species. GA increases the relative amount of the heavy (hybrid) peak in halfseed aleurone layer DNA, further suggesting that the hormone enhances semiconservative replication in halfseeds. DNA methylation was also demonstrated. Over 60% of the radioactivity from [3H-Me]methionine is incorporated into 5-methylcytosine. GA has no effect on the percentage distribution of label among the bases. It was concluded that GA enhances the rate of DNA degradation and DNA synthesis (turnover) in halfseeds, but primarily DNA degradation in isolated aleurone layers. Incorporation by isolated aleurone layers is due to DNA repair. Semiconservative replication apparently plays no physiological role in the hormone response, since both isolated aleurone layers and gamma-irradiated halfseeds respond normally. The hypothesis was advanced that endoreduplication and DNA degradation are means by which the seed stores and mobilizes deoxyribonucleotides for the embryo during

  20. Amino acid racemization in amber-entombed insects: implications for DNA preservation

    NASA Technical Reports Server (NTRS)

    Bada, J. L.; Wang, X. S.; Poinar, H. N.; Paabo, S.; Poinar, G. O.

    1994-01-01

    DNA depurination and amino acid racemization take place at similar rates in aqueous solution at neutral pH. This relationship suggests that amino acid racemization may be useful in accessing the extent of DNA chain breakage in ancient biological remains. To test this suggestion, we have investigated the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 million years. The amino acids present in 40 to 130 million year old amber-entombed insects resemble those in a modern fly and are probably the most ancient, unaltered amino acids found so far on Earth. In comparison to other geochemical environments on the surface of the Earth, the amino acid racemization rate in amber insect inclusions is retarded by a factor of >10(4). These results suggest that in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms.

  1. Ultrasensitive FRET-based DNA sensor using PNA/DNA hybridization.

    PubMed

    Yang, Lan-Hee; Ahn, Dong June; Koo, Eunhae

    2016-12-01

    In the diagnosis of genetic diseases, rapid and highly sensitive DNA detection is crucial. Therefore, many strategies for detecting target DNA have been developed, including electrical, optical, and mechanical methods. Herein, a highly sensitive FRET based sensor was developed by using PNA (Peptide Nucleic Acid) probe and QD, in which red color QDs are hybridized with capture probes, reporter probes and target DNAs by EDC-NHS coupling. The hybridized probe with target DNA gives off fluorescent signal due to the energy transfer from QD to Cy5 dye in the reporter probe. Compared to the conventional DNA sensor using DNA probes, the DNA sensor using PNA probes shows higher FRET factor and efficiency due to the higher reactivity between PNA and target DNA. In addition, to elicit the effect of the distance between the donor and the acceptor, we have investigated two types of the reporter probes having Cy5 dyes attached at the different positions of the reporter probes. Results show that the shorter the distance between QDs and Cy5s, the stronger the signal intensity. Furthermore, based on the fluorescence microscopy images using microcapillary chips, the FRET signal is enhanced to be up to 276% times stronger than the signal obtained using the cuvette by the fluorescence spectrometer. These results suggest that the PNA probe system conjugated with QDs can be used as ultrasensitive DNA nanosensors. PMID:27612755

  2. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  3. Detection of Damaged DNA Bases by DNA Glycosylase Enzymes†

    PubMed Central

    Friedman, Joshua I.; Stivers, James T.

    2010-01-01

    A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly-ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we refer to as the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome, and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location. PMID:20469926

  4. A liquid-crystal-based DNA biosensor for pathogen detection

    NASA Astrophysics Data System (ADS)

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-03-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection.

  5. A liquid-crystal-based DNA biosensor for pathogen detection.

    PubMed

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-01-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection. PMID:26940532

  6. A liquid-crystal-based DNA biosensor for pathogen detection

    PubMed Central

    Khan, Mashooq; Khan, Abdur Rahim; Shin, Jae-Ho; Park, Soo-Young

    2016-01-01

    A liquid-crystal (LC)-filled transmission electron microscopy (TEM) grid cell coated with the cationic surfactant dodecyltrimethylammonium bromide (DTAB), to which a single-stranded deoxyribonucleic acid probe (ssDNAprobe) was adsorbed at the LC/aqueous interface (TEMDTAB/DNA), was applied for the highly specific detection of target DNA molecules. The DTAB-coated E7 (used LC mixture) in the TEM grid (TEMDTAB) exhibited a homeotropic orientation, and changed to a planar orientation upon adsorption of the ssDNAprobe. The TEMDTAB/DNA was then exposed to complementary (target) ssDNA, which resulted in a planar-to-homeotropic configurational change of E7 that could be observed through a polarized optical microscope under crossed polarizers. The optimum adsorption density (2 μM) of ssDNAprobe enabled the detection of ≥0.05 nM complementary ssDNA. This TEMDTAB/DNA biosensor could differentiate complementary ssDNA from mismatched ssDNA as well as double-stranded DNA. It also successfully detected the genomic DNAs of the bacterium Erwinia carotovora and the fungi Rhazictonia solani. Owe to the high specificity, sensitivity, and label-free detection, this biosensor may broaden the applications of LC-based biosensors to pathogen detection. PMID:26940532

  7. Spherical Nucleic Acids: A New Form of DNA

    NASA Astrophysics Data System (ADS)

    Cutler, Joshua Isaac

    Spherical Nucleic Acids (SNAs) are a new class of nucleic acid-based nanomaterials that exhibit unique properties currently being explored in the contexts of gene-based cancer therapies and in the design of programmable nanoparticle-based materials. The properties of SNAs differ from canonical, linear nucleic acids by virtue of their dense packing into an oriented 3-dimensional array. SNAs can be synthesized from a number of useful nanoparticle templates, such as plasmonic gold and silver, magnetic oxides, luminescent semi-conductor quantum dots, and silica. In addition, by crosslinking the oligonucleotides and dissolving the core, they can be made in a hollow form as well. This dissertation describes the evolution of SNAs from initial studies of inorganic nanoparticle-based materials densely functionalized with oligonucleotides to the proving of a hypothesis that their unique properties can be observed in a core-less structure if the nucleic acids are densely packed and highly oriented. Chapter two describes the synthesis of densely functionalized polyvalent oligonucleotide superparamagnetic iron oxide nanoparticles using the copper-catalyzed azide-alkyne cycloaddition reaction. These particles are shown to exhibit cooperative binding in a density- and salt concentration-dependent fashion, with nearly identical behaviors to those of SNA-functionalized gold nanoparticles. Importantly, these particles are the first non-gold particles shown to be capable of entering cells in high numbers via the SNA-mediated cellular uptake pathway, and provided the first evidence that SNA-mediated cellular uptake is core-independent. In the third chapter, a gold nanoparticle catalyzed alkyne cross-linking reaction is described that is capable of forming hollow organic nanoparticles using polymers with alkyne-functionalized backbones. With this method, the alkyne-modified polymers adsorb to the particle surfaces, cross-link on the surface, allowing the gold nanoparticle to be

  8. Protective Effect of Folic Acid on Oxidative DNA Damage

    PubMed Central

    Guo, Xiaojuan; Cui, Huan; Zhang, Haiyang; Guan, Xiaoju; Zhang, Zheng; Jia, Chaonan; Wu, Jia; Yang, Hui; Qiu, Wenting; Zhang, Chuanwu; Yang, Zuopeng; Chen, Zhu; Mao, Guangyun

    2015-01-01

    Abstract Although previous reports have linked DNA damage with both transmissions across generations as well as our own survival, it is unknown how to reverse the lesion. Based on the data from a Randomized, Double-blind, Placebo Controlled Clinical Trial, this study aimed to assess the efficacy of folic acid supplementation (FAS) on DNA oxidative damage reversal. In this randomized clinical trial (RCT), a total of 450 participants were enrolled and randomly assigned to 3 groups to receive folic acid (FA) 0.4 mg/day (low-FA), 0.8 mg/day (high-FA), or placebo (control) for 8 weeks. The urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) and creatinine (Cr) concentration at pre- and post-FAS were measured with modified enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), respectively. A multivariate general linear model was applied to assess the individual effects of FAS and the joint effects between FAS and hypercholesterolemia on oxidative DNA damage improvement. This clinical trial was registered with ClinicalTrials.gov, number NCT02235948. Of the 438 subjects that received FA fortification or placebo, the median (first quartile, third quartile) of urinary 8-OHdG/Cr for placebo, low-FA, and high-FA groups were 58.19 (43.90, 82.26), 53.51 (38.97, 72.74), 54.73 (39.58, 76.63) ng/mg at baseline and 57.77 (44.35, 81.33), 51.73 (38.20, 71.30), and 50.65 (37.64, 76.17) ng/mg at the 56th day, respectively. A significant decrease of urinary 8-OHdG was observed after 56 days FA fortification (P < 0.001). Compared with the placebo, after adjusting for some potential confounding factors, including the baseline urinary 8-OHdG/Cr, the urinary 8-OHdG/Cr concentration significantly decreased after 56 days FAS [β (95% confidence interval) = −0.88 (−1.62, −0.14) and P = 0.020 for low-FA; and β (95% confidence interval) = −2.68 (−3.42, −1.94) and P < 0.001 for high-FA] in a dose-response fashion (Ptrend

  9. Method for sequencing DNA base pairs

    DOEpatents

    Sessler, A.M.; Dawson, J.

    1993-12-14

    The base pairs of a DNA structure are sequenced with the use of a scanning tunneling microscope (STM). The DNA structure is scanned by the STM probe tip, and, as it is being scanned, the DNA structure is separately subjected to a sequence of infrared radiation from four different sources, each source being selected to preferentially excite one of the four different bases in the DNA structure. Each particular base being scanned is subjected to such sequence of infrared radiation from the four different sources as that particular base is being scanned. The DNA structure as a whole is separately imaged for each subjection thereof to radiation from one only of each source. 6 figures.

  10. Unraveling Base Stacking Driving Forces in DNA.

    PubMed

    Mak, Chi H

    2016-07-01

    Base stacking is a key determinant of nucleic acid structures, but the precise origin of the thermodynamic driving force behind the stacking of nucleobases remains open. The rather mild stacking free energy measured experimentally, roughly a kcal/mol depending on the identity of the bases, is physiologically significant because while base stacking confers stability to the genome in its double helix form, the duplex also has to be unwound in order to be replicated or transcribed. A stacking free energy that is either too high or too low will over- or understabilize the genome, impacting the storage of genetic information and also its retrieval. While the molecular origin of stacking driving force has been attributed to many different sources including dispersion, electrostatics, and solvent hydrogen bonding, here we show via a systematic decomposition of the stacking free energy using large-scale computer simulations that the dominant driving force stabilizing base stacking is nonhydrophobic solvent entropy. Counteracting this is the conformational entropic penalty on the sugar-phosphate backbone against stacking, while solvent hydrogen-bonding, charge-charge interactions, and dispersive forces produce only secondary perturbations. Solvent entropic forces and DNA backbone conformational strains therefore work against each other, leading to a very mild composite stacking free energy in agreement with experiments. PMID:27045853

  11. Age dependency of base modification in rabbit liver DNA

    NASA Technical Reports Server (NTRS)

    Yamamoto, O.; Fuji, I.; Yoshida, T.; Cox, A. B.; Lett, J. T.

    1988-01-01

    Age-related modifications of DNA bases have been observed in the liver of the New Zealand white (NZW) rabbit (Oryctolagus cuniculus), a lagomorph with a median life span in captivity of 5-7 yr. The ages of the animals studied ranged from 6 wk to 9 yr. After the DNA had been extracted from the liver cell nuclei and hydrolyzed with acid, the bases were analyzed by column chromatography with Cellulofine gels (GC-15-m). Two peaks in the chromatogram, which eluted before the four DNA bases, contained modified bases. Those materials, which were obtained in relatively large amounts from old animals, were highly fluorescent, and were shown to be crosslinked base products by mass spectrometry. The yield of crosslinked products versus rabbit age (greater than 0.5 yr) can be fitted by an exponential function (correlation coefficient: 0.76 +/- 0.09).

  12. DNA as a Binary Code: How the Physical Structure of Nucleotide Bases Carries Information

    ERIC Educational Resources Information Center

    McCallister, Gary

    2005-01-01

    The DNA triplet code also functions as a binary code. Because double-ring compounds cannot bind to double-ring compounds in the DNA code, the sequence of bases classified simply as purines or pyrimidines can encode for smaller groups of possible amino acids. This is an intuitive approach to teaching the DNA code. (Contains 6 figures.)

  13. Magnetophoretic-based microfluidic device for DNA Concentration.

    PubMed

    Shim, Sangjo; Shim, Jiwook; Taylor, William R; Kosari, Farhad; Vasmatzis, George; Ahlquist, David A; Bashir, Rashid

    2016-04-01

    Nucleic acids serve as biomarkers of disease and it is highly desirable to develop approaches to extract small number of such genomic extracts from human bodily fluids. Magnetic particles-based nucleic acid extraction is widely used for concentration of small amount of samples and is followed by DNA amplification in specific assays. However, approaches to integrate such magnetic particles based capture with micro and nanofluidic based assays are still lacking. In this report, we demonstrate a magnetophoretic-based approach for target-specific DNA extraction and concentration within a microfluidic device. This device features a large chamber for reducing flow velocity and an array of μ-magnets for enhancing magnetic flux density. With this strategy, the device is able to collect up to 95 % of the magnetic particles from the fluidic flow and to concentrate these magnetic particles in a collection region. Then an enzymatic reaction is used to detach the DNA from the magnetic particles within the microfluidic device, making the DNA available for subsequent analysis. Concentrations of over 1000-fold for 90 bp dsDNA molecules is demonstrated. This strategy can bridge the gap between detection of low concentration analytes from clinical samples and a range of micro and nanofluidic sensors and devices including nanopores, nano-cantilevers, and nanowires. PMID:26899965

  14. Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

    PubMed

    Yao, Dongbao; Song, Tingjie; Sun, Xianbao; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures. PMID:26485090

  15. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    NASA Astrophysics Data System (ADS)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  16. Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

    NASA Astrophysics Data System (ADS)

    Chung, Da-Jung; Kim, Ki-Chul; Choi, Seong-Ho

    2011-09-01

    An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5'-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3', and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5'-TTC GAC CTC GGT TAG AAG ACT CAT-3' and two-base mismatched DNA, 5'-TTC GAC AGC GGT TAT AAG ACT CAT-3'.

  17. Base pairing and base mis-pairing in nucleic acids

    NASA Technical Reports Server (NTRS)

    Wang, A. H. J.; Rich, A.

    1986-01-01

    In recent years we have learned that DNA is conformationally active. It can exist in a number of different stable conformations including both right-handed and left-handed forms. Using single crystal X-ray diffraction analysis we are able to discover not only additional conformations of the nucleic acids but also different types of hydrogen bonded base-base interactions. Although Watson-Crick base pairings are the predominant type of interaction in double helical DNA, they are not the only types. Recently, we have been able to examine mismatching of guanine-thymine base pairs in left-handed Z-DNA at atomic resolution (1A). A minimum amount of distortion of the sugar phosphate backbone is found in the G x T pairing in which the bases are held together by two hydrogen bonds in the wobble pairing interaction. Because of the high resolution of the analysis we can visualize water molecules which fill in to accommodate the other hydrogen bonding positions in the bases which are not used in the base-base interactions. Studies on other DNA oligomers have revealed that other types of non-Watson-Crick hydrogen bonding interactions can occur. In the structure of a DNA octamer with the sequence d(GCGTACGC) complexed to an antibiotic triostin A, it was found that the two central AT base pairs are held together by Hoogsteen rather than Watson-Crick base pairs. Similarly, the G x C base pairs at the ends are also Hoogsteen rather than Watson-Crick pairing. Hoogsteen base pairs make a modified helix which is distinct from the Watson-Crick double helix.

  18. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  19. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  20. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  1. Associations between Serum Perfluoroalkyl Acids and LINE-1 DNA Methylation

    PubMed Central

    Watkins, Deborah J.; Wellenius, Gregory A.; Butler, Rondi A.; Bartell, Scott M.; Fletcher, Tony; Kelsey, Karl T.

    2014-01-01

    Perfluoroalkyl acids (PFAAs) are persistent, synthetic compounds that are used in a number of consumer products. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been associated with cardiovascular risk factors, and changes in gene expression and DNA methylation in animals and cellular systems. However, whether PFAA exposure is associated with LINE-1 DNA methylation, a potential marker of cardiovascular risk, in humans remains unknown. We sought to evaluate the cross-sectional associations between serum PFAAs and LINE-1 DNA methylation in a population highly exposed to PFOA. We measured serum PFAAs twice four to five years apart in 685 adult participants (47% male, mean age ± SD=42 ± 11 years). We measured percent LINE-1 DNA methylation in peripheral blood leukocytes at the second time point (follow-up), and estimated absolute differences in LINE-1 methylation associated with an interquartile (IQR) shift in mean PFAA serum levels. IQR increases in mean serum PFOA, PFOS, perfluorononanoic acid (PFNA), and perfluorohexane sulfonate (PFHxS) were associated with differences of −0.04 (p=0.16), 0.20 (p=0.001), 0.06 (p=0.19), and 0.02 (p=0.57), respectively, in % LINE-1 methylation at follow-up after adjustment for potential confounders. We observed a monotonic increase in LINE-1 DNA methylation across tertiles of PFOS and PFNA (ptrend=0.02 for both associations), but not across tertiles of PFOA or PFHxS (ptrend=0.71 and 0.44, respectively). In summary, serum PFOS was associated with LINE-1 methylation, while serum PFOA, PFHxS, and PFNA were not. Additional research is needed to more precisely determine whether these compounds are epigenetically active. PMID:24263140

  2. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  3. The DNA-based human karyotype.

    PubMed

    Mayall, B H; Carrano, A V; Moore, D H; Ashworth, L K; Bennett, D E; Mendelsohn, M L

    1984-07-01

    Image cytometry and computer analysis are used to determine the relative DNA content and the DNA-based centromeric index of the 24 chromosomes of the human karyotype. A two-step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q-band patterns. They then are stained for DNA using gallocyanin-chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA-based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA-based human karyotype. Analysis of the DNA-based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q- or C-band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined. PMID:6205836

  4. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    NASA Astrophysics Data System (ADS)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  5. Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase.

    PubMed Central

    Zyskind, J W; Deen, L T; Smith, D W

    1977-01-01

    Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of Escherichia coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas renitiation was not inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid ((RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initation of DNA replication (origin-RNA). Reinitiation at 30 degrees C was not inhibited by streptolydigin in a stretolydigin-sensitive dnaA muntant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 muntant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribounucleotide polymerization event wheareas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events that occur during initiation of a round of DNA replication, based on results in this and the accompanying paper. PMID:321429

  6. Arachidonic and oleic acid exert distinct effects on the DNA methylome.

    PubMed

    Silva-Martínez, Guillermo A; Rodríguez-Ríos, Dalia; Alvarado-Caudillo, Yolanda; Vaquero, Alejandro; Esteller, Manel; Carmona, F Javier; Moran, Sebastian; Nielsen, Finn C; Wickström-Lindholm, Marie; Wrobel, Katarzyna; Wrobel, Kazimierz; Barbosa-Sabanero, Gloria; Zaina, Silvio; Lund, Gertrud

    2016-05-01

    Abnormal fatty acid metabolism and availability are landmarks of metabolic diseases, which in turn are associated with aberrant DNA methylation profiles. To understand the role of fatty acids in disease epigenetics, we sought DNA methylation profiles specifically induced by arachidonic (AA) or oleic acid (OA) in cultured cells and compared those with published profiles of normal and diseased tissues. THP-1 monocytes were stimulated with AA or OA and analyzed using Infinium HumanMethylation450 BeadChip (Illumina) and Human Exon 1.0 ST array (Affymetrix). Data were corroborated in mouse embryonic fibroblasts. Comparisons with publicly available data were conducted by standard bioinformatics. AA and OA elicited a complex response marked by a general DNA hypermethylation and hypomethylation in the 1-200 μM range, respectively, with a maximal differential response at the 100 μM dose. The divergent response to AA and OA was prominent within the gene body of target genes, where it correlated positively with transcription. AA-induced DNA methylation profiles were similar to the corresponding profiles described for palmitic acid, atherosclerosis, diabetes, obesity, and autism, but relatively dissimilar from OA-induced profiles. Furthermore, human atherosclerosis grade-associated DNA methylation profiles were significantly enriched in AA-induced profiles. Biochemical evidence pointed to β-oxidation, PPAR-α, and sirtuin 1 as important mediators of AA-induced DNA methylation changes. In conclusion, AA and OA exert distinct effects on the DNA methylome. The observation that AA may contribute to shape the epigenome of important metabolic diseases, supports and expands current diet-based therapeutic and preventive efforts. PMID:27088456

  7. DNA Diagnostics: Nanotechnology-enhanced Electrochemical Detection of Nucleic Acids

    PubMed Central

    Wei, Fang; Lillehoj, Peter B.; Ho, Chih-Ming

    2010-01-01

    The detection of mismatched base pairs in DNA plays a crucial role in the diagnosis of genetic-related diseases and conditions, especially for early stage treatment. Among the various biosensors that have been employed for DNA detection, electrochemical sensors show great promise since they are capable of precise DNA recognition and efficient signal transduction. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed DNA detection for point-of-care (POC) clinical diagnostics. PMID:20075759

  8. DNA tetrahedron and star trigon nanostructures for target recycling detection of nucleic acid.

    PubMed

    Li, Yueran; Chen, Xifeng; Wang, Bidou; Liu, Guangxing; Tang, Yuguo; Miao, Peng

    2016-06-01

    Human immunodeficiency virus (HIV) is a retrovirus which attacks the human body's immune system and further leads to acquired immunodeficiency syndrome (AIDS). Nucleic acid detection is of great importance in the medical diagnosis of such diseases. Herein, we develop a simple and enzyme-free electrochemical method for the target recycling detection of nuclei acid. DNA tetrahedron and star trigon nanostructures are designed and constructed on the electrode interface for target capture and signal enrichment. This strategy is convenient and sensitive, with a limit of detection as low as 1 fM, and can also successfully distinguish single-base mismatched DNA. Therefore, the proposed method has a promising potential application for HIV DNA detection. PMID:27170090

  9. Formulation of a Peptide Nucleic Acid Based Nucleic Acid Delivery Construct

    PubMed Central

    Millili, Peter G.; Yin, Daniel H.; Fan, Haihong; Naik, Ulhas P.; Sullivan, Millicent O.

    2010-01-01

    Gene delivery biomaterials need to be designed to efficiently achieve nuclear delivery of plasmid DNA. Polycations have been used to package DNA and other nucleic acids within sub-micron sized particles, offering protection from shear-induced or enzymatic degradation. However, cytotoxicity issues coupled with limited in vivo transfection efficiencies minimize the effectiveness of this approach. In an effort to improve upon existing technologies aimed at delivering nucleic acids, an alternative approach to DNA packaging was explored. Peptide nucleic acids (PNAs) were used to directly functionalize DNA with poly(ethylene glycol) (PEG) chains that provide a steric layer and inhibit multimolecular aggregation during complexation. DNA prePEGylation by this strategy was predicted to enable the formation of more homogeneous and efficiently packaged polyplexes. In this work, DNA-PNA-peptide-PEG (DP3) conjugates were synthesized and self-assembled with 25 kDa poly(ethylenimine) (PEI). Complexes with small standard deviations and average diameters ranging from 30 – 50 nm were created, with minimal dependence of complex size on N:P ratio (PEI amines to DNA phosphates). Furthermore, PEI-DNA interactions were altered by the derivitization strategy, resulting in tighter compaction of the PEI-DP3 complexes in comparison with PEI-DNA complexes. Transfection experiments in Chinese Hamster Ovary (CHO) cells revealed comparable transfection efficiencies but reduced cytotoxicities of the PEI-DP3 complexes relative to PEI-DNA complexes. The enhanced cellular activities of the PEI-DP3 complexes were maintained following the removal of free PEI from the PEI-DP3 formulations, whereas the cellular activity of the conventional PEI-DNA formulations was reduced by free PEI removal. These findings suggest that DNA prePEGylation by the PNA-based strategy might provide a way to circumvent cytotoxicity and formulation issues related to the use of PEI for in vivo gene delivery. PMID:20131756

  10. Interaction of photosensitive surfactant with DNA and poly acrylic acid.

    PubMed

    Zakrevskyy, Yuriy; Cywinski, Piotr; Cywinska, Magdalena; Paasche, Jens; Lomadze, Nino; Reich, Oliver; Löhmannsröben, Hans-Gerd; Santer, Svetlana

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes' properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate - for the first time - complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules. PMID:25669583

  11. Interaction of photosensitive surfactant with DNA and poly acrylic acid

    SciTech Connect

    Zakrevskyy, Yuriy Paasche, Jens; Lomadze, Nino; Santer, Svetlana; Cywinski, Piotr; Cywinska, Magdalena; Reich, Oliver; Löhmannsröben, Hans-Gerd

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes’ properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate – for the first time – complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules.

  12. Molten fatty acid based microemulsions.

    PubMed

    Noirjean, Cecile; Testard, Fabienne; Dejugnat, Christophe; Jestin, Jacques; Carriere, David

    2016-06-21

    We show that ternary mixtures of water (polar phase), myristic acid (MA, apolar phase) and cetyltrimethylammonium bromide (CTAB, cationic surfactant) studied above the melting point of myristic acid allow the preparation of microemulsions without adding a salt or a co-surfactant. The combination of SANS, SAXS/WAXS, DSC, and phase diagram determination allows a complete characterization of the structures and interactions between components in the molten fatty acid based microemulsions. For the different structures characterized (microemulsion, lamellar or hexagonal phases), a similar thermal behaviour is observed for all ternary MA/CTAB/water monophasic samples and for binary MA/CTAB mixtures without water: crystalline myristic acid melts at 52 °C, and a thermal transition at 70 °C is assigned to the breaking of hydrogen bounds inside the mixed myristic acid/CTAB complex (being the surfactant film in the ternary system). Water determines the film curvature, hence the structures observed at high temperature, but does not influence the thermal behaviour of the ternary system. Myristic acid is partitioned in two "species" that behave independently: pure myristic acid and myristic acid associated with CTAB to form an equimolar complex that plays the role of the surfactant film. We therefore show that myristic acid plays the role of a solvent (oil) and a co-surfactant allowing the fine tuning of the structure of oil and water mixtures. This solvosurfactant behaviour of long chain fatty acid opens the way for new formulations with a complex structure without the addition of any extra compound. PMID:27241163

  13. Intelligent DNA machine for the ultrasensitive colorimetric detection of nucleic acids.

    PubMed

    Xu, Jianguo; Qian, Jun; Li, Hongling; Wu, Zai-Sheng; Shen, Weiyu; Jia, Lee

    2016-01-15

    As DNA is employed to serve as a smart building block, an increasing interest has been devoted to the development of different DNA-based machines for the specific purpose, for example, the exploration of inter- or intramolecular interaction. In the current contribution, we developed an intelligent DNA machine and its operation can be designed to execute the ultrasensitive colorimetric detection of target nucleic acids. The DNA machine consists of a hairpin probe (HP) and an assistant template (AT). Using p53 gene as the target model to trigger the molecular machine operation, cyclic nucleic acid strand displacement polymerization (CNDP) was specifically induced, leading to the DNAzyme mediated catalytic reaction for signal readout. Specifically, with the help of polymerase and nickase, one target molecule was able to drive DNA nano-mechanical devices one-by-one through the hybridization/polymerization displacement cycles, and every initiated machine continued to operate, causing the dramatic accumulation of G-quadruplex-contained products. The G-quadruplex structure after binding to hemin could act as a horseradish peroxidase (HRP)-mimicking DNAzyme and catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by H2O2. As a result, an enhanced color change could be detected because of the generation of oxidation product ABTS•(+). In this way, the DNA machine has no any signal loss and enables the quantitative measurement of p53 DNA with a detection limit of 10fM, indicating great promise for unique application in biomedical research and early clinical diagnosis. PMID:26291961

  14. Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase.

    PubMed

    Nabel, Christopher S; Lee, Jae W; Wang, Laura C; Kohli, Rahul M

    2013-08-27

    Activation-induced deaminase (AID), a member of the larger AID/APOBEC family, is the key catalyst in initiating antibody somatic hypermutation and class-switch recombination. The DNA deamination model accounting for AID's functional role posits that AID deaminates genomic deoxycytosine bases within the immunoglobulin locus, activating downstream repair pathways that result in antibody maturation. Although this model is well supported, the molecular basis for AID's selectivity for DNA over RNA remains an open and pressing question, reflecting a broader need to elucidate how AID/APOBEC enzymes engage their substrates. To address these questions, we have synthesized a series of chimeric nucleic acid substrates and characterized their reactivity with AID. These chimeric substrates feature targeted variations at the 2'-position of nucleotide sugars, allowing us to interrogate the steric and conformational basis for nucleic acid selectivity. We demonstrate that modifications to the target nucleotide can significantly alter AID's reactivity. Strikingly, within a substrate that is otherwise DNA, a single RNA-like 2'-hydroxyl substitution at the target cytosine is sufficient to compromise deamination. Alternatively, modifications that favor a DNA-like conformation (or sugar pucker) are compatible with deamination. AID's closely related homolog APOBEC1 is similarly sensitive to RNA-like substitutions at the target cytosine. Inversely, with unreactive 2'-fluoro-RNA substrates, AID's deaminase activity was rescued by introducing a trinucleotide DNA patch spanning the target cytosine and two nucleotides upstream. These data suggest a role for nucleotide sugar pucker in explaining the molecular basis for AID's DNA selectivity and, more generally, suggest how other nucleic acid-modifying enzymes may distinguish DNA from RNA. PMID:23942124

  15. One-stop Genomic DNA Extraction by Salicylic Acid Coated Magnetic Nanoparticles

    PubMed Central

    Zhou, Zhongwu; Kadam, Ulhas; Irudayaraj, Joseph

    2014-01-01

    Salicylic acid coated magnetic nanoparticles were prepared via a modified, one-step synthesis and used for a one-stop extraction of genomic DNA from mammalian cells. The synthesized magnetic particles were used for magnetic separation of cells from the media by non-specific binding of the particles, as well as extraction of genomic DNA from the lysate. The quantity and quality were confirmed by agarose gel electrophoresis and polymerase chain reaction. The entire process of extraction and isolation can be completed within 30 min. Compared to traditional methods based on centrifugation and filtration, the established method is fast, simple, reliable, and environmentally-friendly. PMID:23911528

  16. Power of surface-based DNA computation

    SciTech Connect

    Cai, Weiping; Condon, A.E.; Corn, R.M.

    1997-12-01

    A new model of DNA computation that is based on surface chemistry is studied. Such computations involve the manipulation of DNA strands that are immobilized on a surface, rather than in solution as in the work of Adleman. Surface-based chemistry has been a critical technology in many recent advances in biochemistry and offers several advantages over solution-based chemistry, including simplified handling of samples and elimination of loss of strands, which reduce error in the computation. The main contribution of this paper is in showing that in principle, surface-based DNA chemistry can efficiently support general circuit computation on many inputs in parallel. To do this, an abstract model of computation that allows parallel manipulation of binary inputs is described. It is then shown that this model can be implemented by encoding inputs as DNA strands and repeatedly modifying the strands in parallel on a surface, using the chemical processes of hybridization, exonuclease degradation, polymerase extension, and ligation. Thirdly, it is shown that the model supports efficient circuit simulation in the following sense: exactly those inputs that satisfy a circuit can be isolated and the number of parallel operations needed to do this is proportional to the size of the circuit. Finally, results are presented on the power of the model when another resource of DNA computation is limited, namely strand length. 12 refs.

  17. Duplex-Selective Ruthenium-based DNA Intercalators

    PubMed Central

    Shade, Chad M.; Kennedy, Robert D.; Rouge, Jessica L.; Rosen, Mari S.; Wang, Mary X.; Seo, Soyoung E.; Clingerman, Daniel J.

    2016-01-01

    We report the design and synthesis of small molecules that exhibit enhanced luminescence in the presence of duplex rather than single-stranded DNA. The local environment presented by a well-known [Ru(dipyrido[2,3-a:3',2'-c]phenazine)L2]2+-based DNA intercalator was modified by functionalizing the bipyridine ligands with esters and carboxylic acids. By systematically varying the number and charge of the pendant groups, it was determined that decreasing the electrostatic interaction between the intercalator and the anionic DNA backbone reduced single-strand interactions and translated to better duplex specificity. In studying this class of complexes, a single RuII complex emerged that selectively luminesces in the presence of duplex DNA with little to no background from interacting with single stranded DNA. This complex shows promise as a new dye capable of selectively staining double versus single-stranded DNA in gel electrophoresis, which cannot be done with conventional SYBR dyes. PMID:26119581

  18. Multiphoton excitation of fluorescent DNA base analogs

    NASA Astrophysics Data System (ADS)

    Katilius, Evaldas; Woodbury, Neal W.

    2006-07-01

    Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5×10-50 cm4s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

  19. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results. PMID:26581621

  20. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products. PMID:26556214

  1. Boronic Acid-modified DNA that Changes Fluorescent Properties upon Carbohydrate Binding†

    PubMed Central

    Yang, Xiaochuan; Dai, Chaofeng; Molina, Angie Dayan Calderon

    2010-01-01

    A long wavelength boronic acid-modified TTP (NB-TTP) has been synthesized and enzymatically incorporated into DNA. Such DNA shows intrinsic fluorescent changes upon carbohydrate addition. PMID:20126717

  2. DNA nanotechnology based on i-motif structures.

    PubMed

    Dong, Yuanchen; Yang, Zhongqiang; Liu, Dongsheng

    2014-06-17

    CONSPECTUS: Most biological processes happen at the nanometer scale, and understanding the energy transformations and material transportation mechanisms within living organisms has proved challenging. To better understand the secrets of life, researchers have investigated artificial molecular motors and devices over the past decade because such systems can mimic certain biological processes. DNA nanotechnology based on i-motif structures is one system that has played an important role in these investigations. In this Account, we summarize recent advances in functional DNA nanotechnology based on i-motif structures. The i-motif is a DNA quadruplex that occurs as four stretches of cytosine repeat sequences form C·CH(+) base pairs, and their stabilization requires slightly acidic conditions. This unique property has produced the first DNA molecular motor driven by pH changes. The motor is reliable, and studies show that it is capable of millisecond running speeds, comparable to the speed of natural protein motors. With careful design, the output of these types of motors was combined to drive micrometer-sized cantilevers bend. Using established DNA nanostructure assembly and functionalization methods, researchers can easily integrate the motor within other DNA assembled structures and functional units, producing DNA molecular devices with new functions such as suprahydrophobic/suprahydrophilic smart surfaces that switch, intelligent nanopores triggered by pH changes, molecular logic gates, and DNA nanosprings. Recently, researchers have produced motors driven by light and electricity, which have allowed DNA motors to be integrated within silicon-based nanodevices. Moreover, some devices based on i-motif structures have proven useful for investigating processes within living cells. The pH-responsiveness of the i-motif structure also provides a way to control the stepwise assembly of DNA nanostructures. In addition, because of the stability of the i-motif, this

  3. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  4. Optical detection of PNA/DNA hybridization in resonant porous silicon-based devices

    NASA Astrophysics Data System (ADS)

    Rotiroti, Lucia; Arcari, Paolo; Lamberti, Annalisa; Sanges, Carmen; De Tommasi, Edoardo; Rea, Ilaria; Rendina, Ivo; De Stefano, Luca

    2008-04-01

    The development of label-free optical biosensors could have a great impact on life sciences as well as on screening techniques for medical and environmental applications. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone, resulting in an achiral and uncharged mimic. Due to the uncharged nature of PNA, PNA-DNA duplexes show a better thermal stability respect the DNA-DNA equivalents. In this work, we used an optical biosensor, based on the porous silicon (PSi) nanotechnology, to detect PNA-DNA interactions. PSi optical sensors are based on changes of reflectivity spectrum when they are exposed to the target analytes. The porous silicon surface was chemically modified to covalently link the PNA which acts as a very specific probe for its ligand (cDNA).

  5. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  6. An overhang-based DNA block shuffling method for creating a customized random library

    PubMed Central

    Fujishima, Kosuke; Venter, Chris; Wang, Kendrick; Ferreira, Raphael; Rothschild, Lynn J.

    2015-01-01

    We present an overhang-based DNA block shuffling method to create a customized random DNA library with flexible sequence design and length. Our method enables the efficient and seamless assembly of short DNA blocks with dinucleotide overhangs through a simple ligation process. Next generation sequencing analysis of the assembled DNA library revealed that ligation was accurate, directional and unbiased. This straightforward DNA assembly method should fulfill the versatile needs of both in vivo and in vitro functional screening of random peptides and RNA created with a desired amino acid and nucleotide composition, as well as making highly repetitive gene constructs that are difficult to synthesize de novo. PMID:26010273

  7. PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

    PubMed

    Gonzalez-Hunt, Claudia P; Rooney, John P; Ryde, Ian T; Anbalagan, Charumathi; Joglekar, Rashmi; Meyer, Joel N

    2016-01-01

    Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  8. PCR-based analysis of mitochondrial DNA copy number, mitochondrial DNA damage, and nuclear DNA damage

    PubMed Central

    Gonzalez-Hunt, Claudia P.; Rooney, John P.; Ryde, Ian T.; Anbalagan, Charumathi; Joglekar, Rashmi

    2016-01-01

    Because of the role DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays. PMID:26828332

  9. Biosensors based on DNA-Functionalized Graphene

    NASA Astrophysics Data System (ADS)

    Vishnubhotla, Ramya; Ping, Jinglei; Vrudhula, Amey; Johnson, A. T. Charlie

    Since its discovery, graphene has been used for sensing applications due to its outstanding electrical properties and biocompatibility. Here, we demonstrate the capabilities of field effect transistors (FETs) based on CVD-grown graphene functionalized with commercially obtained DNA oligomers and aptamers for detection of various biomolecular targets (e.g., complementary DNA and small molecule drug targets). Graphene FETs were created with a scalable photolithography process that produces arrays consisting of 50-100 FETs with a layout suitable for multiplexed detection of four molecular targets. FETs were characterized via AFM to confirm the presence of the aptamer. From the measured electrical characteristics, it was determined that binding of molecular targets by the DNA chemical recognition element led to a reproducible, concentration-dependent shift in the Dirac voltage. This biosensor class is potentially suitable for applications in drug detection. This work is funded by NIH through the Center for AIDS Research at the University of Pennsylvania.

  10. DNA-Based Nanostructures: Changes of Mechanical Properties of DNA upon Ligand Binding

    NASA Astrophysics Data System (ADS)

    Nechipurenko, Yury; Grokhovsky, Sergey; Gursky, Georgy; Nechipurenko, Dmitry; Polozov, Robert

    The formation of DNA-based nanostructures involves the binding of different kinds of ligands to DNA as well as the interaction of DNA molecules with each other. Complex formation between ligand and DNA can alter physicochemical properties of the DNA molecule. In the present work, the accessibility of DNA-ligand complexes to cleavage by DNase I are considered, and the exact algorithms for analysis of diagrams of DNase I footprinting for ligand-DNA complexes are obtained. Changes of mechanical properties of the DNA upon ligand binding are also demonstrated by the cleavage patterns generated upon ultrasound irradiation of cis-platin-DNA complexes. Propagation of the mechanical perturbations along DNA in the presence of bound ligands is considered in terms of a string model with a heterogeneity corresponding to the position of a bound ligand on DNA. This model can reproduce qualitatively the cleavage patterns obtained upon ultrasound irradiation of cis-platin-DNA complexes.

  11. Luminescent DNA- and agar-based membranes.

    PubMed

    Leones, R; Fernandes, M; Ferreira, R A S; Cesarino, I; Lima, J F; Carlos, L D; Bermudez, V de Zea; Magon, C J; Donoso, J P; Silva, M M; Pawlicka, A

    2014-09-01

    Luminescent materials containing europium ions are investigated for different optical applications. They can be obtained using bio-macromolecules, which are promising alternatives to synthetic polymers based on the decreasing oil resources. This paper describes studies of the DNA- and Agar-europium triflate luminescent membranes and its potential technological applications are expanded to electroluminescent devices. Polarized optical microscopy demonstrated that the samples are birefringent with submicrometer anisotropy. The X-ray diffraction analysis revealed predominantly amorphous nature of the samples and the atomic force microscopy images showed a roughness of the membranes of 409.0 and 136.1 nm for the samples of DNA10Eu and Agar1.11Eu, respectively. The electron paramagnetic resonance spectra of the DNA(n)Eu membranes with the principal lines at g ≈ 2.0 and g ≈ 4.8 confirmed uniform distribution of rare earth ions in a disordered matrix. Moreover, these strong and narrow resonance lines for the samples of DNA(n)Eu when compared to the Agar(n)Eu suggested a presence of paramagnetic radicals arising from the DNA matrix. The emission spectra suggested that the Eu3+ ions occupy a single local environment in both matrices and the excitation spectra monitored around the Eu emission lines pointed out that the Eu3+ ions in the Agar host were mainly excited via the broad band component rather than by direct intra-4f(6) excitation, whereas the opposite case occurred for the DNA-based sample. PMID:25924317

  12. All-atom polarizable force field for DNA based on the classical Drude oscillator model.

    PubMed

    Savelyev, Alexey; MacKerell, Alexander D

    2014-06-15

    Presented is a first generation atomistic force field (FF) for DNA in which electronic polarization is modeled based on the classical Drude oscillator formalism. The DNA model is based on parameters for small molecules representative of nucleic acids, including alkanes, ethers, dimethylphosphate, and the nucleic acid bases and empirical adjustment of key dihedral parameters associated with the phosphodiester backbone, glycosidic linkages, and sugar moiety of DNA. Our optimization strategy is based on achieving a compromise between satisfying the properties of the underlying model compounds in the gas phase targeting quantum mechanical (QM) data and reproducing a number of experimental properties of DNA duplexes in the condensed phase. The resulting Drude FF yields stable DNA duplexes on the 100-ns time scale and satisfactorily reproduce (1) the equilibrium between A and B forms of DNA and (2) transitions between the BI and BII substates of B form DNA. Consistency with the gas phase QM data for the model compounds is significantly better for the Drude model as compared to the CHARMM36 additive FF, which is suggested to be due to the improved response of the model to changes in the environment associated with the explicit inclusion of polarizability. Analysis of dipole moments associated with the nucleic acid bases shows the Drude model to have significantly larger values than those present in CHARMM36, with the dipoles of individual bases undergoing significant variations during the MD simulations. Additionally, the dipole moment of water was observed to be perturbed in the grooves of DNA. PMID:24752978

  13. All-Atom Polarizable Force Field for DNA Based on the Classical Drude Oscillator Model

    PubMed Central

    Savelyev, Alexey; MacKerell, Alexander D.

    2014-01-01

    Presented is a first generation atomistic force field for DNA in which electronic polarization is modeled based on the classical Drude oscillator formalism. The DNA model is based on parameters for small molecules representative of nucleic acids, including alkanes, ethers, dimethylphosphate, and the nucleic acid bases and empirical adjustment of key dihedral parameters associated with the phosphodiester backbone, glycosidic linkages and sugar moiety of DNA. Our optimization strategy is based on achieving a compromise between satisfying the properties of the underlying model compounds in the gas phase targeting QM data and reproducing a number of experimental properties of DNA duplexes in the condensed phase. The resulting Drude force field yields stable DNA duplexes on the 100 ns time scale and satisfactorily reproduces (1) the equilibrium between A and B forms of DNA and (2) transitions between the BI and BII sub-states of B form DNA. Consistency with the gas phase QM data for the model compounds is significantly better for the Drude model as compared to the CHARMM36 additive force field, which is suggested to be due to the improved response of the model to changes in the environment associated with the explicit inclusion of polarizability. Analysis of dipole moments associated with the nucleic acid bases shows the Drude model to have significantly larger values than those present in CHARMM36, with the dipoles of individual bases undergoing significant variations during the MD simulations. Additionally, the dipole moment of water was observed to be perturbed in the grooves of DNA. PMID:24752978

  14. Escherichia coli DnaB Helicase–DnaC Protein Complex: Allosteric Effects of the Nucleotides on the Nucleic Acid Binding and the Kinetic Mechanism of NTP Hydrolysis. 3†

    PubMed Central

    Roychowdhury, Anasuya; Szymanski, Michal R.; Jezewska, Maria J.; Bujalowski, Wlodzimierz

    2011-01-01

    Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB–DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB–DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the antagonistic allosteric effect of NTP and NDP on the ssDNA affinity of the enzyme. The protein changes the engagement of the DNA-binding subsites of the helicase in interactions with the nucleic acid, depending on the structure of the phosphate group of the present nucleotide cofactor and profoundly affects the structure of the bound DNA. Moreover, the ssDNA affinity of the helicase in the DnaB–DnaC complex is under the control of the nucleotide-binding site of the DnaC protein. The protein does not affect the NTP hydrolysis mechanism of the helicase. Nevertheless, the rate of the chemical step is diminished in the DnaB–DnaC complex. In the tertiary DnaB–DnaC–ssDNA complex, the ssDNA changes the internal dynamics between intermediates of the pyrimidine cofactor, in a manner independent of the base composition of the DNA, while the hydrolysis step of the purine cofactor is specifically stimulated by the homoadenosine ssDNA. The significance of these results for functional activities of the DnaB–DnaC complex is discussed. PMID:19432487

  15. Communication: Electron ionization of DNA bases.

    PubMed

    Rahman, M A; Krishnakumar, E

    2016-04-28

    No reliable experimental data exist for the partial and total electron ionization cross sections for DNA bases, which are very crucial for modeling radiation damage in genetic material of living cell. We have measured a complete set of absolute partial electron ionization cross sections up to 500 eV for DNA bases for the first time by using the relative flow technique. These partial cross sections are summed to obtain total ion cross sections for all the four bases and are compared with the existing theoretical calculations and the only set of measured absolute cross sections. Our measurements clearly resolve the existing discrepancy between the theoretical and experimental results, thereby providing for the first time reliable numbers for partial and total ion cross sections for these molecules. The results on fragmentation analysis of adenine supports the theory of its formation in space. PMID:27131520

  16. Communication: Electron ionization of DNA bases

    NASA Astrophysics Data System (ADS)

    Rahman, M. A.; Krishnakumar, E.

    2016-04-01

    No reliable experimental data exist for the partial and total electron ionization cross sections for DNA bases, which are very crucial for modeling radiation damage in genetic material of living cell. We have measured a complete set of absolute partial electron ionization cross sections up to 500 eV for DNA bases for the first time by using the relative flow technique. These partial cross sections are summed to obtain total ion cross sections for all the four bases and are compared with the existing theoretical calculations and the only set of measured absolute cross sections. Our measurements clearly resolve the existing discrepancy between the theoretical and experimental results, thereby providing for the first time reliable numbers for partial and total ion cross sections for these molecules. The results on fragmentation analysis of adenine supports the theory of its formation in space.

  17. Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S-23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene.

    PubMed

    Trcek, Janja

    2005-10-01

    Acetic acid bacteria (AAB) are well known for oxidizing different ethanol-containing substrates into various types of vinegar. They are also used for production of some biotechnologically important products, such as sorbose and gluconic acids. However, their presence is not always appreciated since certain species also spoil wine, juice, beer and fruits. To be able to follow AAB in all these processes, the species involved must be identified accurately and quickly. Because of inaccuracy and very time-consuming phenotypic analysis of AAB, the application of molecular methods is necessary. Since the pairwise comparison among the 16S rRNA gene sequences of AAB shows very high similarity (up to 99.9%) other DNA-targets should be used. Our previous studies showed that the restriction analysis of 16S-23S rDNA internal transcribed spacer region is a suitable approach for quick affiliation of an acetic acid bacterium to a distinct group of restriction types and also for quick identification of a potentially novel species of acetic acid bacterium (Trcek & Teuber 2002; Trcek 2002). However, with the exception of two conserved genes, encoding tRNAIle and tRNAAla, the sequences of 16S-23S rDNA are highly divergent among AAB species. For this reason we analyzed in this study a gene encoding PQQ-dependent ADH as a possible DNA-target. First we confirmed the expression of subunit I of PQQ-dependent ADH (AdhA) also in Asaia, the only genus of AAB which exhibits little or no ADH-activity. Further we analyzed the partial sequences of adhA among some representative species of the genera Acetobacter, Gluconobacter and Gluconacetobacter. The conserved and variable regions in these sequences made possible the construction of A. acetispecific oligonucleotide the specificity of which was confirmed in PCR-reaction using 45 well-defined strains of AAB as DNA-templates. The primer was also successfully used in direct identification of A. aceti from home made cider vinegar as well as for

  18. Chaos-based image encryption using a hybrid genetic algorithm and a DNA sequence

    NASA Astrophysics Data System (ADS)

    Enayatifar, Rasul; Abdullah, Abdul Hanan; Isnin, Ismail Fauzi

    2014-05-01

    The paper studies a recently developed evolutionary-based image encryption algorithm. A novel image encryption algorithm based on a hybrid model of deoxyribonucleic acid (DNA) masking, a genetic algorithm (GA) and a logistic map is proposed. This study uses DNA and logistic map functions to create the number of initial DNA masks and applies GA to determine the best mask for encryption. The significant advantage of this approach is improving the quality of DNA masks to obtain the best mask that is compatible with plain images. The experimental results and computer simulations both confirm that the proposed scheme not only demonstrates excellent encryption but also resists various typical attacks.

  19. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  20. Direct Detection and Sequencing of Damaged DNA Bases

    PubMed Central

    2011-01-01

    Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

  1. Simulation and analysis of an evolutionary model of deoxyribonucleic acid (DNA). Master's thesis

    SciTech Connect

    McNally, R.E.

    1983-09-01

    A Monte Carlo simulation model was developed in order to evaluate model predictions with expectations of the evolutionary hypothesis of nearly neutral point mutations. The beta chain of hemoglobin was chosen as the strand of deoxyribonucleic acid (DNA) to be analyzed due to the extensive characterization of point mutations along the 146 amino acids of the protein chain. The nucleotide sequences of human, rabbit and a hypothetical ancestral hemoglobin were used as a starting point in the simulation. Three models of point mutations were tested. Equiprobable mutation from one nucleotide to any of the remaining three nucleotides composing DNA was one model. The second model incorporated observed first order probability of transition from each nucleotide to the remaining three nucleotides composing DNA using observed probabilities from three independent assessments. The third model was an Ising type model employing a probability of nucleotide change based on the nucleotide composition of the nearest neighbors. Use of these models resulted in evidence to suggest that five methods of simulating the mutations in an evolutionary system produced results that primarily differed in the way in which nulceotide changes resulted in a pattern of amino acid changes.

  2. Relationship inference based on DNA mixtures.

    PubMed

    Kaur, Navreet; Bouzga, Mariam M; Dørum, Guro; Egeland, Thore

    2016-03-01

    Today, there exists a number of tools for solving kinship cases. But what happens when information comes from a mixture? DNA mixtures are in general rarely seen in kinship cases, but in a case presented to the Norwegian Institute of Public Health, sample DNA was obtained after a rape case that resulted in an unwanted pregnancy and abortion. The only available DNA from the fetus came in form of a mixture with the mother, and it was of interest to find the father of the fetus. The mother (the victim), however, refused to give her reference data and so commonly used methods for paternity testing were no longer applicable. As this case illustrates, kinship cases involving mixtures and missing reference profiles do occur and make the use of existing methods rather inconvenient. We here present statistical methods that may handle general relationship inference based on DNA mixtures. The basic idea is that likelihood calculations for mixtures can be decomposed into a series of kinship problems. This formulation of the problem facilitates the use of kinship software. We present the freely available R package relMix which extends on the R version of Familias. Complicating factors like mutations, silent alleles, and θ-correction are then easily handled for quite general family relationships, and are included in the statistical methods we develop in this paper. The methods and their implementations are exemplified on the data from the rape case. PMID:26541994

  3. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    ABSTRACT

    Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  4. Use of an Acid-Base Table.

    ERIC Educational Resources Information Center

    Willis, Grover; And Others

    1986-01-01

    Identifies several ways in which an acid-base table can provide students with information about chemical reactions. Cites examples of the chart's use and includes a table which indicates the strengths of some common acids and bases. (ML)

  5. The Acid-Base Titration of a Very Weak Acid: Boric Acid

    ERIC Educational Resources Information Center

    Celeste, M.; Azevedo, C.; Cavaleiro, Ana M. V.

    2012-01-01

    A laboratory experiment based on the titration of boric acid with strong base in the presence of d-mannitol is described. Boric acid is a very weak acid and direct titration with NaOH is not possible. An auxiliary reagent that contributes to the release of protons in a known stoichiometry facilitates the acid-base titration. Students obtain the…

  6. Excess Electron Localization in Solvated DNA Bases

    SciTech Connect

    Smyth, Maeve; Kohanoff, Jorge

    2011-06-10

    We present a first-principles molecular dynamics study of an excess electron in condensed phase models of solvated DNA bases. Calculations on increasingly large microsolvated clusters taken from liquid phase simulations show that adiabatic electron affinities increase systematically upon solvation, as for optimized gas-phase geometries. Dynamical simulations after vertical attachment indicate that the excess electron, which is initially found delocalized, localizes around the nucleobases within a 15 fs time scale. This transition requires small rearrangements in the geometry of the bases.

  7. An Electrochemical DNA Microbiosensor Based on Succinimide-Modified Acrylic Microspheres

    PubMed Central

    Ulianas, Alizar; Heng, Lee Yook; Hanifah, Sharina Abu; Ling, Tan Ling

    2012-01-01

    An electrochemical microbiosensor for DNA has been fabricated based on new acrylic microspheres modified with reactive N-acryloxysuccinimide (NAS) functional groups. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized in an emulsion form with a simple one-step photopolymerization technique. Aminated DNA probe was attached to the succinimde functional group of the acrylic microspheres via covalent bonding. The hybridization of the immobilized DNA probe with the complementary DNA was studied by differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, pH, type of ions, buffer concentrations, ionic strength, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a linear response range to target DNA over a wide concentration range of 1.0 × 10−16 and 1.0 × 10−8 M with a lower limit of detection (LOD) of 9.46 × 10−17 M (R2 = 0.97). This DNA microbiosensor showed good reproducibility with 2.84% RSD (relative standard deviation) (n = 3). Application of the NAS-modified acrylic microspheres in the construction of DNA microbiosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices. PMID:22778594

  8. Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

    NASA Astrophysics Data System (ADS)

    Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

    2014-01-01

    DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

  9. Chiral separation of amino acids in ultrafiltration through DNA-immobilized cellulose membranes

    NASA Astrophysics Data System (ADS)

    Higuchi, Akon; Hayashi, Akiyuki; Kanda, Naoki; Sanui, Kohei; Kitamura, Hanako

    2005-04-01

    Ultrafiltration experiments for the chiral separation of racemic tryptophan, phenylglycine and phenylalanine were investigated through immobilized DNA membranes having various pore sizes. L-tryptophan preferentially permeated through immobilized DNA membranes with a pore size<2.0 nm (molecular weight cut-off (MWCO)<5000) while D-tryptophan preferentially permeated through immobilized DNA membranes with a pore size>2.0 nm (MWCO>5000). These results are completely opposite tendency in the ultrafiltration of racemic phenylalanine through the immobilized DNA membranes. This may be originated from the different interaction between DNA and tryptophan compared to that between DNA and phenylalanine. However, in both cases the pore size of the immobilized DNA membranes regulated preferential permeation of the enantiomer through the membranes. The immobilized DNA membranes are categorized as channel type membranes and not as affinity membranes. Chiral separation models were proposed from using the chiral separation results of racemic amino acids, preferential adsorption of amino acid enantiomers and EPMA results.

  10. Radiation-induced degradation of DNA bases

    NASA Astrophysics Data System (ADS)

    Douki, T.; Delatour, T.; Martini, R.; Cadet, J.

    1999-01-01

    Radio-induced degradation of DNA involves radical processes. A series of lesions among the major bases degradation products has been measured in isolated DNA exposed to gamma radiation in aerated aqueous solution. Degradation can be accounted for by the formation of hydroxyl radicals upon radiolysis of water (indirect effect). The four bases are degraded in high yield. Direct effect has been mimicked by photo-induced electron abstraction from the bases producing their radical cation. Quantification of the modified bases showed that guanine is the preferential target. This can be explained by its lower oxidation potential and charge transfer phenomena. La décomposition radio-induite de l'ADN fait intervenir des processus radicalaires. Une série de lésions choisies parmi les produits majeurs de dégradation des bases a été mesurée dans de l'ADN isolé exposé au rayonnement en solution aqueuse aérée. Les modifications sont alors dues aux radicaux hydroxyles produits par la radiolyse de l'eau (effet indirect) et les quatre bases sont efficacement dégradées. L'arrachement d'électrons aux bases par photosensibilisation pour produire leur radical cation, a été utilisé comme modèle de l'effet direct. La quantification des bases modifiées montre que la guanine est préférentiellement dégradée. Cette observation peut s'expliquer par le plus faible potentiel d'oxydation de cette base ainsi que par les phénomènes de transfert de charge vers les guanines.

  11. A simple silica-based method for metagenomic DNA extraction from soil and sediments.

    PubMed

    Rojas-Herrera, R; Narváez-Zapata, J; Zamudio-Maya, M; Mena-Martínez, M E

    2008-09-01

    A new method is described for extraction of metagenomic DNA from soil and sediments which is based on DNA adsorption to silica without the use of phenol, ethanol precipitation or a cesium chloride gradient. High-quality DNA was obtained, and PCR inhibition was overcome by adding bovine serum albumin and adjusting magnesium concentration. By using PCR-DGGE with Firmicutes and lactic acid bacteria-specific primers the extracted metagenomic DNA was shown to contain a mixture of bacterial genomes. This method can be used for screening bacterial diversity in soil and sediment samples. PMID:18373226

  12. Automated DNA Base Pair Calling Algorithm

    1999-07-07

    The procedure solves the problem of calling the DNA base pair sequence from two channel electropherogram separations in an automated fashion. The core of the program involves a peak picking algorithm based upon first, second, and third derivative spectra for each electropherogram channel, signal levels as a function of time, peak spacing, base pair signal to noise sequence patterns, frequency vs ratio of the two channel histograms, and confidence levels generated during the run. Themore » ratios of the two channels at peak centers can be used to accurately and reproducibly determine the base pair sequence. A further enhancement is a novel Gaussian deconvolution used to determine the peak heights used in generating the ratio.« less

  13. DNA-based diagnosis of lymphatic filariasis.

    PubMed

    Nuchprayoon, Surang

    2009-09-01

    Lymphatic filariasis (LF) is still a major public health problem. The disease is ranked by the World Health Organization (WHO) as the second leading cause of permanent and long-term disability, and has been targeted for elimination by 2020. Effective diagnosis LF is required for treatment of infected individuals, for epidemiological assessment and for monitoring of the control program. Conventional diagnosis of LF depends on detection of microfilariae (Mf) in blood specimens, which has low sensitivity and specificity. Detection of specific circulating filarial antigens is regarded by WHO as the 'gold standard' for diagnosis of LF. However, the limitations of the antigen tests are cost and inconsistent availability. Although anti-filarial IgG4 antibody levels are associated with active LF infections, however, cross-reactivity with other filarial parasites is common. Not as sensitive as antigen tests, DNA-based techniques have been developed to diagnose and differentiate filarial parasites in humans, animal reservoir hosts, and mosquito vectors. These include DNA hybridization, polymerase chain reaction (PCR) amplification using specific primers (eg Ssp I repeat, pWb12 repeat, pWb-35 repeat, and LDR repeat for Wuchereria bancrofti and Hha I repeat, glutathione peroxidase gene, mitochondrial DNA for Brugia malayi), and universal primers, multiplex-PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-enzyme linked immunosorbent assay (PCR-ELISA), as well as quantitative PCR. Furthermore, because bancroftian filariasis is endemic on the Thai-Myanmar border, the potential now exists for a re-emergence of bancroftian filariasis in Thailand, and random amplified polymorphic DNA (RAPD) analysis has proved effective to differentiate Thai and Myanmar strains of W. bancrofti. PMID:19842372

  14. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    PubMed

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  15. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates.

    PubMed

    Güixens-Gallardo, Pedro; Hocek, Michal; Perlíková, Pavla

    2016-01-15

    A simple and elegant method for inhibition of non-templated nucleotide addition by DNA polymerases and for following DNA 3'-heterogeneity in enzymatic DNA synthesis by primer extension (PEX) is described. When template bearing ortho-twisted intercalating nucleic acid (ortho-TINA) at the 5'-end is used, non-templated nucleotide addition is reduced in both the A- and B-family DNA polymerases (KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-) and Taq). Formation of a single oligonucleotide product was observed with ortho-TINA modified template and KOD XL, KOD (exo-), Bst 2.0, Deep Vent (exo-) and Taq DNA polymerases. This approach can be applied to the synthesis of both unmodified and base-modified oligonucleotides. PMID:26707394

  16. Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

    SciTech Connect

    James, Conrad D.; Pohl, Kenneth Roy; Derzon, Mark Steven; McClain, Jaime; Achyuthan, Komandoor

    2006-11-01

    Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.

  17. Chip-based sequencing nucleic acids

    DOEpatents

    Beer, Neil Reginald

    2014-08-26

    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  18. DNA Polymorphism Among American Watermelon Cultivars Based on DNA Methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    American watermelon heirlooms are diverse in their growth habits, fruit qualities and responses to biotic and abiotic stress. Wide ranging DNA marker tools resolved a narrow molecular diversity among these collections. The current research explored additional insights such as extent of diversity a...

  19. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  20. Direct detection of circulating free DNA extracted from serum samples of breast cancer using locked nucleic acid molecular beacon.

    PubMed

    Gui, Zhen; Wang, Quanbo; Li, Jinchang; Zhu, Mingchen; Yu, Lili; Xun, Tang; Yan, Feng; Ju, Huangxian

    2016-07-01

    As an emerging noninvasive blood biomarker, circulating free DNA (cfDNA) can be utilized to assess diagnosis, progression and evaluate prognosis of cancer. However, cfDNAs are not "naked", they can be part of complexes, or are bound to the surface of the cells via proteins, which make the detection more challenging. Here, a simple method for the detection of Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) DNA exacted from serum of breast cancer (BC) has been developed using a novel locked nucleic acid molecular beacon (LNA-MB). In order to enhance the stability and detection efficiency of the probe in biofluids, we design a shared-stem molecular beacon containing a 27-mer loop and a 4-mer stem with DNA/LNA alternating bases. The fluorescence is released in the presence of target. The detection procedure is simple and can be completed within 1h. This method shows a sensitive response to UHRF1 DNA with a dynamic range of 3 orders of magnitude. The limit of detection is 11nM (S/N=3) with excellent selectivity. It can discriminate UHRF1 DNA from three-base mismatched DNA with a high specificity. More importantly, this method can distinguish the expression of serum UHRF1 DNA among 5 breast cancer patients and 5 healthy controls. The mentioned superiority may suggest that this assay can be served as a promising noninvasive detection tool for early BC diagnosis and monitoring. PMID:27154709

  1. Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via Replica Molding for Facile Nucleic Acid Hybridization Assays

    PubMed Central

    Lewis, Christina L.; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-01-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of probe and target DNA, femtomole sensitivity, and sequence specificity. Combined these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  2. Fabrication of uniform DNA-conjugated hydrogel microparticles via replica molding for facile nucleic acid hybridization assays.

    PubMed

    Lewis, Christina L; Choi, Chang-Hyung; Lin, Yan; Lee, Chang-Soo; Yi, Hyunmin

    2010-07-01

    We identify and investigate several critical parameters in the fabrication of single-stranded DNA conjugated poly(ethylene glycol) (PEG) microparticles based on replica molding (RM) for highly uniform and robust nucleic acid hybridization assays. The effects of PEG-diacrylate, probe DNA, and photoinitiator concentrations on the overall fluorescence and target DNA penetration depth upon hybridization are examined. Fluorescence and confocal microscopy results illustrate high conjugation capacity of the probe and target DNA, femtomole sensitivity, and sequence specificity. Combined, these findings demonstrate a significant step toward simple, robust, and scalable procedures to manufacture highly uniform and high-capacity hybridization assay particles in a well-controlled manner by exploiting many advantages that the batch processing-based RM technique offers. We envision that the results presented here may be readily applied to rapid and high-throughput hybridization assays for a wide variety of applications in bioprocess monitoring, food safety, and biological threat detection. PMID:20527819

  3. Biodegradation of DNA and nucleotides to nucleosides and free bases.

    PubMed

    Kruszewska, Hanna; Misicka, Aleksandra; Chmielowiec, Urszula

    2004-01-01

    Thirty-two different microorganisms were examined in order to check their ability to degrade an exogenous DNA. Bacteria from species: Stenotrophomonas maltophilia, Brevundimonas diminuta, Bacillus subtilis, Mycobacterium butyricum and fungus Fusarium moniliforme were capable to degrade DNA to nucleic bases or their derivatives. Degradation of DNA by S. maltophilia resulted in formation of free bases, such as hypoxanthine, thymine, uracil and xanthine. The optimum concentration of DNA seemed to be 3 mg ml(-1). The mode of degradation of DNA nucleotides depended on the type of nucleotide and its concentration, but nucleic bases or their derivatives were always formed at the end of the reaction process. PMID:14751311

  4. Site-Selective Binding of Nanoparticles to Double-Stranded DNA via Peptide Nucleic Acid "Invasion"

    SciTech Connect

    Stadler, A.L.; van der Lelie, D.; Sun, D.; Maye, M. M.; Gang, O.

    2011-04-01

    We demonstrate a novel method for by-design placement of nano-objects along double-stranded (ds) DNA. A molecular intercalator, designed as a peptide nucleic acid (PNA)-DNA chimera, is able to invade dsDNA at the PNA-side due to the hybridization specificity between PNA and one of the duplex strands. At the same time, the single-stranded (ss) DNA tail of the chimera, allows for anchoring of nano-objects that have been functionalized with complementary ssDNA. The developed method is applied for interparticle attachment and for the fabrication of particle clusters using a dsDNA template. This method significantly broadens the molecular toolbox for constructing nanoscale systems by including the most conventional not yet utilized DNA motif, double helix DNA.

  5. Interaction of Ku protein and DNA-dependent protein kinase catalytic subunit with nucleic acids.

    PubMed Central

    Dynan, W S; Yoo, S

    1998-01-01

    The Ku protein-DNA-dependent protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku protein or DNA-PKcs. PMID:9512523

  6. Selection of Natural and Base-Modified DNA Aptamers for a Camptothecin Derivative.

    PubMed

    Fujita, Hiroto; Kuwahara, Masayasu

    2016-01-01

    Nucleic acid aptamers for small molecules are currently being developed and have a potential role in diverse applications including biosensing, diagnostics, and therapeutics involving low-molecular-weight biomarkers and drugs. To enhance and broaden their functions through chemical modification, systematic evolution of ligands by exponential enrichment (SELEX) selection has been attempted with modified DNA/RNA libraries. Recently, we demonstrated the superior efficacy of base modification for affinity enhancement and the usefulness of unnatural nucleic acid libraries for development of small-molecule aptamers. In this unit, we describe construction of a modified DNA library that includes (E)-5-(2-(N-(2-(N(6) -adeninyl)ethyl))carbamylvinyl)uracil bases and acquisition of high-affinity camptothecin-binding DNA aptamers, in addition to those of the corresponding natural DNA library and aptamers, using the SELEX method. © 2016 by John Wiley & Sons, Inc. PMID:27248786

  7. Novel materials based on DNA-CTMA and lanthanide (Ce(3+) , Pr(3+) ).

    PubMed

    Lazar, Cosmina Andreea; Kajzar, François; Mihaly, Maria; Rogozea, Adina Elena; Petcu, Adina Roxana; Olteanu, Nicoleta Liliana; Rau, Ileana

    2016-09-01

    New, deoxyribonucleic acid (DNA) based compounds, functionalized with hexadecyltrimethylammonium chloride (CTMA) and lanthanide hydroxide nanoparticles were synthesized. The spectral measurements suggest that between the DNA-CTMA complex and the lanthanide (III) ions a chemical interaction takes place. The obtained materials exhibit an improved fluorescence efficiency, showing a potential interest for application in photonics, and more particularly, in light emitting devices. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 613-617, 2016. PMID:27120012

  8. Physics of base-pairing dynamics in DNA

    NASA Astrophysics Data System (ADS)

    Manghi, Manoel; Destainville, Nicolas

    2016-05-01

    As a key molecule of life, Deoxyribo-Nucleic Acid (DNA) is the focus of numbers of investigations with the help of biological, chemical and physical techniques. From a physical point of view, both experimental and theoretical works have brought quantitative insights into DNA base-pairing dynamics that we review in this Report, putting emphasis on theoretical developments. We discuss the dynamics at the base-pair scale and its pivotal coupling with the polymer one, with a polymerization index running from a few nucleotides to tens of kilo-bases. This includes opening and closure of short hairpins and oligomers as well as zipping and unwinding of long macromolecules. We review how different physical mechanisms are either used by Nature or utilized in biotechnological processes to separate the two intertwined DNA strands, by insisting on quantitative results. They go from thermally-assisted denaturation bubble nucleation to force- or torque-driven mechanisms. We show that the helical character of the molecule, possibly supercoiled, can play a key role in many denaturation and renaturation processes. We categorize the mechanisms according to the relative timescales associated with base-pairing and chain orientational degrees of freedom such as bending and torsional elastic ones. In some specific situations, these chain orientational degrees of freedom can be integrated out, and the quasi-static approximation is valid. The complex dynamics then reduces to the diffusion in a low-dimensional free-energy landscape. In contrast, some important cases of experimental interest necessarily appeal to far-from-equilibrium statistical mechanics and hydrodynamics.

  9. Phosphonic acid based exchange resins

    DOEpatents

    Horwitz, E. Philip; Alexandratos, Spiro D.; Gatrone, Ralph C.; Chiarizia, Ronato

    1995-01-01

    An ion exchange resin for extracting metal ions from a liquid waste stream. An ion exchange resin is prepared by copolymerizing a vinylidene diphosphonic acid with styrene, acrylonitrile and divinylbenzene.

  10. Phosphonic acid based exchange resins

    DOEpatents

    Horwitz, E.P.; Alexandratos, S.D.; Gatrone, R.C.; Chiarizia, R.

    1995-09-12

    An ion exchange resin is described for extracting metal ions from a liquid waste stream. An ion exchange resin is prepared by copolymerizing a vinylidene diphosphonic acid with styrene, acrylonitrile and divinylbenzene. 10 figs.

  11. A comprehensive classification of nucleic acid structural families based on strand direction and base pairing.

    PubMed Central

    Lavery, R; Zakrzewska, K; Sun, J S; Harvey, S C

    1992-01-01

    We propose a classification of DNA structures formed from 1 to 4 strands, based only on relative strand directions, base to strand orientation and base pairing geometries. This classification and its associated notation enable all nucleic acids to be grouped into structural families and bring to light possible structures which have not yet been observed experimentally. It also helps in understanding transitions between families and can assist in the design of multistrand structures. PMID:1383936

  12. Synthesis of nucleoside and nucleotide conjugates of bile acids, and polymerase construction of bile acid-functionalized DNA.

    PubMed

    Ikonen, Satu; Macícková-Cahová, Hana; Pohl, Radek; Sanda, Miloslav; Hocek, Michal

    2010-03-01

    Aqueous Sonogashira cross-coupling reactions of 5-iodopyrimidine or 7-iodo-7-deazaadenine nucleosides with bile acid-derived terminal acetylenes linked via an ester or amide tether gave the corresponding bile acid-nucleoside conjugates. Analogous reactions of halogenated nucleoside triphosphates gave directly bile acid-modified dNTPs. Enzymatic incorporation of these modified nucleotides to DNA was successfully performed using Phusion polymerase for primer extension. One of the dNTPs (dCTP bearing cholic acid) was also efficient for PCR amplification. PMID:20165813

  13. A gold nanoparticle based approach for screening triplex DNA binders.

    PubMed

    Han, Min Su; Lytton-Jean, Abigail K R; Mirkin, Chad A

    2006-04-19

    Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change do not occur in the presence of duplex DNA binders and therefore provide a significantly better screening process for triplex DNA binding molecules compared to standard methods. PMID:16608320

  14. A Gold Nanoparticle Based Approach for Screening Triplex DNA Binders

    PubMed Central

    Han, Min Su; Lytton-Jean, Abigail K. R.; Mirkin, Chad A.

    2011-01-01

    Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change does not occur in the presence of duplex DNA binders and therefore provides a significantly better screening process for triplex DNA binding molecules compared to standard methods. PMID:16608320

  15. Substitution of DNA-Contacting Amino Acids with Functional Variants in the Gata-1 Zinc Finger: A Structurally and Phylogenetically Guided Mutagenesis

    PubMed Central

    Vonderfecht, Tyson R.; Schroyer, Daniel L.; Schenck, Brandy L.; McDonough, Virginia M.; Pikaart, Michael J.

    2008-01-01

    DNA binding functionality among transcription factor proteins is afforded by a number of structural motifs, such as the helix-turn-helix, helix-loop-helix, and zinc finger domains. The common thread among these diverse structures is their sequence-specific binding to essential promoter or other genetic regulatory sequences with high selectivity and affinity. One such motif, present in a wide range of organisms from bacteria to vertebrates, is the Gata-type zinc finger. This family of DNA-binding proteins is characterized by the presence of one or two (Cys)4 metal binding sites which recognize the protein’s eponymous binding site, GATA. Unlike other conserved DNA binding domains, Gata proteins appear to be restricted to binding consensus GATA sequences, or near variations, in DNA. Since the architecture of the Gata finger seems built around recognizing this particular sequence, we set out to define the allowable range of amino acid substitutions along the DNA-binding surface of a Gata finger that could continue to support sequence specific DNA binding activity. Accordingly, we set up a one-hybrid screen in yeast based on the chicken Gata-1 C-terminal zinc finger. Mutant libraries were generated at five amino acids identified in the Gata-DNA structure as likely to mediate sequence-specific contacts between the Gata finger and DNA. These libraries were designed to give as exhaustive amino acid coverage as possible such that almost all alternative amino acids were screened at each of the five probed positions. Screening and characterization of these libraries revealed several functional amino acid substitutions at two leucines which contact the DNA at the 3’ and 5’ flanks of the GATA binding site, but no functional substituents for amino acids near the core of the binding site. This pattern is consistent with amino acid sequences of known DNA-binding Gata fingers. PMID:18328814

  16. A Novel Bio-Sensor Based on DNA Strand Displacement

    PubMed Central

    Shi, Xiaolong; Wang, Zhiyu; Deng, Chenyan; Song, Tao; Pan, Linqiang; Chen, Zhihua

    2014-01-01

    DNA strand displacement technology performs well in sensing and programming DNA segments. In this work, we construct DNA molecular systems based on DNA strand displacement performing computation of logic gates. Specifically, a class of so-called “DNA neurons” are achieved, in which a “smart” way inspired by biological neurons encoding information is developed to encode and deliver information using DNA molecules. The “DNA neuron” is bistable, that is, it can sense DNA molecules as input signals, and release “negative” or “positive” signals DNA molecules. We design intelligent DNA molecular systems that are constructed by cascading some particularly organized “DNA neurons”, which could perform logic computation, including AND, OR, XOR logic gates, automatically. Both simulation results using visual DSD (DNA strand displacement) software and experimental results are obtained, which shows that the proposed systems can detect DNA signals with high sensitivity and accretion; moreover, the systems can process input signals automatically with complex nonlinear logic. The method proposed in this work may provide a new way to construct a sensitive molecular signal detection system with neurons spiking behavior in vitro, and can be used to develop intelligent molecular processing systems in vivo. PMID:25303242

  17. The Kidney and Acid-Base Regulation

    ERIC Educational Resources Information Center

    Koeppen, Bruce M.

    2009-01-01

    Since the topic of the role of the kidneys in the regulation of acid base balance was last reviewed from a teaching perspective (Koeppen BM. Renal regulation of acid-base balance. Adv Physiol Educ 20: 132-141, 1998), our understanding of the specific membrane transporters involved in H+, HCO , and NH transport, and especially how these…

  18. Students' Alternate Conceptions on Acids and Bases

    ERIC Educational Resources Information Center

    Pan, Hanqing; Henriques, Laura

    2015-01-01

    Knowing what students bring to the classroom can and should influence how we teach them. This study is a review of the literature associated with secondary and postsecondary students' ideas about acids and bases. It was found that there are six types of alternate ideas about acids and bases that students hold. These are: macroscopic properties of…

  19. The Conjugate Acid-Base Chart.

    ERIC Educational Resources Information Center

    Treptow, Richard S.

    1986-01-01

    Discusses the difficulties that beginning chemistry students have in understanding acid-base chemistry. Describes the use of conjugate acid-base charts in helping students visualize the conjugate relationship. Addresses chart construction, metal ions, buffers and pH titrations, and the organic functional groups and nonaqueous solvents. (TW)

  20. qPCR-based mitochondrial DNA quantification: influence of template DNA fragmentation on accuracy.

    PubMed

    Jackson, Christopher B; Gallati, Sabina; Schaller, André

    2012-07-01

    Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λnDNA) and mtDNA (λmtDNA) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number. PMID:22683632

  1. High Fidelity of Base Paring by 2-Selenothymidine in DNA

    SciTech Connect

    Hassan, A.; Sheng, J; Zhang, W; Huang, Z

    2010-01-01

    The base pairs are the contributors to the sequence-dependent recognition of nucleic acids, genetic information storage, and high fidelity of DNA polymerase replication. However, the wobble base pairing, where T pairs with G instead of A, reduces specific base-pairing recognition and compromises the high fidelity of the enzymatic polymerization. Via the selenium atomic probing at the 2-position of thymidine, we have investigated the wobble discrimination by manipulating the steric and electronic effects at the 2-exo position, providing a unique chemical strategy to enhance the base pair specificity. We report here the first synthesis of the novel 2-Se-thymidine ({sup Se}T) derivative, its phosphoramidite, and the Se-DNAs. Our biophysical and structural studies of the 2-Se-T DNAs reveal that the bulky 2-Se atom with a weak hydrogen-bonding ability can largely increase mismatch discriminations (including T/G wobble and T/C mismatched base pairs) while maintaining the {sup Se}T/A virtually identical to the native T/A base pair. The 2-Se atom bulkiness and the electronic effect are probably the main factors responsible for the discrimination against the formation of the wobble {sup Se}T/G base pair. Our investigations provide a potential novel tool to investigate the specific recognition of base pairs, which is the basis of high fidelity during replication, transcription, and translation. Furthermore, this Se-atom-specific substitution and probing are useful for X-ray crystal structure and function studies of nucleic acids.

  2. First Principles Dynamics of Photoexcited DNA and RNA Bases

    SciTech Connect

    Hudock, Hanneli R.; Levine, Benjamin G.; Thompson, Alexis L.; Martinez, Todd J.

    2007-12-26

    The reaction dynamics of excited electronic states in nucleic acid bases is a key process in DNA photodamage. Recent ultrafast spectroscopy experiments have shown multi-component decays of excited uracil and thymine, tentatively assigned to nonadiabatic transitions involving multiple electronic states. Using both quantum chemistry and first principles quantum molecular dynamics methods we show that a true minimum on the bright S{sub 2} electronic state is responsible for the first step which occurs on a femtosecond timescale. Thus the observed femtosecond decay does not correspond to surface crossing as previously thought. We suggest that subsequent barrier crossing to the minimal energy S{sub 2}/S{sub 1} conical intersection is responsible for the picosecond decay.

  3. A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments

    PubMed Central

    Natarajan, Vengadesh Perumal; Zhang, Xinxu; Morono, Yuki; Inagaki, Fumio; Wang, Fengping

    2016-01-01

    Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment. PMID:27446026

  4. A Modified SDS-Based DNA Extraction Method for High Quality Environmental DNA from Seafloor Environments.

    PubMed

    Natarajan, Vengadesh Perumal; Zhang, Xinxu; Morono, Yuki; Inagaki, Fumio; Wang, Fengping

    2016-01-01

    Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment. PMID:27446026

  5. Internalization of Locked Nucleic Acids/DNA Hybrid Oligomers into Escherichia coli.

    PubMed

    Traglia, German M; Sala, Carol Davies; Fuxman Bass, Juan I; Soler-Bistué, Alfonso J C; Zorreguieta, Angeles; Ramírez, María Soledad; Tolmasky, Marcelo E

    2012-10-01

    Delivery inside the cells is essential for practical application of antisense technologies. The hybrid locked nucleic acid (LNA)/DNA CAAGTACTGTTCCACCA (LNA residues are underlined) was labeled by conjugation to Alexa Fluor 488 (fLNA/DNA) and tested to determine its ability to penetrate Escherichia coli cells and reach the cytoplasm. Flow cytometry analysis showed that the fLNA/DNA was associated with 14% of cells from a stationary phase culture, while association with a labeled isosequential oligodeoxynucleotide was negligible. Laser scanning confocal microscopy confirmed that the fLNA/DNA was located inside the cytoplasm. PMID:23515318

  6. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  7. Breaking the dogma: PCB-derived semiquinone free radicals do not form covalent adducts with DNA, GSH, and amino acids.

    PubMed

    Wangpradit, Orarat; Rahaman, Asif; Mariappan, S V Santhana; Buettner, Garry R; Robertson, Larry W; Luthe, Gregor

    2016-02-01

    Covalent bond formations of free radical metabolites with biomolecules like DNA and proteins are thought to constitute a major mechanism of toxicity and carcinogenesis. Glutathione (GSH) is generally accepted as a radical scavenger protecting the cell. In the present study, we investigated a semiquinone radical (SQ(●-)) metabolite of the semivolatile 4-chlorobiphenyl, using electron paramagnetic resonance spectroscopy, and oxygen consumption. Proton nuclear magnetic resonance ((1)H NMR) and liquid chromatography-mass spectrometry (LC-MS) were also employed to elucidate the radical interaction with DNA, amino acids, and GSH. We found that DNA and oligonucleotides stabilized SQ(●-) by electron delocalization in the π-stacking system, resulting in persistent radical intercalated, rather than forming a covalent bond with SQ(●-). This finding was strongly supported by the semiempirical calculation of the semioccupied molecular orbital and the linear combination of the atomic orbitals, indicating 9.8 kcal mol(-1) energy gain. The insertion of SQ(●-) into the DNA strand may result in DNA strand breaks and interruption of DNA replication process or even activate radical mediated secondary reactions. The presence of amino acids resulted in a decrease of the electron paramagnetic resonance (EPR) signal of SQ(●-) and correlated with their isoelectric points. The pH shifts the equilibrium of the dianions of hydroquinone and influenced indirectly the formation of SQ(●-). Similar findings were observed with GSH and Cys. GSH and Cys functioned as indirect radical scavengers; their activities depend on their chemical equilibria with the corresponding quinones, and their further reaction via Michael addition. The generally accepted role of GSH as radical scavenger in biological systems should be reconsidered based upon these findings, questioning the generally accepted view of radical interaction of semiquinones with biologically active compounds, like DNA, amino acids

  8. Nucleic acid-8-methoxypsoralen crosslinks bind monoclonal anti-Z-DNA antibody.

    PubMed

    Arif, Z; Ali, R

    1996-11-01

    Native calf thymus DNA and poly(dA-dT).poly(dA-dT) were photo-adducted with 8-methoxypsoralen and characterized by thermal denaturation (Tm) and hydroxyapatite column chromatography. The data demonstrated the formation of interstrand photo-crosslinks. It has been shown by competition ELISA and band shift assays that crosslinked species of DNA-8-MOP and poly(dA-dT)-8-MOP photoadducts recognize previously defined monoclonal anti-Z-DNA antibody (Z22). The results indicate the possible presence of Z- or Z-like epitopes on nucleic acid-8-MOP crosslinks as Z22 antibody does not recognize other nucleic acid conformations. These studies also point out that conformational changes in DNA arising from the photo-addition could induce antibodies to DNA or could cause autoimmune disease. PMID:8955875

  9. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

  10. Dihydrobetulinic Acid Induces Apoptosis in Leishmania donovani by Targeting DNA Topoisomerase I and II: Implications in Antileishmanial Therapy

    PubMed Central

    Chowdhury, Arnab Roy; Mandal, Suparna; Goswami, Anindya; Ghosh, Monidipa; Mandal, Labanya; Chakraborty, Debabani; Ganguly, Agneyo; Tripathi, Gayatri; Mukhopadhyay, Sibabrata; Bandyopadhyay, Santu; Majumder, Hemanta K

    2003-01-01

    Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 μM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs. PMID:12765337

  11. Quantitative and qualitative validations of a sonication-based DNA extraction approach for PCR-based molecular biological analyses.

    PubMed

    Dai, Xiaohu; Chen, Sisi; Li, Ning; Yan, Han

    2016-05-15

    The aim of this study was to comprehensively validate the sonication-based DNA extraction method, in hope of the replacement of the so-called 'standard DNA extraction method' - the commercial kit method. Microbial cells in the digested sludge sample, containing relatively high amount of PCR-inhibitory substances, such as humic acid and protein, were applied as the experimental alternatives. The procedure involving solid/liquid separation of sludge sample and dilution of both DNA templates and inhibitors, the minimum templates for PCR-based analyses, and the in-depth understanding from the bias analysis by pyrosequencing technology were obtained and confirmed the availability of the sonication-based DNA extraction method. PMID:26774955

  12. An Evolution Based Biosensor Receptor DNA Sequence Generation Algorithm

    PubMed Central

    Kim, Eungyeong; Lee, Malrey; Gatton, Thomas M.; Lee, Jaewan; Zang, Yupeng

    2010-01-01

    A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements. PMID:22315543

  13. Molecular analysis of two cDNA clones encoding acidic class I chitinase in maize.

    PubMed Central

    Wu, S; Kriz, A L; Widholm, J M

    1994-01-01

    The cloning and analysis of two different cDNA clones encoding putative maize (Zea mays L.) chitinases obtained by polymerase chain reaction (PCR) and cDNA library screening is described. The cDNA library was made from poly(A)+ RNA from leaves challenged with mercuric chloride for 2 d. The two clones, pCh2 and pCh11, appear to encode class I chitinase isoforms with cysteine-rich domains (not found in pCh11 due to the incomplete sequence) and proline-/glycine-rich or proline-rich hinge domains, respectively. The pCh11 clone resembles a previously reported maize seed chitinase; however, the deduced proteins were found to have acidic isoelectric points. Analysis of all monocot chitinase sequences available to date shows that not all class I chitinases possess the basic isoelectric points usually found in dicotyledonous plants and that monocot class II chitinases do not necessarily exhibit acidic isoelectric points. Based on sequence analysis, the pCh2 protein is apparently synthesized as a precursor polypeptide with a signal peptide. Although these two clones belong to class I chitinases, they share only about 70% amino acid homology in the catalytic domain region. Southern blot analysis showed that pCh2 may be encoded by a small gene family, whereas pCh11 was single copy. Northern blot analysis demonstrated that these genes are differentially regulated by mercuric chloride treatment. Mercuric chloride treatment caused rapid induction of pCh2 from 6 to 48 h, whereas pCh11 responded only slightly to the same treatment. During seed germination, embryos constitutively expressed both chitinase genes and the phytohormone abscisic acid had no effect on the expression. The fungus Aspergillus flavus was able to induce both genes to comparable levels in aleurone layers and embryos but not in endosperm tissue. Maize callus growth on the same plate with A. flavus for 1 week showed induction of the transcripts corresponding to pCh2 but not to pCh11. These studies indicate that

  14. Direct Quantification of DNA Base Composition by Surface-Enhanced Raman Scattering Spectroscopy.

    PubMed

    Morla-Folch, Judit; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2016-08-01

    Design of ultrasensitive DNA sensors based on the unique physical properties of plasmonic nanostructures has become one of the most exciting areas in nanomedicine. However, despite the vast number of proposed applications, the determination of the base composition in nucleic acids, a fundamental parameter in genomic analyses and taxonomic classification, is still restricted to time-consuming and poorly sensitive conventional methods. Herein, we demonstrate the possibility of determining the base composition in single- and double-stranded DNA by using a simple, low-cost, high-throughput, and label-free surface-enhanced Raman scattering (SERS) method in combination with cationic nanoparticles. PMID:27441814

  15. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  16. Fluorescent sensors based on boronic acids

    NASA Astrophysics Data System (ADS)

    Cooper, Christopher R.; James, Tony D.

    1999-05-01

    Sensor systems have long been needed for detecting the presence in solution of certain chemically or biologically important species. Sensors are used in a wide range of applications from simple litmus paper that shows a single color change in acidic or basic environments to complex biological assays that use enzymes, antibodies and antigens to display binding events. With this work the use of boronic acids in the design and synthesis of sensors for saccharides (diols) will be presented. The fluorescent sensory systems rely on photoinduced electron transfer (PET) to modulate the observed fluorescence. When saccharides form cyclic boronate esters with boronic acids, the Lewis acidity of the boronic acid is enhanced and therefore the Lewis acid-base interaction between the boronic acid and a neighboring amine is strengthened. The strength of this acid-base interaction modulates the PET from the amine (acting as a quencher) to anthracene (acting as a fluorophore). These compounds show increased fluorescence at neutral pH through suppression of the PET from nitrogen to anthracene on saccharide binding. The general strategy for the development of saccharide selective systems will be discussed. The potential of the boronic acid based systems will be illustrated using the development of glucose and glucosamine selective fluorescent sensors as examples.

  17. Complexities of the DNA Base Excision Repair Pathway for Repair of Oxidative DNA Damage

    PubMed Central

    Mitra, Sankar; Boldogh, Istvan; Izumi, Tadahide; Hazra, Tapas K.

    2016-01-01

    Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase β or replicative polymerases δ and ε. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation. PMID:11746753

  18. Reliability and Efficiency of a DNA-Based Computation

    NASA Astrophysics Data System (ADS)

    Deaton, R.; Garzon, M.; Murphy, R. C.; Rose, J. A.; Franceschetti, D. R.; Stevens, S. E., Jr.

    1998-01-01

    DNA-based computing uses the tendency of nucleotide bases to bind (hybridize) in preferred combinations to do computation. Depending on reaction conditions, oligonucleotides can bind despite noncomplementary base pairs. These mismatched hybridizations are a source of false positives and negatives, which limit the efficiency and scalability of DNA-based computing. The ability of specific base sequences to support error-tolerant Adleman-style computation is analyzed, and criteria are proposed to increase reliability and efficiency. A method is given to calculate reaction conditions from estimates of DNA melting.

  19. Uracil misincorporation into DNA and folic acid supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Folate deficiency decreases thymidylate synthesis from deoxyuridylate, which results in an imbalance of deoxyribonucleotide that may lead to excessive uracil misincorporation (UrMis) into DNA during replication and repair. OBJECTIVE: We evaluated the relation between UrMis in different ...

  20. Role of amidation in bile acid effect on DNA synthesis by regenerating mouse liver.

    PubMed

    Barbero, E R; Herrera, M C; Monte, M J; Serrano, M A; Marin, J J

    1995-06-01

    Effect of bile acids on DNA synthesis by the regenerating liver was investigated in mice in vivo after partial hepatectomy (PH). Radioactivity incorporation into DNA after [14C]thymidine intraperitoneal administration peaked at 48 h after PH. At this time a significant taurocholate-induced dose-dependent reduction in DNA synthesis without changes in total liver radioactivity content was found (half-maximal effect at approximately 0.1 mumol/g body wt). Effect of taurocholate (0.5 mumol/g body wt) was mimicked by chocolate, ursodeoxycholate, deoxycholate, dehydrocholate, tauroursodeoxycholate, taurochenodeoxycholate, and taurodeoxycholate. In contrast, chenodeoxycholate, glycocholate, glycochenodeoxycholate, glycoursodeoxycholate, glycodeoxycholate, 5 beta-cholestane, bromosulfophthalein, and free taurine lacked this effect. No relationship between hydrophobic-hydrophilic balance and inhibitory effect was observed. Analysis by high-performance liquid chromatography indicated that inhibition of thymidine incorporation into DNA was not accompanied by an accumulation of phosphorylated DNA precursors in the liver but rather by a parallel increase in nucleotide catabolism. Bile acid-induced modifications in DNA synthesis were observed in vivo even in the absence of changes in toxicity tests, which suggests that the inhibitory effect shared by most unconjugated and tauroconjugated bile acids but not by glycoconjugated bile acids should be accounted for by mechanisms other than nonselective liver cell injury. PMID:7611405

  1. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

    PubMed Central

    Sirois, Cherilyn M.; Jin, Tengchuan; Miller, Allison L.; Bertheloot, Damien; Nakamura, Hirotaka; Horvath, Gabor L.; Mian, Abubakar; Jiang, Jiansheng; Schrum, Jacob; Bossaller, Lukas; Pelka, Karin; Garbi, Natalio; Brewah, Yambasu; Tian, Jane; Chang, ChewShun; Chowdhury, Partha S.; Sims, Gary P.; Kolbeck, Roland; Coyle, Anthony J.; Humbles, Alison A.

    2013-01-01

    Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo. PMID:24081950

  2. Base-acid hybrid water electrolysis.

    PubMed

    Chen, Long; Dong, Xiaoli; Wang, Fei; Wang, Yonggang; Xia, Yongyao

    2016-02-21

    A base-acid hybrid electrolytic system with a low onset voltage of 0.78 V for water electrolysis was developed by using a ceramic Li-ion exchange membrane to separate the oxygen-evolving reaction (OER) in a basic electrolyte solution containing the Li-ion and hydrogen-evolving reaction (HER) in an acidic electrolyte solution. PMID:26804323

  3. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1987-10-07

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  4. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Moyzis, Robert K.; Ratliff, Robert L.; Shera, E. Brooks; Stewart, Carleton C.

    1990-01-01

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

  5. Method for rapid base sequencing in DNA and RNA

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff, R.L.; Shera, E.B.; Stewart, C.C.

    1990-10-09

    A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed. 2 figs.

  6. Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

    SciTech Connect

    Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

    2002-03-01

    Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appears to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.

  7. A wavelet-based feature vector model for DNA clustering.

    PubMed

    Bao, J P; Yuan, R Y

    2015-01-01

    DNA data are important in the bioinformatic domain. To extract useful information from the enormous collection of DNA sequences, DNA clustering is often adopted to efficiently deal with DNA data. The alignment-free method is a very popular way of creating feature vectors from DNA sequences, which are then used to compare DNA similarities. This paper proposes a wavelet-based feature vector (WFV) model, which is also an alignment-free method. From the perspective of signal processing, a DNA sequence is a sequence of digital signals. However, most traditional alignment-free models only extract features in the time domain. The WFV model uses discrete wavelet transform to adaptively yield feature vectors with a fixed dimension based on the features in both the time and frequency domains. The level of wavelet transform is adjusted according to the length of the DNA sequence rather than a fixed manually set value. The WFV model prefers a 32-dimension feature vector, which greatly promotes system performance. We compared the WFV model with the other five alignment-free models, i.e., k-tuple, DMK, TSM, AMI, and CV, on several large-scale DNA datasets on the DNA clustering application by means of the K-means algorithm. The experimental results showed that the WFV model outperformed the other models in terms of both the clustering results and the running time. PMID:26782569

  8. DNA fingerprinting of lactic acid bacteria in sauerkraut fermentations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies using traditional biochemical methods to study the ecology of commercial sauerkraut fermentations revealed that four lactic acid bacteria species, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis were the primary microorganisms in...

  9. Characterization of the tunneling conductance across DNA bases

    SciTech Connect

    Zikic, Radomir; Krstic, Predrag S; Zhang, Xiaoguang; Fuentes-Cabrera, Miguel A; Wells, Jack C; Zhao, Xiongce

    2006-01-01

    Characterization of the electrical properties of the DNA bases, Adenine, Cytosine, Guanine and Thymine, besides building the basic knowledge on these fundamental constituents of a DNA, is a crucial step in developing a DNA sequencing technology. We present a first-principles study of the current-voltage characteristics of nucleotide-like molecules of the DNA bases, placed in a 1.5 nm gap formed between gold nanoelectrodes. The quantum transport calculations in the tunneling regime are shown to vary strongly with the electrode-molecule geometry and the choice of the DFT exchangecorrelation functionals. Analysis of the results in the zero-bias limit indicates that distinguishable current-voltage characteristics of different DNA bases are dominated by the geometrical conformations of the bases and nanoelectrodes.

  10. UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke.

    PubMed

    Lee, Jae Young; Yang, Wei

    2006-12-29

    Helicases use the energy derived from nucleoside triphosphate hydrolysis to unwind double helices in essentially every metabolic pathway involving nucleic acids. Earlier crystal structures have suggested that DNA helicases translocate along a single-stranded DNA in an inchworm fashion. We report here a series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates. These structures reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand. Thus DNA unwinding is achieved by a two-part power stroke in a combined wrench-and-inchworm mechanism. The rotational angle and translational distance of DNA define the unwinding step to be 1 base pair per ATP hydrolyzed. Finally, a gateway for ssDNA translocation and an alternative strand-displacement mode may explain the varying step sizes reported previously. PMID:17190599

  11. Nucleic acid-based nanoengineering: novel structures for biomedical applications

    PubMed Central

    Li, Hanying; LaBean, Thomas H.; Leong, Kam W.

    2011-01-01

    Nanoengineering exploits the interactions of materials at the nanometre scale to create functional nanostructures. It relies on the precise organization of nanomaterials to achieve unique functionality. There are no interactions more elegant than those governing nucleic acids via Watson–Crick base-pairing rules. The infinite combinations of DNA/RNA base pairs and their remarkable molecular recognition capability can give rise to interesting nanostructures that are only limited by our imagination. Over the past years, creative assembly of nucleic acids has fashioned a plethora of two-dimensional and three-dimensional nanostructures with precisely controlled size, shape and spatial functionalization. These nanostructures have been precisely patterned with molecules, proteins and gold nanoparticles for the observation of chemical reactions at the single molecule level, activation of enzymatic cascade and novel modality of photonic detection, respectively. Recently, they have also been engineered to encapsulate and release bioactive agents in a stimulus-responsive manner for therapeutic applications. The future of nucleic acid-based nanoengineering is bright and exciting. In this review, we will discuss the strategies to control the assembly of nucleic acids and highlight the recent efforts to build functional nucleic acid nanodevices for nanomedicine. PMID:23050076

  12. Nucleic acid-based tissue biomarkers of urologic malignancies.

    PubMed

    Dietrich, Dimo; Meller, Sebastian; Uhl, Barbara; Ralla, Bernhard; Stephan, Carsten; Jung, Klaus; Ellinger, Jörg; Kristiansen, Glen

    2014-08-01

    Molecular biomarkers play an important role in the clinical management of cancer patients. Biomarkers allow estimation of the risk of developing cancer; help to diagnose a tumor, ideally at an early stage when cure is still possible; and aid in monitoring disease progression. Furthermore, they hold the potential to predict the outcome of the disease (prognostic biomarkers) and the response to therapy (predictive biomarkers). Altogether, biomarkers will help to avoid tumor-related deaths and reduce overtreatment, and will contribute to increased survival and quality of life in cancer patients due to personalized treatments. It is well established that the process of carcinogenesis is a complex interplay between genomic predisposition, acquired somatic mutations, epigenetic changes and genomic aberrations. Within this complex interplay, nucleic acids, i.e. RNA and DNA, play a fundamental role and therefore represent ideal candidates for biomarkers. They are particularly promising candidates because sequence-specific hybridization and amplification technologies allow highly accurate and sensitive assessment of these biomarker levels over a broad dynamic range. This article provides an overview of nucleic acid-based biomarkers in tissues for the management of urologic malignancies, i.e. tumors of the prostate, testis, kidney, penis, urinary bladder, renal pelvis, ureter and other urinary organs. Special emphasis is put on genomic, transcriptomic and epigenomic biomarkers (SNPs, mutations [genomic and mitochondrial], microsatellite instabilities, viral and bacterial DNA, DNA methylation and hydroxymethylation, mRNA expression, and non-coding RNAs [lncRNA, miRNA, siRNA, piRNA, snRNA, snoRNA]). Due to the multitude of published biomarker candidates, special focus is given to the general applicability of different molecular classes as biomarkers and some particularly promising nucleic acid biomarkers. Furthermore, specific challenges regarding the development and clinical

  13. The DNA binding specificity of the basic region of the yeast transcriptional activator GCN4 can be changed by substitution of a single amino acid.

    PubMed Central

    Suckow, M; von Wilcken-Bergmann, B; Müller-Hill, B

    1993-01-01

    The X-ray structure of a GCN4 DNA complex (1) shows, that specific DNA binding of the GCN4 basic region is mediated by a complicated network of base pair and DNA backbone contacts. According to the X-ray structure, alanine -14 of the basic region of GCN4 (we define the first leucine of the leucine zipper as +1) makes a hydrophobic contact to the methyl group of the thymine next to the center of the GCN4 binding site 5' ATGACTCAT 3'. We tested the DNA binding properties of the nineteen derivatives of GCN4, which carry all possible amino acids in position -14 of the basic region. Substitution of alanine -14 of GCN4 by either asparagine or cysteine changes the DNA binding specificity. Serine in this position broadens the specificity for position 1 of the target, whereas other amino acids either retain or decrease GCN4 specificity. Images PMID:8502548

  14. Ultra-sensitive detection of zinc oxide nanowires using a quartz crystal microbalance and phosphoric acid DNA

    NASA Astrophysics Data System (ADS)

    Jang, Kuewhan; You, Juneseok; Park, Chanhoo; Park, Hyunjun; Choi, Jaeyeong; Choi, Chang-Hwan; Park, Jinsung; Lee, Howon; Na, Sungsoo

    2016-09-01

    Recent advancements of nanomaterials have inspired numerous scientific and industrial applications. Zinc oxide nanowires (ZnO NWs) is one of the most important nanomaterials due to their extraordinary properties. However, studies performed over the past decade have reported toxicity of ZnO NWs. Therefore, there has been increasing demand for effective detection of ZnO NWs. In this study, we propose a method for the detection of ZnO NW using a quartz crystal microbalance (QCM) and DNA probes. The detection method is based on the covalent interaction between ZnO NWs and the phosphoric acid group of single-stranded DNA (i.e., linker DNA), and DNA hybridization between the linker DNA and the probe DNA strand on the QCM electrode. Rapid, high sensitivity, in situ detection of ZnO NWs was demonstrated for the first time. The limit of detection was 10‑4 μg ml‑1 in deionized water, which represents a sensitivity that is 100000 times higher than the toxic ZnO NW concentration level. Moreover, the selectivity of the ZnO NW detection method was demonstrated by comparison with other types of nanowires and the method was able to detect ZnO NWs in tap water sensitively even after stored for 14 d in a refrigerator. The performance of our proposed method was sufficient to achieve detection of ZnO NW in the ‘real-world’ environment.

  15. Ultra-sensitive detection of zinc oxide nanowires using a quartz crystal microbalance and phosphoric acid DNA.

    PubMed

    Jang, Kuewhan; You, Juneseok; Park, Chanhoo; Park, Hyunjun; Choi, Jaeyeong; Choi, Chang-Hwan; Park, Jinsung; Lee, Howon; Na, Sungsoo

    2016-09-01

    Recent advancements of nanomaterials have inspired numerous scientific and industrial applications. Zinc oxide nanowires (ZnO NWs) is one of the most important nanomaterials due to their extraordinary properties. However, studies performed over the past decade have reported toxicity of ZnO NWs. Therefore, there has been increasing demand for effective detection of ZnO NWs. In this study, we propose a method for the detection of ZnO NW using a quartz crystal microbalance (QCM) and DNA probes. The detection method is based on the covalent interaction between ZnO NWs and the phosphoric acid group of single-stranded DNA (i.e., linker DNA), and DNA hybridization between the linker DNA and the probe DNA strand on the QCM electrode. Rapid, high sensitivity, in situ detection of ZnO NWs was demonstrated for the first time. The limit of detection was 10(-4) μg ml(-1) in deionized water, which represents a sensitivity that is 100000 times higher than the toxic ZnO NW concentration level. Moreover, the selectivity of the ZnO NW detection method was demonstrated by comparison with other types of nanowires and the method was able to detect ZnO NWs in tap water sensitively even after stored for 14 d in a refrigerator. The performance of our proposed method was sufficient to achieve detection of ZnO NW in the 'real-world' environment. PMID:27479871

  16. A pressure cooking-based DNA extraction from archival formalin fixed, paraffin embedded tissue

    PubMed Central

    Chung, Joon-Yong; Yi, Joo Mi; Xie, Ran; Brown, Victoria; Lee, Olivia; Ahuja, Nita; Braunschweig, Till; Hewitt, Stephen M.

    2012-01-01

    As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high quality DNA extraction from archival FFPE tissue specimen remains complex and time consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit; O.D 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with MSP can be used for epigenetic studies with the advantages of rapidity and high quality, and may contribute to the development of biomarkers in clinical studies. PMID:22449494

  17. Beyond DNA origami: A look on the bright future of nucleic acid nanotechnology

    PubMed Central

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J.

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA ”staples”. This revolutionary approach has led to the creation of a multitude of 2D and 3D scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. PMID:22131292

  18. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli. PMID:15304751

  19. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    SciTech Connect

    Jackson, Christopher B.; Gallati, Sabina; Schaller, Andre

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  20. Formation of Silver Nanoclusters from a DNA Template Containing Ag(I)-Mediated Base Pairs

    PubMed Central

    Léon, J. Christian; Stegemann, Linda; Peterlechner, Martin; Litau, Stefanie; Wilde, Gerhard; Strassert, Cristian A.; Müller, Jens

    2016-01-01

    A series of DNA double helices containing different numbers of silver(I)-mediated base pairs involving the artificial nucleobases imidazole or 2-methylimidazole has been applied for the generation of DNA-templated silver nanoclusters. The original Ag(I)-containing nucleic acids as well as the resulting nanoclusters and nanoparticles have been characterized by means of UV/Vis spectroscopy, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and transmission electron microscopy (TEM). The results show for the first time that metal-mediated base pairs can be used for the templated growth of metal nanoclusters. PMID:27034627

  1. Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination.

    PubMed

    Henley, Robert Y; Vazquez-Pagan, Ana G; Johnson, Michael; Kanavarioti, Anastassia; Wanunu, Meni

    2015-01-01

    Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation. PMID:26657869

  2. Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination

    PubMed Central

    Henley, Robert Y.; Vazquez-Pagan, Ana G.; Johnson, Michael; Kanavarioti, Anastassia; Wanunu, Meni

    2015-01-01

    Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore. While this approach has shown great promise, accuracy is not optimal because the four bases are chemically comparable to one another, leading to small differences in current obstruction. Nucleobase-specific chemical tagging can be a viable approach to enhancing the contrast between different bases in the sequence. Herein we show that covalent modification of one or both of the pyrimidine bases by an osmium bipyridine complex leads to measureable differences in the blockade amplitudes of DNA molecules. We qualitatively determine the degree of osmylation of a DNA strand by passing it through a solid-state nanopore, and are thus able to gauge T and C base content. In addition, we show that osmium bipyridine reacts with dsDNA, leading to substantially different current blockade levels than exhibited for bare dsDNA. This work serves as a proof of principle for nanopore sequencing and mapping via base-specific DNA osmylation. PMID:26657869

  3. A novel chaotic based image encryption using a hybrid model of deoxyribonucleic acid and cellular automata

    NASA Astrophysics Data System (ADS)

    Enayatifar, Rasul; Sadaei, Hossein Javedani; Abdullah, Abdul Hanan; Lee, Malrey; Isnin, Ismail Fauzi

    2015-08-01

    Currently, there are many studies have conducted on developing security of the digital image in order to protect such data while they are sending on the internet. This work aims to propose a new approach based on a hybrid model of the Tinkerbell chaotic map, deoxyribonucleic acid (DNA) and cellular automata (CA). DNA rules, DNA sequence XOR operator and CA rules are used simultaneously to encrypt the plain-image pixels. To determine rule number in DNA sequence and also CA, a 2-dimension Tinkerbell chaotic map is employed. Experimental results and computer simulations, both confirm that the proposed scheme not only demonstrates outstanding encryption, but also resists various typical attacks.

  4. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  5. Associations between whole peripheral blood fatty acids and DNA methylation in humans

    PubMed Central

    de la Rocha, Carmen; Pérez-Mojica, J. Eduardo; León, Silvia Zenteno-De; Cervantes-Paz, Braulio; Tristán-Flores, Fabiola E.; Rodríguez-Ríos, Dalia; Molina-Torres, Jorge; Ramírez-Chávez, Enrique; Alvarado-Caudillo, Yolanda; Carmona, F. Javier; Esteller, Manel; Hernández-Rivas, Rosaura; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5’UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage. PMID:27181711

  6. Associations between whole peripheral blood fatty acids and DNA methylation in humans.

    PubMed

    de la Rocha, Carmen; Pérez-Mojica, J Eduardo; León, Silvia Zenteno-De; Cervantes-Paz, Braulio; Tristán-Flores, Fabiola E; Rodríguez-Ríos, Dalia; Molina-Torres, Jorge; Ramírez-Chávez, Enrique; Alvarado-Caudillo, Yolanda; Carmona, F Javier; Esteller, Manel; Hernández-Rivas, Rosaura; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio; Lund, Gertrud

    2016-01-01

    Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage. PMID:27181711

  7. DNA nanostructures based biosensor for the determination of aromatic compounds.

    PubMed

    Gayathri, S Baby; Kamaraj, P; Arthanareeswari, M; Devikala, S

    2015-10-15

    Graphite electrode was modified using multi-walled carbon nanotubes (MWCNT), chitosan (CS), glutaraldehyde (GTA) and DNA nanostructures (nsDNA). DNA nanostructures of 50 nm in size were produced from single DNA template sequence using a simple two step procedure and were confirmed using TEM and AFM analysis. The modified electrode was applied to the electrochemical detection of aromatic compounds using EIS. The modified electrode was characterized using differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For comparison, electrochemical results derived from single stranded (50 bp length) and double stranded (50 bp length) DNA based biosensors were used. The results indicate that the modified electrode prior to nsDNA immobilization provides a viable platform that effectively promotes electron transfer between nsDNA and the electrode. The mode of binding between the nsDNA and aromatic compounds was investigated using EIS, indicating that the dominant interaction is non-covalent. nsDNA based biosensor was observed to act as an efficient biosensor in selective and sensitive identification of aromatic compounds. PMID:25982727

  8. A Direct, Biomass-Based Synthesis of Benzoic Acid: Formic Acid-Mediated Deoxygenation of the Glucose-Derived Materials Quinic Acid and Shikimic Acid

    SciTech Connect

    Arceo, Elena; Ellman, Jonathan; Bergman, Robert

    2010-05-03

    An alternative biomass-based route to benzoic acid from the renewable starting materials quinic acid and shikimic acid is described. Benzoic acid is obtained selectively using a highly efficient, one-step formic acid-mediated deoxygenation method.

  9. Natural DNA-modified graphene/Pd nanoparticles as highly active catalyst for formic acid electro-oxidation and for the Suzuki reaction.

    PubMed

    Qu, Konggang; Wu, Li; Ren, Jinsong; Qu, Xiaogang

    2012-09-26

    Natural DNA has been considered as a building block for developing novel functional materials. It is abundant, renewable, and biodegradable and has a well-defined structure and conformation with many unique features, which are difficult to find in other polymers. Herein, calf thymus DNA modified graphene/Pd nanoparticle (DNA-G-Pd) hybrid materials are constructed for the first time using DNA as a mediator, and the prepared DNA-G-Pd hybrid shows high catalytic activity for fuel cell formic acid electro-oxidation and for organic Suzuki reaction. The main advantages of using DNA are not only because the aromatic nucleobases in DNA can interact through π-π stacking with graphene basal surface but also because they can chelate Pd via dative bonding in such defined sites along the DNA lattice. Our results indicate that isolated, homogeneous, and ultrafine spherical Pd nanoparticles are densely in situ decorated on DNA-modified graphene surfaces with high stability and dispersibility. The prepared DNA-G-Pd hybrid has much greater activity and durability for formic acid electro-oxidation than the commercial Pd/C catalyst and polyvinylpyrrolidone-mediated graphene/Pd nanoparticle (PVP-G-Pd) hybrid used for direct formic acid fuel cells (DFAFCs). Besides, the DNA-G-Pd hybrid can also be an efficient and recyclable catalyst for the organic Suzuki reaction in aqueous solution under aerobic conditions without any preactivation. Since DNA can chelate various transition metal cations, this proof-of-concept protocol provides the possibility for the tailored design of other novel catalytic materials based on graphene with full exploitation of their properties. PMID:22973944

  10. Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles.

    PubMed

    Jin, Mingliang; Liu, Xia; van den Berg, Albert; Zhou, Guofu; Shui, Lingling

    2016-08-19

    Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10(-18) mol l(-1) for t-DNA has been achieved. PMID:27378514

  11. Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Jin, Mingliang; Liu, Xia; van den Berg, Albert; Zhou, Guofu; Shui, Lingling

    2016-08-01

    Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10‑18 mol l‑1 for t-DNA has been achieved.

  12. Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy

    PubMed Central

    Firozi, P; Zhang, W; Chen, L; Quiocho, FA; Worley, KC; Templeton, NS

    2012-01-01

    Polysaccharide contaminants in plasmid DNA, including current good manufacturing practices (cGMP) clinical preparations, must be removed to provide the greatest safety and efficacy for use in gene therapy and other clinical applications. We developed assays and methods for the detection and removal of these polysaccharides, our Super Clean DNA (SC-DNA) process, and have shown that these contaminants in plasmid DNA preparations are responsible for toxicity observed post-injection in animals. Furthermore, these contaminants limit the efficacy of low and high doses of plasmid DNA administered by numerous delivery routes. In particular, colanic acid (CA) that is mainly long-chained, branched and has high molecular weight (MW) is most refractory when complexed to cationic delivery vehicles and injected intravenously (IV). Because CA is often extremely large and tightly intertwined with DNA, it must be degraded, in order, to be effectively removed. We have produced a recombinant, truncated colanic acid degrading enzyme (CAE) that successfully accomplishes this task. Initially, we isolated a newly identified CAE from a bacteriophage that required truncation for proper folding while retaining its full enzymatic activity during production. Any plasmid DNA preparation can be digested with CAE and further purified, providing a critical advance to non-viral gene therapy. PMID:20664542

  13. The impact of α-hydrazino acids embedded in short fluorescent peptides on peptide interactions with DNA and RNA.

    PubMed

    Suć, Josipa; Tumir, Lidija-Marija; Glavaš-Obrovac, Ljubica; Jukić, Marijana; Piantanida, Ivo; Jerić, Ivanka

    2016-06-01

    A series of novel hydrazino-based peptidomimetics and analogues comprising N-terminal lysine and C-terminal phenanthridinyl-l-alanine were prepared. The presented results demonstrate the up to now unknown possibility to finely modulate peptide interactions with DNA/RNA by α-hydrazino group insertion and how the different positioning of two α-hydrazino groups in peptides controls binding to various double stranded and single stranded DNA and RNA. All peptidomimetics bind with 1-10 micromolar affinity to ds-DNA/RNA, whereby the binding mode is a combination of electrostatic interactions and hydrophobic interactions within DNA/RNA grooves. Insertion of the α-hydrazino group into the peptide systematically decreased its fluorimetric response to DNA/RNA binding in the order: mono-hydrazino < alternating-hydrazino < sequential-hydrazino group. Binding studies of ss-polynucleotides suggest intercalation of phenanthridine between polynucleotide bases, whereby affinity and fluorimetric response decrease with the number of α-hydrazino groups in the peptide sequence. Particularly interesting was the interaction of two sequential α-hydrazino acids-peptidomimetic with poly rG, characterised by a specific strong increase of CD bands, while all other peptide/ssRNA combinations gave only a CD-band decrease. All mentioned interactions could also be reversibly controlled by adjusting the pH, due to the protonation of the fluorophore. PMID:27161341

  14. DNA Targeting Sequence Improves Magnetic Nanoparticle-Based Plasmid DNA Transfection Efficiency in Model Neurons

    PubMed Central

    Vernon, Matthew M.; Dean, David A.; Dobson, Jon

    2015-01-01

    Efficient non-viral plasmid DNA transfection of most stem cells, progenitor cells and primary cell lines currently presents an obstacle for many applications within gene therapy research. From a standpoint of efficiency and cell viability, magnetic nanoparticle-based DNA transfection is a promising gene vectoring technique because it has demonstrated rapid and improved transfection outcomes when compared to alternative non-viral methods. Recently, our research group introduced oscillating magnet arrays that resulted in further improvements to this novel plasmid DNA (pDNA) vectoring technology. Continued improvements to nanomagnetic transfection techniques have focused primarily on magnetic nanoparticle (MNP) functionalization and transfection parameter optimization: cell confluence, growth media, serum starvation, magnet oscillation parameters, etc. Noting that none of these parameters can assist in the nuclear translocation of delivered pDNA following MNP-pDNA complex dissociation in the cell’s cytoplasm, inclusion of a cassette feature for pDNA nuclear translocation is theoretically justified. In this study incorporation of a DNA targeting sequence (DTS) feature in the transfecting plasmid improved transfection efficiency in model neurons, presumably from increased nuclear translocation. This observation became most apparent when comparing the response of the dividing SH-SY5Y precursor cell to the non-dividing and differentiated SH-SY5Y neuroblastoma cells. PMID:26287182

  15. Sequence-specific DNA damage induced by ultraviolet A-irradiated folic acid via its photolysis product.

    PubMed

    Hirakawa, Kazutaka; Suzuki, Hiroyuki; Oikawa, Shinji; Kawanishi, Shosuke

    2003-02-15

    DNA damage mediated by photosensitizers participates in solar carcinogenesis. Fluorescence measurement and high-performance liquid chromatography analysis demonstrated that photoirradiated folic acid, one of the photosensitizers in cells, generates pterine-6-carboxylic acid (PCA). Experiments using 32P-labeled DNA fragments obtained from a human gene showed that ultraviolet A-irradiated folic acid or PCA caused DNA cleavage specifically at consecutive G residues in double-stranded DNA after Escherichia coli formamidopyrimidine-DNA glycosylase or piperidine treatment. The amount of 8-oxo-7,8-dihydro-2(')-deoxyguanosine formed through this DNA photoreaction in double-stranded DNA exceeded that in single-stranded DNA. Kinetic studies suggested that DNA damage is caused mainly by photoexcited PCA generated from folic acid rather than by folic acid itself. In conclusion, photoirradiated folic acid generates PCA, which induces DNA photooxidation specifically at consecutive G residues through electron transfer. Excess intake of folic acid supplements may increase a risk of skin cancer by solar ultraviolet light. PMID:12573286

  16. Size-Expanded yDNA bases: An Ab Initio Study

    SciTech Connect

    Fuentes-Cabrera, Miguel A; Sumpter, Bobby G; Lipkowski, Pawel; Wells, Jack C

    2006-01-01

    xDNA and yDNA are new classes of synthetic nucleic acids characterized by having base-pairs with one of the bases larger than the natural congeners. Here these larger bases are called x- and y-bases. We recently investigated and reported the structural and electronic properties of the x-bases (Fuentes-Cabrera et al. J. Phys. Chem. B 2005, 109, 21135-21139). Here we extend this study by investigating the structure and electronic properties of the y-bases. These studies are framed within our interest that xDNA and yDNA could function as nanowires, for they could have smaller HOMO-LUMO gaps than natural DNA. The limited amount of experimental structural data in these synthetic duplexes makes it necessary to first understand smaller models and, subsequently, to use that information to build larger models. In this paper, we report the results on the chemical and electronic structure of the y-bases. In particular, we predict that the y-bases have smaller HOMO-LUMO gaps than their natural congeners, which is an encouraging result for it indicates that yDNA could have a smaller HOMO-LUMO gap than natural DNA. Also, we predict that the y-bases are less planar than the natural ones. Particularly interesting are our results corresponding to yG. Our studies show that yG is unstable because it is less aromatic and has a Coulombic repulsion that involves the amino group, as compared with a more stable tautomer. However, yG has a very small HOMO-LUMO gap, the smallest of all the size-expanded bases we have considered. The results of this study provide useful information that may allow the synthesis of an yG-mimic that is stable and has a small HOMO-LUMO gap.

  17. Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

    PubMed Central

    Kuramae, Eiko E.; Verbruggen, Erik; Hillekens, Remy; de Hollander, Mattias; Röling, Wilfred F. M.; van der Heijden, Marcel G. A.; Kowalchuk, George A.

    2013-01-01

    We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time. PMID:23875012

  18. Multiple Base Substitution Corrections in DNA Sequence Evolution

    NASA Astrophysics Data System (ADS)

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

    We discuss the Jukes and Cantor's one-parameter model and Kimura's two-parameter model unability to describe evolution of asymmetric DNA molecules. The standard distance measure between two DNA sequences, which is the number of substitutions per site, should include the effect of multiple base substitutions separately for each type of the base. Otherwise, the respective tables of substitutions cannot reconstruct the asymmetric DNA molecule with respect to the composition. Basing on Kimura's neutral theory, we have derived a linear law for the correlation of the mean survival time of nucleotides under constant mutation pressure and their fraction in the genome. According to the law, the corrections to Kimura's theory have been discussed to describe evolution of genomes with asymmetric nucleotide composition. We consider the particular case of the strongly asymmetric Borrelia burgdorferi genome and we discuss in detail the corrections, which should be introduced into the distance measure between two DNA sequences to include multiple base substitutions.

  19. [Kidney, Fluid, and Acid-Base Balance].

    PubMed

    Shioji, Naohiro; Hayashi, Masao; Morimatsu, Hiroshi

    2016-05-01

    Kidneys play an important role to maintain human homeostasis. They contribute to maintain body fluid, electrolytes, and acid-base balance. Especially in fluid control, we, physicians can intervene body fluid balance using fluid resuscitation and diuretics. In recent years, one type of fluid resuscitation, hydroxyl ethyl starch has been extensively studied in the field of intensive care. Although their effects on fluid resuscitation are reasonable, serious complications such as kidney injury requiring renal replacement therapy occur frequently. Now we have to pay more attention to this important complication. Another topic of fluid management is tolvaptan, a selective vasopressin-2 receptor antagonist Recent randomized trial suggested that tolvaptan has a similar supportive effect for fluid control and more cost effective compared to carperitide. In recent years, Stewart approach is recognized as one important tool to assess acid-base balance in critically ill patients. This approach has great value, especially to understand metabolic components in acid-base balance. Even for assessing the effects of kidneys on acid-base balance, this approach gives us interesting insight. We should appropriately use this new approach to treat acid-base abnormality in critically ill patients. PMID:27319095

  20. DNA sequence analysis with droplet-based microfluidics

    PubMed Central

    Abate, Adam R.; Hung, Tony; Sperling, Ralph A.; Mary, Pascaline; Rotem, Assaf; Agresti, Jeremy J.; Weiner, Michael A.; Weitz, David A.

    2014-01-01

    Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence. PMID:24185402

  1. Operation of a DNA-Based Autocatalytic Network in Serum

    NASA Astrophysics Data System (ADS)

    Graugnard, Elton; Cox, Amber; Lee, Jeunghoon; Jorcyk, Cheryl; Yurke, Bernard; Hughes, William L.

    The potential for inferring the presence of cancer by the detection of miRNA in human blood has motivated research into the design and operation of DNA-based chemical amplifiers that can operate in bodily fluids. As a first step toward this goal, we have tested the operation of a DNA-based autocatalytic network in human serum and mouse serum. With the addition of sodium dodecyl sulfate to prevent degradation by nuclease activity, the network was found to operate successfully with both DNA and RNA catalysts.

  2. [Determination of body fluid based on analysis of nucleic acids].

    PubMed

    Korabečná, Marie

    2015-01-01

    Recent methodological approaches of molecular genetics allow isolation of nucleic acids (DNA and RNA) from negligible forensic samples. Analysis of these molecules may be used not only for individual identification based on DNA profiling but also for the detection of origin of the body fluid which (alone or in mixture with other body fluids) forms the examined biological trace. Such an examination can contribute to the evaluation of procedural, technical and tactical value of the trace. Molecular genetic approaches discussed in the review offer new possibilities in comparison with traditional spectrum of chemical, immunological and spectroscopic tests especially with regard to the interpretation of mixtures of biological fluids and to the confirmatory character of the tests. Approaches based on reverse transcription of tissue specific mRNA and their subsequent polymerase chain reaction (PCR) and fragmentation analysis are applicable on samples containing minimal amounts of biological material. Methods for body fluid discrimination based on examination of microRNA in samples provided so far confusing results therefore further development in this field is needed. The examination of tissue specific methylation of nucleotides in selected gene sequences seems to represent a promising enrichment of the methodological spectrum. The detection of DNA sequences of tissue related bacteria has been established and it provides satisfactory results mainly in combination with above mentioned methodological approaches. PMID:26419517

  3. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

    NASA Astrophysics Data System (ADS)

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  4. Artifacts associated with the measurement of oxidized DNA bases.

    PubMed Central

    Cadet, J; Douki, T; Ravanat, J L

    1997-01-01

    In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay. Images Figure 1. PMID:9349826

  5. cDNA-derived amino-acid sequence of a land turtle (Geochelone carbonaria) beta-chain hemoglobin.

    PubMed

    Bordin, S; Meza, A N; Saad, S T; Ogo, S H; Costa, F F

    1997-06-01

    The cDNA sequence encoding the turtle Geochelone carbonaria beta-chain was determinated. The isolation of hemoglobin mRNA was based on degenerate primers' PCR in combination with 5'- and 3'-RACE protocol. The full length cDNA is 615 bp with the ATG start codon at position 53 and TGA stop codon at position 495; The AATAAA polyadenylation signal is found at position 599. The deduced polypeptyde contains 146 amino-acid residues. The predicted amino acid sequence shares 83% identity with the beta-globin of a related specie, the aquatic turtle C. p. belli. Otherwise, identity is higher when compared with chicken beta-Hb (80%) than with other reptilian orders (Squamata, 69%, and Crocodilia, 61%). Compared with human HbA, there is 67% identity, and at least three amino acid substitutions could be of some functional significance (Glu43 beta-->Ser, His116 beta-->Thr and His143 beta-->Leu). To our knowledge this represents the first cDNA sequence of a reptile globin gene described. PMID:9238523

  6. Amino acid sequence of the serine-repeat antigen (SERA) of Plasmodium falciparum determined from cloned cDNA.

    PubMed

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1988-09-01

    We report the isolation of cDNA clones for a Plasmodium falciparum gene that encodes the complete amino acid sequence of a previously identified exported blood stage antigen. The Mr of this antigen protein had been determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis analysis, by different workers, to be 113,000, 126,000, and 140,000. We show, by cDNA nucleotide sequence analysis, that this antigen gene encodes a 989 amino acid protein (111 kDa) that contains a potential signal peptide, but not a membrane anchor domain. In the FCR3 strain the serine content of the protein was 11%, of which 57% of the serine residues were localized within a 201 amino acid sequence that included 35 consecutive serine residues. The protein also contained three possible N-linked glycosylation sites and numerous possible O-linked glycosylation sites. The mRNA was abundant during late trophozoite-schizont parasite stages. We propose to identity this antigen, which had been called p126, by the acronym SERA, serine-repeat antigen, based on its complete structure. The usefulness of the cloned cDNA as a source of a possible malaria vaccine is considered in view of the previously demonstrated ability of the antigen to induce parasite-inhibitory antibodies and a protective immune response in Saimiri monkeys. PMID:2847041

  7. Label-free detection of DNA single-base mismatches using a simple reflectance-based optical technique.

    PubMed

    Nava, G; Ceccarello, E; Giavazzi, F; Salina, M; Damin, F; Chiari, M; Buscaglia, M; Bellini, T; Zanchetta, G

    2016-05-21

    Rapid and quantitative detection of the binding of nucleic acids to surface-immobilized probes remains a challenge in many biomedical applications. We investigated the hybridization of a set of fully complementary and defected 12-base long DNA oligomers by using the Reflective Phantom Interface (RPI), a recently developed multiplexed label-free detection technique. Based on the simple measurement of reflected light intensity, this technology enables to quantify the hybridization directly as it occurs on the surface with a sensitivity of 10 pg mm(-2). We found a strong effect of single-base mismatches and of their location on hybridization kinetics and equilibrium binding. In line with previous studies, we found that DNA-DNA binding is weaker on a surface than in the bulk. Our data indicate that this effect is a consequence of weak nonspecific binding of the probes to the surface. PMID:27122358

  8. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

    PubMed

    Komor, Alexis C; Kim, Yongjoo B; Packer, Michael S; Zuris, John A; Liu, David R

    2016-05-19

    Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations. PMID:27096365

  9. DNA Aptamer Generation by Genetic Alphabet Expansion SELEX (ExSELEX) Using an Unnatural Base Pair System.

    PubMed

    Kimoto, Michiko; Matsunaga, Ken-ichiro; Hirao, Ichiro

    2016-01-01

    Genetic alphabet expansion of DNA using unnatural base pair systems is expected to provide a wide variety of novel tools and methods. Recent rapid progress in this area has enabled the creation of several types of unnatural base pairs that function as a third base pair in polymerase reactions. Presently, a major topic is whether the genetic alphabet expansion system actually increases nucleic acid functionalities. We recently applied our unnatural base pair system to in vitro selection (SELEX), using a DNA library containing four natural bases and an unnatural base, and succeeded in the generation of high-affinity DNA aptamers that specifically bind to target proteins. Only a few hydrophobic unnatural bases greatly augmented the affinity of the aptamers. Here, we describe a new approach (genetic alphabet Expansion SELEX, ExSELEX), using our hydrophobic unnatural base pair system for high affinity DNA aptamer generation. PMID:26552815

  10. DNA immobilization on a polypyrrole nanofiber modified electrode and its interaction with salicylic acid/aspirin.

    PubMed

    Yousef Elahi, M; Bathaie, S Z; Kazemi, S H; Mousavi, M F

    2011-04-15

    A double-stranded calf thymus DNA (dsDNA) was physisorbed onto a polypyrrole (PPy) nanofiber film that had been electrochemically deposited onto a Pt electrode. The surface morphology of the polymeric film was characterized using scanning electron microscopy (SEM). The electrochemical characteristics of the PPy film and the DNA deposited onto the PPy modified electrode were investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). Then the interaction of DNA with salicylic acid (SA) and acetylsalicylic acid (ASA), or aspirin, was studied on the electrode surface with DPV. An increase in the DPV current was observed due to the oxidation of guanine, which decreased with the increasing concentrations of the ligands. The interactions of SA and ASA with the DNA follow the saturation isotherm behavior. The binding constants of these interactions were 1.15×10(4)M for SA and 7.46×10(5)M for ASA. The numbers of binding sites of SA and ASA on DNA were approximately 0.8 and 0.6, respectively. The linear dynamic ranges of the sensors were 0.1-2μM (r(2)=0.996) and 0.05-1mM (r(2)=0.996) with limits of detection of 8.62×10(-1) and 5.24×10(-6)μM for SA and ASA, respectively. PMID:21236237

  11. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    EPA Science Inventory

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin.

    Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  12. DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles.

    PubMed

    Liu, Pei; Wang, Dandan; Zhou, Yunlei; Wang, Haiyan; Yin, Huanshun; Ai, Shiyun

    2016-06-15

    DNA methyltransferase (MTase) has received a large amount of attention due to its catalyzation of DNA methylation in both eukaryotes and prokaryotes, which has a close relationship to cancer and bacterial diseases. Herein, a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine (polyA) DNA with a gold nanoparticles functioned glassy carbon electrode (AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue (MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in real biological matrices and inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery. PMID:26807517

  13. Nanobiodevices for fast DNA separation and detection toward nanopore-based DNA sequencing

    NASA Astrophysics Data System (ADS)

    Kaji, Noritada; Yasui, Takao; Baba, Yoshinobu

    2014-03-01

    There is an increasing demand for using micro- and nanofabricated structures as tools for separation, manipulation, detection and analysis of biomolecules such as DNA and proteins. So far, we have developed fabrication techniques for constructing several types of nanostructures on quartz substrate for biomolecules separation, e.g., nanopillar and nanowall array structures, and demonstrated their analytical performances. Some important findings were that the nanopillar array pattern could control the DNA separation mode and electroosmotic flows in the nanopillar array structures were reduced according to the nanopillar spacing. Since these small confined nanospaces are suitable for manipulating biomolecules at a single molecule level, several approaches have been tried to analyze DNA denaturation and DNA-protein interactions in parallel. However, it is difficult to say that the observed phenomena reflect an intrinsic DNA property or DNA-protein interaction manner because all these approaches requires fluorescently labeled DNA molecules for observation. To address these issues, we are trying to develop a novel nanostructure-based and label-free detection system to integrate a biomolecule separation media and a detection system on a single chip.

  14. A CLIQUE algorithm using DNA computing techniques based on closed-circle DNA sequences.

    PubMed

    Zhang, Hongyan; Liu, Xiyu

    2011-07-01

    DNA computing has been applied in broad fields such as graph theory, finite state problems, and combinatorial problem. DNA computing approaches are more suitable used to solve many combinatorial problems because of the vast parallelism and high-density storage. The CLIQUE algorithm is one of the gird-based clustering techniques for spatial data. It is the combinatorial problem of the density cells. Therefore we utilize DNA computing using the closed-circle DNA sequences to execute the CLIQUE algorithm for the two-dimensional data. In our study, the process of clustering becomes a parallel bio-chemical reaction and the DNA sequences representing the marked cells can be combined to form a closed-circle DNA sequences. This strategy is a new application of DNA computing. Although the strategy is only for the two-dimensional data, it provides a new idea to consider the grids to be vertexes in a graph and transform the search problem into a combinatorial problem. PMID:21511001

  15. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    PubMed

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  16. The cell as the smallest DNA-based molecular computer.

    PubMed

    Ji, S

    1999-10-01

    The pioneering work of Adleman (1994) demonstrated that DNA molecules in test tubes can be manipulated to perform a certain type of mathematical computation. This has stimulated a theoretical interest in the possibility of constructing DNA-based molecular computers. To gauge the practicality of realizing such microscopic computers, it was thought necessary to learn as much as possible from the biology of the living cell--presently the only known DNA-based molecular computer in existence. Here the recently developed theoretical model of the living cell (the Bhopalator) and its associated theories (e.g. cell language), principles, laws and concepts (e.g. conformons, IDS's) are briefly reviewed and summarized in the form of a set of five laws of 'molecular semiotics' (synonyms include 'microsemiotics', 'cellular semiotics', or 'cytosemiotics') the study of signs mediating measurement, computation, and communication on the cellular and molecular levels. Hopefully, these laws will find practical applications in designing DNA-based computing systems. PMID:10636037

  17. Charge Transport across DNA-Based Three-Way Junctions.

    PubMed

    Young, Ryan M; Singh, Arunoday P N; Thazhathveetil, Arun K; Cho, Vincent Y; Zhang, Yuqi; Renaud, Nicolas; Grozema, Ferdinand C; Beratan, David N; Ratner, Mark A; Schatz, George C; Berlin, Yuri A; Lewis, Frederick D; Wasielewski, Michael R

    2015-04-22

    DNA-based molecular electronics will require charges to be transported from one site within a 2D or 3D architecture to another. While this has been shown previously in linear, π-stacked DNA sequences, the dynamics and efficiency of charge transport across DNA three-way junction (3WJ) have yet to be determined. Here, we present an investigation of hole transport and trapping across a DNA-based three-way junction systems by a combination of femtosecond transient absorption spectroscopy and molecular dynamics simulations. Hole transport across the junction is proposed to be gated by conformational fluctuations in the ground state which bring the transiently populated hole carrier nucleobases into better aligned geometries on the nanosecond time scale, thus modulating the π-π electronic coupling along the base pair sequence. PMID:25822073

  18. Carbon-based electrode materials for DNA electroanalysis.

    PubMed

    Kato, Dai; Niwa, Osamu

    2013-01-01

    This review addresses recent studies of newly developed carbon-based electrode materials and their use for DNA electroanalysis. Recently, new carbon materials including carbon nanotubes (CNT), graphene and diamond-based nanocarbon electrodes have been actively developed as sensing platforms for biomolecules, such as DNA and proteins. Electrochemical techniques using these new material-based electrodes can provide very simple and inexpensive sensing platforms, and so are expected to be used as one of the "post-light" DNA analysis methods, which include coulometric detection, amperometric detection with electroactive tags or intercalators, and potentiometric detection. DNA electroanalysis using these new carbon materials is summarized in view of recent advances on electrodes. PMID:23574663

  19. Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

    PubMed Central

    Moreno, Pedro M. D.; Geny, Sylvain; Pabon, Y. Vladimir; Bergquist, Helen; Zaghloul, Eman M.; Rocha, Cristina S. J.; Oprea, Iulian I.; Bestas, Burcu; Andaloussi, Samir EL; Jørgensen, Per T.; Pedersen, Erik B.; Lundin, Karin E.; Zain, Rula; Wengel, Jesper; Smith, C. I. Edvard

    2013-01-01

    In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON—bisLNA—with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson–Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes. PMID:23345620

  20. Highly sensitive DNA sensor based on polypyrrole nanowire

    NASA Astrophysics Data System (ADS)

    Mai, Anh Tuan; Duc, Thanh Pham; Thi, Xuan Chu; Nguyen, Minh Hieu; Nguyen, Hoang Hai

    2014-08-01

    This paper describes the development of a DNA sensor based on polypyrrole nanowire. By using potentiostatic technique, in the presence of gelatin as the soft mold, the polypyrrole nanowires were synthesized on the surface of the micro-sensor. The surface enhanced Raman spectroscopy shows that the Nsbnd H ends of the polypyrrole nanowires orientate upward from the surface facilitating the DNA probe immobilization through the simple linkage with the phosphate groups of the probe DNA. The label-free signal readout was carried out by lock-in amplifier technique. The response time of the DNA sensor is 10 s and the measurement time was 5 min. The lowest detectable concentration of Escherichia coli DNA was 0.1 nM.

  1. Introduction to DNA-Based Genetic Diagnostics

    PubMed Central

    Glickman, Richard M.; Phillips, M. Ann; Glickman, Barry W.

    1988-01-01

    Molecular biology and recombinant DNA technology are beginning to have an effect on the medical health care field, particularly in the area of clinical genetics. Dramatic improvements in the prerequisite technology are in the process of being transferred from the research lab to routine clinical laboratories. The general practitioner, along with his genetic diagnostic colleagues, can soon expect to have access to accurate and reliable diagnostic assays for a wide variety of genetic disorders. This article introduces the basic technologies involved in genetic diagnostics and provides an indication both of their limitations and their future potential. PMID:21253094

  2. Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

    PubMed

    Ito, Tomoko; Iida-Tanaka, Naoko; Koyama, Yoshiyuki

    2008-05-01

    Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells. PMID:18446606

  3. Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids.

    PubMed

    Kato, Takuma; Yamashita, Hiroko; Misawa, Takashi; Nishida, Koyo; Kurihara, Masaaki; Tanaka, Masakazu; Demizu, Yosuke; Oba, Makoto

    2016-06-15

    Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery. PMID:27132868

  4. Determination of thymine glycol residues in irradiated or oxidized DNA by formation of methylglyceric acid

    SciTech Connect

    Schellenberg, K.A.; Shaeffer, J.

    1986-05-01

    Treatment of DNA solutions with X-irradiation various oxidants including hydrogen peroxide plus ferrous ion, hydrogen peroxide plus copper ion and ascorbate, permanganate, or sonication in the presence of dissolved oxygen all produced varying amounts of thymine glycol residues. After denaturing the DNA with heat, the glycol residues were reduced and labeled at the 6 position with tritium- labeled sodium borohydride. Subsequent reaction with anhydrous methanolic HCl gave a quantitative yield of the methyl ester of methylglyceric acid, which was determined by thin layer chromatography. The method, developed using thymidine as a model, was used to ascertain the requirements for glycol formation in DNA. It was shown that hydroxyl radical generating systems, permanganate, X-irradiation, or sonication in presence of oxygen were required, but hydrogen peroxide in the absence of iron or copper and ascorbate was inactive. Application to determination of DNA damage in vivo is being explored.

  5. A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.

    PubMed

    Burrell, Matthew R; Burton, Nicolas P; Maxwell, Anthony

    2010-01-01

    We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase. PMID:19997889

  6. Gelatin- and DNA-based ionic conducting membranes for electrochromic devices

    NASA Astrophysics Data System (ADS)

    Pawlicka, A.; Firmino, A.; Vieira, D.; Sentanin, F.; Grote, J. G.; Kajzar, F.

    2009-09-01

    Gelatin and DNA are abundant natural products with very good biodegradation properties and can be used to obtain acetic acid or LiClO4-based gel polymer electrolytes (GPEs) with high ionic conductivity and good stability. This article presents the results of the ionic conductivity measurements of GPEs membranes based on crosslinked and plasticized gelatin and on plasticized DNA as well as on inserted/extracted charge density of electrochemical devices (ECDs) obtained with these samples. The membranes were analyzed by impedance spectroscopy, UV-Vis spectroscopy and the ECDs by charge density measurements, respectively. At room temperature the measured ionic conductivity of the membranes is in the range of 10-4-10-5 S/cm. It obeys predominantly an Arrhenius relationship in function of temperature. The ECD with red gelatin changed the color from red to deep red and the ECD with DNA-based electrolyte changes from transparent to blue. The inserted charge density values of these ECDs were of -3.0 mC/cm2 for the device with red gelatin and -6.6 mC/cm2 for the ECD with DNA-based electrolyte. The reverse potential application promoted a charge extraction and, as consequence, bleaching of the devices. Good ionic conductivity results combined with transparency and good adhesion to the electrodes and promising preliminary results of small ECDs have shown that gelatin and DNA-based GPEs are very promising materials to be used as gel polymer electrolytes in electrochromic devices.

  7. cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins.

    PubMed

    Iwanami, Kentaro; Mita, Hajime; Yamamoto, Yasuhiko; Fujise, Yoshihiro; Yamada, Tadasu; Suzuki, Tomohiko

    2006-10-01

    We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level. PMID:16962803

  8. Standardization of DNA extraction from methanol acetic acid fixed cytogenetic cells of cattle and buffalo.

    PubMed

    Kotikalapudi, Rosaiah; Patel, Rajesh K; Katragadda, Sanghamitra

    2013-12-01

    The aim of the study is to standardize the simple method for extracting DNA from cells fixed in fixative (3:1 ratio of methanol and acetic acid glacial) mostly used for chromosomal studies in cattle and buffaloes. These fixed cells were stored for more than 6 months at refrigerated temperature. The fixed cells were washed 2-3 times by the ice cold 1x Phosphate Buffer Saline (PBS) with pH 7.4, so that effect of fixative may be eliminated. The genomic DNA was extracted by adding cell lysis and nucleus lysis buffers. The quality and quantity of DNA were estimated. The readings of nano drop and agarose gel electrophoresis indicate good quality DNA isolated with a rapid and simple protocol routinely using in our laboratory. The method enables us to study the DNA of a cattle and buffaloes after completing cytogenetic investigation or in cases where DNA samples are otherwise not available. This protocol may be useful for molecular analysis of DNA from fixed cells palettes. PMID:24506057

  9. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    PubMed

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems. PMID:26095642

  10. Responsive DNA-based hydrogels and their applications

    PubMed Central

    Xiong, Xiangling; Zhou, Cuisong; Wu, Cuichen; Zhu, Guizhi; Chen, Zhuo; Tan, Weihong

    2015-01-01

    The term hydrogel describes a type of soft and wet material formed by crosslinked hydrophilic polymers. The distinct feature of hydrogels is their ability to absorb a large amount of water and swell. The properties of a hydrogel are usually determined by the type of polymer and crosslinker, the degree of crosslinking, and the water content. However, a group of hydrogels, called “smart hydrogels”, changes properties in response to environmental changes or external stimuli. Recently, DNA or DNA-inspired responsive hydrogels have attracted considerable attention in construction of smart hydrogels because of the intrinsic advantages of DNA. As a biological polymer, DNA is hydrophilic, biocompatible, and highly programmable by Watson-Crick base pairing. DNA can form a hydrogel by itself under certain conditions, and it can also be incorporated into synthetic polymers to form DNA-hybrid hydrogels. Functional DNAs, such as aptamers and DNAzymes, provide additional molecular recognition capabilities and versatility. In this review, we discuss DNA-based hydrogels in terms of their stimulus response, as well as their applications. PMID:23857726

  11. Optical fibre-based detection of DNA hybridization.

    PubMed

    Hine, Anna V; Chen, Xianfeng; Hughes, Marcus D; Zhou, Kaiming; Davies, Edward; Sugden, Kate; Bennion, Ian; Zhang, Lin

    2009-04-01

    A dual-peak LPFG (long-period fibre grating), inscribed in an optical fibre, has been employed to sense DNA hybridization in real time, over a 1 h period. One strand of the DNA was immobilized on the fibre, while the other was free in solution. After hybridization, the fibre was stripped and repeated detection of hybridization was achieved, so demonstrating reusability of the device. Neither strand of DNA was fluorescently or otherwise labelled. The present paper will provide an overview of our early-stage experimental data and methodology, examine the potential of fibre gratings for use as biosensors to monitor both nucleic acid and other biomolecular interactions and then give a summary of the theory and fabrication of fibre gratings from a biological standpoint. Finally, the potential of improving signal strength and possible future directions of fibre grating biosensors will be addressed. PMID:19290879

  12. Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals.

    PubMed Central

    Simandan, T; Sun, J; Dix, T A

    1998-01-01

    DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO. ) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO. oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO. radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO.) oxidations in cells. ROO. radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO. radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO. oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO. in cellular systems. PMID:9761719

  13. Proton exchange in acid-base complexes induced by reaction coordinates with heavy atom motions

    NASA Astrophysics Data System (ADS)

    Alavi, Saman; Taghikhani, Mahdi

    2012-06-01

    We extend previous work on nitric acid-ammonia and nitric acid-alkylamine complexes to illustrate that proton exchange reaction coordinates involve the rocking motion of the base moiety in many double hydrogen-bonded gas phase strong acid-strong base complexes. The complexes studied involve the biologically and atmospherically relevant glycine, formic, acetic, propionic, and sulfuric acids with ammonia/alkylamine bases. In these complexes, the magnitude of the imaginary frequencies associated with the proton exchange transition states are <400 cm-1. This contrasts with widely studied proton exchange reactions between symmetric carboxylic acid dimers or asymmetric DNA base pair and their analogs where the reaction coordinate is localized in proton motions and the magnitude of the imaginary frequencies for the transition states are >1100 cm-1. Calculations on complexes of these acids with water are performed for comparison. Variations of normal vibration modes along the reaction coordinate in the complexes are described.

  14. DNA barcode-based molecular identification system for fish species.

    PubMed

    Kim, Sungmin; Eo, Hae-Seok; Koo, Hyeyoung; Choi, Jun-Kil; Kim, Won

    2010-12-01

    In this study, we applied DNA barcoding to identify species using short DNA sequence analysis. We examined the utility of DNA barcoding by identifying 53 Korean freshwater fish species, 233 other freshwater fish species, and 1339 saltwater fish species. We successfully developed a web-based molecular identification system for fish (MISF) using a profile hidden Markov model. MISF facilitates efficient and reliable species identification, overcoming the limitations of conventional taxonomic approaches. MISF is freely accessible at http://bioinfosys.snu.ac.kr:8080/MISF/misf.jsp . PMID:21110132

  15. A Novel Image Encryption Algorithm Based on DNA Subsequence Operation

    PubMed Central

    Zhang, Qiang; Xue, Xianglian; Wei, Xiaopeng

    2012-01-01

    We present a novel image encryption algorithm based on DNA subsequence operation. Different from the traditional DNA encryption methods, our algorithm does not use complex biological operation but just uses the idea of DNA subsequence operations (such as elongation operation, truncation operation, deletion operation, etc.) combining with the logistic chaotic map to scramble the location and the value of pixel points from the image. The experimental results and security analysis show that the proposed algorithm is easy to be implemented, can get good encryption effect, has a wide secret key's space, strong sensitivity to secret key, and has the abilities of resisting exhaustive attack and statistic attack. PMID:23093912

  16. A novel image encryption algorithm based on DNA subsequence operation.

    PubMed

    Zhang, Qiang; Xue, Xianglian; Wei, Xiaopeng

    2012-01-01

    We present a novel image encryption algorithm based on DNA subsequence operation. Different from the traditional DNA encryption methods, our algorithm does not use complex biological operation but just uses the idea of DNA subsequence operations (such as elongation operation, truncation operation, deletion operation, etc.) combining with the logistic chaotic map to scramble the location and the value of pixel points from the image. The experimental results and security analysis show that the proposed algorithm is easy to be implemented, can get good encryption effect, has a wide secret key's space, strong sensitivity to secret key, and has the abilities of resisting exhaustive attack and statistic attack. PMID:23093912

  17. An Optimal Seed Based Compression Algorithm for DNA Sequences

    PubMed Central

    Gopalakrishnan, Gopakumar; Karunakaran, Muralikrishnan

    2016-01-01

    This paper proposes a seed based lossless compression algorithm to compress a DNA sequence which uses a substitution method that is similar to the LempelZiv compression scheme. The proposed method exploits the repetition structures that are inherent in DNA sequences by creating an offline dictionary which contains all such repeats along with the details of mismatches. By ensuring that only promising mismatches are allowed, the method achieves a compression ratio that is at par or better than the existing lossless DNA sequence compression algorithms. PMID:27555868

  18. A Rewritable, Random-Access DNA-Based Storage System

    PubMed Central

    Tabatabaei Yazdi, S. M. Hossein; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-01-01

    We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications. PMID:26382652

  19. A Rewritable, Random-Access DNA-Based Storage System.

    PubMed

    Yazdi, S M Hossein Tabatabaei; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-01-01

    We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications. PMID:26382652

  20. A Rewritable, Random-Access DNA-Based Storage System

    NASA Astrophysics Data System (ADS)

    Tabatabaei Yazdi, S. M. Hossein; Yuan, Yongbo; Ma, Jian; Zhao, Huimin; Milenkovic, Olgica

    2015-09-01

    We describe the first DNA-based storage architecture that enables random access to data blocks and rewriting of information stored at arbitrary locations within the blocks. The newly developed architecture overcomes drawbacks of existing read-only methods that require decoding the whole file in order to read one data fragment. Our system is based on new constrained coding techniques and accompanying DNA editing methods that ensure data reliability, specificity and sensitivity of access, and at the same time provide exceptionally high data storage capacity. As a proof of concept, we encoded parts of the Wikipedia pages of six universities in the USA, and selected and edited parts of the text written in DNA corresponding to three of these schools. The results suggest that DNA is a versatile media suitable for both ultrahigh density archival and rewritable storage applications.

  1. Subpicosecond Excited State Lifetimes in DNA Polymers Require Unstacked Bases

    NASA Astrophysics Data System (ADS)

    de La Harpe, Kimberly; Su, Charlene; Kohler, Bern

    2009-06-01

    The femtosecond lifetimes of excited states of monomeric DNA bases are the result of nuclear motions that lead to one or more conical intersections (CIs). Surprisingly, femtosecond pump-probe experiments reveal that excitations in DNA base polymers, including ones with genomic or 'natural' sequences of the four bases, decay at least an order of magnitude more slowly. Although the reasons for this dramatic change in photophysics are unclear, evidence strongly suggests that the long-lived states are exciplexes formed when an electron is partially transferred from one base to its π-stacked neighbor. Experiments also show that monomer-like subpicosecond decay to the ground state is frequently observed in many DNA oligomers and polymers in addition to exciplex formation. We will present results from high-temperature and other experiments suggesting that monomer-like, CI-mediated dynamics are only possible when unstacked bases are present.

  2. Construction of a fuzzy and Boolean logic gates based on DNA.

    PubMed

    Zadegan, Reza M; Jepsen, Mette D E; Hildebrandt, Lasse L; Birkedal, Victoria; Kjems, Jørgen

    2015-04-17

    Logic gates are devices that can perform logical operations by transforming a set of inputs into a predictable single detectable output. The hybridization properties, structure, and function of nucleic acids can be used to make DNA-based logic gates. These devices are important modules in molecular computing and biosensing. The ideal logic gate system should provide a wide selection of logical operations, and be integrable in multiple copies into more complex structures. Here we show the successful construction of a small DNA-based logic gate complex that produces fluorescent outputs corresponding to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics. PMID:25565140

  3. Heterogeneous base distribution in mitochondrial DNA of Neurospora crassa.

    PubMed Central

    Terpstra, P; Holtrop, M; Kroon, A

    1977-01-01

    The mitochondrial DNA of Neurospora crassa has a heterogeneous intramolecular base distribution. A contiguous piece, representing at least 30% of the total genome, has a G+C content that is 6% lower than the overall G+C content of the DNA. The genes for both ribosomal RNAs are contained in the remaining, relatively G+C rich, part of the genome. PMID:141040

  4. Homogeneous detection of unamplified genomic DNA sequences based on colorimetric scatter of gold nanoparticle probes

    PubMed Central

    Storhoff, James J; Lucas, Adam D; Garimella, Viswanadham; Bao, Y Paul; Müller, Uwe R

    2005-01-01

    Nucleic acid diagnostics is dominated by fluorescence-based assays that use complex and expensive enzyme-based target or signal-amplification procedures1–6. Many clinical diagnostic applications will require simpler, inexpensive assays that can be done in a screening mode. We have developed a ‘spot-and-read’ colorimetric detection method for identifying nucleic acid sequences based on the distance-dependent optical properties of gold nanoparticles. In this assay, nucleic acid targets are recognized by DNA-modified gold probes, which undergo a color change that is visually detectable when the solutions are spotted onto an illuminated glass waveguide. This scatter-based method enables detection of zeptomole quantities of nucleic acid targets without target or signal amplification when coupled to an improved hybridization method that facilitates probe-target binding in a homogeneous format. In comparison to a previously reported absorbance-based method7, this method increases detection sensitivity by over four orders of magnitude. We have applied this method to the rapid detection of mecA in methicillin-resistant Staphylococcus aureus genomic DNA samples. PMID:15170215

  5. Student Concept Changes in Acids and Bases.

    ERIC Educational Resources Information Center

    Ye, Renmin; Wells, Raymond R.

    This study focuses on student concept changes in acids and bases. Variables include field dependent level, personal independence level, interest in science or chemistry, teaching strategy, and student gender. This study of Grade 10 students (N=81) provides information relevant to secondary school chemistry learning, teaching, and concept change.…

  6. Jigsaw Cooperative Learning: Acid-Base Theories

    ERIC Educational Resources Information Center

    Tarhan, Leman; Sesen, Burcin Acar

    2012-01-01

    This study focused on investigating the effectiveness of jigsaw cooperative learning instruction on first-year undergraduates' understanding of acid-base theories. Undergraduates' opinions about jigsaw cooperative learning instruction were also investigated. The participants of this study were 38 first-year undergraduates in chemistry education…

  7. DNA damage and oxidative stress induced by acetylsalicylic acid in Daphnia magna.

    PubMed

    Gómez-Oliván, Leobardo Manuel; Galar-Martínez, Marcela; Islas-Flores, Hariz; García-Medina, Sandra; SanJuan-Reyes, Nely

    2014-08-01

    Acetylsalicylic acid is a nonsteroidal anti-inflammatory widely used due to its low cost and high effectiveness. This compound has been found in water bodies worldwide and is toxic to aquatic organisms; nevertheless its capacity to induce oxidative stress in bioindicators like Daphnia magna remains unknown. This study aimed to evaluate toxicity in D. magna induced by acetylsalicylic acid in water, using oxidative stress and DNA damage biomarkers. An acute toxicity test was conducted in order to determine the median lethal concentration (48-h LC50) and the concentrations to be used in the subsequent subacute toxicity test in which the following biomarkers were evaluated: lipid peroxidation, oxidized protein content, activity of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, and level of DNA damage. Lipid peroxidation level and oxidized protein content were significantly increased (p<0.05), and antioxidant enzymes significantly altered with respect to controls; while the DNA damage were significantly increased (p<0.05) too. In conclusion, acetylsalicylic acid induces oxidative stress and DNA damage in D. magna. PMID:24747829

  8. Computational investigation of CNT-based DNA polymerase nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    2015-03-01

    DNA polymerases are important enzymes that replicate DNA molecules with very low error rates - about one error in 105 bases. Recently, it was found that the replication process can be electrically monitored by attaching a Klenow fragment of polymerase I to the surface of a carbon nanotube and monitoring the current along the tube [1]. In this talk, we report results from computational studies on DNA polymerase nanocircuits. We have first performed classical molecular dynamics (MD) calculations to get snapshots of different enzymatic stages, particularly the open state (no DNA binding) and the closed state (DNA double helix binding). We then used density functional theory (DFT) and Keldysh non-equilibrium Green's function (NEGF) formalism to calculate transmission coefficients and currents for each enzymatic state. Our results show that the transmission spectrum and the currents change significantly when the enzyme moves from the open to the closed state. While the initial experiments did not show signal differences between dissimilar bases, the theoretical work in progress is investigating conditions where bases might have distinct signatures, which would allow for DNA sequencing.

  9. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  10. DNA from uncultured organisms as a source of 2,5-diketo-L-gluconic acid reductases.

    SciTech Connect

    Eschenfeldt, W. H.; Stols, L.; Rosenbaum, H.; Khambatta, Z. S.; Quaite, E. R.; Wu, S.; Kilgore, D. C.; Trent, J. D.; Donnelly, M. I.; Genencor International; Eastman Chemical Company

    2001-09-01

    Total DNA of a population of uncultured organisms was extracted from soil samples, and by using PCR methods, the genes encoding two different 2,5-diketo-D-gluconic acid reductases (DKGRs) were recovered. Degenerate PCR primers based on published sequence information gave internal gene fragments homologous to known DKGRs. Nested primers specific for the internal fragments were combined with random primers to amplify flanking gene fragments from the environmental DNA, and two hypothetical full-length genes were predicted from the combined sequences. Based on these predictions, specific primers were used to amplify the two complete genes in single PCRs. These genes were cloned and expressed in Escherichia coli. The purified gene products catalyzed the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid. Compared to previously described DKGRs isolated from Corynebacterium spp., these environmental reductases possessed some valuable properties. Both exhibited greater than 20-fold-higher k{sub cat}/K{sub m} values than those previously determined, primarily as a result of better binding of substrate. The K{sub m} values for the two new reductases were 57 and 67 {mu}M, versus 2 and 13 mM for the Corynebacterium enzymes. Both environmental DKGRs accepted NADH as well as NADPH as a cosubstrate; other DKGRs and most related aldo-keto reductases use only NADPH. In addition, one of the new reductases was more thermostable than known DKGRs.

  11. DNA-based methods of geochemical prospecting

    DOEpatents

    Ashby, Matthew

    2011-12-06

    The present invention relates to methods for performing surveys of the genetic diversity of a population. The invention also relates to methods for performing genetic analyses of a population. The invention further relates to methods for the creation of databases comprising the survey information and the databases created by these methods. The invention also relates to methods for analyzing the information to correlate the presence of nucleic acid markers with desired parameters in a sample. These methods have application in the fields of geochemical exploration, agriculture, bioremediation, environmental analysis, clinical microbiology, forensic science and medicine.

  12. Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence.

    PubMed

    Qiu, Wanwei; Xu, Hui; Takalkar, Sunitha; Gurung, Anant S; Liu, Bin; Zheng, Yafeng; Guo, Zebin; Baloda, Meenu; Baryeh, Kwaku; Liu, Guodong

    2015-02-15

    In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1 nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40 pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5 nM, Mao et al. Anal. Chem. 2009, 81, 1660-1668). Another important feature is that the preparation of MWCNT-DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT-antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents. PMID:25262062

  13. Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

    PubMed

    Shlyahovsky, Bella; Li, Yang; Lioubashevski, Oleg; Elbaz, Johann; Willner, Itamar

    2009-07-28

    A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold. PMID:19507821

  14. Immobilization of DNA via oligonucleotides containing an aldehyde or carboxylic acid group at the 5' terminus.

    PubMed Central

    Kremsky, J N; Wooters, J L; Dougherty, J P; Meyers, R E; Collins, M; Brown, E L

    1987-01-01

    A general method for the immobilization of DNA through its 5'-end has been developed. A synthetic oligonucleotide, modified at its 5'-end with an aldehyde or carboxylic acid, was attached to latex microspheres containing hydrazide residues. Using T4 polynucleotide ligase and an oligonucleotide splint, a single stranded 98mer was efficiently joined to the immobilized synthetic fragment. After impregnation of the latex microspheres with the fluorescent dye, Nile Red and attachment of an aldehyde 16mer, 5 X 10(5) bead-DNA conjugates could be detected with a conventional fluorimeter. Images PMID:3562241

  15. Development of an electrochemical biosensor methods based on acrylic microsphere for the determination of Arowana DNA hybridization

    NASA Astrophysics Data System (ADS)

    Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Chiang, Chew Poh

    2015-09-01

    An electrochemical method of Arowana DNA determination based of N-acrylosuccinimide (NAS) modified acrylic microsphere was fabricated. Hydrophobic succinimide functional group containing poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized with a simple one-step photopolymerization pocedure. Aminated DNA probe was covalently bonded to the succinimde functional group of the acrylic microspheres. The hybridization of the immobilized DNA probe with the complementary DNA was determined by the differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a wide linear response range to target DNA is 1.0 × 10-16 and 1.0 × 10-8 M with a lower limit of detection (LOD) of 9.46 × 10-17 M (R2 = 0.99) were calculated. This biosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices.

  16. 8-oxoguanine DNA glycosylase-1 driven DNA base excision repair: role in asthma pathogenesis

    PubMed Central

    Ba, Xueqing; Aguilera Aguirre, Leopoldo; Sur, Sanjiv; Boldogh, Istvan

    2015-01-01

    Purpose of review To provide both an overview and evidence of the potential etiology of oxidative DNA base damage and repair-signaling in chronic inflammation and histological changes associated with asthma. Recent findings Asthma is initiated/maintained by immunological, genetic/epigenetic and environmental factors. It is a world-wide health problem, as current therapies suppress symptoms rather than prevent/reverse the disease, largely due to gaps in understanding its molecular mechanisms. Inflammation, oxidative stress and DNA damage are inseparable phenomena, but their molecular roles in asthma pathogenesis are unclear. It was found that among oxidatively modified DNA bases, 8-oxoguanine (8-oxoG) is one of the most abundant, and its levels in DNA and body fluids are considered a biomarker of ongoing asthmatic processes. Free 8-oxoG forms a complex with 8-oxoguanine DNA glycosylase-1 (OGG1) and activates RAS-family GTPases that induce gene expression to mobilize innate and adaptive immune systems, along with genes regulating airway hyperplasia, hyper-responsiveness and lung remodeling in atopic and non-atopic asthma. Summary DNA’s integrity must be maintained to prevent mutation, so its continuous repair and downstream signaling “fuels” chronic inflammatory processes in asthma, and forms the basic mechanism whose elucidation will allow the development of new drug targets for the prevention/reversal of lung diseases. PMID:25486379

  17. RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology

    PubMed Central

    Chícharo, Maria Alexandra; Chícharo, Luis

    2008-01-01

    Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival) will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems. PMID:19325815

  18. Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland.

    PubMed

    Roberto, Patrícia G; Kashima, Simone; Soares, Andreimar M; Chioato, Lucimara; Faça, Victor M; Fuly, André L; Astolfi-Filho, Spartaco; Pereira, José O; França, Suzelei C

    2004-09-01

    The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities. PMID:15294287

  19. Binding-Induced DNA Nanomachines Triggered by Proteins and Nucleic Acids.

    PubMed

    Zhang, Hongquan; Lai, Maode; Zuehlke, Albert; Peng, Hanyong; Li, Xing-Fang; Le, X Chris

    2015-11-23

    We introduce the concept and operation of a binding-induced DNA nanomachine that can be activated by proteins and nucleic acids. This new type of nanomachine harnesses specific target binding to trigger assembly of separate DNA components that are otherwise unable to spontaneously assemble. Three-dimensional DNA tracks of high density are constructed on gold nanoparticles functionalized with hundreds of single-stranded oligonucleotides and tens of an affinity ligand. A DNA swing arm, free in solution, is linked to a second affinity ligand. Binding of a target molecule to the two ligands brings the swing arm to AuNP and initiates autonomous, stepwise movement of the swing arm around the AuNP surface. The movement of the swing arm, powered by enzymatic cleavage of conjugated oligonucleotides, cleaves hundreds of oligonucleotides in response to a single binding event. We demonstrate three nanomachines that are specifically activated by streptavidin, platelet-derived growth factor, and the Smallpox gene. Substituting the ligands enables the nanomachine to respond to other molecules. The new nanomachines have several unique and advantageous features over DNA nanomachines that rely on DNA self-assembly. PMID:26457803

  20. Monitoring DNA polymerase with nanotube-based nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    DNA polymerases play an important role in the process of life by accurately and efficiently replicating our genetic information. They use a single-stranded DNA as a template and incorporate nucleotides to create the full, double-stranded DNA. Recent experiments have successfully monitored this process by attaching a Klenow fragment of polymerase I to a carbon nanotube and measuring the current along the tube. Follow-up experiments have shown promise for distinguishing between DNA base pairs when nucleotide analogs are used, thus opening a new avenue for DNA sequencing. In this talk, we present results from computational studies on DNA polymerase I nanocircuits. The enzyme was first equilibrated in molecular dynamics and then density functional theory and Keldysh non-equilibrium Green's function methods were used to calculate the ballistic transmission coefficients and currents for different enzymatic states. Our results show significant change in current when the enzyme alternates between open (idle) and closed (synthesizing) states. We can also differentiate between some template bases when modified nucleotides and gate scanning are used.

  1. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles

    PubMed Central

    2016-01-01

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  2. Ultrasensitive electrochemical cocaine biosensor based on reversible DNA nanostructure.

    PubMed

    Sheng, Qinglin; Liu, Ruixiao; Zhang, Sai; Zheng, Jianbin

    2014-01-15

    We proposed an ultrasensitive electrochemical cocaine biosensor based on the three-dimensional (3D) DNA structure conversion of nanostructure from Triangular Pyramid Frustum (TPFDNA) to Equilateral Triangle (ETDNA). The presence of cocaine triggered the aptamer-composed DNA nanostructure change from "Close" to "Open", leading to obvious faradaic impedance changes. The unique properties with excellent stability and specific rigid structure of the 3D DNA nanostructure made the biosensing functions stable, sensitive, and regenerable. The Faradaic impedance responses were linearly related to cocaine concentration between 1.0 nM and 2.0 μM with a correlation coefficient of 0.993. The limit of detection was calculated to be 0.21 nM following IUPAC recommendations (3Sb/b). It is expected that the distinctive features of DNA nanostructure would make it potentially advantageous for a broad range of biosensing, bionanoelectronics, and therapeutic applications. PMID:23962705

  3. Magnetic Propulsion of Microswimmers with DNA-Based Flagellar Bundles.

    PubMed

    Maier, Alexander M; Weig, Cornelius; Oswald, Peter; Frey, Erwin; Fischer, Peer; Liedl, Tim

    2016-02-10

    We show that DNA-based self-assembly can serve as a general and flexible tool to construct artificial flagella of several micrometers in length and only tens of nanometers in diameter. By attaching the DNA flagella to biocompatible magnetic microparticles, we provide a proof of concept demonstration of hybrid structures that, when rotated in an external magnetic field, propel by means of a flagellar bundle, similar to self-propelling peritrichous bacteria. Our theoretical analysis predicts that flagellar bundles that possess a length-dependent bending stiffness should exhibit a superior swimming speed compared to swimmers with a single appendage. The DNA self-assembly method permits the realization of these improved flagellar bundles in good agreement with our quantitative model. DNA flagella with well-controlled shape could fundamentally increase the functionality of fully biocompatible nanorobots and extend the scope and complexity of active materials. PMID:26821214

  4. Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase.

    PubMed

    Abedinia, M; Layfield, R; Jones, S M; Nixon, P F; Mattick, J S

    1992-03-31

    Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wernicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species. PMID:1567394

  5. Partial base flipping is sufficient for strand slippage near DNA duplex termini.

    PubMed

    Banavali, Nilesh K

    2013-06-01

    Strand slippage is a structural mechanism by which insertion-deletion (indel) mutations are introduced during replication by polymerases. Three-dimensional atomic-resolution structural pathways are still not known for the decades-old template slippage description. The dynamic nature of the process and the higher energy intermediates involved increase the difficulty of studying these processes experimentally. In the present study, restrained and unrestrained molecular dynamics simulations, carried out using multiple nucleic acid force fields, are used to demonstrate that partial base-flipping can be sufficient for strand slippage at DNA duplex termini. Such strand slippage can occur in either strand, i.e. near either the 3' or the 5' terminus of a DNA strand, which suggests that similar structural flipping mechanisms can cause both primer and template slippage. In the repetitive mutation hot-spot sequence studied, non-canonical base-pairing with exposed DNA groove atoms of a neighboring G:C base-pair stabilizes a partially flipped state of the cytosine. For its base-pair partner guanine, a similar partially flipped metastable intermediate was not detected, and the propensity for sustained slippage was also found to be lower. This illustrates that a relatively small metastable DNA structural distortion in polymerase active sites could allow single base insertion or deletion mutations to occur, and stringent DNA groove molecular recognition may be required to maintain intrinsic DNA polymerase fidelity. The implications of a close relationship between base-pair dissociation, base unstacking, and strand slippage are discussed in the context of sequence dependence of indel mutations. PMID:23692220

  6. Evolutionary patterns of DNA base composition and correlation to polymorphisms in DNA repair systems

    PubMed Central

    Li, Xianran; Scanlon, Michael J.; Yu, Jianming

    2015-01-01

    DNA base composition is a fundamental genome feature. However, the evolutionary pattern of base composition and its potential causes have not been well understood. Here, we report findings from comparative analysis of base composition at the whole-genome level across 2210 species, the polymorphic-site level across eight population comparison sets, and the mutation-site level in 12 mutation-tracking experiments. We first demonstrate that base composition follows the individual-strand base equality rule at the genome, chromosome and polymorphic-site levels. More intriguingly, clear separation of base-composition values calculated across polymorphic sites was consistently observed between basal and derived groups, suggesting common underlying mechanisms. Individuals in the derived groups show an A&T-increase/G&C-decrease pattern compared with the basal groups. Spontaneous and induced mutation experiments indicated these patterns of base composition change can emerge across mutation sites. With base-composition across polymorphic sites as a genome phenotype, genome scans with human 1000 Genomes and HapMap3 data identified a set of significant genomic regions enriched with Gene Ontology terms for DNA repair. For three DNA repair genes (BRIP1, PMS2P3 and TTDN), ENCODE data provided evidence for interaction between genomic regions containing these genes and regions containing the significant SNPs. Our findings provide insights into the mechanisms of genome evolution. PMID:25765652

  7. The current state of eukaryotic DNA base damage and repair.

    PubMed

    Bauer, Nicholas C; Corbett, Anita H; Doetsch, Paul W

    2015-12-01

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. PMID:26519467

  8. The current state of eukaryotic DNA base damage and repair

    PubMed Central

    Bauer, Nicholas C.; Corbett, Anita H.; Doetsch, Paul W.

    2015-01-01

    DNA damage is a natural hazard of life. The most common DNA lesions are base, sugar, and single-strand break damage resulting from oxidation, alkylation, deamination, and spontaneous hydrolysis. If left unrepaired, such lesions can become fixed in the genome as permanent mutations. Thus, evolution has led to the creation of several highly conserved, partially redundant pathways to repair or mitigate the effects of DNA base damage. The biochemical mechanisms of these pathways have been well characterized and the impact of this work was recently highlighted by the selection of Tomas Lindahl, Aziz Sancar and Paul Modrich as the recipients of the 2015 Nobel Prize in Chemistry for their seminal work in defining DNA repair pathways. However, how these repair pathways are regulated and interconnected is still being elucidated. This review focuses on the classical base excision repair and strand incision pathways in eukaryotes, considering both Saccharomyces cerevisiae and humans, and extends to some important questions and challenges facing the field of DNA base damage repair. PMID:26519467

  9. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    SciTech Connect

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-12-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA{sup III}); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA{sup III} [URO-MSC(+)] and after subsequent removal of MMA{sup III} [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA{sup III}, URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA{sup III}. URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA{sup III} exposure, suggesting the presence of MMA{sup III} is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA{sup III} results in: the induction of DNA damage that remains elevated upon removal of MMA{sup III}; increased levels of ROS that play a role in MMA{sup III} induced-DNA damage; and decreased PARP activity in the presence of MMA{sup III}.

  10. A CCD-based system for the detection of DNA in electrophoresis gels by UV absorption

    NASA Astrophysics Data System (ADS)

    Mahon, Alex R.; MacDonald, John H.; Ott, Robert J.; Mainwood, Alison

    1999-06-01

    A method and apparatus for the detection and quantification of large fragments of unlabelled nucleic acids in agarose gels is presented. The technique is based on ultraviolet (UV) absorption by nucleotides. A deuterium source illuminates individual sample lanes of an electrophoresis gel via an array of optical fibres. As DNA bands pass through the illuminated region of the gel the amount of UV light transmitted is reduced because of absorption by the DNA. During electrophoresis the regions of DNA are detected on-line using a UV-sensitive charge coupled device (CCD). As the absorption coefficient is proportional to the mass of DNA the technique is inherently quantitative. The mass of DNA in a region of the gel is approximately proportional to the integrated signal in the corresponding section of the CCD image. This system currently has a detection limit of less than 1.25 ng compared with 2-10 ng for the most popular conventional technique, ethidium bromide (EtBr) staining. In addition the DNA sample remains in its native state. The removal of the carcinogenic dye from the detection procedure greatly reduces associated biological hazards.

  11. The first proton sponge-based amino acids: synthesis, acid-base properties and some reactivity.

    PubMed

    Ozeryanskii, Valery A; Gorbacheva, Anastasia Yu; Pozharskii, Alexander F; Vlasenko, Marina P; Tereznikov, Alexander Yu; Chernov'yants, Margarita S

    2015-08-21

    The first hybrid base constructed from 1,8-bis(dimethylamino)naphthalene (proton sponge or DMAN) and glycine, N-methyl-N-(8-dimethylamino-1-naphthyl)aminoacetic acid, was synthesised in high yield and its hydrobromide was structurally characterised and used to determine the acid-base properties via potentiometric titration. It was found that the basic strength of the DMAN-glycine base (pKa = 11.57, H2O) is on the level of amidine amino acids like arginine and creatine and its structure, zwitterionic vs. neutral, based on the spectroscopic (IR, NMR, mass) and theoretical (DFT) approaches has a strong preference to the zwitterionic form. Unlike glycine, the DMAN-glycine zwitterion is N-chiral and is hydrolytically cleaved with the loss of glycolic acid on heating in DMSO. This reaction together with the mild decarboxylative conversion of proton sponge-based amino acids into 2,3-dihydroperimidinium salts under air-oxygen was monitored with the help of the DMAN-alanine amino acid. The newly devised amino acids are unique as they combine fluorescence, strongly basic and redox-active properties. PMID:26159785

  12. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    PubMed

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection. PMID:26202628

  13. Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects

    PubMed Central

    Guan, Zheng; Liu, Jinchuan; Bai, Wenhui; Lv, Zhenzhen; Jiang, Xiaoling; Yang, Shuming; Chen, Ailiang; Lv, Guiyuan

    2014-01-01

    The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg·mL−1 Lambda DNA, and 50 nM target ssDNA in 5 µg·mL−1 Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes. PMID:25285445

  14. Label-free and sensitive fluorescent detection of sequence-specific single-strand DNA based on S1 nuclease cleavage effects.

    PubMed

    Guan, Zheng; Liu, Jinchuan; Bai, Wenhui; Lv, Zhenzhen; Jiang, Xiaoling; Yang, Shuming; Chen, Ailiang; Lv, Guiyuan

    2014-01-01

    The ability to detect sequence-specific single-strand DNA (ssDNA) in complex, contaminant-ridden samples, using a fluorescent method directly without a DNA extraction and PCR step could simplify the detection of pathogens in the field and in the clinic. Here, we have demonstrated a simple label-free sensing strategy to detect ssDNA by employing its complementary ssDNA, S1 nuclease and nucleic acid fluorescent dyes. Upon clearing away redundant complementary ssDNA and possibly mismatched double strand DNA by using S1 nuclease, the fluorescent signal-to-noise ratio could be increased dramatically. It enabled the method to be adaptable to three different types of DNA fluorescent dyes and the ability to detect target ssDNA in complex, multicomponent samples, like tissue homogenate. The method can distinguish a two-base mismatch from avian influenza A (H1N1) virus. Also, it can detect the appearance of 50 pM target ssDNA in 0.5 µg · mL(-1) Lambda DNA, and 50 nM target ssDNA in 5 µg · mL(-1) Lambda DNA or in tissue homogenate. It is facile and cost-effective, and could be easily extended to detect other ssDNA with many common nucleic acid fluorescent dyes. PMID:25285445

  15. DNA binding, photoactivated DNA cleavage and cytotoxic activity of Cu(II) and Co(II) based Schiff-base azo photosensitizers

    NASA Astrophysics Data System (ADS)

    Pradeepa, S. M.; Bhojya Naik, H. S.; Vinay Kumar, B.; Indira Priyadarsini, K.; Barik, Atanu; Prabhakara, M. C.

    2015-04-01

    A new class of Cu(II) and Co(II) complexes of azo-containing Schiff base of the type [Cu(L1)2] and [Co(L1)2], where L1 = 4-[(E)-{2-hydroxy-3-[(E)-(4-bromophenyl)diazenyl]benzylidene}amino]benzoic acid have been synthesized and characterized. Extension of conjugation and the presence of free carboxylic acid group of the ligand L1 increased the wavelength of the complexes from visible region to the near IR region (620-850 nm). The Cu(II) and Co(II) complexes interacted with CT-DNA via intercalative mode with the respective Kb value of 3.2 × 104 M-1 and 2.9 × 104 M-1 and acted as proficient photocleavers of SC pUC19 DNA in UV-A light, forming 1O2 as the reactive oxygen species with the quantum yield of 0.38 and 0.36, respectively. Furthermore, the Cu(II) and Co(II) complexes showed photocytotoxicity toward two selected tumor cell lines MCF-7 and A549 by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method, and the Cu(II) complex exhibits higher photocytotoxicity than Co(II) complex against each of the selected cell lines, this result is identical with their DNA binding ability order.

  16. DNA base damage by reactive oxygen species, oxidizing agents, and UV radiation.

    PubMed

    Cadet, Jean; Wagner, J Richard

    2013-02-01

    Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand cross-links, purine 5',8-cyclonucleosides, DNA-protein adducts and interstrand cross-links formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addition, recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed. PMID:23378590

  17. Gibberellic-acid-induced cell elongation in pea epicotyls: Effect on polyploidy and DNA content.

    PubMed

    Boeken, G; Van Oostveldt, P

    1977-01-01

    In gibberellic-acid(GA3)-treated epicotyls of dwarf peas (Pisum sativum L.) grown in the light, DNA (per cell and per epicotyl) is followed. Histofluorometric DNA determinations show that GA3-promoted cell elongation is not accompanied by increased endomitosis, but chemical estimations show an increased DNA content per epicotyl. This difference must therefore be the result of increased mitotic activity in the GA3-treated tissue. Epicotyls of seedlings grown with or without cotyledons under continuous light with GA3 are tetraploid, as are those of ecotylized embryos grown in darkness. These epicotyls reach no more than half the length of octaploid epicotyls of seedlings grown in darkness. This result provides evidence for a relationship between polyploidy and final possible cell length. PMID:24419898

  18. A MEMS-based miniature DNA analysis system

    SciTech Connect

    Northrup, M.A.; Gonzalez, C.; Hadley, D.

    1995-04-25

    We detail the design and development of a miniature thermal cycling instrument for performing the polymerase chain reaction (PCR) that uses microfabricated, silicon-based reaction chambers. The MEMS-based, battery-operated instrument shows significant improvements over commercial thermal cycling instrumentation. Several different biological systems have been amplified and verified with the miniature PCR instrument including the Human Immunodeficiency Virus; both cloned and genomic DNA templates of {beta} globin; and the genetic disease, Cystic Fibrosis from human DNA. The miniaturization of a PCR thermal cycler is the initial module of a fully-integrated portable, low-power, rapid, and highly efficient bioanalytical instrument.

  19. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  20. Electrochemical DNA Hybridization Sensors Based on Conducting Polymers

    PubMed Central

    Rahman, Md. Mahbubur; Li, Xiao-Bo; Lopa, Nasrin Siraj; Ahn, Sang Jung; Lee, Jae-Joon

    2015-01-01

    Conducting polymers (CPs) are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective. PMID:25664436

  1. Charge Transport in DNA with Five Base Pairs

    NASA Astrophysics Data System (ADS)

    Lee, Sunhee; Hedin, Eric; Joe, Yong

    2008-10-01

    Recently, much interest has arisen in the process of charge transport through DNA due to its fundamental roles in biological processes and in possible novel molecular electronics. We investigate quantum mechanical electron transmission along the long axis of the DNA molecule using a one-dimensional tight-binding model. In this system, we consider a single central conduction channel in which individual sites represent a base-pair formed by either AT (TA) or GC (CG) pairs coupled via hydrogen bonds. The sites are linked by a hopping amplitude, or quantum overlap integral. The sugar-phosphate backbone and the hopping amplitude between each site of the base and the backbone are incorporated into an energy-dependent on-site potential in the main DNA site. For the sake of simplicity, a simple DNA molecule segment with five base pairs is studied, and the transmission for different values of on-site energy is calculated to determine the influence of mismatch (impurity) effects in the DNA sequence. Finally, we present results for the temperature dependence of the transmission, and the current-voltage characteristics in order to examine the extent and efficiency of charge migration. *One of the authors (E.R.H) is partially supported by a grant from the Center for Energy Research, Education, and Service (CERES) at Ball State University.

  2. Single-molecule derivation of salt dependent base-pair free energies in DNA.

    PubMed

    Huguet, Josep M; Bizarro, Cristiano V; Forns, Núria; Smith, Steven B; Bustamante, Carlos; Ritort, Felix

    2010-08-31

    Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol(-1) precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our unique energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies, and entropies in conditions not accessible by bulk methodologies. PMID:20716688

  3. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944

  4. Single-molecule derivation of salt dependent base-pair free energies in DNA

    PubMed Central

    Huguet, Josep M.; Bizarro, Cristiano V.; Forns, Núria; Smith, Steven B.; Bustamante, Carlos; Ritort, Felix

    2010-01-01

    Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol-1 precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our unique energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies, and entropies in conditions not accessible by bulk methodologies. PMID:20716688

  5. A new ellagic acid glycoside and DNA topoisomerase IB inhibitory activity of saponins from Putranjiva roxburghii.

    PubMed

    Kumar, Ashish; Chowdhury, Somenath Roy; Chakrabarti, Tulika; Majumdarb, Hemanta K; Jha, Tarun; Mukhopadhyay, Sibabrata

    2014-05-01

    Chemical investigation of the stem bark and leaves of Putranjiva roxburghii has resulted in the isolation of a new ellagic acid glycoside (5) along with four saponins (1-4). The structures of the isolated compounds were established by detailed spectral analysis. Incidentally putranoside-A methyl ester (4) has been isolated for the first time from this species and the saponins (1-4) exhibited potent DNA topoisomerase IB inhibitory activity. PMID:25026719

  6. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  7. Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".

    PubMed

    Çağlayan, Melike; Wilson, Samuel H

    2015-12-01

    DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511

  8. DNA-based species detection capabilities using laser transmission spectroscopy

    PubMed Central

    Mahon, A. R.; Barnes, M. A.; Li, F.; Egan, S. P.; Tanner, C. E.; Ruggiero, S. T.; Feder, J. L.; Lodge, D. M.

    2013-01-01

    Early detection of invasive species is critical for effective biocontrol to mitigate potential ecological and economic damage. Laser transmission spectroscopy (LTS) is a powerful solution offering real-time, DNA-based species detection in the field. LTS can measure the size, shape and number of nanoparticles in a solution and was used here to detect size shifts resulting from hybridization of the polymerase chain reaction product to nanoparticles functionalized with species-specific oligonucleotide probes or with the species-specific oligonucleotide probes alone. We carried out a series of DNA detection experiments using the invasive freshwater quagga mussel (Dreissena bugensis) to evaluate the capability of the LTS platform for invasive species detection. Specifically, we tested LTS sensitivity to (i) DNA concentrations of a single target species, (ii) the presence of a target species within a mixed sample of other closely related species, (iii) species-specific functionalized nanoparticles versus species-specific oligonucleotide probes alone, and (iv) amplified DNA fragments versus unamplified genomic DNA. We demonstrate that LTS is a highly sensitive technique for rapid target species detection, with detection limits in the picomolar range, capable of successful identification in multispecies samples containing target and non-target species DNA. These results indicate that the LTS DNA detection platform will be useful for field application of target species. Additionally, we find that LTS detection is effective with species-specific oligonucleotide tags alone or when they are attached to polystyrene nanobeads and with both amplified and unamplified DNA, indicating that the technique may also have versatility for broader applications. PMID:23015524

  9. Exploring the DNA binding mode of transition metal based biologically active compounds

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.

    2012-01-01

    Few novel 4-aminoantipyrine derived Schiff bases and their metal complexes were synthesized and characterized. Their structural features and other properties were deduced from the elemental analysis, magnetic susceptibility and molar conductivity as well as from mass, IR, UV-vis, 1H NMR and EPR spectral studies. The binding of the complexes with CT-DNA was analyzed by electronic absorption spectroscopy, viscosity measurement, and cyclic voltammetry. The interaction of the metal complexes with DNA was also studied by molecular modeling with special reference to docking. The experimental and docking results revealed that the complexes have the ability of interaction with DNA of minor groove binding mode. The intrinsic binding constants ( Kb) of the complexes with CT-DNA were found out which show that they are minor groove binders. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the pUC19 DNA in the presence of AH 2 (ascorbic acid). Moreover, the oxidative cleavage studies using distamycin revealed the minor groove binding for the newly synthesized 4-aminoantipyrine derived Schiff bases and their metal complexes. Evaluation of antibacterial activity of the complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, and Klebsiella pneumoniae exhibited that the complexes have potent biocidal activity than the free ligands.

  10. Flow-cytometry-based DNA hybidization and polymorphism analysis

    NASA Astrophysics Data System (ADS)

    Cai, Hong; Kommander, Kristina; White, P. S.; Nolan, John P.

    1998-05-01

    Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  11. Trial watch: Naked and vectored DNA-based anticancer vaccines

    PubMed Central

    Bloy, Norma; Buqué, Aitziber; Aranda, Fernando; Castoldi, Francesca; Eggermont, Alexander; Cremer, Isabelle; Sautès-Fridman, Catherine; Fucikova, Jitka; Galon, Jérôme; Spisek, Radek; Tartour, Eric; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

    2015-01-01

    One type of anticancer vaccine relies on the administration of DNA constructs encoding one or multiple tumor-associated antigens (TAAs). The ultimate objective of these preparations, which can be naked or vectored by non-pathogenic viruses, bacteria or yeast cells, is to drive the synthesis of TAAs in the context of an immunostimulatory milieu, resulting in the (re-)elicitation of a tumor-targeting immune response. In spite of encouraging preclinical results, the clinical efficacy of DNA-based vaccines employed as standalone immunotherapeutic interventions in cancer patients appears to be limited. Thus, efforts are currently being devoted to the development of combinatorial regimens that allow DNA-based anticancer vaccines to elicit clinically relevant immune responses. Here, we discuss recent advances in the preclinical and clinical development of this therapeutic paradigm. PMID:26155408

  12. Finding human promoter groups based on DNA physical properties

    NASA Astrophysics Data System (ADS)

    Zeng, Jia; Cao, Xiao-Qin; Zhao, Hongya; Yan, Hong

    2009-10-01

    DNA rigidity is an important physical property originating from the DNA three-dimensional structure. Although the general DNA rigidity patterns in human promoters have been investigated, their distinct roles in transcription are largely unknown. In this paper, we discover four highly distinct human promoter groups based on similarity of their rigidity profiles. First, we find that all promoter groups conserve relatively rigid DNAs at the canonical TATA box [a consensus TATA(A/T)A(A/T) sequence] position, which are important physical signals in binding transcription factors. Second, we find that the genes activated by each group of promoters share significant biological functions based on their gene ontology annotations. Finally, we find that these human promoter groups correlate with the tissue-specific gene expression.

  13. Chem I Supplement: Emphasis on Acids and Bases

    ERIC Educational Resources Information Center

    Journal of Chemical Education Staff

    1977-01-01

    Provides supplementary notes on acids and bases suitable for secondary school chemistry instruction, including acidity in solid and natural waters, acidity balance in body chemistry, acid and basic foods, pH values of common fluids, examples of drugs, and commercial preparation of nitric acid. (SL)

  14. Implementing a two-layer feed-forward catalytic DNA circuit for enzyme-free and colorimetric detection of nucleic acids.

    PubMed

    Ravan, Hadi

    2016-03-01

    In the present study, a highly sensitive and specific bio-sensing platform for enzyme-free and colorimetric detection of nucleic acids has been developed. The biosensor is composed of two DNA nanostructures and two fuel strands that construct the foundation of a feed-forward catalytic DNA circuit. Upon binding the target strand to a specific DNA nanostructure, the circuit is run in order that at the end a hemin-binding aptamer, with the ability to convert a colorless substrate into a colored substance is released. Based on this strategy, 4 pM of the target DNA can be easily detected in serum samples by naked eyes after only a two-hour incubation with the circuit; meanwhile, if the incubation time is extended to 3 h, the biosensor can detect 1 pM of the target DNA. Besides the elevated sensitivity, the circuit can truly discriminate a spurious target containing one nucleotide mismatch with high specificity. Overall, the enzyme-free catalytic DNA circuit can be used as a sensitive alternative method to enzyme-based biosensors for the specific and cost-effective detection of nucleic acids. PMID:26873470

  15. Chemical Morphing of DNA Containing Four Noncanonical Bases.

    PubMed

    Eremeeva, Elena; Abramov, Michail; Margamuljana, Lia; Rozenski, Jef; Pezo, Valerie; Marlière, Philippe; Herdewijn, Piet

    2016-06-20

    The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo. PMID:27159019

  16. Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

    PubMed

    McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

    2016-01-01

    FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets. PMID:27583575

  17. Thermodynamics and Kinetics of DNA Tile-Based Self-Assembly

    NASA Astrophysics Data System (ADS)

    Jiang, Shuoxing

    Deoxyribonucleic acid (DNA) has emerged as an attractive building material for creating complex architectures at the nanometer scale that simultaneously affords versatility and modularity. Particularly, the programmability of DNA enables the assembly of basic building units into increasingly complex, arbitrary shapes or patterns. With the expanding complexity and functionality of DNA toolboxes, a quantitative understanding of DNA self-assembly in terms of thermodynamics and kinetics, will provide researchers with more subtle design guidelines that facilitate more precise spatial and temporal control. This dissertation focuses on studying the physicochemical properties of DNA tile-based self-assembly process by recapitulating representative scenarios and intermediate states with unique assembly pathways. First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed "multitile" arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step. The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be

  18. Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications.

    PubMed

    Li, Juan; Mo, Liuting; Lu, Chun-Hua; Fu, Ting; Yang, Huang-Hao; Tan, Weihong

    2016-03-01

    Hydrogels are crosslinked hydrophilic polymers that can absorb a large amount of water. By their hydrophilic, biocompatible and highly tunable nature, hydrogels can be tailored for applications in bioanalysis and biomedicine. Of particular interest are DNA-based hydrogels owing to the unique features of nucleic acids. Since the discovery of the DNA double helical structure, interest in DNA has expanded beyond its genetic role to applications in nanotechnology and materials science. In particular, DNA-based hydrogels present such remarkable features as stability, flexibility, precise programmability, stimuli-responsive DNA conformations, facile synthesis and modification. Moreover, functional nucleic acids (FNAs) have allowed the construction of hydrogels based on aptamers, DNAzymes, i-motif nanostructures, siRNAs and CpG oligodeoxynucleotides to provide additional molecular recognition, catalytic activities and therapeutic potential, making them key players in biological analysis and biomedical applications. To date, a variety of applications have been demonstrated with FNA-based hydrogels, including biosensing, environmental analysis, controlled drug release, cell adhesion and targeted cancer therapy. In this review, we focus on advances in the development of FNA-based hydrogels, which have fully incorporated both the unique features of FNAs and DNA-based hydrogels. We first introduce different strategies for constructing DNA-based hydrogels. Subsequently, various types of FNAs and the most recent developments of FNA-based hydrogels for bioanalytical and biomedical applications are described with some selected examples. Finally, the review provides an insight into the remaining challenges and future perspectives of FNA-based hydrogels. PMID:26758955

  19. DNA methylation detection based on difference of base content

    NASA Astrophysics Data System (ADS)

    Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

    2016-04-01

    Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

  20. An unprecedented nucleic acid capture mechanism for excision of DNA damage

    SciTech Connect

    Rubinson, Emily H.; Prakasha Gowda, A.S.; Spratt, Thomas E.; Gold, Barry; Eichmanbrand, Brandt F.

    2010-11-18

    DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson-Crick base pairs without duplex intercalation.

  1. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone, B.L.; Hammond, M.L.; Simpson, D.J.

    1995-04-11

    A method is described for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand. 4 figures.

  2. Method for rapid base sequencing in DNA and RNA with two base labeling

    DOEpatents

    Jett, James H.; Keller, Richard A.; Martin, John C.; Posner, Richard G.; Marrone, Babetta L.; Hammond, Mark L.; Simpson, Daniel J.

    1995-01-01

    Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

  3. Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

    PubMed Central

    Prinsen, C F; Veerkamp, J H

    1998-01-01

    We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect. PMID:9425108

  4. Magnetic bead-based nucleic acid purification kit: Clinical application and performance evaluation in stool specimens.

    PubMed

    Yoon, Jihoon G; Kang, Jin Seok; Hwang, Seung Yong; Song, Jaewoo; Jeong, Seok Hoon

    2016-05-01

    Two different methods - the semi-automated magnetic bead-based kit (SK, Stool DNA/RNA Purification kit®) and the manual membrane column-based kit (QS, QIAamp® DNA Stool Mini kit) - for purifying nucleic acids from clinical stool samples were compared and evaluated. The SK kit was more user-friendly than QS due to the reduced manual processing, partial automation, and short turnaround time with half cost. Furthermore, SK produced high yields in both DNA and RNA extractions but poor purity in RNA extraction. In the assessment of rotavirus and Clostridium difficile infection, both kits had equivalent or more sensitive performance compared with the standard method. Although SK showed some interference and inhibition in nucleic acid extraction, the performance, including the repeatability, linearity, analytical sensitivity, and matrix effect, was sufficient for routine clinical use. PMID:27030641

  5. Natural versus artificial creation of base pairs in DNA: origin of nucleobases from the perspectives of unnatural base pair studies.

    PubMed

    Hirao, Ichiro; Kimoto, Michiko; Yamashige, Rie

    2012-12-18

    Since life began on Earth, the four types of bases (A, G, C, and T(U)) that form two sets of base pairs have remained unchanged as the components of nucleic acids that replicate and transfer genetic information. Throughout evolution, except for the U to T modification, the four base structures have not changed. This constancy within the genetic code raises the question of how these complicated nucleotides were generated from the molecules in a primordial soup on the early Earth. At some prebiotic stage, the complementarity of base pairs might have accelerated the generation and accumulation of nucleotides or oligonucleotides. We have no clues whether one pair of nucleobases initially appeared on the early Earth during this process or a set of two base pairs appeared simultaneously. Recently, researchers have developed new artificial pairs of nucleobases (unnatural base pairs) that function alongside the natural base pairs. Some unnatural base pairs in duplex DNA can be efficiently and faithfully amplified in a polymerase chain reaction (PCR) using thermostable DNA polymerases. The addition of unnatural base pair systems could expand the genetic alphabet of DNA, thus providing a new mechanism for the generation novel biopolymers by the site-specific incorporation of functional components into nucleic acids and proteins. Furthermore, the process of unnatural base pair development might provide clues to the origin of the natural base pairs in a primordial soup on the early Earth. In this Account, we describe the development of three representative types of unnatural base pairs that function as a third pair of nucleobases in PCR and reconsider the origin of the natural nucleic acids. As researchers developing unnatural base pairs, they use repeated "proof of concept" experiments. As researchers design new base pairs, they improve the structures that function in PCR and eliminate those that do not. We expect that this process is similar to the one functioning in the

  6. A 265-base DNA sequencing read by capillary electrophoresis with no separation matrix.

    PubMed

    Albrecht, Jennifer Coyne; Lin, Jennifer S; Barron, Annelise E

    2011-01-15

    Electrophoretic DNA sequencing without a polymer matrix is currently possible only with the use of some kind of "drag-tag" as a mobility modifier. In free-solution conjugate electrophoresis (FSCE), a drag-tag attached to each DNA fragment breaks linear charge-to-friction scaling, enabling size-based separation in aqueous buffer alone. Here we report a 265-base read for free-solution DNA sequencing by capillary electrophoresis using a random-coil protein drag-tag of unprecedented length and purity. We identified certain methods of protein expression and purification that allow the production of highly monodisperse drag-tags as long as 516 amino acids, which are almost charge neutral (+1 to +6) and yet highly water-soluble. Using a four-color LIF detector, 265 bases could be read in 30 min with a 267-amino acid drag-tag, on par with the average read of current next-gen sequencing systems. New types of multichannel systems that allow much higher throughput electrophoretic sequencing should be much more accessible in the absence of a requirement for viscous separation matrix. PMID:21182303

  7. Local alignment of two-base encoded DNA sequence

    PubMed Central

    Homer, Nils; Merriman, Barry; Nelson, Stanley F

    2009-01-01

    Background DNA sequence comparison is based on optimal local alignment of two sequences using a similarity score. However, some new DNA sequencing technologies do not directly measure the base sequence, but rather an encoded form, such as the two-base encoding considered here. In order to compare such data to a reference sequence, the data must be decoded into sequence. The decoding is deterministic, but the possibility of measurement errors requires searching among all possible error modes and resulting alignments to achieve an optimal balance of fewer errors versus greater sequence similarity. Results We present an extension of the standard dynamic programming method for local alignment, which simultaneously decodes the data and performs the alignment, maximizing a similarity score based on a weighted combination of errors and edits, and allowing an affine gap penalty. We also present simulations that demonstrate the performance characteristics of our two base encoded alignment method and contrast those with standard DNA sequence alignment under the same conditions. Conclusion The new local alignment algorithm for two-base encoded data has substantial power to properly detect and correct measurement errors while identifying underlying sequence variants, and facilitating genome re-sequencing efforts based on this form of sequence data. PMID:19508732

  8. Molecular cloning and characterization of a human cDNA and gene encoding a novel acid ceramidase-like protein.

    PubMed

    Hong, S B; Li, C M; Rhee, H J; Park, J H; He, X; Levy, B; Yoo, O J; Schuchman, E H

    1999-12-01

    Computer-assisted database analysis of sequences homologous to human acid ceramidase (ASAH) revealed a 1233-bp cDNA (previously designated cPj-LTR) whose 266-amino-acid open reading frame had approximately 36% identity with the ASAH polypeptide. Based on this high degree of homology, we undertook further molecular characterization of cPj-LTR and now report the full-length cDNA sequence, complete gene structure (renamed human ASAHL since it is a human acid ceramidase-like sequence), chromosomal location, primer extension and promoter analysis, and transient expression results. The full-length human ASAHL cDNA was 1825 bp and contained an open-reading frame encoding a 359-amino-acid polypeptide that was 33% identical and 69% similar to the ASAH polypeptide over its entire length. Numerous short regions of complete identity were observed between these two sequences and two sequences obtained from the Caenorhabditis elegans genome database. The 30-kb human ASAHL genomic sequence contained 11 exons, which ranged in size from 26 to 671 bp, and 10 introns, which ranged from 150 bp to 6.4 kb. The gene was localized to the chromosomal region 4q21.1 by fluorescence in situ hybridization analysis. Northern blotting experiments revealed a major 2.0-kb ASAHL transcript that was expressed at high levels in the liver and kidney, but at relatively low levels in other tissues such as the lung, heart, and brain. Sequence analysis of the 5'-flanking region of the human ASAHL gene revealed a putative promoter region that lacked a TATA box and was GC rich, typical features of a housekeeping gene promoter, as well as several tissue-specific and/or hormone-induced transcription regulatory sites. 5'-Deletion analysis localized the promoter activity to a 1. 1-kb fragment within this region. A major transcription start site also was located 72 bp upstream from the ATG translation initiation site by primer extension analysis. Expression analysis of a green fluorescence protein/ASAHL fusion

  9. Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis

    PubMed Central

    Ennahar, Saïd; Cai, Yimin; Fujita, Yasuhito

    2003-01-01

    A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Weissella. Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality. PMID:12514026

  10. 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

  11. MUTYH DNA glycosylase: the rationale for removing undamaged bases from the DNA

    PubMed Central

    Markkanen, Enni; Dorn, Julia; Hübscher, Ulrich

    2013-01-01

    Maintenance of genetic stability is crucial for all organisms in order to avoid the onset of deleterious diseases such as cancer. One of the many proveniences of DNA base damage in mammalian cells is oxidative stress, arising from a variety of endogenous and exogenous sources, generating highly mutagenic oxidative DNA lesions. One of the best characterized oxidative DNA lesion is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can give rise to base substitution mutations (also known as point mutations). This mutagenicity is due to the miscoding potential of 8-oxo-G that instructs most DNA polymerases (pols) to preferentially insert an Adenine (A) opposite 8-oxo-G instead of the appropriate Cytosine (C). If left unrepaired, such A:8-oxo-G mispairs can give rise to CG→AT transversion mutations. A:8-oxo-G mispairs are proficiently recognized by the MutY glycosylase homologue (MUTYH). MUTYH can remove the mispaired A from an A:8-oxo-G, giving way to the canonical base-excision repair (BER) that ultimately restores undamaged Guanine (G). The importance of this MUTYH-initiated pathway is illustrated by the fact that biallelic mutations in the MUTYH gene are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). In this review, we will focus on MUTYH, from its discovery to the most recent data regarding its cellular roles and interaction partners. We discuss the involvement of the MUTYH protein in the A:8-oxo-G BER pathway acting together with pol λ, the pol that can faithfully incorporate C opposite 8-oxo-G and thus bypass this lesion in a correct manner. We also outline the current knowledge about the regulation of MUTYH itself and the A:8-oxo-G repair pathway by posttranslational modifications (PTM). Finally, to achieve a clearer overview of the literature, we will briefly touch on the rather confusing MUTYH nomenclature. In short, MUTYH is a unique DNA glycosylase that catalyzes the excision of an undamaged base from DNA. PMID

  12. Technological applications arising from the interactions of DNA bases with metal ions.

    PubMed

    Park, Ki Soo; Park, Hyun Gyu

    2014-08-01

    An intense interest has grown in the unique interactions of nucleic acids with metal ions, which lead to the formation of metal-base pairs and the generation of fluorescent nanomaterials. In this review, different types of metal-base pairs, especially those formed from naturally occurring nucleosides, are described with emphasis also being given to recent advances made in employing these complexes to govern enzymatic reactions. The review also contains a comprehensive description of DNA-templated inorganic nanomaterials such as silver nanoclusters which possess excellent fluorescence properties. Finally, a summary is given about how these materials have led to recent advances in the field of nanobiotechnology. PMID:24832070

  13. Sensitive Zn(2+) sensor based on biofunctionalized nanopores via combination of DNAzyme and DNA supersandwich structures.

    PubMed

    Liu, Nannan; Hou, Ruizuo; Gao, Pengcheng; Lou, Xiaoding; Xia, Fan

    2016-06-21

    The sensitivity of detection based on biofunctionalized nanopores is limited since the target-to-signal ratio is 1 : 1. Isothermal amplification is a promising amplification strategy at constant temperature due to its easy operation, quick results, PCR-like sensitivity, low cost and energy efficiency. In the present work, the isothermally amplified detection of Zn(2+) is achieved by using a DNA supersandwich structure and Zn(2+)-requiring DNAzymes. The DNA supersandwich structures, due to the multiple amplification of nucleic acids, heavily plug the nanopore. Simultaneously, the DNA supersandwich structures bond with the sessile probe (SP) of the substrate in the nanopore which partially hybridizes with DNAzymes. In the presence of Zn(2+), the Zn(2+)-requiring DNAzyme cleaves the SP into two fragments, while the DNA supersandwich structures are peeled off and the ionic pathway is unimpeded. A steep drop and a sequential complete recovery of the current occur in the I-V plot when the DNA supersandwich structures are decorated and peeled off. In the present system, the reliable detection limit of Zn(2+) is as low as 1 nM. Discrimination between different types of ions (Cu(2+), Hg(2+), Pb(2+)) is achieved. PMID:26911926

  14. Sensitive Detection of ssDNA Using an LRET-Based Upconverting Nanohybrid Material.

    PubMed

    Jesu Raj, Joe Gerald; Quintanilla, Marta; Mahmoud, Khaled A; Ng, Andy; Vetrone, Fiorenzo; Zourob, Mohammed

    2015-08-26

    Water-dispersible, optical hybrid nanoparticles are preferred materials for DNA biosensing due to their biocompatibility. Upconverting nanoparticles are highly desirable optical probes in sensors and bioimaging owing to their sharp emission intensity in the visible region. We herein report a highly sensitive ss-DNA detection based on an energy transfer system that uses a nanohybrid material synthesized by doping NaYF4:Tm(3+)/Yb(3+) upconverting nanoparticles (UCNPs) on silica coated polystyrene-co-acrylic acid (PSA) nanoparticles (PSA/SiO2) as the donor, and gold nanoparticles (AuNPs) decorated with Ir(III) complex as the acceptor. UCNPs tagged on PSA/SiO2 and the cyclometalated Ir(III)/AuNP conjugates were then linked through the ss-DNA sequence. Sequential addition of the target DNA to the probe molecular beacon complex resulted in the separation of the optical nanohybrid material and the quencher, leading to a measurable increase in the blue fluorescence emission intensity. Our results have shown a linear relationship between the fluorescence intensity and target DNA concentration down to the picomolar. PMID:26280649

  15. Phosphorescent quantum dots/ethidium bromide nanohybrids based on photoinduced electron transfer for DNA detection

    NASA Astrophysics Data System (ADS)

    Bi, Lin; Yu, Yuan-Hua

    2015-04-01

    Mercaptopropionic acid-capped Mn-doped ZnS quantum dots/ethidium bromide (EB) nanohybrids were constructed for photoinduced electron transfer (PIET) and then used as a room-temperature phosphorescence (RTP) probe for DNA detection. EB could quench the RTP of Mn-doped ZnS QDs by PIET, thereby forming Mn-doped ZnS QDs/EB nanohybrids and storing RTP. Meanwhile, EB could be inserted into DNA and EB could be competitively desorbed from the surface of Mn-doped ZnS QDs by DNA, thereby releasing the RTP of Mn-doped ZnS QDs. Based on this mechanism, a RTP sensor for DNA detection was developed. Under optimal conditions, the detection limit for DNA was 0.045 mg L-1, the relative standard deviation was 1.7%, and the method linear ranged from 0.2 to 20 mg L-1. The proposed method was applied to biological fluids, in which satisfactory results were obtained.

  16. Simultaneous detection of RNA and DNA targets based on multiplex isothermal amplification.

    PubMed

    Dobnik, David; Morisset, Dany; Lenarčič, Rok; Ravnikar, Maja

    2014-04-01

    The detection of pathogenic microorganisms present in food, feed, plant, and other samples is important for providing safe food as well as for preventing the spread of microbes. The genome of pathogens is made of DNA or RNA, therefore a multiplex diagnostics tool would ideally be able to amplify and detect both RNA and DNA targets in parallel. With this goal we have developed an isothermal nucleic acid sequence based amplification [NASBA] implemented microarray analysis (NAIMA) procedure, suitable for the simultaneous multiplex amplification of RNA and DNA targets, coupled with the detection on ArrayTubes. The method is demonstrated to be very sensitive and specific for the detection of two economically important quarantine plant pathogens of potato, the potato spindle tuber viroid (RNA target) and Ralstonia solanacearum (DNA target). Because of its isothermal amplification and simple detection equipment, the method is also applicable for on-site analyses. NAIMA can be used in any domain where there is the need to detect RNA and DNA targets simultaneously. PMID:24625323

  17. A Simple DNA-based Electrochemical Biosensor for Highly Sensitive Detection of Ciprofloxacin Using Disposable Graphene.

    PubMed

    Lim, Syazana A; Ahmed, Minhaz U

    2016-01-01

    In this work we exploited the electrostatic interaction of double stranded DNA (dsDNA) with drug components to construct a simple, but highly sensitive, DNA-electrochemical sensor for detecting ciprofloxacin. The following straightforward three-step procedure was performed to determine ciprofloxacin: (i) dsDNA-layer immobilization on the surface of the working graphene-modified screen-printed carbon electrode; (ii) dsDNA-ciprofloxacin interaction for 2 min; and (iii) electrochemical measurement using square-wave voltammetry. An increased oxidation of the guanine component was observed, at +1.0 V, as a result of the electrostatic interaction of positively charged ciprofloxacin with the negatively charged nucleic acid sugar phosphate. Based on the International Conference on Harmonization Guidelines, a linear relationship between the guanine oxidation peak and ciprofloxacin concentration (0.1 to 100 μM) was obtained with a detection limit of 0.1 μM. Our developed sensor is straightforward to construct and use, requiring no multi-step time-consuming preconditioning of electrodes. It is highly sensitive and selective in the detection of ciprofloxacin, and has the potential to be useful in the future fabrication of rapid and portable on-site food safety analysis devices. PMID:27302591

  18. The Bronsted-Lowery Acid-Base Concept.

    ERIC Educational Resources Information Center

    Kauffman, George B.

    1988-01-01

    Gives the background history of the simultaneous discovery of acid-base relationships by Johannes Bronsted and Thomas Lowry. Provides a brief biographical sketch of each. Discusses their concept of acids and bases in some detail. (CW)

  19. An Introductory Laboratory Exercise for Acids and Bases.

    ERIC Educational Resources Information Center

    Miller, Richard; Silberman, Robert

    1986-01-01

    Discusses an acid-base neutralization exercise requiring groups of students to determine: (1) combinations of solutions giving neutralization; (2) grouping solutions as acids or bases; and (3) ranking groups in order of concentration. (JM)

  20. Mathematical modeling of acid-base physiology

    PubMed Central

    Occhipinti, Rossana; Boron, Walter F.

    2015-01-01

    pH is one of the most important parameters in life, influencing virtually every biological process at the cellular, tissue, and whole-body level. Thus, for cells, it is critical to regulate intracellular pH (pHi) and, for multicellular organisms, to regulate extracellular pH (pHo). pHi regulation depends on the opposing actions of plasma-membrane transporters that tend to increase pHi, and others that tend to decrease pHi. In addition, passive fluxes of uncharged species (e.g., CO2, NH3) and charged species (e.g., HCO3− , NH4+) perturb pHi. These movements not only influence one another, but also perturb the equilibria of a multitude of intracellular and extracellular buffers. Thus, even at the level of a single cell, perturbations in acid-base reactions, diffusion, and transport are so complex that it is impossible to understand them without a quantitative model. Here we summarize some mathematical models developed to shed light onto the complex interconnected events triggered by acids-base movements. We then describe a mathematical model of a spherical cell–which to our knowledge is the first one capable of handling a multitude of buffer reaction–that our team has recently developed to simulate changes in pHi and pHo caused by movements of acid-base equivalents across the plasma membrane of a Xenopus oocyte. Finally, we extend our work to a consideration of the effects of simultaneous CO2 and HCO3− influx into a cell, and envision how future models might extend to other cell types (e.g., erythrocytes) or tissues (e.g., renal proximal-tubule epithelium) important for whole-body pH homeostasis. PMID:25617697

  1. Photocurable bioadhesive based on lactic acid.

    PubMed

    Marques, D S; Santos, J M C; Ferreira, P; Correia, T R; Correia, I J; Gil, M H; Baptista, C M S G

    2016-01-01

    Novel photocurable and low molecular weight oligomers based on l-lactic acid with proven interest to be used as bioadhesive were successfully manufactured. Preparation of lactic acid oligomers with methacrylic end functionalizations was carried out in the absence of catalyst or solvents by self-esterification in two reaction steps: telechelic lactic acid oligomerization with OH end groups and further functionalization with methacrylic anhydride. The final adhesive composition was achieved by the addition of a reported biocompatible photoinitiator (Irgacure® 2959). Preliminary in vitro biodegradability was investigated by hydrolytic degradation in PBS (pH=7.4) at 37 °C. The adhesion performance was evaluated using glued aminated substrates (gelatine pieces) subjected to pull-to-break test. Surface energy measured by contact angles is lower than the reported values of the skin and blood. The absence of cytoxicity was evaluated using human fibroblasts. A notable antimicrobial behaviour was observed using two bacterial models (Staphylococcus aureus and Escherichia coli). The cured material exhibited a strong thrombogenic character when placed in contact with blood, which can be predicted as a haemostatic effect for bleeding control. This novel material was subjected to an extensive characterization showing great potential for bioadhesive or other biomedical applications where biodegradable and biocompatible photocurable materials are required. PMID:26478350

  2. Bipolar Membranes for Acid Base Flow Batteries

    NASA Astrophysics Data System (ADS)

    Anthamatten, Mitchell; Roddecha, Supacharee; Jorne, Jacob; Coughlan, Anna

    2011-03-01

    Rechargeable batteries can provide grid-scale electricity storage to match power generation with consumption and promote renewable energy sources. Flow batteries offer modular and flexible design, low cost per kWh and high efficiencies. A novel flow battery concept will be presented based on acid-base neutralization where protons (H+) and hydroxyl (OH-) ions react electrochemically to produce water. The large free energy of this highly reversible reaction can be stored chemically, and, upon discharge, can be harvested as usable electricity. The acid-base flow battery concept avoids the use of a sluggish oxygen electrode and utilizes the highly reversible hydrogen electrode, thus eliminating the need for expensive noble metal catalysts. The proposed flow battery is a hybrid of a battery and a fuel cell---hydrogen gas storing chemical energy is produced at one electrode and is immediately consumed at the other electrode. The two electrodes are exposed to low and high pH solutions, and these solutions are separated by a hybrid membrane containing a hybrid cation and anion exchange membrane (CEM/AEM). Membrane design will be discussed, along with ion-transport data for synthesized membranes.

  3. A novel Lie algebra of the genetic code over the Galois field of four DNA bases.

    PubMed

    Sánchez, Robersy; Grau, Ricardo; Morgado, Eberto

    2006-07-01

    Starting from the four DNA bases order in the Boolean lattice, a novel Lie Algebra of the genetic code is proposed. Here, the main partitions of the genetic code table were obtained as equivalent classes of quotient spaces of the genetic code vector space over the Galois field of the four DNA bases. The new algebraic structure shows strong connections among algebraic relationships, codon assignments and physicochemical properties of amino acids. Moreover, a distance defined between codons expresses a physicochemical meaning. It was also noticed that the distance between wild type and mutant codons tends to be small in mutational variants of four genes: human phenylalanine hydroxylase, human beta-globin, HIV-1 protease and HIV-1 reverse transcriptase. These results strongly suggest that deterministic rules in genetic code origin must be involved. PMID:16780898

  4. Evaluation of DNA typing as a positive identification method for soft and hard tissues immersed in strong acids.

    PubMed

    Robino, C; Pazzi, M; Di Vella, G; Martinelli, D; Mazzola, L; Ricci, U; Testi, R; Vincenti, M

    2015-11-01

    Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes. PMID:26195111

  5. Recovery Based Nanowire Field-Effect Transistor Detection of Pathogenic Avian Influenza DNA

    NASA Astrophysics Data System (ADS)

    Lin, Chih-Heng; Chu, Chia-Jung; Teng, Kang-Ning; Su, Yi-Jr; Chen, Chii-Dong; Tsai, Li-Chu; Yang, Yuh-Shyong

    2012-02-01

    Fast and accurate diagnosis is critical in infectious disease surveillance and management. We proposed a DNA recovery system that can easily be adapted to DNA chip or DNA biosensor for fast identification and confirmation of target DNA. This method was based on the re-hybridization of DNA target with a recovery DNA to free the DNA probe. Functionalized silicon nanowire field-effect transistor (SiNW FET) was demonstrated to monitor such specific DNA-DNA interaction using high pathogenic strain virus hemagglutinin 1 (H1) DNA of avian influenza (AI) as target. Specific electric changes were observed in real-time for AI virus DNA sensing and device recovery when nanowire surface of SiNW FET was modified with complementary captured DNA probe. The recovery based SiNW FET biosensor can be further developed for fast identification and further confirmation of a variety of influenza virus strains and other infectious diseases.

  6. Functional nucleic-acid-based sensors for environmental monitoring.

    PubMed

    Sett, Arghya; Das, Suradip; Bora, Utpal

    2014-10-01

    Efforts to replace conventional chromatographic methods for environmental monitoring with cheaper and easy to use biosensors for precise detection and estimation of hazardous environmental toxicants, water or air borne pathogens as well as various other chemicals and biologics are gaining momentum. Out of the various types of biosensors classified according to their bio-recognition principle, nucleic-acid-based sensors have shown high potential in terms of cost, sensitivity, and specificity. The discovery of catalytic activities of RNA (ribozymes) and DNA (DNAzymes) which could be triggered by divalent metallic ions paved the way for their extensive use in detection of heavy metal contaminants in environment. This was followed with the invention of small oligonucleotide sequences called aptamers which can fold into specific 3D conformation under suitable conditions after binding to target molecules. Due to their high affinity, specificity, reusability, stability, and non-immunogenicity to vast array of targets like small and macromolecules from organic, inorganic, and biological origin, they can often be exploited as sensors in industrial waste management, pollution control, and environmental toxicology. Further, rational combination of the catalytic activity of DNAzymes and RNAzymes along with the sequence-specific binding ability of aptamers have given rise to the most advanced form of functional nucleic-acid-based sensors called aptazymes. Functional nucleic-acid-based sensors (FNASs) can be conjugated with fluorescent molecules, metallic nanoparticles, or quantum dots to aid in rapid detection of a variety of target molecules by target-induced structure switch (TISS) mode. Although intensive research is being carried out for further improvements of FNAs as sensors, challenges remain in integrating such bio-recognition element with advanced transduction platform to enable its use as a networked analytical system for tailor made analysis of environmental

  7. An electrochemical DNA sensor based on polyaniline/graphene: high sensitivity to DNA sequences in a wide range.

    PubMed

    Zheng, Qing; Wu, Hao; Shen, Zongxu; Gao, Wenyu; Yu, Yu; Ma, Yuehui; Guang, Weijun; Guo, Quangui; Yan, Rui; Wang, Junzhong; Ding, Kejian

    2015-10-01

    A label-free electrochemical DNA sensor was fabricated by deposition of polyaniline and pristine graphene nanosheet (P/G(ratios)) composites in different mass ratios, DNA probe and bovine serum albumin (BSA) layer by layer on the surface of a glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS) was employed to monitor every step of fabrication of P/G(ratio)-based DNA sensors and to evaluate the detection results in terms of the hybridization of complementary DNA, mutant DNA and non-complementary DNA. The results illustrate that the P/G(ratio)-based DNA sensor could highly efficiently detect complementary DNA from 0.01 pm to 1 μm and discriminate single-nucleotide polymorphisms (SNPs). In the process of detection, double-stranded DNA (dsDNA), resulting from hybridization of a DNA probe, escaping from or remaining on the sensor surface, was monitored by changing the ratio of polyaniline (PANI) to graphene, which was decided by the competition between the electrostatic interaction and Brownian motion. PMID:26309910

  8. DNA/polyethyleneimine/hyaluronic acid small complex particles and tumor suppression in mice.

    PubMed

    Ito, Tomoko; Yoshihara, Chieko; Hamada, Katsuyuki; Koyama, Yoshiyuki

    2010-04-01

    The highest barriers for non-viral vectors to an efficient in vivo gene transfection would be (1) non-specific interaction with biological molecules, and (2) large size of the DNA complex particles. Protective coating of the DNA/polyethyleneimine (PEI) complexes by hyaluronic acid (HA) effectively diminished the adverse interactions with biological molecules. Here we found HA also protected the DNA/PEI complexes against aggregation and inactivation through lyophilization-and-rehydration procedures. It allows us to prepare the concentrated very small DNA complex particles (<70 nm) suspension by preparing the complexes at highly diluted conditions, followed by lyophilized-and-rehydrated to a small volume. In vivo gene expression efficiency of the small complex was examined with mice subcutaneously inoculated with B16 melanoma cells. These formulations showed high reporter-gene expression level in tumor after intravenous injection into tumor-bearing mice. Small complex was then made of the plasmid encoding GM-CSF gene, and injected into the mice bearing subcutaneous solid B16 tumor. After intravenous injection, it induced apparent tumor growth suppression in 50% of the mice. Notably, significant therapeutic effect was detected in the mice that received intratumoral injection, and 75% of the mice were completely cured with disappearance of tumor. PMID:20047759

  9. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  10. UV-Light-Induced Improvement of Fluorescence Quantum Yield of DNA-Templated Gold Nanoclusters: Application to Ratiometric Fluorescent Sensing of Nucleic Acids.

    PubMed

    Li, Zong-Yu; Wu, Yun-Tse; Tseng, Wei-Lung

    2015-10-28

    The use of DNA as a template has been demonstrated as an effective method for synthesizing different-sized silver nanoclusters. Although DNA-templated silver nanoclusters show outstanding performance as fluorescent probes for chemical sensing and cellular imaging, the synthesis of DNA-stabilized gold nanoclusters (AuNCs) with high fluorescence intensity remains a challenge. Here a facile, reproducible, scalable, NaBH4-free, UV-light-assisted method was developed to prepare AuNCs using repeats of 30 adenosine nucleotides (A30). The maximal fluorescence of A30-stabilized AuNCs appeared at 475 nm with moderate quantum yield, two fluorescence lifetimes, and a small amount of Au(+) on the surface of the Au core. Results of size-exclusion chromatography revealed that A30-stabilized AuNCs were more compact than A30. A series of control experiments showed that UV light played a dual role in the reduction of gold-ion precursors and the decomposition of citrate ions. A30 also acted as a stabilizer to prevent the aggregation of AuNCs. In addition, single-stranded DNA (ssDNA) consisting of an AuNC-nucleation sequence and a hybridization sequence was utilized to develop a AuNC-based ratiometric fluorescent probe in the presence of the double-strand-chelating dye SYBR Green I (SG). Under conditions of single-wavelength excitation, the combination of AuNC/SG-bearing ssDNA and perfectly matched DNA emitted fluorescence at 475 and 525 nm, respectively. The formed AuNC/SG-bearing ssDNA enabled the sensitive, selective, and ratiometric detection of specific nucleic acid targets. Finally, the AuNC-based ratiometric probes were successfully applied to determine specific nucleic acid targets in human serum. PMID:26443919

  11. Nanoinformatics and DNA-based computing: catalyzing nanomedicine.

    PubMed

    Maojo, Victor; Martin-Sanchez, Fernando; Kulikowski, Casimir; Rodriguez-Paton, Alfonso; Fritts, Martin

    2010-05-01

    Five decades of research and practical application of computers in biomedicine has given rise to the discipline of medical informatics, which has made many advances in genomic and translational medicine possible. Developments in nanotechnology are opening up the prospects for nanomedicine and regenerative medicine where informatics and DNA computing can become the catalysts enabling health care applications at sub-molecular or atomic scales. Although nanomedicine promises a new exciting frontier for clinical practice and biomedical research, issues involving cost-effectiveness studies, clinical trials and toxicity assays, drug delivery methods, and the implementation of new personalized therapies still remain challenging. Nanoinformatics can accelerate the introduction of nano-related research and applications into clinical practice, leading to an area that could be called "translational nanoinformatics." At the same time, DNA and RNA computing presents an entirely novel paradigm for computation. Nanoinformatics and DNA-based computing are together likely to completely change the way we model and process information in biomedicine and impact the emerging field of nanomedicine most strongly. In this article, we review work in nanoinformatics and DNA (and RNA)-based computing, including applications in nanopediatrics. We analyze their scientific foundations, current research and projects, envisioned applications and potential problems that might arise from them. PMID:20118825

  12. Prognostic value of DNA repair based stratification of hepatocellular carcinoma.

    PubMed

    Lin, Zhuo; Xu, Shi-Hao; Wang, Hai-Qing; Cai, Yi-Jing; Ying, Li; Song, Mei; Wang, Yu-Qun; Du, Shan-Jie; Shi, Ke-Qing; Zhou, Meng-Tao

    2016-01-01

    Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival (p = 0.004, hazard ratio (HR): 2.989) and tumor-free survival (p = 0.049, HR: 3.366) in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. PMID:27174663

  13. Prognostic value of DNA repair based stratification of hepatocellular carcinoma

    PubMed Central

    Lin, Zhuo; Xu, Shi-Hao; Wang, Hai-Qing; Cai, Yi-Jing; Ying, Li; Song, Mei; Wang, Yu-Qun; Du, Shan-Jie; Shi, Ke-Qing; Zhou, Meng-Tao

    2016-01-01

    Aberrant activation of DNA repair is frequently associated with tumor progression and response to therapy in hepatocellular carcinoma (HCC). Bioinformatics analyses of HCC data in the Cancer Genome Atlas (TCGA) were performed to define DNA repair based molecular classification that could predict the prognosis of patients with HCC. Furthermore, we tested its predictive performance in 120 independent cases. Four molecular subgroups were identified on the basis of coordinate DNA repair cluster (CDRC) comprising 15 genes in TCGA dataset. Increasing expression of CDRC genes were significantly associated with TP53 mutation. High CDRC was significantly correlated with advanced tumor grades, advanced pathological stage and increased vascular invasion rate. Multivariate Cox regression analysis indicated that the molecular subgrouping was an independent prognostic parameter for both overall survival (p = 0.004, hazard ratio (HR): 2.989) and tumor-free survival (p = 0.049, HR: 3.366) in TCGA dataset. Similar results were also obtained by analyzing the independent cohort. These data suggest that distinct dysregulation of DNA repair constituents based molecular classes in HCC would be useful for predicting prognosis and designing clinical trials for targeted therapy. PMID:27174663

  14. Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

    PubMed Central

    Lui, Clarissa; Cady, Nathaniel C.; Batt, Carl A.

    2009-01-01

    The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform. PMID:22412335

  15. Transcutaneous DNA immunization following waxing-based hair depilation

    PubMed Central

    Sloat, Brian R.; Kiguchi, Kaoru; Xiao, Gang; DiGiovanni, John; Maury, Wendy; Cui, Zhengrong

    2011-01-01

    Transcutaneous DNA immunization is an attractive immunization approach. Previously, we reported that transcutaneous immunization by applying plasmid DNA onto a skin area wherein the hair follicles had been induced into growth stage by ‘cold’ waxing-based hair plucking significantly enhanced the resultant immune responses. In the present study, using a plasmid that encodes the Bacillus anthracis protective antigen (PA63) gene fragment, it was shown that the anti-PA63 antibody responses induced by applying the plasmid onto a skin area where the hair was plucked by ‘warm’ waxing were significantly stronger than by ‘cold’ waxing, very likely because the ‘warm’ waxing-based hair depilation significantly i) enhanced the uptake (or retention) of the plasmid in the application area and ii) enhanced the expression of the transfected gene in the follicular and interfollicular epidermis in the skin. The antibody response induced by transcutaneous DNA immunization was hair cycle dependent, because the plasmid needed to be applied within 5 days after the hair plucking to induce a strong antibody response. The antibody responses were not affected by whether the expressed PA63 protein, as an antigen, was secreted or cell surface bound. Finally, this strategy of enhancing the immune responses induced by transcutaneous DNA immunization following ‘warm’ waxing-based hair depilation was not limited to the PA63 as an antigen, because immunization with a plasmid that encodes the HIV-1 env gp160 gene induced a strong anti-gp160 response as well. Transcutaneous DNA immunization by modifying the hair follicle cycle may hold a great promise in inducing strong and functional immune responses. PMID:21907253

  16. Monitoring of the retinoic acid receptor-retinoid X receptor dimerization upon DNA binding by native mass spectrometry.

    PubMed

    Nguyen-Huynh, Nha-Thi; Osz, Judit; Peluso-Iltis, Carole; Rochel, Natacha; Potier, Noëlle; Leize-Wagner, Emmanuelle

    2016-03-01

    Identifying protein-DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor-retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR-RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR-RXR heterodimerization. PMID:26558701

  17. Teaching Acid/Base Physiology in the Laboratory

    ERIC Educational Resources Information Center

    Friis, Ulla G.; Plovsing, Ronni; Hansen, Klaus; Laursen, Bent G.; Wallstedt, Birgitta

    2010-01-01

    Acid/base homeostasis is one of the most difficult subdisciplines of physiology for medical students to master. A different approach, where theory and practice are linked, might help students develop a deeper understanding of acid/base homeostasis. We therefore set out to develop a laboratory exercise in acid/base physiology that would provide…

  18. Using Willie's Acid-Base Box for Blood Gas Analysis

    ERIC Educational Resources Information Center

    Dietz, John R.

    2011-01-01

    In this article, the author describes a method developed by Dr. William T. Lipscomb for teaching blood gas analysis of acid-base status and provides three examples using Willie's acid-base box. Willie's acid-base box is constructed using three of the parameters of standard arterial blood gas analysis: (1) pH; (2) bicarbonate; and (3) CO[subscript…

  19. A novel electrochemical DNA biosensor construction based on layered CuS-graphene composite and Au nanoparticles.

    PubMed

    Xu, Chun-Xuan; Zhai, Qiu-Ge; Liu, Yu-Jie; Huang, Ke-Jing; Lu, Lu; Li, Ke-Xin

    2014-11-01

    A novel CuS-graphene (CuS-Gr) composite was synthesized to achieve excellent electrochemical properties for application as a DNA electrochemical biosensor. CuS-Gr composite was prepared by a hydrothermal method, in which two-dimensional graphene served as a two-dimensional conductive skeleton to support CuS nanoparticles. A sensitive electrochemical DNA biosensor was fabricated by immobilizing single-stranded DNA (ss-DNA) labeled at the 5' end using 6-mercapto-1-hexane (HS-ssDNA) on the surface of Au nanoparticles (AuNPs) to form ssDNA-S-AuNPs/CuS-Gr, and hybridization sensing was done in phosphate buffer. Cyclic voltammetry and electrochemical impedance spectroscopy were performed for the characterization of the modified electrodes. Differential pulse voltammetry was applied to monitor the DNA hybridization using an [Fe(CN)6](3-/4-) solution as a probe. Under optimum conditions, the biosensor developed exhibited a good linear relationship between the current and the logarithm of the target DNA concentration ranging from 0.001 to 1 nM, with a low detection limit of 0.1 pM (3σ/S). The biosensor exhibited high selectivity to differentiate one-base-mismatched DNA and three-base-mismatched DNA. The results indicated that the sensing platform based on CuS-Gr provides a stable and conductive interface for electrochemical detection of DNA hybridization, and could easily be extended to the detection of other nucleic acids. PMID:24894519

  20. Fluorescence-based DNA minisequence analysis for detection of known single-base changes in genomic DNA.

    PubMed

    Kobayashi, M; Rappaport, E; Blasband, A; Semeraro, A; Sartore, M; Surrey, S; Fortina, P

    1995-06-01

    We describe a rapid, automated method for direct detection of known single-base changes in genomic DNA. Fluorescence-based DNA minisequence analysis is employed in a template-dependent reaction which involves a single nucleotide extension of an oligonucleotide primer by the correct fluorescently-tagged dideoxynucleotide chain terminator. Detection following electrophoresis on denaturing acrylamide gels is facilitated by alkaline phosphatase treatment of reaction products after extension followed by isopropanol precipitation of the dye-tagged, single-base-extended primer to remove unincorporated deoxynucleotides. Fluorescence analysis of the incorporated dye tag reveals the identity of the template nucleotide immediately 3' to the primer site. This technique does not require radioactivity or biotinylated PCR product, relies on the incorporation of a single dideoxynucleotide terminator to extend the primer by one nucleotide and takes advantage of the sensitivity of fluorescent terminators developed for automated DNA sequence analysis. As a demonstration, we have applied the assay to human genomic DNA for detection of the sickle mutation in the beta-globin gene, and have also examined feasibility for simultaneous delineation using a multiplex-like strategy in a single gel-lane of some of the most common beta-thalassemia mutations in the Mediterranean basin. PMID:7477010

  1. 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

  2. Rapid DNA Sequencing by Direct Nanoscale Reading of Nucleotide Bases on Individual DNA Chains

    SciTech Connect

    Lee, James Weifu; Meller, Amit

    2007-01-01

    Since the independent invention of DNA sequencing by Sanger and by Gilbert 30 years ago, it has grown from a small scale technique capable of reading several kilobase-pair of sequence per day into today's multibillion dollar industry. This growth has spurred the development of new sequencing technologies that do not involve either electrophoresis or Sanger sequencing chemistries. Sequencing by Synthesis (SBS) involves multiple parallel micro-sequencing addition events occurring on a surface, where data from each round is detected by imaging. New High Throughput Technologies for DNA Sequencing and Genomics is the second volume in the Perspectives in Bioanalysis series, which looks at the electroanalytical chemistry of nucleic acids and proteins, development of electrochemical sensors and their application in biomedicine and in the new fields of genomics and proteomics. The authors have expertly formatted the information for a wide variety of readers, including new developments that will inspire students and young scientists to create new tools for science and medicine in the 21st century. Reviews of complementary developments in Sanger and SBS sequencing chemistries, capillary electrophoresis and microdevice integration, MS sequencing and applications set the framework for the book.

  3. The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering

    NASA Astrophysics Data System (ADS)

    Zhou, Ying-lin; Li, Yuan-zong

    2004-01-01

    For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 μg ml -1 for herring sperm DNA (hsDNA), 0.02-10.0 μg ml -1 for calf thymus DNA (ctDNA), 0.02-20.0 μg ml -1 for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml -1, respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2×10 5 M -1. IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.

  4. Identification and Optimization of Anthranilic Acid Based Inhibitors of Replication Protein A.

    PubMed

    Patrone, James D; Pelz, Nicholas F; Bates, Brittney S; Souza-Fagundes, Elaine M; Vangamudi, Bhavatarini; Camper, Demarco V; Kuznetsov, Alexey G; Browning, Carrie F; Feldkamp, Michael D; Frank, Andreas O; Gilston, Benjamin A; Olejniczak, Edward T; Rossanese, Olivia W; Waterson, Alex G; Chazin, Walter J; Fesik, Stephen W

    2016-04-19

    Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that initiates the DNA damage response pathway through protein-protein interactions (PPIs) mediated by its 70N domain. The identification and use of chemical probes that can specifically disrupt these interactions is important for validating RPA as a cancer target. A high-throughput screen (HTS) to identify new chemical entities was conducted, and 90 hit compounds were identified. From these initial hits, an anthranilic acid based series was optimized by using a structure-guided iterative medicinal chemistry approach to yield a cell-penetrant compound that binds to RPA70N with an affinity of 812 nm. This compound, 2-(3- (N-(3,4-dichlorophenyl)sulfamoyl)-4-methylbenzamido)benzoic acid (20 c), is capable of inhibiting PPIs mediated by this domain. PMID:26748787

  5. [Progress in biotransformation of bio-based lactic acid ].

    PubMed

    Gao, Chao; Ma, Cuiqing; Xu, Ping

    2013-10-01

    Fermentative production of lactic acid, an important bio-based chemicals, has made considerable progress. In addition to the food industry and production of polylactic acid, lactic acid also can be used as an important platform chemical for the production of acrylic acid, pyruvic acid, 1,2-propanediol, and lactic acid esters. This article summarizes the recent progress in biocatalytic production of lactic acid derivatives by dehydration, dehydrogenation, reduction, and esterification. Trends in the biotransformation of lactic acid are also discussed. PMID:24432656

  6. The amplification effect of functionalized gold nanoparticles on the binding of anticancer drug dacarbazine to DNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Shen, Qin; Wang, Xuemei; Fu, Degang

    2008-11-01

    The promising application of functionalized gold nanoparticles to amplify the performance of biosensors and relevant biomolecular recognition processes has been explored in this paper. Our observations illustrate the apparent enhancement effect of the gold nanoparticles on the electrochemical response of the anticancer drug dacarbazine (DTIC) binding to DNA and DNA bases, indicating that these functionalized gold nanoparticles could readily facilitate the specific interactions between DTIC and DNA/DNA bases. This raises the potential valuable applications of these biocompatible nanoparticles in the promising biosensors and biomedical engineering.

  7. Locked nucleic acid based beacons for surface interaction studies and biosensor development

    PubMed Central

    Martinez, Karen; Estevez, M.-Carmen; Wu, Yanrong; Phillips, Joseph A.; Medley, Colin D.; Tan, Weihong

    2011-01-01

    DNA sensors and microarrays permit fast, simple and real-time detection of nucleic acids through the design and use of increasingly sensitive, selective and robust molecular probes. Specifically, molecular beacons (MBs) have been employed for this purpose; however, their potential in the development of solid-surface-based biosensors has not been fully realized. This is mainly a consequence of the beacon’s poor stability due to the hairpin structure once immobilized onto a solid surface, commonly resulting in a low signal enhancement. Here, we report the design of a new MB that overcomes some of the limitations of MBs for surface immobilization. Essentially, this new design adds locked nucleic acid bases (LNAs) to the beacon structure, resulting in a LNA molecular beacon (LMB) with robust stability after surface immobilization. To test the efficacy of LMBs against that of regular molecular beacons (RMBs), the properties of selectivity, sensitivity, thermal stability, hybridization kinetics and robustness for the detection of target sequences were compared and evaluated. A 25-fold enhancement was achieved for the LMB on surface with detection limits reaching the low nanomolar range. In addition, the LMB-based biosensor was shown to possess better stability, reproducibility, selectivity and robustness when compared to the RMB. Therefore, as an alternative to conventional DNA and as a prospective tool for use in both DNA microarrays and biosensors, these results demonstrate the potential of the locked nucleic acid bases for nucleic acid design for surface immobilization. PMID:19351140

  8. Development of a Nucleic Acid Extraction Procedure for Simultaneous Recovery of DNA and RNA from Diverse Microbes in Water

    PubMed Central

    Hill, Vincent R.; Narayanan, Jothikumar; Gallen, Rachel R.; Ferdinand, Karen L.; Cromeans, Theresa; Vinjé, Jan

    2015-01-01

    Drinking and environmental water samples contain a diverse array of constituents that can interfere with molecular testing techniques, especially when large volumes of water are concentrated to the small volumes needed for effective molecular analysis. In this study, a suite of enteric viruses, bacteria, and protozoan parasites were seeded into concentrated source water and finished drinking water samples, in order to investigate the relative performance of nucleic acid extraction techniques for molecular testing. Real-time PCR and reverse transcription-PCR crossing threshold (CT) values were used as the metrics for evaluating relative performance. Experimental results were used to develop a guanidinium isothiocyanate-based lysis buffer (UNEX buffer) that enabled effective simultaneous extraction and recovery of DNA and RNA from the suite of study microbes. Procedures for bead beating, nucleic acid purification, and PCR facilitation were also developed and integrated in the protocol. The final lysis buffer and sample preparation procedure was found to be effective for a panel of drinking water and source water concentrates when compared to commercial nucleic acid extraction kits. The UNEX buffer-based extraction protocol enabled PCR detection of six study microbes, in 100 L finished water samples from four drinking water treatment facilities, within three CT values (i.e., within 90% difference) of the reagent-grade water control. The results from this study indicate that this newly formulated lysis buffer and sample preparation procedure can be useful for standardized molecular testing of drinking and environmental waters. PMID:26016775

  9. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

    PubMed

    Rohrman, Brittany; Richards-Kortum, Rebecca

    2015-02-01

    Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads. PMID:25560368

  10. Evidence that 3-hydroxy-3-methylglutaric and 3-methylglutaric acids induce DNA damage in rat striatum.

    PubMed

    da Rosa, Mateus Struecker; Scaini, Giselli; Damiani, Adriani Paganini; Longaretti, Luiza Martins; Pereira, Maiara; Seminotti, Bianca; Zapelini, Hugo Galvane; Schuck, Patrícia Fernanda; Streck, Emílio Luiz; de Andrade, Vanessa Moraes; Wajner, Moacir; Leipnitz, Guilhian

    2015-08-01

    3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a rare autosomal recessive disorderaffecting the final step of leucine degradation and ketogenesis and biochemically characterized by the predominant accumulation of 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA) acids in biological fluids and tissues of affected patients. Considering that previous studies reported that HMG and MGA have pro oxidant properties, the present study evaluated the ex vivo and in vitro effects of HMG and MGA on frequency and index of DNA damage in cerebral cortex and striatum of young rats. The ex vivo effects of both organic acids on 8-hydroxy-2'-deoxyguanosine (OHdG) levels and their in vitro effects on 2',7'-dichlorofluorescin (DCFH) oxidation and glutathione (GSH) concentrations in rat striatum were also determined. We also investigated the ex vivo effects of both organic acids on 8-hydroxy-2'-deoxyguanosine (OHdG) levels in rat striatum. In the ex vivo experiments, DNA damage was determined in striatum homogenates prepared 30 min after a single intrastriatal administration of HMG or MGA. On the other hand, the in vitro evaluation was performed after an incubation of rat cerebral cortex or striatum homogenates or slices in the presence of HMG or MGA during 1 h at 37 °C. We observed that the intrastriatal administration of HMG and MGA increased the frequency and the index of DNA damage, as well as OHdG staining in rat striatum. We also verified that MGA, but not HMG, increased DNA damage frequency and index in vitro in striatum of rats. In contrast, no alterations were verified in vitro in cerebral cortex. Finally, we found that HMG and MGA increased DCFH oxidation and decreased GSH concentrations in rat striatum. Therefore, it may be presumed that DNA damage provoked by HMG and MGA possibly via reactive species generation is involved, at least in part, in the pathophysiology of brain injury, particularly in the striatum of HL-deficient patients. PMID:25939283

  11. Running DNA Mini-Gels in 20 Minutes or Less Using Sodium Boric Acid Buffer

    ERIC Educational Resources Information Center

    Jenkins, Kristin P.; Bielec, Barbara

    2006-01-01

    Providing a biotechnology experience for students can be challenging on several levels, and time is a real constraint for many experiments. Many DNA based methods require a gel electrophoresis step, and although some biotechnology procedures have convenient break points, gel electrophoresis does not. In addition to the time required for loading…

  12. A sticker-based model for DNA computation.

    PubMed

    Roweis, S; Winfree, E; Burgoyne, R; Chelyapov, N V; Goodman, M F; Rothemund, P W; Adleman, L M

    1998-01-01

    We introduce a new model of molecular computation that we call the sticker model. Like many previous proposals it makes use of DNA strands as the physical substrate in which information is represented and of separation by hybridization as a central mechanism. However, unlike previous models, the stickers model has a random access memory that requires no strand extension and uses no enzymes; also (at least in theory), its materials are reusable. The paper describes computation under the stickers model and discusses possible means for physically implementing each operation. Finally, we go on to propose a specific machine architecture for implementing the stickers model as a microprocessor-controlled parallel robotic workstation. In the course of this development a number of previous general concerns about molecular computation (Smith, 1996; Hartmanis, 1995; Linial et al., 1995) are addressed. First, it is clear that general-purpose algorithms can be implemented by DNA-based computers, potentially solving a wide class of search problems. Second, we find that there are challenging problems, for which only modest volumes of DNA should suffice. Third, we demonstrate that the formation and breaking of covalent bonds is not intrinsic to DNA-based computation. Fourth, we show that a single essential biotechnology, sequence-specific separation, suffices for constructing a general-purpose molecular computer. Concerns about errors in this separation operation and means to reduce them are addressed elsewhere (Karp et al., 1995; Roweis and Winfree, 1999). Despite these encouraging theoretical advances, we emphasize that substantial engineering challenges remain at almost all stages and that the ultimate success or failure of DNA computing will certainly depend on whether these challenges can be met in laboratory investigations. PMID:10072080

  13. Identifying a base in a nucleic acid

    DOEpatents

    Fodor, Stephen P. A.; Lipshutz, Robert J.; Huang, Xiaohua

    2005-02-08

    Devices and techniques for hybridization of nucleic acids and for determining the sequence of nucleic acids. Arrays of nucleic acids are formed by techniques, preferably high resolution, light-directed techniques. Positions of hybridization of a target nucleic acid are determined by, e.g., epifluorescence microscopy. Devices and techniques are proposed to determine the sequence of a target nucleic acid more efficiently and more quickly through such synthesis and detection techniques.

  14. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    SciTech Connect

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

    2010-08-01

    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  15. Nanoparticles based DNA conjugates for detection of pathogenic microorganisms

    NASA Astrophysics Data System (ADS)

    Jamdagni, Pragati; Khatri, Poonam; Rana, J. S.

    2016-01-01

    Infectious diseases have been on rise in the recent past. Early diagnosis plays a role as important as proper treatment and prophylaxis. The current practices of detection are time consuming which may result in unnecessary delays in treatment. Advances in nanodiagnostic approaches have been in focus lately. The rising interest and better understanding of nanoparticles have led to opening up of new frontiers in the concerned area. Optical properties of nanoparticles are being exploited to design detection systems that can provide fast, one-step and reliable results. Based on conserved DNA sequences unique to the target organism, the results offer accuracy comparable to conventional tests. Further, visual or spectrophotometric analysis omits the need of costly apparatus for result interpretation. The present review aims at putting together the information on nanoparticles based DNA conjugate systems for detection of pathogenic microorganisms.

  16. A dynamic bead-based microarray for parallel DNA detection

    NASA Astrophysics Data System (ADS)

    Sochol, R. D.; Casavant, B. P.; Dueck, M. E.; Lee, L. P.; Lin, L.

    2011-05-01

    A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening.

  17. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization.

    PubMed

    Addamiano, Claudia; Gerland, Béatrice; Payrastre, Corinne; Escudier, Jean-Marc

    2016-01-01

    Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5'-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5'-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected. PMID:27563857

  18. Impact of boric acid exposure at different concentrations on testicular DNA and male rats fertility.

    PubMed

    El-Dakdoky, Mai H; Abd El-Wahab, Hanan M F

    2013-06-01

    The aim of this study was to investigate the consequences of exposure to three levels of boric acid (BA) on male rats reproduction, fertility and progeny outcome, with emphasis on testicular DNA level and quality. Adult male rats (12 weeks old) were treated orally with 125, 250 and 500 mg/kg bwt/d of BA for 60 d. The results indicated that BA administration at 125 mg/kg bwt had no adverse effects on fertility, sperm characteristics or prenatal development of the impregnated females. However, at dose 250 mg, BA treatment significantly increased serum nitric oxide, testosterone, estradiol levels and testicular boron and calcium levels and also significantly reduced serum arginase activity, sperm quality and testicular DNA content with minor DNA fragmentation. The impact of BA exposure at dose 250 mg on male rats fertility was translated into increases in pre-implantation loss with a resulting decrease in the number of live fetuses/litter. In addition to the significant alteration of biochemical measurements, observed at dose 250 mg, administration of BA at 500 mg caused testicular atrophy, severe damage of spermatogenesis, spermiation failure and significant reduction of Mg and Zn testicular levels. None of the male rats, treated with 500 mg/kg bwt, could impregnate untreated females, suggesting the occurrence of definitive loss of fertility. In conclusion, BA impaired fertility, in a dose-dependant manner, by targeting the highly proliferative cells, the germ cells, through decreasing DNA synthetic rate rather than the induction of DNA damage. PMID:23301826

  19. Suberoylanilide Hydroxyamic Acid Modification of Chromatin Architecture Affects DNA Break Formation and Repair

    SciTech Connect

    Singh, Sheetal; Le Hongan; Shih, S.-J.; Ho, Bay; Vaughan, Andrew T.

    2010-02-01

    Purpose: Chromatin-modifying compounds that inhibit the activity of histone deacetylases have shown potency as radiosensitizers, but the action of these drugs at a molecular level is not clear. Here we investigated the effect of suberoylanilide hydroxyamic acid (SAHA) on DNA breaks and their repair and induction of rearrangements. Methods and Materials: The effect of SAHA on both clonogenic survival and repair was assessed using cell lines SCC-25, MCF7, and TK6. In order to study unique DNA double-strand breaks, anti-CD95 antibody was employed to introduce a DNA double-strand break at a known location within the 11q23 region. The effects of SAHA on DNA cleavage and rearrangements were analyzed by ligation-mediated PCR and inverse PCR, respectively. Results: SAHA acts as radiosensitizer at 1 {mu}M, with dose enhancement factors (DEFs) at 10% survival of: SCC-25 - 1.24 +- 0.05; MCF7 - 1.16 +- 0.09 and TK6 - 1.17 +- 0.05, and it reduced the capacity of SCC-25 cells to repair radiation induced lesions. Additionally, SAHA treatment diffused site-specific fragmentation over at least 1 kbp in TK6 cells. Chromosomal rearrangements produced in TK6 cells exposed to SAHA showed a reduction in microhomology at the breakpoint between 11q23 and partner chromosomes. Conclusions: SAHA shows efficacy as a radiosensitizer at clinically obtainable levels. In its presence, targeted DNA strand breaks occur over an expanded region, indicating increased chromatin access. The rejoining of such breaks is degraded by SAHA when measured as rearrangements at the molecular level and rejoining that contributes to cell survival.

  20. CD44-Targeted Hyaluronic Acid-Coated Redox-Responsive Hyperbranched Poly(amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery.

    PubMed

    Gu, Jijin; Chen, Xinyi; Ren, Xiaoqing; Zhang, Xiulei; Fang, Xiaoling; Sha, Xianyi

    2016-07-20

    Hyaluronic acid (HA), which can specifically bind to CD44 receptor, is a specific ligand for targeting to CD44-overexpressing cancer cells. The current study aimed to develop ternary nanoassemblies based on HA-coating for targeted gene delivery to CD44-positive tumors. A novel reducible hyperbranched poly(amido amine) (RHB) was assembled with plasmid DNA (pDNA) to form RHB/pDNA nanoassemblies. HA/RHB/pDNA nanoassemblies were fabricated by coating HA on the surface of the RHB/pDNA nanoassembly core through electrostatic interaction. After optimization, HA/RHB/pDNA nanoassemblies were spherical, core-shell nanoparticles with nanosize (187.6 ± 11.4 nm) and negative charge (-9.1 ± 0.3 mV). The ternary nanoassemblies could efficiently protect the condensed pDNA from enzymatic degradation by DNase I, and HA could significantly improve the stability of nanoassemblies in the sodium heparin solution or serum in vitro. As expected, HA significantly decreased the cytotoxicity of RHB/pDNA nanoassemblies due to the negative surface charges. Moreover, it revealed that HA/RHB/pDNA nanoassemblies showed higher transfection activity than RHB/pDNA nanoassemblies in B16F10 cells, especially in the presence of serum in vitro. Because of the active recognition between HA and CD44 receptor, there was significantly different transfection efficiency between B16F10 (CD44+) and NIH3T3 (CD44-) cells after treatment with HA/RHB/pDNA nanoassemblies. In addition, the cellular targeting and transfection activity of HA/RHB/pDNA nanoassemblies were further evaluated in vivo. The results indicated that the interaction between HA and CD44 receptor dramatically improved the accumulation of HA/RHB/pDNA nanoassemblies in CD44-positive tumor, leading to higher gene expression than RHB/pDNA nanoassemblies. Therefore, HA/RHB/pDNA ternary nanoassemblies may be a potential gene vector for delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo. PMID:27311558

  1. Guanine base stacking in G-quadruplex nucleic acids.

    PubMed

    Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2013-02-01

    G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5'-5' manner based on different accessible tetrad stacking modes at the stacking interfaces of 5'-5' and 3'-3' stacked G-quadruplexes. PMID:23268444

  2. Sensitive and visual detection of sequence-specific DNA-binding protein via a gold nanoparticle-based colorimetric biosensor.

    PubMed

    Ou, Li-Juan; Jin, Pei-Yan; Chu, Xia; Jiang, Jian-Hui; Yu, Ru-Qin

    2010-07-15

    A novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNA-binding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. Interestingly, we disclosed a new finding that binding of target proteins to their binding sequences in the aggregated AuNP network rendered a stable and long-period protection of DNA. Unlike conventional fluorescence assays merely based on temporal protection of DNA from Exo III digestion, the stable protection afforded a static color transition indicator for DNA-protein interactions with no time-dependent monitoring required in the assay. Therefore, it furnished the developed strategy with improved technical robustness and operational convenience. Furthermore, we introduced thioctic acid as a stable anchor for tethering DNA on AuNPs. This DNA-tethering protocol circumvented the interferences from thiol compounds in common enzymatic systems. The Exo III protection-based colorimetric biosensor was demonstrated using a model target of TATA binding protein, a key transcriptional factor involving in various transcriptional regulatory networks. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM. PMID:20565105

  3. DNA biosensors based on layer-by-layer self-assembled multilayer films of carbon nanotubes and gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Xiao, Yiyun; Dai, Zhao; Zhang, Jimei; Pang, Jiechun; Xu, Shichao; Zheng, Guo

    2009-07-01

    A novel DNA biosensor based on layer-by-layer self-assembled multi-walled carbon nanotubes (MWNTs) and gold nano-particles (GNPs) was presented in this paper, in which the probe HS-ssDNA oligonucleotides, MWNTs and GNPs were all covalently immobilized by chemical Au-Sulphide bonding. Firstly, the super short MWNTs were prepared and modified with thio groups which could be self-assembled onto the surface of Au elcetrode by Au-sulphide bonding, then the GNPs were chemically adhered to the surfaces of MWNTs by forming Au-sulphide bonding again, at last the selfassamble of probe DNA oligonucleotides were also covalently immobilized via Au-sulphide bonding between thio groups at the ends of the DNA oligonucleotides and GNPs. Hybridization between the probe HS-ssDNA oligonucleotides and target DNA oligonucleotides was confirmed by the changes in the voltammetric peak of an anionic intercalator, anthraquinone-2,6-disulfonic acid (AQDS) as a hybridization indicator. The cyclic voltammetric and differential pulse voltammetry responses demonstrated that the DNA biosensors based on Layer-by-layer self-assembled multilayer films of MWNTs and NGPs offer a higher hybridization efficiency and selectivity compared to those based on only random MWNTs or GNPs.

  4. Electric-field induced mutation of DNA: a theoretical investigation of the GC base pair.

    PubMed

    Cerón-Carrasco, José P; Jacquemin, Denis

    2013-04-01

    It is known that intense external electric fields affect the proton transfer (PT) reactions in simple chemical systems, such as hydrated chlorhydric acid or formic acid dimer. Accordingly, electric fields might be used to modulate the PT reactions responsible for the spontaneous mutation mechanism in DNA. In this contribution, we investigate the effect of these fields on the tautomeric equilibria of the guanine-cytosine (GC) base pair in order to gain further insight into this hypothesis. This task is performed with both density functional theory (DFT) and second-order Møller-Plesset (MP2) approaches. Our results demonstrate that electric fields not only drastically alter the rate constants of PT but also tune the mechanism of the PT reactions in the GC base pair. PMID:23338206

  5. Arduino-based automation of a DNA extraction system.

    PubMed

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile. PMID:26409535

  6. APE1-mediated DNA damage repair provides survival advantage for esophageal adenocarcinoma cells in response to acidic bile salts.

    PubMed

    Hong, Jun; Chen, Zheng; Peng, Dunfa; Zaika, Alexander; Revetta, Frank; Washington, M Kay; Belkhiri, Abbes; El-Rifai, Wael

    2016-03-29

    Chronic Gastroesophageal Reflux Disease (GERD) is the main risk factor for the development of Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Accordingly, EAC cells are subjected to high levels of oxidative stress and subsequent DNA damage. In this study, we investigated the expression and role of Apurinic/apyrimidinic endonuclease 1 (APE1) protein in promoting cancer cell survival by counteracting the lethal effects of acidic bile salts (ABS)-induced DNA damage. Immunohistochemistry analysis of human tissue samples demonstrated overexpression of APE1 in more than half of EACs (70 of 130), as compared to normal esophagus and non-dysplastic BE samples (P < 0.01). To mimic in vivo conditions, we treated in vitro cell models with a cocktail of ABS. The knockdown of endogenous APE1 in EAC FLO-1 cells significantly increased oxidative DNA damage (P < 0.01) and DNA single- and double-strand breaks (P < 0.01), whereas overexpression of APE1 in EAC OE33 cells reversed these effects. Annexin V/PI staining indicated that the APE1 expression in OE33 cells protects against ABS-induced apoptosis. In contrast, knockdown of endogenous APE1 in FLO-1 cells increased apoptosis under the same conditions. Mechanistic investigations indicated that the pro-survival function of APE1 was associated with the regulation of stress response c-Jun N-terminal protein kinase (JNK) and p38 kinases. Pharmacological inhibition of APE1 base excision repair (BER) function decreased cell survival and enhanced activation of JNK and p38 kinases by ABS. Our findings suggest that constitutive overexpression of APE1 in EAC may be an adaptive pro-survival mechanism that protects against the genotoxic lethal effects of bile reflux episodes. PMID:26934647

  7. APE1-mediated DNA damage repair provides survival advantage for esophageal adenocarcinoma cells in response to acidic bile salts

    PubMed Central

    Hong, Jun; Chen, Zheng; Peng, Dunfa; Zaika, Alexander; Revetta, Frank; Washington, M. Kay; Belkhiri, Abbes; El-Rifai, Wael

    2016-01-01

    Chronic Gastroesophageal Reflux Disease (GERD) is the main risk factor for the development of Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Accordingly, EAC cells are subjected to high levels of oxidative stress and subsequent DNA damage. In this study, we investigated the expression and role of Apurinic/apyrimidinic endonuclease 1 (APE1) protein in promoting cancer cell survival by counteracting the lethal effects of acidic bile salts (ABS)-induced DNA damage. Immunohistochemistry analysis of human tissue samples demonstrated overexpression of APE1 in more than half of EACs (70 of 130), as compared to normal esophagus and non-dysplastic BE samples (P < 0.01). To mimic in vivo conditions, we treated in vitro cell models with a cocktail of ABS. The knockdown of endogenous APE1 in EAC FLO-1 cells significantly increased oxidative DNA damage (P < 0.01) and DNA single- and double-strand breaks (P < 0.01), whereas overexpression of APE1 in EAC OE33 cells reversed these effects. Annexin V/PI staining indicated that the APE1 expression in OE33 cells protects against ABS-induced apoptosis. In contrast, knockdown of endogenous APE1 in FLO-1 cells increased apoptosis under the same conditions. Mechanistic investigations indicated that the pro-survival function of APE1 was associated with the regulation of stress response c-Jun N-terminal protein kinase (JNK) and p38 kinases. Pharmacological inhibition of APE1 base excision repair (BER) function decreased cell survival and enhanced activation of JNK and p38 kinases by ABS. Our findings suggest that constitutive overexpression of APE1 in EAC may be an adaptive pro-survival mechanism that protects against the genotoxic lethal effects of bile reflux episodes. PMID:26934647

  8. Free energy analysis and mechanism of base pair stacking in nicked DNA.

    PubMed

    Häse, Florian; Zacharias, Martin

    2016-09-01

    The equilibrium of stacked and unstacked base pairs is of central importance for all nucleic acid structure formation processes. The stacking equilibrium is influenced by intramolecular interactions between nucleosides but also by interactions with the solvent. Realistic simulations on nucleic acid structure formation and flexibility require an accurate description of the stacking geometry and stability and its sequence dependence. Free energy simulations have been conducted on a series of double stranded DNA molecules with a central strand break (nick) in one strand. The change in free energy upon unstacking was calculated for all ten possible base pair steps using umbrella sampling along a center-of-mass separation coordinate and including a comparison of different water models. Comparison to experimental studies indicates qualitative agreement of the stability order but a general overestimation of base pair stacking interactions in the simulations. A significant dependence of calculated nucleobase stacking free energies on the employed water model was observed with the tendency of stacking free energies being more accurately reproduced by more complex water models. The simulation studies also suggest a mechanism of stacking/unstacking that involves significant motions perpendicular to the reaction coordinate and indicate that the equilibrium nicked base pair step may slightly differ from regular B-DNA geometry in a sequence-dependent manner. PMID:27407106

  9. Free energy analysis and mechanism of base pair stacking in nicked DNA

    PubMed Central

    Häse, Florian; Zacharias, Martin

    2016-01-01

    The equilibrium of stacked and unstacked base pairs is of central importance for all nucleic acid structure formation processes. The stacking equilibrium is influenced by intramolecular interactions between nucleosides but also by interactions with the solvent. Realistic simulations on nucleic acid structure formation and flexibility require an accurate description of the stacking geometry and stability and its sequence dependence. Free energy simulations have been conducted on a series of double stranded DNA molecules with a central strand break (nick) in one strand. The change in free energy upon unstacking was calculated for all ten possible base pair steps using umbrella sampling along a center-of-mass separation coordinate and including a comparison of different water models. Comparison to experimental studies indicates qualitative agreement of the stability order but a general overestimation of base pair stacking interactions in the simulations. A significant dependence of calculated nucleobase stacking free energies on the employed water model was observed with the tendency of stacking free energies being more accurately reproduced by more complex water models. The simulation studies also suggest a mechanism of stacking/unstacking that involves significant motions perpendicular to the reaction coordinate and indicate that the equilibrium nicked base pair step may slightly differ from regular B-DNA geometry in a sequence-dependent manner. PMID:27407106

  10. Genome defense against exogenous nucleic acids in eukaryotes by non-coding DNA occurs through CRISPR-like mechanisms in the cytosol and the bodyguard protection in the nucleus.

    PubMed

    Qiu, Guo-Hua

    2016-01-01

    In this review, the protective function of the abundant non-coding DNA in the eukaryotic genome is discussed from the perspective of genome defense against exogenous nucleic acids. Peripheral non-coding DNA has been proposed to act as a bodyguard that protects the genome and the central protein-coding sequences from ionizing radiation-induced DNA damage. In the proposed mechanism of protection, the radicals generated by water radiolysis in the cytosol and IR energy are absorbed, blocked and/or reduced by peripheral heterochromatin; then, the DNA damage sites in the heterochromatin are removed and expelled from the nucleus to the cytoplasm through nuclear pore complexes, most likely through the formation of extrachromosomal circular DNA. To strengthen this hypothesis, this review summarizes the experimental evidence supporting the protective function of non-coding DNA against exogenous nucleic acids. Based on these data, I hypothesize herein about the presence of an additional line of defense formed by small RNAs in the cytosol in addition to their bodyguard protection mechanism in the nucleus. Therefore, exogenous nucleic acids may be initially inactivated in the cytosol by small RNAs generated from non-coding DNA via mechanisms similar to the prokaryotic CRISPR-Cas system. Exogenous nucleic acids may enter the nucleus, where some are absorbed and/or blocked by heterochromatin and others integrate into chromosomes. The integrated fragments and the sites of DNA damage are removed by repetitive non-coding DNA elements in the heterochromatin and excluded from the nucleus. Therefore, the normal eukaryotic genome and the central protein-coding sequences are triply protected by non-coding DNA against invasion by exogenous nucleic acids. This review provides evidence supporting the protective role of non-coding DNA in genome defense. PMID:27036064

  11. A Graphene-Based Biosensing Platform Based on Regulated Release of an Aptameric DNA Biosensor.

    PubMed

    Mao, Yu; Chen, Yongli; Li, Song; Lin, Shuo; Jiang, Yuyang

    2015-01-01

    A novel biosensing platform was developed by integrating an aptamer-based DNA biosensor with graphene oxide (GO) for rapid and facile detection of adenosine triphosphate (ATP, as a model target). The DNA biosensor, which is locked by GO, is designed to contain two sensing modules that include recognition site for ATP and self-replication track that yields the nicking domain for Nt.BbvCI. By taking advantage of the different binding affinity of single-stranded DNA, double-stranded DNA and aptamer-target complex toward GO, the DNA biosensor could be efficiently released from GO in the presence of target with the help of a complementary DNA strand (CPDNA) that partially hybridizes to the DNA biosensor. Then, the polymerization/nicking enzyme synergetic isothermal amplification could be triggered, leading to the synthesis of massive DNA amplicons, thus achieving an enhanced sensitivity with a wide linear dynamic response range of four orders of magnitude and good selectivity. This biosensing strategy expands the applications of GO-DNA nanobiointerfaces in biological sensing, showing great potential in fundamental research and biomedical diagnosis. PMID:26569239

  12. A Graphene-Based Biosensing Platform Based on Regulated Release of an Aptameric DNA Biosensor

    PubMed Central

    Mao, Yu; Chen, Yongli; Li, Song; Lin, Shuo; Jiang, Yuyang

    2015-01-01

    A novel biosensing platform was developed by integrating an aptamer-based DNA biosensor with graphene oxide (GO) for rapid and facile detection of adenosine triphosphate (ATP, as a model target). The DNA biosensor, which is locked by GO, is designed to contain two sensing modules that include recognition site for ATP and self-replication track that yields the nicking domain for Nt.BbvCI. By taking advantage of the different binding affinity of single-stranded DNA, double-stranded DNA and aptamer-target complex toward GO, the DNA biosensor could be efficiently released from GO in the presence of target with the help of a complementary DNA strand (CPDNA) that partially hybridizes to the DNA biosensor. Then, the polymerization/nicking enzyme synergetic isothermal amplification could be triggered, leading to the synthesis of massive DNA amplicons, thus achieving an enhanced sensitivity with a wide linear dynamic response range of four orders of magnitude and good selectivity. This biosensing strategy expands the applications of GO-DNA nanobiointerfaces in biological sensing, showing great potential in fundamental research and biomedical diagnosis. PMID:26569239

  13. Direct assay of radiation-induced DNA base lesions in mammalian cells

    SciTech Connect

    Not Available

    1992-01-01

    Adenine (Ade), 2'-deoxyadenosine (dAdo), 5'-deoxyadenosine monophosphate (dAUT), single stranded poly adenylic acid [poly (dA)], double stranded deoxyadenylic-thymidylic acid [ds poly (dA-T)] and salmon testis DNA were irradiated with 500 Gy under oxic and anoxic conditions. The major damage products were analyzed by BPLC with optical detection and quantitated in terms of the percentage of the adenosine in each model compound found as a specific damage product. Outside of the Ade free base, 8-OH-dAdo was the major oxic damage product from each model compound. The type and quantity of the major damage products depended on the sequence and conformation of the model compounds under anoxic conditions. When dAdo and dAMP were irradiated under anoxic conditions, the major damage product was either the R or S isomer of 8,5'cdAdo and little Ade or [alpha]-dAdo was observed. However, when poly(dA), poly(dA-dT), and salmon testis DNA were [gamma]-irradiated under nitrogen, the major deoxyadenosine damage product was identified as the [alpha]-anomer of deoxyadenosine. No [alpha]-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the [alpha]-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.

  14. DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation

    PubMed Central

    Cadet, Jean; Wagner, J. Richard

    2013-01-01

    Emphasis has been placed in this article dedicated to DNA damage on recent aspects of the formation and measurement of oxidatively generated damage in cellular DNA in order to provide a comprehensive and updated survey. This includes single pyrimidine and purine base lesions, intrastrand cross-links, purine 5′,8-cyclonucleosides, DNA–protein adducts and interstrand cross-links formed by the reactions of either the nucleobases or the 2-deoxyribose moiety with the hydroxyl radical, one-electron oxidants, singlet oxygen, and hypochlorous acid. In addition, recent information concerning the mechanisms of formation, individual measurement, and repair-rate assessment of bipyrimidine photoproducts in isolated cells and human skin upon exposure to UVB radiation, UVA photons, or solar simulated light is critically reviewed. PMID:23378590

  15. Base Pair Fraying in Molecular Dynamics Simulations of DNA and RNA.

    PubMed

    Zgarbová, Marie; Otyepka, Michal; Šponer, Jiří; Lankaš, Filip; Jurečka, Petr

    2014-08-12

    Terminal base pairs of DNA and RNA molecules in solution are known to undergo frequent transient opening events (fraying). Accurate modeling of this process is important because of its involvement in nucleic acid end recognition and enzymatic catalysis. In this article, we describe fraying in molecular dynamics simulations with the ff99bsc0, ff99bsc0χOL3, and ff99bsc0χOL4 force fields, both for DNA and RNA molecules. Comparison with the experiment showed that while some features of fraying are consistent with the available data, others indicate potential problems with the force field description. In particular, multiple noncanonical structures are formed at the ends of the DNA and RNA duplexes. Among them are tWC/sugar edge pair, C-H edge/Watson-Crick pair, and stacked geometries, in which the terminal bases are stacked above each other. These structures usually appear within the first tens to hundreds of nanoseconds and substantially limit the usefulness of the remaining part of the simulation due to geometry distortions that are transferred to several neighboring base pairs ("end effects"). We show that stability of the noncanonical structures in ff99bsc0 may be partly linked to inaccurate glycosidic (χ) torsion potentials that overstabilize the syn region and allow for rapid anti to syn transitions. The RNA refined glycosidic torsion potential χOL3 provides an improved description and substantially more stable MD simulations of RNA molecules. In the case of DNA, the χOL4 correction gives only partial improvement. None of the tested force fields provide a satisfactory description of the terminal regions, indicating that further improvement is needed to achieve realistic modeling of fraying in DNA and RNA molecules. PMID:26588288

  16. Physiological roles of acid-base sensors.

    PubMed

    Levin, Lonny R; Buck, Jochen

    2015-01-01

    Acid-base homeostasis is essential for life. The macromolecules upon which living organisms depend are sensitive to pH changes, and physiological systems use the equilibrium between carbon dioxide, bicarbonate, and protons to buffer their pH. Biological processes and environmental insults are constantly challenging an organism's pH; therefore, to maintain a consistent and proper pH, organisms need sensors that measure pH and that elicit appropriate responses. Mammals use multiple sensors for measuring both intracellular and extracellular pH, and although some mammalian pH sensors directly measure protons, it has recently become apparent that many pH-sensing systems measure pH via bicarbonate-sensing soluble adenylyl cyclase. PMID:25340964

  17. Polymer gel dosimeter based on itaconic acid.

    PubMed

    Mattea, Facundo; Chacón, David; Vedelago, José; Valente, Mauro; Strumia, Miriam C

    2015-11-01

    A new polymeric dosimeter based on itaconic acid and N, N'-methylenebisacrylamide was studied. The preparation method, compositions of monomer and crosslinking agent and the presence of oxygen in the dosimetric system were analyzed. The resulting materials were irradiated with an X-ray tube at 158cGy/min, 226cGymin and 298cGy/min with doses up to 1000Gy. The dosimeters presented a linear response in the dose range 75-1000Gy, sensitivities of 0.037 1/Gyat 298cGy/min and an increase in the sensitivity with lower dose rates. One of the most relevant outcomes in this study was obtaining different monomer to crosslinker inclusion in the formed gel for the dosimeters where oxygen was purged during the preparation method. This effect has not been reported in other typical dosimeters and could be attributed to the large differences in the reactivity among these species. PMID:26275817

  18. Silica nanoparticles as a delivery system for nucleic acid-based reagents

    PubMed Central

    Hom, Christopher; Lu, Jie

    2010-01-01

    The transport of nucleic acid-based reagents is predicated upon developing structurally stable delivery systems that can preferentially bind and protect DNA and RNA, and release their cargo upon reaching their designated sites. Recent advancements in tailoring the size, shape, and external surface functionalization of silica materials have led to increased biocompatibility and efficiency of delivery. In this Feature Article, we highlight recent research progress in the use of silica nanoparticles as a delivery vehicle for nucleic acid-based reagents. PMID:20740060

  19. Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

    PubMed

    Stachler, Matthew; Jia, Yonghui; Sharaf, Nematullah; Wade, Jacqueline; Longtine, Janina; Garcia, Elizabeth; Sholl, Lynette M

    2015-01-01

    Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas. PMID:25221956

  20. Comparison of the levels of 8-hydroxyguanine in DNA as measured by gas chromatography mass spectrometry following hydrolysis of DNA by Escherichia coli Fpg protein or formic acid

    PubMed Central

    Rodriguez, Henry; Jurado, Juan; Laval, Jacques; Dizdaroglu, Miral

    2000-01-01

    8-hydroxyguanine (8-OH-Gua) is one of many lesions generated in DNA by oxidative processes including free radicals. It is the most extensively investigated lesion, due to its miscoding properties and its potential role in mutagenesis, carcinogenesis and aging, and also to the existence of analytical methods using HPLC and gas chromatography mass spectrometry (GC/MS). Some studies raised the possibility of artifacts generated during sample preparation. We investigated several experimental conditions in order to eliminate possible artifacts during the measurement of 8-OH-Gua by GC/MS. Derivatization has been reported to produce artifacts by oxidation of guanine to 8-OH-Gua in acid-hydrolysates of DNA, although the extent of artifacts seems to depend on experimental conditions. For removal of 8-OH-Gua from DNA, we used either formic acid hydrolysis or specific enzymatic hydrolysis with Escherichia coli Fpg protein. Derivatization of enzyme-hydrolysates should not generate additional 8-OH-Gua because of the absence of guanine, which is not released by the enzyme, whereas guanine released by acid may be oxidized to yield 8-OH-Gua. The measurement of 8-OH-Gua in calf thymus DNA by GC/isotope-dilution MS (GC/IDMS) using these two different hydrolyses yielded similar levels of 8-OH-Gua. This indicated that no artifacts occurred during derivatization of acid-hydrolysates of DNA. Pyridine instead of acetonitrile and room temperature were used during derivatization. Pyridine reduced the level of 8-OH-Gua, when compared with acetonitrile, indicating its potential to prevent oxidation. Two different stable-isotope labeled analogs of 8-OH-Gua used as internal standards for GC/IDMS analysis yielded similar results. A comparison of the present results with the results of recent trials by the European Standards Committee for Oxidative DNA Damage (ESCODD) is also presented. PMID:10908368

  1. Base sequence effects on interactions of aromatic mutagens with DNA

    SciTech Connect

    Geacintov, N.E.

    1992-09-30

    The chemical binding of bulky, mutagenic and carcinogenic polynuclear aromatic compounds to certain base-sequences in genomic DNA is known to inhibit DNA replication, and to induce mutations and cancer. In particular, sequences that contain multiple consecutive guanines appear to be hot spots of mutation. The objectives of this research are to determine how the base sequence around the mutagen-modified target bases influences the local DNA conformation and gives rise to mispairing of bases, or deletions, near the lesion. Oligonucleotides containing one, two, or three guanines were synthesized and chemically reacted with the mutagen anti-7,8-dihydroxy-9,10-epoxy-benzo(a)pyrene (BPDE), one of the most mutagenic and tumorigenic metabolites of benzo(a)pyrene. Adducts are formed in which only one of the guanines is modified by trans or cis addition to the exocyclic amino group. The BPDE-oligonucleotides are separated chromatographically, and the site of modification is established by Maxam-Gilbert high resolution gel electrophoresis techniques. The thermodynamic properties of duplexes using complementary, or partially complementary strands were examined. In the latter, the base opposite the modified guanine was varied in order to investigate the probability of mispairing of the modified G with A,T and G. The successful synthesis of stereospecific and site-specific mutagen-oligonucleotide adducts opens new possibilities for correlating adduct structure-biological activity relationships, and thus lead to a better understanding of base-sequence effects in mutagenesis induced by energy-related bulky polynuclear aromatic chemicals.

  2. Ellagic acid metabolism and binding to DNA in organ explant cultures of the rat.

    PubMed

    Teel, R W; Martin, R M; Allahyari, R

    1987-08-01

    Ellagic acid (EA) is a plant phenolic compound with postulated antimutagenic and anticarcinogenic activity. In this study, explants of esophagus, forestomach, colon, bladder, trachea, lung and liver from male Sprague-Dawley rats (130-140 g) were incubated in culture medium containing [3H]EA (20 microM, 4.5 microCi/ml) for 24 h at 37 degrees C. After extraction, purification and quantitation of explant DNA significant differences in the binding of EA to the DNA was observed. The most binding occurred in esophagus and the least in lung. Analysis of the organsoluble fraction of the culture medium by high performance liquid chromatography yielded 3 metabolites of EA. None of the metabolites were identified. Elution of water-soluble metabolites from an alumina column showed that there were sulfate ester, glucuronide and glutathione conjugates of EA in the explant culture medium from all the organs. The profile of water-soluble conjugates was very similar between colon and forestomach and between trachea and lung. These results indicate that EA binds to DNA in different tissues and that tissues metabolize EA to both organosoluble and water-soluble products. PMID:3621152

  3. Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

    PubMed Central

    Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2012-01-01

    A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. Addition of thermostable RNase H resulted in the cleavage of the RNA loop which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9x above background), resulting in a ~2–2.8 fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time PCR reactions by measuring enhancement of donor fluorescence upon R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  4. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

    Jacroux, Thomas; Rieck, Daniel C; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The addition of thermostable RNase H resulted in the cleavage of the RNA loop, which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9× above background), resulting in an approximately 2- to 2.8-fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time polymerase chain reactions by measuring enhancement of donor fluorescence on R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  5. Validation and scale-up of plasmid DNA purification by phenyl-boronic acid chromatography.

    PubMed

    Gomes, A Gabriela; Azevedo, Ana M; Aires-Barros, M Raquel; Prazeres, D Miguel F

    2012-11-01

    This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm(3) of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm(3) /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93-96% of pDNA was recovered in the flow through while 66-71% of impurities remained bound (~2.5-fold purification). The entire sequence of loading, washing, elution, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75-15.2 cm(3) ) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plasmids. The column productivity at large scale was 4 dm(3) of alkaline lysate per hour per dm(3) of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration. PMID:23175141

  6. [Release of Extracellular DNA after Administration of Radioprotective Combination of α-Tocopherol and Ascorbic Acid].

    PubMed

    Vasilyeval, I N; Bespalov, V G

    2015-01-01

    Radioprotective and apoptotic activities of α-tocopherol acetate (vitamin E) and ascorbic acid (vitamin C) have been studied in 180 Wistar male rats. Rats were administered a single oral dose with vitamin E, vitamin C or their combination at prophylactic doses before or after the single whole body exposure to irradiation at the doses of 2 or 8 Gy. The radioprotective effect was evaluated by the frequency of chromosomal aberrations at metaphase plates of the bone marrow cells, apoptotic--by the level of circulating low-molecular-weight DNA (ImwDNA) in the blood plasma of irradiated rats. Administration of the combination of vitamins E and C before and after the irradiation at the dose of 2 Gy reduced the number of the cells with chromosomal aberrations thus providing the radioprotective effect, but separately administration of these vitamins did not show the significant radioprotective activity. Administration of the combination of vitamins E and C before irradiation with 8 Gy increased the lmwDNA in blood thus providing the apoptotic effect. So, synergy of radioprotective activities has been revealed in vitamins E and C action at prophylactic doses. Radioprotective effect of the combination of vitamins E and C can be associated with the apoptotic activity and can be explained by elimination of the least viable irradiated cells from the cell population. PMID:26863779

  7. A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequences Using Flow Cytometry

    PubMed Central

    Spiro, Alexander; Lowe, Mary; Brown, Drew

    2000-01-01

    A new multiplexed, bead-based method which utilizes nucleic acid hybridizations on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences is described. The method consists of three elements: beads (5.6-μm diameter) with oligomer capture probes attached to the surface, three fluorophores for multiplexed detection, and flow cytometry instrumentation. Two fluorophores are impregnated within each bead in varying amounts to create different bead types, each associated with a unique probe. The third fluorophore is a reporter. Following capture of fluorescent cDNA sequences from environmental samples, the beads are analyzed by flow cytometric techniques which yield a signal intensity for each capture probe proportional to the amount of target sequences in the analyte. In this study, a direct hybrid capture assay was developed and evaluated with regard to sequence discrimination and quantitation of abundances. The target sequences (628 to 728 bp in length) were obtained from the 16S/23S intergenic spacer region of microorganisms collected from polluted groundwater at the nuclear waste site in Hanford, Wash. A fluorescence standard consisting of beads with a known number of fluorescent DNA molecules on the surface was developed, and the resolution, sensitivity, and lower detection limit for measuring abundances were determined. The results were compared with those of a DNA microarray using the same sequences. The bead method exhibited far superior sequence discrimination and possesses features which facilitate accurate quantitation. PMID:11010868

  8. Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

    PubMed Central

    Iyer, Lakshminarayan M.; Zhang, Dapeng; Maxwell Burroughs, A.; Aravind, L.

    2013-01-01

    Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems

  9. Semiconductor-based DNA sequencing of histone modification states.

    PubMed

    Cheng, Christine S; Rai, Kunal; Garber, Manuel; Hollinger, Andrew; Robbins, Dana; Anderson, Scott; Macbeth, Alyssa; Tzou, Austin; Carneiro, Mauricio O; Raychowdhury, Raktima; Russ, Carsten; Hacohen, Nir; Gershenwald, Jeffrey E; Lennon, Niall; Nusbaum, Chad; Chin, Lynda; Regev, Aviv; Amit, Ido

    2013-01-01

    The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues. PMID:24157732

  10. caos software for use in character-based DNA barcoding.

    PubMed

    Sarkar, Indra Neil; Planet, Paul J; Desalle, Rob

    2008-11-01

    The success of character-based DNA barcoding depends on the efficient identification of diagnostic character states from molecular sequences that have been organized hierarchically (e.g. according to phylogenetic methods). Similarly, the reliability of these identified diagnostic character states must be assessed according to their ability to diagnose new sequences. Here, a set of software tools is presented that implement the previously described Characteristic Attribute Organization System for both diagnostic identification and diagnostic-based classification. The software is publicly available from http://sarkarlab.mbl.edu/CAOS. PMID:21586014

  11. Semiconductor-based DNA sequencing of histone modification states

    PubMed Central

    Cheng, Christine S.; Rai, Kunal; Garber, Manuel; Hollinger, Andrew; Robbins, Dana; Anderson, Scott; Macbeth, Alyssa; Tzou, Austin; Carneiro, Mauricio O.; Raychowdhury, Raktima; Russ, Carsten; Hacohen, Nir; Gershenwald, Jeffrey E.; Lennon, Niall; Nusbaum, Chad; Chin, Lynda; Regev, Aviv; Amit, Ido

    2013-01-01

    The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues. PMID:24157732

  12. Magnetophoresis of flexible DNA-based dumbbell structures

    NASA Astrophysics Data System (ADS)

    Babić, B.; Ghai, R.; Dimitrov, K.

    2008-02-01

    Controlled movement and manipulation of magnetic micro- and nanostructures using magnetic forces can give rise to important applications in biomedecine, diagnostics, and immunology. We report controlled magnetophoresis and stretching, in aqueous solution, of a DNA-based dumbbell structure containing magnetic and diamagnetic microspheres. The velocity and stretching of the dumbbell were experimentally measured and correlated with a theoretical model based on the forces acting on individual magnetic beads or the entire dumbbell structures. The results show that precise and predictable manipulation of dumbbell structures is achievable and can potentially be applied to immunomagnetic cell separators.

  13. Ligand-activated PPARα-dependent DNA demethylation regulates the fatty acid β-oxidation genes in the postnatal liver.

    PubMed

    Ehara, Tatsuya; Kamei, Yasutomi; Yuan, Xunmei; Takahashi, Mayumi; Kanai, Sayaka; Tamura, Erina; Tsujimoto, Kazutaka; Tamiya, Takashi; Nakagawa, Yoshimi; Shimano, Hitoshi; Takai-Igarashi, Takako; Hatada, Izuho; Suganami, Takayoshi; Hashimoto, Koshi; Ogawa, Yoshihiro

    2015-03-01

    The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage-specific DNA demethylation of a particular metabolic pathway. PMID:25311726

  14. Abietadiene synthase from grand fir (Abies grandis). cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis.

    PubMed

    Vogel, B S; Wildung, M R; Vogel, G; Croteau, R

    1996-09-20

    (-)-Abietic acid, the principal diterpenoid resin acid of the wound-induced oleoresin secreted by grand fir (Abies grandis), is synthesized by the cyclization of geranylgeranyl diphosphate to (-)-abieta-7(8),13(14)-diene, followed by sequential three-step oxidation of the C-18 methyl group of the olefin to a carboxyl function. The enzyme catalyzing the cyclization reaction, abietadiene synthase, was purified from stems of wounded grand fir saplings and was digested with trypsin. Amino acid sequence information from the resulting peptides allowed construction of degenerate oligonucleotide primers, which amplified a 551-base pair fragment from a wound-induced stem cDNA library. This hybridization probe was then utilized to screen the wound-induced stem cDNA library, from which three cDNA clones were isolated that were functionally expressed in Escherichia coli, thereby confirming that a single protein catalyzes the complex, multistep cyclization of geranylgeranyl diphosphate to abietadiene. cDNA isolate Ac22.1, which yielded the highest expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino acid open reading frame that included a putative plastidial transit peptide. Deduced amino acid sequence comparison to other terpene cyclases revealed an amino-terminal region of the abietadiene synthase, which resembles those of enzymes that employ substrate double bond protonation to initiate the carbocationic reaction cascade, and a carboxyl-terminal region of the synthase, which resembles those of enzymes that employ ionization of the substrate allylic diphosphate ester function to initiate the cyclization reaction. This apparent fusion of segments of the two distinct terpenoid cyclase types is consistent with the novel mechanism of the bifunctional abietadiene synthase in catalyzing both protonation-initiated and ionization-initiated cyclization steps. PMID:8798524

  15. Structure-based model for light-harvesting properties of nucleic acid nanostructures

    PubMed Central

    Pan, Keyao; Boulais, Etienne; Yang, Lun; Bathe, Mark

    2014-01-01

    Programmed self-assembly of DNA enables the rational design of megadalton-scale macromolecular assemblies with sub-nanometer scale precision. These assemblies can be programmed to serve as structural scaffolds for secondary chromophore molecules with light-harvesting properties. Like in natural systems, the local and global spatial organization of these synthetic scaffolded chromophore systems plays a crucial role in their emergent excitonic and optical properties. Previously, we introduced a computational model to predict the large-scale 3D solution structure and flexibility of nucleic acid nanostructures programmed using the principle of scaffolded DNA origami. Here, we use Förster resonance energy transfer theory to simulate the temporal dynamics of dye excitation and energy transfer accounting both for overall DNA nanostructure architecture as well as atomic-level DNA and dye chemical structure and composition. Results are used to calculate emergent optical properties including effective absorption cross-section, absorption and emission spectra and total power transferred to a biomimetic reaction center in an existing seven-helix double stranded DNA-based antenna. This structure-based computational framework enables the efficient in silico evaluation of nucleic acid nanostructures for diverse light-harvesting and photonic applications. PMID:24311563

  16. Structure-based model for light-harvesting properties of nucleic acid nanostructures.

    PubMed

    Pan, Keyao; Boulais, Etienne; Yang, Lun; Bathe, Mark

    2014-02-01

    Programmed self-assembly of DNA enables the rational design of megadalton-scale macromolecular assemblies with sub-nanometer scale precision. These assemblies can be programmed to serve as structural scaffolds for secondary chromophore molecules with light-harvesting properties. Like in natural systems, the local and global spatial organization of these synthetic scaffolded chromophore systems plays a crucial role in their emergent excitonic and optical properties. Previously, we introduced a computational model to predict the large-scale 3D solution structure and flexibility of nucleic acid nanostructures programmed using the principle of scaffolded DNA origami. Here, we use Förster resonance energy transfer theory to simulate the temporal dynamics of dye excitation and energy transfer accounting both for overall DNA nanostructure architecture as well as atomic-level DNA and dye chemical structure and composition. Results are used to calculate emergent optical properties including effective absorption cross-section, absorption and emission spectra and total power transferred to a biomimetic reaction center in an existing seven-helix double stranded DNA-based antenna. This structure-based computational framework enables the efficient in silico evaluation of nucleic acid nanostructures for diverse light-harvesting and photonic applications. PMID:24311563

  17. An impedance-based integrated biosensor for suspended DNA characterization

    PubMed Central

    Ma, Hanbin; Wallbank, Richard W. R.; Chaji, Reza; Li, Jiahao; Suzuki, Yuji; Jiggins, Chris; Nathan, Arokia

    2013-01-01

    Herein, we describe a novel integrated biosensor for performing dielectric spectroscopy to analyze biological samples. We analyzed biomolecule samples with different concentrations and demonstrated that the solution's impedance is highly correlated with the concentration, indicating that it may be possible to use this sensor as a concentration sensor. In contrast with standard spectrophotometers, this sensor offers a low-cost and purely electrical solution for the quantitative analysis of biomolecule solutions. In addition to determining concentrations, we found that the sample solution impedance is highly correlated with the length of the DNA fragments, indicating that the sizes of PCR products could be validated with an integrated chip-based, sample-friendly system within a few minutes. The system could be the basis of a rapid, low-cost platform for DNA characterization with broad applications in cancer and genetic disease research. PMID:24060937

  18. Nanopore-based Fourth-generation DNA Sequencing Technology

    PubMed Central

    Feng, Yanxiao; Zhang, Yuechuan; Ying, Cuifeng; Wang, Deqiang; Du, Chunlei

    2015-01-01

    Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications. PMID:25743089

  19. Nanopore-based fourth-generation DNA sequencing technology.

    PubMed

    Feng, Yanxiao; Zhang, Yuechuan; Ying, Cuifeng; Wang, Deqiang; Du, Chunlei

    2015-02-01

    Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein. Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale. In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications. PMID:25743089

  20. The in Silico Insight into Carbon Nanotube and Nucleic Acid Bases Interaction

    PubMed Central

    Karimi, Ali Asghar; Ghalandari, Behafarid; Tabatabaie, Seyed Saleh; Farhadi, Mohammad

    2016-01-01

    Background To explore practical applications of carbon nanotubes (CNTs) in biomedical fields the properties of their interaction with biomolecules must be revealed. Recent years, the interaction of CNTs with biomolecules is a subject of research interest for practical applications so that previous research explored that CNTs have complementary structure properties with single strand DNA (ssDNA). Objectives Hence, the quantum mechanics (QM) method based on ab initio was used for this purpose. Therefore values of binding energy, charge distribution, electronic energy and other physical properties of interaction were studied for interaction of nucleic acid bases and SCNT. Materials and Methods In this study, the interaction between nucleic acid bases and a (4, 4) single-walled carbon nanotube (SCNT) were investigated through calculations within quantum mechanics (QM) method at theoretical level of Hartree-Fock (HF) method using 6-31G basis set. Hence, the physical properties such as electronic energy, total dipole moment, charge distributions and binding energy of nucleic acid bases interaction with SCNT were investigated based on HF method. Results It has been found that the guanine base adsorption is bound stronger to the outer surface of nanotube in comparison to the other bases, consistent with the recent theoretical studies. In the other words, the results explored that guanine interaction with SCNT has optimum level of electronic energy so that their interaction is stable. Also, the calculations illustrated that SCNT interact to nucleic acid bases by noncovalent interaction because of charge distribution an electrostatic area is created in place of interaction. Conclusions Consequently, small diameter SCNT interaction with nucleic acid bases is noncovalent. Also, the results revealed that small diameter SCNT interaction especially SCNT (4, 4) with nucleic acid bases can be useful in practical application area of biomedical fields such detection and drug delivery.

  1. Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers

    PubMed Central

    AL-Huqail, Asma A.; Abdelhaliem, Ekram

    2015-01-01

    The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (Rf), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors. PMID:26180815

  2. Multiphoton excitation fluorescence correlation spectroscopy of fluorescent DNA base analogs

    NASA Astrophysics Data System (ADS)

    Katilius, Evaldas; Woodbury, Neal W.

    2004-06-01

    Two- and three-photon excitation was used to investigate the properties of two fluorescent DNA base analogs: 2-aminopurine and 6-methylisoxanthopterin. 2-aminopurine is a widely used fluorescent analog of the DNA base adenine. Three-photon excitation of 2-aminopurine is achievable by using intense femtosecond laser pulses in 850-950 nm spectral region. Interestingly, the three-photon excitation spectrum is blue-shifted relative to the three-times-wavelength single-photon excitation spectrum. The maximum of the absorbance band in the UV is at 305 nm, while the three-photon excitation spectrum has a maximum at around 880 nm. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2-aminopurine for DNA-protein interaction studies. However, due to relatively small three-photon absorption cross-section, a good signal-to-noise fluorescence correlation curves take very long time to obtain. Fluorescence properties of 6-methylisoxanthopterin, the fluorescent analog of guanine, were investigated using two-photon excitation. This molecule has the lowest energy absorption band centered around 350 nm, thus, two-photon excitation is attainable using 700 to 760 nm output of Ti-sapphire laser. The excitation spectrum of this molecule in the infrared well matches the doubled-wavelength single-photon excitation spectrum in the UV. The high fluorescence quantum yield of 6-methylisoxanthopterin allows efficient fluorescence correlation measurements and makes this molecule a very good candidate for using in in vitro DNA-protein interaction studies.

  3. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

    PubMed

    Maciąg-Dorszyńska, Monika; Węgrzyn, Grzegorz; Guzow-Krzemińska, Beata

    2014-04-01

    Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus. PMID:24571086

  4. Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

    PubMed Central

    Horn, T; Chang, C A; Urdea, M S

    1997-01-01

    The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology. PMID:9365266

  5. DNA Music.

    ERIC Educational Resources Information Center

    Miner, Carol; della Villa, Paula

    1997-01-01

    Describes an activity in which students reverse-translate proteins from their amino acid sequences back to their DNA sequences then assign musical notes to represent the adenine, guanine, cytosine, and thymine bases. Data is obtained from the National Institutes of Health (NIH) on the Internet. (DDR)

  6. Duplex DNA-Invading γ-Modified Peptide Nucleic Acids Enable Rapid Identification of Bloodstream Infections in Whole Blood

    PubMed Central

    Nölling, Jörk; Rapireddy, Srinivas; Amburg, Joel I.; Crawford, Elizabeth M.; Prakash, Ranjit A.; Rabson, Arthur R.

    2016-01-01

    ABSTRACT Bloodstream infections are a leading cause of morbidity and mortality. Early and targeted antimicrobial intervention is lifesaving, yet current diagnostic approaches fail to provide actionable information within a clinically viable time frame due to their reliance on blood culturing. Here, we present a novel pathogen identification (PID) platform that features the use of duplex DNA-invading γ-modified peptide nucleic acids (γPNAs) for the rapid identification of bacterial and fungal pathogens directly from blood, without culturing. The PID platform provides species-level information in under 2.5 hours while reaching single-CFU-per-milliliter sensitivity across the entire 21-pathogen panel. The clinical utility of the PID platform was demonstrated through assessment of 61 clinical specimens, which showed >95% sensitivity and >90% overall correlation to blood culture findings. This rapid γPNA-based platform promises to improve patient care by enabling the administration of a targeted first-line antimicrobial intervention. PMID:27094328

  7. Characterization of nucleic acids by tandem mass spectrometry - The second decade (2004-2013): From DNA to RNA and modified sequences.

    PubMed

    Schürch, Stefan

    2016-07-01

    Nucleic acids play key roles in the storage and processing of genetic information, as well as in the regulation of cellular processes. Consequently, they represent attractive targets for drugs against gene-related diseases. On the other hand, synthetic oligonucleotide analogues have found application as chemotherapeutic agents targeting cellular DNA and RNA. The development of effective nucleic acid-based chemotherapeutic strategies requires adequate analytical techniques capable of providing detailed information about the nucleotide sequences, the presence of structural modifications, the formation of higher-order structures, as well as the interaction of nucleic acids with other cellular components and chemotherapeutic agents. Due to the impressive technical and methodological developments of the past years, tandem mass spectrometry has evolved to one of the most powerful tools supporting research related to nucleic acids. This review covers the literature of the past decade devoted to the tandem mass spectrometric investigation of nucleic acids, with the main focus on the fundamental mechanistic aspects governing the gas-phase dissociation of DNA, RNA, modified oligonucleotide analogues, and their adducts with metal ions. Additionally, recent findings on the elucidation of nucleic acid higher-order structures by tandem mass spectrometry are reviewed. © 2014 Wiley Periodicals, Inc., Mass Spec Rev 35:483-523, 2016. PMID:25288464

  8. Renal acid-base metabolism after ischemia.

    PubMed

    Holloway, J C; Phifer, T; Henderson, R; Welbourne, T C

    1986-05-01

    The response of the kidney to ischemia-induced cellular acidosis was followed over the immediate one hr post-ischemia reflow period. Clearance and extraction experiments as well as measurement of cortical intracellular pH (pHi) were performed on Inactin-anesthetized Sprague-Dawley rats. Arteriovenous concentration differences and para-aminohippurate extraction were obtained by cannulating the left renal vein. Base production was monitored as bicarbonate released into the renal vein and urine; net base production was related to the renal handling of glutamine and ammonia as well as to renal oxygen consumption and pHi. After a 15 min control period, the left renal artery was snared for one-half hr followed by release and four consecutive 15 min reflow periods. During the control period, cortical cell pHi measured by [14C]-5,5-Dimethyl-2,4-Oxazolidinedione distribution was 7.07 +/- 0.08, and Q-O2 was 14.1 +/- 2.2 micromoles/min; neither net glutamine utilization nor net bicarbonate generation occurred. After 30 min of ischemia, renal tissue pH fell to 6.6 +/- 0.15. However, within 45 min of reflow, cortical cell pH returned and exceeded the control value, 7.33 +/- 0.06 vs. 7.15 +/- 0.08. This increase in pHi was associated with a significant rise in cellular metabolic rate, Q-O2 increased to 20.3 +/- 6.4 micromoles/min. Corresponding with cellular alkalosis was a net production of bicarbonate and a net ammonia uptake and glutamine release; urinary acidification was abolished. These results are consistent with a nonexcretory renal metabolic base generating mechanism governing cellular acid base homeostasis following ischemia. PMID:3723929

  9. Aromaticity-induced changes in the electronic properties of size-expanded DNA bases: Case of xC.

    SciTech Connect

    Fuentes-Cabrera, Miguel A; Lipkowski, Pawel; Huertas, Oscar; Sumpter, Bobby G; Orozco, Modesto; Luque, Javier; Wells, Jack C; Leszczynski, Jerzy

    2006-01-01

    Size-expanded DNA bases are analogues of natural bases that can be described as a synthesis between benzene and a natural base. Size-expanded bases have been combined with natural bases to form xDNA and yDNA, a new class of synthetic nucleic acids. We are interested in xDNA and yDNA because they might function as molecular wires. Recently, we also became intrigued by the possibility of altering the electronic conductivity of xDNA and yDNA by means of structural changes in the constituent bases. This possibility appeared after we noticed that the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) gap of the base yG can be increased dramatically, {approx}0.73 eV, by changing the aromaticity of its benzene ring. Therefore, if one is able to alter the HOMO-LUMO gap of size-expanded bases, it should be possible to change the electronic conductivity of xDNAs and yDNAs as well. In the present work, we extend our study on aromaticity-induced changes on the electronic properties of size-expanded bases by investigating the HOMO-LUMO gap of all possible tautomers of xC. We have found that, as for yG, the HOMO-LUMO gap of xC can be modified by {approx} 0.74 eV, and that this can be accomplished by changing the aromaticity of its benzene ring.

  10. Pre-Incubation of Auric Acid with DNA Is Unnecessary for the Formation of DNA-Templated Gold Nanoclusters.

    PubMed

    Chen, Yang; Tao, Guangyu; Lin, Ruoyun; Pei, Xiaojing; Liu, Feng; Li, Na

    2016-06-01

    The rationale for the preparation of DNA-templated gold nanoclusters (DNA-Au NCs) has not been well understood, thereby slowing down the advancement of the synthesis and applications of DNA-Au NCs. The interaction between metal ions and the DNA template seems to be the key factor for the successful preparation of DNA-templated metal nanoclusters. With the help of circular dichroism in this contribution, we put efforts into interrogating the necessity of pre-incubation of HAuCl4 with poly-adenine template in the formation of Au NCs by citrate reduction. Our results revealed that the pre-incubation of HAuCl4 with poly-adenine is not favorable for the formation of Au NCs, which is distinctly different from the formation process for silver nanoclusters. It is our hope that this study can provide guidance in the preparation of Au NCs with more DNA templates. PMID:27060903

  11. Evaluating the effects of galbanic acid on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

    PubMed Central

    Shirani, Kobra; Behravan, Javad; Mosaffa, Fatemeh; Iranshahi, Mehrdad; Mehmankhah, Babak; Razavi-Azarkhiavi, Kamal; Karimi, Gholamreza

    2014-01-01

    Objective: Ferula szowitsiana has been widely used for medicinal purposes around the world. The anti-oxidant effect of F. szowitsiana had been proved. The current study aims to determine the protective effects of galbanic acid, a sesquiterpene coumarin from F. szowitsiana, against hydrogen peroxide (H2O2) - induced oxidative DNA damage in human lymphocytes. Materials and Methods: Human lymphocytes were incubated with H2O2 (0, 25, 50, 100, and 200 µM), galbanic acid (200 and 400 µM) and a combination of galbanic acid (200 and 400 µM) and H2O2 (25 µM) at 4 C for 30 minutes. Solvents of galbanic acid without H2O2 were used as negative controls. Results: The findings of this study demonstrated that H2O2 exposure leads to a significant concentration-dependent increase in DNA damage. Galbanic acid did not cause DNA damage compared with the control cells. Data showed that galbanic acid does not have a protective effect against H2O2-induced oxidative DNA damage in human lymphocytes. Conclusion: According to the results, it is concluded that the capability of F. szowitsiana in reducing reactive oxygen species and the anti-inflammatory property of its methanolic extract may be due to its other ingredients. PMID:25386396

  12. Hydroxyl radical mediated DNA base modification by manmade mineral fibres.

    PubMed Central

    Leanderson, P; Söderkvist, P; Tagesson, C

    1989-01-01

    Manmade mineral fibres (MMMFs) were examined for their ability to hydroxylate 2-deoxyguanosine (dG) to 8-hydroxydeoxyguanosine (8-OH-dG), a reaction that is mediated by hydroxyl radicals. It appeared that (1) catalase and the hydroxyl radical scavengers, dimethylsulphoxide and sodium benzoate, inhibited the hydroxylation, whereas Fe2+ and H2O2 potentiated it; (2) pretreatment of MMMFs with the iron chelator, deferoxamine, or with extensive heat (200-400 degrees C), attenuated the hydroxylation; (3) the hydroxylation obtained by various MMMFs varied considerably; (4) there was no apparent correlation between the hydroxylation and the surface area of different MMMFs, although increasing the surface area of a fibre by crushing it increased its hydroxylating capacity; and (5) there was good correlation between the hydroxylation of dG residues in DNA and the hydroxylation of pure dG in solution for the 16 different MMMFs investigated. These findings indicate that MMMFs cause a hydroxyl radical mediated DNA base modification in vitro and that there is considerable variation in the reactivity of different fibre species. The DNA modifying ability seems to depend on physical or chemical characteristics, or both, of the fibre. PMID:2765416

  13. Applications of nanoparticles for DNA based rabies vaccine.

    PubMed

    Shah, Muhammad Ali A; Khan, Sajid Umar; Ali, Zeeshan; Yang, Haowen; Liu, Keke; Mao, Lanlan

    2014-01-01

    Rabies is a fatal encephalomyelitis. Most cases occur in developing countries and are transmitted by dogs. The cell culture vaccines as associated with high cost; therefore, have not replaced the unsafe brain-derived vaccines. In the developing countries these brain-derived rabies vaccines still can be seen in action. Moreover, there will be a need for vaccines against rabies-related viruses against which classical vaccines are not always effective. The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost-effective therapy indicate the need for alternate control strategies. DNA vaccines have emerged as the safest vaccines and best remedy for complicated diseases like hepatitis, HIV, and rabies. A number of recombinant DNA vaccines are now being developed against several diseases such as AIDS and malaria. Therefore, it can be a valuable alternative for the production of cheaper rabies vaccines against its larger spectrum of viruses. In this review we report published data on DNA-based immunization with sequences encoding rabies with special reference to nanotechnology. PMID:24730305

  14. Electrochemical DNA biosensor based on the BDD nanograss array electrode

    PubMed Central

    2013-01-01

    Background The development of DNA biosensor has attracted considerable attention due to their potential applications, including gene analysis, clinical diagnostics, forensic study and more medical applications. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry in this study. Results Electrochemical DNA biosensor was developed based on the BDD film electrode (fBDD) and BDD nanograss array electrode (nBDD). In comparison with fBDD and AuNPs/CA/fBDD electrode, the lower semicircle diameter of electrochemical impedance spectroscopy obtained on nBDD and AuNPs/CA/nBDD electrode indicated that the presence of nanograss array improved the reactive site, reduced the interfacial resistance, and made the electron transfer easier. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry. Conclusions The experimental results demonstrated that the prepared AuNPs/CA/nBDD electrode was suitable for DNA hybridization with favorable performance of faster response, higher sensitivity, lower detection limit and satisfactory selectivity, reproducibility and stability. PMID:23575250

  15. Identification and characterization of 2nd generation Invader Locked Nucleic Acids (LNAs) for mixed-sequence recognition of double-stranded DNA

    PubMed Central

    Sau, Sujay P.; Madsen, Andreas S.; Podbevsek, Peter; Andersen, Nicolai K.; Kumar, T. Santhosh; Andersen, Sanne; Rathje, Rie L.; Anderson, Brooke A.; Guenther, Dale C.; Karmakar, Saswata; Kumar, Pawan; Plavec, Janez; Wengel, Jesper

    2013-01-01

    Development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate and modify genes. Progress has been made with triplex-forming oligonucleotides, PNAs, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome limitations observed with the classic approaches. In 2005, we introduced Invader Locked Nucleic Acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with ‘+1 interstrand zippers’ of 2’-N-(pyren-1-yl)methyl-2’-amino-α-L-LNA monomers. Despite promising preliminary results, progress has been slow due to the synthetic complexity of the building blocks. Here, we describe a study that led to the identification of two simpler classes of Invader monomers. We compare thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2’-amino-α-L-LNA, 2’-N-methyl-2’-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2’-O-(pyren-1-yl)methyl-RNA or 2’-N-methyl-2’-N-(pyren-1-yl)methyl-2’-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA-recognition for applications in molecular biology and nucleic acid diagnostics. PMID:24032477

  16. DNA recognition by peptide nucleic acid-modified PCFs: from models to real samples

    NASA Astrophysics Data System (ADS)

    Selleri, S.; Coscelli, E.; Poli, F.; Passaro, D.; Cucinotta, A.; Lantano, C.; Corradini, R.; Marchelli, R.

    2010-04-01

    The increased concern, emerged in the last few years, on food products safety has stimulated the research on new techniques for traceability of raw food materials. DNA analysis is one of the most powerful tools for the certification of food quality, and it is presently performed through the polymerase chain reaction technique. Photonic crystal fibers, due to the presence of an array of air holes running along their length, can be exploited for performing DNA recognition by derivatizing hole surfaces and checking hybridization of complementary nucledotide chains in the sample. In this paper the application of a suspended core photonic crystal fiber in the recognition of DNA sequences is discussed. The fiber is characterized in terms of electromagnetic properties by means of a full-vector modal solver based on the finite element method. Then, the performances of the fiber in the recognition of mall synthetic oligonucleotides are discussed, together with a test of the possibility to extend this recognition to samples of DNA of applicative interest, such as olive leaves.

  17. Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA

    PubMed Central

    Christensen, Ulf B.; Pedersen, Erik B.

    2002-01-01

    We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4′-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9°C per modification when the IPN was added as a dangling end and up to 6.7°C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8°C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes. PMID:12433995

  18. Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

    PubMed Central

    Ho, Dominik; Dose, Christian; Albrecht, Christian H.; Severin, Philip; Falter, Katja; Dervan, Peter B.; Gaub, Hermann E.

    2009-01-01

    Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes. PMID:19486688

  19. Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.

    PubMed Central

    Frommer, W B; Hummel, S; Riesmeier, J W

    1993-01-01

    To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism. Images Fig. 1 PMID:8327465

  20. Detection of base-pair mismatches in DNA using graphene-based nanopore device

    NASA Astrophysics Data System (ADS)

    Kundu, Sourav; Karmakar, S. N.

    2016-04-01

    We present a unique way to detect base-pair mismatches in DNA, leading to a different epigenetic disorder by the method of nanopore sequencing. Based on a tight-binding formulation of a graphene-based nanopore device, using the Green’s function approach we study the changes in the electronic transport properties of the device as we translocate a double-stranded DNA through the nanopore embedded in a zigzag graphene nanoribbon. In the present work we are not only successful in detecting the usual AT and GC pairs but also a set of possible mismatches in the complementary base pairing.

  1. An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    PubMed Central

    Kimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, Ichiro

    2009-01-01

    Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies. PMID:19073696

  2. Electrochemical detection of DNA 3'-phosphatases based on surface-extended DNA nanotail strategy.

    PubMed

    Wu, Dan; Li, Chao; Hu, Xiaolu; Mao, Xiaoxia; Li, Genxi

    2016-06-14

    Determination of DNA dephosphorylation is of great value due to its vital role in many cellular processes. Here we report a surface-extended DNA nanotail strategy for simple and ultrasensitive detection of DNA 3'-phosphatases by terminal deoxynucleotidyl transferase (TdT) mediated signal amplification. In this work, DNA probes labeled with thiols at their 5' terminals and phosphoryls at 3' terminals are immobilized on gold electrode and are used as substrates for DNA 3'-phosphatases, taking T4 polynucleotide kinase phosphatase (T4PNKP) as an example. T4PNKP can catalyze the dephosphorylation reaction of the substrate DNA, followed by the formation of a long DNA strand by TdT on its 3' terminal hydroxyl, leading to an evident chronocoulometry signal enhancement. The proposal presents a considerable analytical performance with low detection limit and wide linear range, making it promise to be applied in the fields of DNA dephosphorylation related processes, drug discovery, and clinical diagnostics. PMID:27181641

  3. MPG-based nanoparticle: An efficient delivery system for enhancing the potency of DNA vaccine expressing HPV16E7.

    PubMed

    Saleh, Tayebeh; Bolhassani, Azam; Shojaosadati, Seyed Abbas; Aghasadeghi, Mohammad Reza

    2015-06-22

    DNA vaccines against human papillomavirus (HPV) type 16 have not been successful in clinical trials, due to the lack of an appropriate delivery system. In this study, a peptide-based gene delivery system, MPG, which forms stable non-covalent nanoparticles with nucleic acids, was used for in vitro and in vivo delivery of HPV16 E7 DNA as a model antigen. The results demonstrated that at Nitrogen/Phosphate (N/P) ratio over 10:1, this peptide can effectively condense plasmid DNA into stable nanoparticles with an average size of 180-210nm and a positive surface charge. The transfection efficiency of MPG-based nanoparticles was shown to be comparable with Polyethyleneimine (PEI). The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent delivery system in DNA vaccine formulations. Immunization with MPG/E7DNA nanoparticles at an N/P ratio of 10:1 induced a stronger Th1 cellular immune response with a predominant interferon-γ (IFN-γ) profile than those induced by E7DNA alone in a murine tumor model. These findings suggest that MPG peptide as a novel gene delivery system could have promising applications in improving HPV therapeutic vaccines. PMID:26001433

  4. Base-by-Base Counting of Nucleotide Incorporations by DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Turvey, Mackenzie W.; Gul, O. Tolga; Pugliese, Kaitlin M.; Marushchak, Denys O.; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Phillip G.

    Previously, the catalytic cycle of DNA polymerase has been recorded by tethering single polymerase molecules to single-walled carbon nanotube field effect transistors (FETs). As the polymerase incorporates nucleotides into a single-stranded DNA template, it generates electrical signals in the SWCNT-FET. Here, we investigate the accuracy of this electronic method by using low concentrations (<10 nM) of DNA template, such that the signal consists of long, diffusion-limited pauses interrupted by template binding and a burst of nucleotide incorporation events. By counting the events generated by as few as 10 template molecules, template length has been correctly determined with <1 base pair resolution. Furthermore, differing template lengths can be identified and correctly enumerated in solutions containing mixtures of templates. Processivity of the Klenow Fragment of DNA polymerase currently limits read lengths to 50-100 base pairs, but the FET technique should work equally well with longer-processivity polymerases.

  5. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-01

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner. PMID:27198220

  6. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution

    NASA Astrophysics Data System (ADS)

    Booth, Michael J.; Marsico, Giovanni; Bachman, Martin; Beraldi, Dario; Balasubramanian, Shankar

    2014-05-01

    Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

  7. Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

    PubMed

    Mizushina, Y; Kasai, N; Sugawara, F; Iida, A; Yoshida, H; Sakaguchi, K

    2001-11-01

    The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar. PMID:11686928

  8. DNA based electrolyte/separator for lithium battery application

    NASA Astrophysics Data System (ADS)

    Kumar, Jitendra; Ouchen, Fahima; Smarra, Devin A.; Subramanyam, Guru; Grote, James G.

    2015-09-01

    In this study, we demonstrated the use of DNA-CTMA (DC) in combination with PolyVinylidene Fluoride (PVDF) as a host matrix or separator for Lithium based electrolyte to form solid polymer/gel like electrolyte for potential application in Li-ion batteries. The addition of DC provided a better thermal stability of the composite electrolyte as shown by the thermos-gravimetric analysis (TGA). The AC conductivity measurements suggest that the addition of DC to the gel electrolyte had no effect on the overall ionic conductivity of the composite. The obtained films are flexible with high mechanical stretch-ability as compared to the gel type electrolytes only.

  9. A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

    PubMed Central

    Eggleston, A K; Rahim, N A; Kowalczykowski, S C

    1996-01-01

    We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids. PMID:8614617

  10. The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

    PubMed Central

    Bernad, A; Lázaro, J M; Salas, M; Blanco, L

    1990-01-01

    The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site. We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser). Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation. Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site. Images PMID:2191296

  11. An Investigation into the Association between DNA Damage and Dietary Fatty Acid in Men with Prostate Cancer

    PubMed Central

    Bishop, Karen S.; Erdrich, Sharon; Karunasinghe, Nishi; Han, Dug Yeo; Zhu, Shuotun; Jesuthasan, Amalini; Ferguson, Lynnette R.

    2015-01-01

    Prostate cancer is a growing problem in New Zealand and worldwide, as populations adopt a Western style dietary pattern. In particular, dietary fat is believed to be associated with oxidative stress, which in turn may be associated with cancer risk and development. In addition, DNA damage is associated with the risk of various cancers, and is regarded as an ideal biomarker for the assessment of the influence of foods on cancer. In the study presented here, 20 men with prostate cancer adhered to a modified Mediterranean style diet for three months. Dietary records, blood fatty acid levels, prostate specific antigen, C-reactive protein and DNA damage were assessed pre- and post-intervention. DNA damage was inversely correlated with dietary adherence (p = 0.013) and whole blood monounsaturated fatty acids (p = 0.009) and oleic acid (p = 0.020). DNA damage was positively correlated with the intake of dairy products (p = 0.043), red meat (p = 0.007) and whole blood omega-6 polyunsaturated fatty acids (p = 0.015). Both the source and type of dietary fat changed significantly over the course of the dietary intervention. Levels of DNA damage were correlated with various dietary fat sources and types of dietary fat. PMID:25580814

  12. Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

    PubMed Central

    Fairhead, H; Setlow, B; Setlow, P

    1993-01-01

    The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A

  13. Gold nanoparticle based signal enhancement liquid crystal biosensors for DNA hybridization assays.

    PubMed

    Yang, Shengyuan; Liu, Yanmei; Tan, Hui; Wu, Chao; Wu, Zhaoyang; Shen, Guoli; Yu, Ruqin

    2012-03-18

    A novel signal enhanced liquid crystal biosensor based on using AuNPs for highly sensitive DNA detection has been developed. This biosensor not only significantly decreases the detection limit, but also offers a simple detection process and shows a good selectivity to distinguish perfectly matched target DNA from two-base mismatched DNA. PMID:22302154

  14. Metal Ion Sensors Based on DNAzymes and Related DNA Molecules

    NASA Astrophysics Data System (ADS)

    Zhang, Xiao-Bing; Kong, Rong-Mei; Lu, Yi

    2011-07-01

    Metal ion sensors are an important yet challenging field in analytical chemistry. Despite much effort, only a limited number of metal ion sensors are available for practical use because sensor design is often a trial-and-error-dependent process. DNAzyme-based sensors, in contrast, can be developed through a systematic selection that is generalizable for a wide range of metal ions. Here, we summarize recent progress in the design of DNAzyme-based fluorescent, colorimetric, and electrochemical sensors for metal ions, such as Pb2+, Cu2+, Hg2+, and UO22+. In addition, we also describe metal ion sensors based on related DNA molecules, including T-T or C-C mismatches and G-quadruplexes.

  15. Metal Ion Sensors Based on DNAzymes and Related DNA Molecules

    PubMed Central

    Kong, Rong-Mei

    2011-01-01

    Metal ion sensors are an important yet challenging field in analytical chemistry. Despite much effort, only a limited number of metal ion sensors are available for practical use because sensor design is often a trial-and-error-dependent process. DNAzyme-based sensors, in contrast, can be developed through a systematic selection that is generalizable for a wide range of metal ions. Here, we summarize recent progress in the design of DNAzyme-based fluorescent, colorimetric, and electrochemical sensors for metal ions, such as Pb2+, Cu2+, Hg2+, and UO22+ In addition, we also describe metal ion sensors based on related DNA molecules, including T-T or C-C mismatches and G-quadruplexes. PMID:21370984

  16. Effect of pollution on DNA damage and essential fatty acid profile in Cirrhinus mrigala from River Chenab

    NASA Astrophysics Data System (ADS)

    Hussain, Bilal; Sultana, Tayyaba; Sultana, Salma; Al-Ghanim, K. A.; Mahboob, Shahid

    2016-05-01

    The objective of this study was to evaluate the effect of anthropogenic pollution on DNA damage and the fatty acid profile of the bottom dweller fish (Cirrhinus mrigala), collected from the River Chenab, in order to assess the effect of the toxicants on the quality of the fish meat. The levels of Cd, Hg, Cu, Mn, Zn, Pb, Cr and Sn and of phenols from this river were significantly higher than the permissible limits set by the USEPA. Comet assays showed DNA damage in Cirrhinus mrigala collected from three different sampling sites in the polluted area of the river. Significant differences were observed for DNA damage through comet assay in fish collected from polluted compared to control sites. No significant differences were observed for DNA damage between farmed and fish collected from upstream. The micronucleus assay showed similar trends. Fish from the highly polluted sites showed less number of fatty acids and more saturated fatty acids in their meat compared to fish from less polluted areas. Several fatty acids were missing in fish with higher levels of DNA in comet tail and micronucleus induction. Long-chain polyunsaturated fatty acids, eicosapentaenoic acid (20:5n-3) was found missing in the fish from polluted environment while it was found in considerable amount in farmed fish 7.8±0.4%. Docosahexaenoic acid (22:6n-3) also showed significant differences as 0.1±0.0 and 7.0±0.1% respectively, in wild polluted and farmed fishes.

  17. Protective effect of borage seed oil and gamma linolenic acid on DNA: in vivo and in vitro studies.

    PubMed

    Tasset-Cuevas, Inmaculada; Fernández-Bedmar, Zahira; Lozano-Baena, María Dolores; Campos-Sánchez, Juan; de Haro-Bailón, Antonio; Muñoz-Serrano, Andrés; Alonso-Moraga, Angeles

    2013-01-01

    Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells. PMID:23460824

  18. Effect of Varying Magnetic Fields on Targeted Gene Delivery of Nucleic Acid-Based Molecules.

    PubMed

    Oral, Ozlem; Cıkım, Taha; Zuvin, Merve; Unal, Ozlem; Yagci-Acar, Havva; Gozuacik, Devrim; Koşar, Ali

    2015-11-01

    Several physical methods have been developed to introduce nucleic acid expression vectors into mammalian cells. Magnetic transfection (magnetofection) is one such transfection method, and it involves binding of nucleic acids such as DNA, RNA or siRNA to magnetic nanoparticles followed by subsequent exposure to external magnetic fields. However, the challenge between high efficiency of nucleic acid uptake by cells and toxicity was not totally resolved. Delivery of nucleic acids and their transport to the target cells require carefully designed and controlled systems. In this study, we introduced a novel magnetic system design providing varying magnet turn speeds and magnetic field directions. The system was tested in the magnetofection of human breast (MCF-7), prostate (DU-145, PC-3) and bladder (RT-4) cancer cell lines using green fluorescent protein DNA as a reporter. Polyethylenimine coated superparamagnetic iron oxide nanoparticles (SPIONs) were used as nucleic acid carriers. Adsorption of PEI on SPION improved the cytocompatibility dramatically. Application of external magnetic field increased intracellular uptake of nanoparticles and transfection efficiency without any additional cytotoxicity. We introduce our novel magnetism-based method as a promising tool for enhanced nucleic acid delivery into mammalian cells. PMID:25963582

  19. DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.

    PubMed

    Olson, Jeff; Whitney, Duncan H; Durkee, Kristine; Shuber, Anthony P

    2005-09-01

    We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation. A study was run comparing paired sample aliquots. Quantitative PCR data for matched aliquots was used to determine first the effect of sample incubation on total recovery and integrity of DNA, then the effect of stabilization buffer addition to stool on recoverable DNA, and finally the impact of buffer addition on assay sensitivity. Comparison of quantitative PCR data for paired aliquots shows that the amount of recoverable human DNA is negatively affected by storing stool samples (N = 43) at room temperature for > or =36 hours (P = 0.0018). However, the addition of stabilization buffer leads to a significant increase in recovery of DNA (P = 0.010), compared with samples incubated without buffer. Whereas the DNA Integrity Assay (DIA) is found to be sensitive to DNA degradation (sensitivity was reduced by 82%; P = 0.0002), point mutation marker sensitivity is more refractory. Overall, DNA can be stabilized by addition of buffer to the sample, leading to increased assay sensitivity. PMID:16106201

  20. History of medical understanding and misunderstanding of Acid base balance.

    PubMed

    Aiken, Christopher Geoffrey Alexander

    2013-09-01

    To establish how controversies in understanding acid base balance arose, the literature on acid base balance was reviewed from 1909, when Henderson described how the neutral reaction of blood is determined by carbonic and organic acids being in equilibrium with an excess of mineral bases over mineral acids. From 1914 to 1930, Van Slyke and others established our acid base principles. They recognised that carbonic acid converts into bicarbonate all non-volatile mineral bases not bound by mineral acids and determined therefore that bicarbonate represents the alkaline reserve of the body and should be a physiological constant. They showed that standard bicarbonate is a good measure of acidosis caused by increased production or decreased elimination of organic acids. However, they recognised that bicarbonate improved low plasma bicarbonate but not high urine acid excretion in diabetic ketoacidosis, and that increasing pCO2 caused chloride to shift into cells raising plasma titratable alkali. Both indicate that minerals influence pH. In 1945 Darrow showed that hyperchloraemic metabolic acidosis in preterm infants fed milk with 5.7 mmol of chloride and 2.0 mmol of sodium per 100 kcal was caused by retention of chloride in excess of sodium. Similar findings were made but not recognised in later studies of metabolic acidosis in preterm infants. Shohl in 1921 and Kildeberg in 1978 presented the theory that carbonic and organic acids are neutralised by mineral base, where mineral base is the excess of mineral cations over anions and organic acid is the difference between mineral base, bicarbonate and protein anion. The degree of metabolic acidosis measured as base excess is determined by deviation in both mineral base and organic acid from normal. PMID:24179938

  1. Determination of Base Binding Strength and Base Stacking Interaction of DNA Duplex Using Atomic Force Microscope

    PubMed Central

    Zhang, Tian-biao; Zhang, Chang-lin; Dong, Zai-li; Guan, Yi-fu

    2015-01-01

    As one of the most crucial properties of DNA, the structural stability and the mechanical strength are attracting a great attention. Here, we take advantage of high force resolution and high special resolution of Atom Force Microscope and investigate the mechanical force of DNA duplexes. To evaluate the base pair hydrogen bond strength and base stacking force in DNA strands, we designed two modes (unzipping and stretching) for the measurement rupture forces. Employing k-means clustering algorithm, the ruptured force are clustered and the mean values are estimated. We assessed the influence of experimental parameters and performed the force evaluation for DNA duplexes of pure dG/dC and dA/dT base pairs. The base binding strength of single dG/dC and single dA/dT were estimated to be 20.0 ± 0.2 pN and 14.0 ± 0.3 pN, respectively, and the base stacking interaction was estimated to be 2.0 ± 0.1 pN. Our results provide valuable information about the quantitative evaluation of the mechanical properties of the DNA duplexes. PMID:25772017

  2. Development of a small gantry robotic workcell for deoxyribonucleic acid (DNA) filter array construction

    SciTech Connect

    Beugelsdijk, T.J.; Hollen, R.M.; Snider, K.T.

    1990-01-01

    At Los Alamos National Laboratory, we have constructed a primary cosmid library of human chromosome 16. This library consists of an 11-fold representation of the chromosome and is arrayed in microtiter plate format. A need has arisen in the large scale physical mapping of this chromosome, to array spots of DNA from each of these colonies onto filter media for hybridization studies. We are currently developing a small gantry robot-based workcell to array small spots of DNA in an interleaved format. This allows for the construction of a high spot density format filter array. This paper will discuss the features incorporated into this workcell for the handling of thousands of colonies and their automatic tracking and positioning onto the filter. 7 refs., 3 figs., 1 tab.

  3. Suberoylanilide Hydroxamic Acid (SAHA) enhances olaparib activity by targeting homologous recombination DNA repair in ovarian cancer

    PubMed Central

    Konstantinopoulos, Panagiotis A.; Wilson, Andrew J.; Saskowski, Jeanette; Wass, Erica; Khabele, Dineo

    2015-01-01

    Objectives Approximately 50% of serous epithelial ovarian cancers (EOC) contain molecular defects in homologous recombination (HR) DNA repair pathways. Poly(ADP-ribose) polymerase inhibitors (PARPi) have efficacy in HR-deficient, but not HR-proficient, EOC tumors as a single agent. Our goal was to determine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize HR-proficient ovarian cancer cells to the PARPi AZD-2281 (olaparib). Methods Ovarian cancer cell lines (SKOV-3, OVCAR-8, NCI/ADR-Res, UWB1.289 BRCA1null and UWB1.289 + BRCA1 wild-type) were treated with saline vehicle, olaparib, SAHA or olaparib/SAHA. Sulforhodamine B (SRB) assessed cytotoxicity and immunofluorescence and Western blot assays assessed markers of apoptosis (cleaved PARP) and DNA damage (pH2AX and RAD51). Drug effects were also tested in SKOV-3 xenografts in Nude mice. Affymetrix microarray experiments were performed in vehicle and SAHA-treated SKOV-3 cells. Results In a microarray analysis, SAHA induced coordinated down-regulation of HR pathway genes, including RAD51 and BRCA1. Nuclear co-expression of RAD51 and pH2AX, a marker of efficient HR repair, was reduced approximately 40% by SAHA treatment alone and combined with olaparib. SAHA combined with olaparib induced apoptosis and pH2AX expression to a greater extent than either drug alone. Olaparib reduced cell viability at increasing concentrations and SAHA enhanced these effects in 4 of 5 cell lines, including BRCA1 null and wild-type cells, in vitro and in SKOV-3 xenografts in vivo. Conclusions These results provide preclinical rationale for targeting DNA damage response pathways by combining small molecule PARPi with HDACi as a mechanism for reducing HR efficiency in ovarian cancer. PMID:24631446

  4. Protection against radiation-induced DNA damage by amino acids: a DFT study.

    PubMed

    Jena, N R; Mishra, P C; Suhai, S

    2009-04-23

    Direct and indirect radiation-induced DNA damage is associated with the formation of radical cations (G(+)) and radical anions (G(-)) of guanine, respectively. Deprotonation of G(+) and dehydrogenation of G(-) generate guanine neutral radical [G(-H)] and guanine anion [G(-H)(-)], respectively. These products are of worrisome concern, as they are involved in reactions that are related to certain lethal diseases. It has been observed that guanyl radicals can be repaired by amino acids having strong reducing properties that are believed to be the residues of DNA-bound proteins such as histones. As a result, repair of G(-H) and G(-H)(-) by the amino acids cysteine and tyrosine has been studied here in detail by density functional theory in both the gas phase and aqueous medium using the polarized continuum and Onsager solvation models of self-consistent reaction field theory. Solvation in aqueous medium using three explicit water molecules was also studied. Four equivalent tautomers of each the above radical and anion that will be formed through proton and hydrogen loss from all of the nitrogen centers of guanine radical cation and guanine radical anion, respectively, were considered in the present study. It was found that in both the gas phase and aqueous medium, normal guanine can be retrieved from its radical-damaged form by a hydrogen-atom-transfer (HT) mechanism. Normal guanine can also be retrieved from its anionic damaged form in both the gas phase and aqueous medium through a two-electron-coupled proton-transfer (TECPT) mechanism or a one-step hydrogen-atom- and electron-transfer (OSHET) mechanism. The present results are discussed in light of the experimental findings. PMID:19334703

  5. Identification of chebulinic acid as potent natural inhibitor of M. tuberculosis DNA gyrase and molecular insights into its binding mode of action.

    PubMed

    Patel, Kunal; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Wahi, Divya; Jain, Ritu; Bharadvaja, Navneeta; Grover, Abhinav

    2015-12-01

    Drug resistant tuberculosis has threatened all the advances that have been made in TB control at the global stage in the last few decades. DNA gyrase enzymes are an excellent target for antibacterial drug discovery as they are involved in essential functions like DNA replication. Here we report, a successful application of high throughput virtual screening (HTVS) to identify an inhibitor of Mycobacterium DNA gyrase targeting the wild type and the most prevalent three double mutants of quinolone resistant DNA gyrase namely A90V+D94G, A74S+D94G and A90V+S91P. HTVS of 179.299 compounds gave five compounds with significant binding affinity. Extra presicion (XP) docking and MD simulations gave a clear view of their interaction pattern. Among them, chebulinic acid (CA), a phytocompound obtained from Terminalia chebula was the most potent inhibitor with significantly high XP docking score, -14.63, -16.46, -15.94 and -15.11 against wild type and three variants respectively. Simulation studies for a period of 16 ns indicated stable DNA gyrA-CA complex formation. This stable binding would result in inhibition of the enzyme by two mechanisms. Firstly, binding of CA causes displacement of catalytic Tyr129 away from its target DNA-phosphate molecule from 1.6 Å to 3.8-7.3 Å and secondly, by causing steric hindrance to the binding of DNA strand at DNA binding site of enzyme. The combined effect would result in loss of cleavage and religation activity of enzyme leading to bactericidal effect on tuberculosis. This phytocompound displays desirable quality for carrying forward as a lead compound for anti-tuberculosis drug development. The results presented here are solely based on computations and need to be validated experimentally in order to assert the proposed mechanism of action. PMID:26410242

  6. DNA methylation patterns are associated with n-3 fatty acid intake in Yup'ik people.

    PubMed

    Aslibekyan, Stella; Wiener, Howard W; Havel, Peter J; Stanhope, Kimber L; O'Brien, Diane M; Hopkins, Scarlett E; Absher, Devin M; Tiwari, Hemant K; Boyer, Bert B

    2014-04-01

    A large body of evidence links a high dietary intake of n-3 (ω-3) polyunsaturated fatty acids (PUFAs) with improved cardiometabolic outcomes. Recent studies suggested that the biologic processes underlying the observed associations may involve epigenetic changes, specifically DNA methylation. To evaluate changes in methylation associated with n-3 PUFA intake, we conducted an epigenome-wide methylation association study of long-chain n-3 PUFA intake and tested associations between the diabetes- and cardiovascular disease-related traits. We assessed DNA methylation at ∼470,000 cytosine-phosphate-guanine (CpG) sites in a cross-sectional study of 185 Yup'ik Alaska Native individuals representing the top and bottom deciles of PUFA intake. Linear regression models were used to test for the associations of interest, adjusting for age, sex, and community group. We identified 27 differentially methylated CpG sites at biologically relevant regions that reached epigenome-wide significance (P < 1 × 10⁻⁷). Specifically, regions on chromosomes 3 (helicase-like transcription factor), 10 (actin α 2 smooth muscle/Fas cell surface death receptor), and 16 (protease serine 36/C16 open reading frame 67) each harbored 2 significant correlates of n-3 PUFA intake. In conclusion, we present promising evidence of association between several biologically relevant epigenetic markers and long-term intake of marine-derived n-3 PUFAs. PMID:24477300

  7. Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors

    SciTech Connect

    Chang, C.; Kokontis, J.; Liao, S. )

    1988-10-01

    Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural Ars. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.

  8. DNA bases assembled on the Au(110)/electrolyte interface: a combined experimental and theoretical study.

    PubMed

    Salvatore, Princia; Nazmutdinov, Renat R; Ulstrup, Jens; Zhang, Jingdong

    2015-02-19

    Among the low-index single-crystal gold surfaces, the Au(110) surface is the most active toward molecular adsorption and the one with fewest electrochemical adsorption data reported. Cyclic voltammetry (CV), electrochemically controlled scanning tunneling microscopy (EC-STM), and density functional theory (DFT) calculations have been employed in the present study to address the adsorption of the four nucleobases adenine (A), cytosine (C), guanine (G), and thymine (T), on the Au(110)-electrode surface. Au(110) undergoes reconstruction to the (1 × 3) surface in electrochemical environment, accompanied by a pair of strong voltammetry peaks in the double-layer region in acid solutions. Adsorption of the DNA bases gives featureless voltammograms with lower double-layer capacitance, suggesting that all the bases are chemisorbed on the Au(110) surface. Further investigation of the surface structures of the adlayers of the four DNA bases by EC-STM disclosed lifting of the Au(110) reconstruction, specific molecular packing in dense monolayers, and pH dependence of the A and G adsorption. DFT computations based on a cluster model for the Au(110) surface were performed to investigate the adsorption energy and geometry of the DNA bases in different adsorbate orientations. The optimized geometry is further used to compute models for STM images which are compared with the recorded STM images. This has provided insight into the physical nature of the adsorption. The specific orientations of A, C, G, and T on Au(110) and the nature of the physical adsorbate/surface interaction based on the combination of the experimental and theoretical studies are proposed, and differences from nucleobase adsorption on Au(111)- and Au(100)-electrode surfaces are discussed. PMID:25611676

  9. DNA-based nanoparticle composite materials for EMI shielding

    NASA Astrophysics Data System (ADS)

    Zang, De Yu; Grote, James

    2012-03-01

    Composite materials, such as polymer-matrix containing conductive fillers, are very attractive for shielding electromagnetic interference (EMI) due to their high shielding efficiency and seamlessness, processability, flexibility, light-weight and low-cost. Here, we report a development of novel, DNA-based EMI-shielding materials (DESM), consisting of DNA and metal nanoparticles. It has been shown that a thin DESM layer (typically ~30 - 50 μm) could block EMI radiations up to 60 dB effectively over an RF frequency range from KHz to tens GHz, exhibiting excellent EMI shielding efficiency. A wide selection of metal nanoparticle fillers for DESM has been tested for their performance in EMI shielding efficiency. Among them, silver and carbon-based nanoparticles have demonstrated the best performance and were selected for further investigation. The silver-doped DESM films could be also non-conductive while their EMI shielding efficiency is still well-preserved. The nonconductive DESM could have a great potential in the microelectronics industries for EMI shielding on electronic devices and circuit boards.

  10. Intelligent DNA-based molecular diagnostics using linked genetic markers

    SciTech Connect

    Pathak, D.K.; Perlin, M.W.; Hoffman, E.P.

    1994-12-31

    This paper describes a knowledge-based system for molecular diagnostics, and its application to fully automated diagnosis of X-linked genetic disorders. Molecular diagnostic information is used in clinical practice for determining genetic risks, such as carrier determination and prenatal diagnosis. Initially, blood samples are obtained from related individuals, and PCR amplification is performed. Linkage-based molecular diagnosis then entails three data analysis steps. First, for every individual, the alleles (i.e., DNA composition) are determined at specified chromosomal locations. Second, the flow of genetic material among the individuals is established. Third, the probability that a given individual is either a carrier of the disease or affected by the disease is determined. The current practice is to perform each of these three steps manually, which is costly, time consuming, labor-intensive, and error-prone. As such, the knowledge-intensive data analysis and interpretation supersede the actual experimentation effort as the major bottleneck in molecular diagnostics. By examining the human problem solving for the task, we have designed and implemented a prototype knowledge-based system capable of fully automating linkage-based molecular diagnostics in X-linked genetic disorders, including Duchenne Muscular Dystrophy (DMD). Our system uses knowledge-based interpretation of gel electrophoresis images to determine individual DNA marker labels, a constraint satisfaction search for consistent genetic flow among individuals, and a blackboard-style problem solver for risk assessment. We describe the system`s successful diagnosis of DMD carrier and affected individuals from raw clinical data.

  11. Acid-Base Pairs in Lewis Acidic Zeolites Promote Direct Aldol Reactions by Soft Enolization.

    PubMed

    Lewis, Jennifer D; Van de Vyver, Stijn; Román-Leshkov, Yuriy

    2015-08-17

    Hf-, Sn-, and Zr-Beta zeolites catalyze the cross-aldol condensation of aromatic aldehydes with acetone under mild reaction conditions with near quantitative yields. NMR studies with isotopically labeled molecules confirm that acid-base pairs in the Si-O-M framework ensemble promote soft enolization through α-proton abstraction. The Lewis acidic zeolites maintain activity in the presence of water and, unlike traditional base catalysts, in acidic solutions. PMID:26138135

  12. Nucleobase-Based Barbiturates: Their Protective Effect against DNA Damage Induced by Bleomycin-Iron, Antioxidant, and Lymphocyte Transformation Assay

    PubMed Central

    Dhorajiya, Bhaveshkumar D.; Dholakiya, Bharatkumar Z.; Ibrahim, Ahmed S.; Badria, Farid A.

    2014-01-01

    A number of nucleobase-based barbiturates have been synthesized by combination of nucleic acid bases and heterocyclic amines and barbituric acid derivatives through green and efficient multicomponent route and one pot reaction. This approach was accomplished efficiently using aqueous medium to give the corresponding products in high yield. The newly synthesized compounds were characterized by spectral analysis (FT-IR, 1H NMR, 13C NMR, HMBC, and UV spectroscopy) and elemental analysis. Representative of all synthesized compounds was tested and ev