Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

ERIC Educational Resources Information Center

Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

2010-01-01

This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

Influence of backwashing on the microbial community in a biofilm developed on biological activated carbon used in a drinking water treatment plant.

PubMed

Kasuga, I; Shimazaki, D; Kunikane, S

2007-01-01

The influence of backwashing on the biofilm community developed on biological activated carbon (BAC) used in a drinking water treatment plant was investigated by means of bacterial cell enumeration and terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting analysis of bacterial and eukaryotic ribosomal RNA genes (rDNA). After backwashing, the attached bacterial abundance in the top layer of the BAC bed decreased to 64% of that before backwashing. The community level changes caused by backwashing were examined through the T-RFLP profiles. In the bacterial 16S rDNA analysis, the relative abundances of some terminal-restriction fragments (T-RFs) including the Planctomycetes-derived fragment increased; however, the relative abundances of some T-RFs including the Betaproteobacteria-derived fragments decreased. In the eukaryotic 18S rDNA analysis, the relative abundances of some T-RFs including the protozoan Cercozoa-derived fragments increased; however, the relative abundances of some T-RFs including the metazoan Chaetonotus- and Paratripyla-derived fragments decreased. The T-RFLP analysis suggests that backwashing can cause changes in the relative compositions of microorganisms in a BAC biofilm in the top layer of the bed.

Pretreatment of low dose radiation reduces radiation-induced apoptosis in mouse lymphoma (EL4) cells.

PubMed

Kim, J H; Hyun, S J; Yoon, M Y; Ji, Y H; Cho, C K; Yoo, S Y

1997-06-01

Induction of an adaptive response to ionizing radiation in mouse lymphoma (EL4) cells was studied by using cell survival fraction and apoptotic nucleosomal DNA fragmentation as biological end points. Cells in early log phase were pre-exposed to low dose of gamma-rays (0.01 Gy) 4 or 20 hrs prior to high dose gamma-ray (4, 8 and 12 Gy for cell survival fraction analysis; 8 Gy for DNA fragmentation analysis) irradiation. Then cell survival fractions and the extent of DNA fragmentation were measured. Significant adaptive response, increase in cell survival fraction and decrease in the extent of DNA fragmentation were induced when low and high dose gamma-ray irradiation time interval was 4 hr. Addition of protein or RNA synthesis inhibitor, cycloheximide or 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRFB), respectively during adaptation period, the period from low dose gamma-ray irradiation to high dose gamma-ray irradiation, was able to inhibit the induction of adaptive response, which is the reduction of the extent DNA fragmentation in irradiated EL4 cells. These data suggest that the induction of adaptive response to ionizing radiation in EL4 cells required both protein and RNA synthesis.

[Fabrications of a poly (methyl methacrylate) (PMMA) microfluidic chip-based DNA analysis device].

PubMed

Du, Xiao-Guang

2009-12-01

A DNA analysis device based on poly(methyl methacrylate) (PMMA) microfluidic chips was developed. A PMMA chip with cross microchannels was fabricated by a simple hot embossing. Microchannels were modified with a static adsorptive coating method using 2% hydroxyethyl cellulose. A high-voltage power unit, variable in the range 0-1 800 V, was used for on-chip DNA sample injection and gel electrophoretic separation. High speed, high resolution DNA analysis was obtained with the home-built PMMA chip in a sieving matrix containing 2% hydroxyethyl cellulose with a blue intercalating dye, TO-PRO-3 (TP3), by using diode laser induced fluorescence detection based on optical fibers with a 670 nm long-pass filter. The DNA analysis device was applied for the separation of phiX-174/HaeIII DNA digest sample with 11 fragments ranging from 72 to 1 353 bp. A separation efficiency of 1.14 x 10(6) plates/m was obtained for the 603 bp fragments, while the R of 271/281 bp fragments was 1.2. The device was characterized by simple design, low cost for fabrication and operation, reusable PMMA chips, and good reproducibility. A portable microfluidic device for DNA analysis can be developed for clinical diagnosis and disease screening.

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

PubMed

Dragan, Anatoliy I; Golberg, Karina; Elbaz, Amit; Marks, Robert; Zhang, Yongxia; Geddes, Chris D

2011-03-07

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Copyright © 2010 Elsevier B.V. All rights reserved.

DNA fragment sizing and sorting by laser-induced fluorescence

DOEpatents

Hammond, Mark L.; Jett, James H.; Keller, Richard A.; Marrone, Babetta L.; Martin, John C.

1996-01-01

A method is provided for sizing DNA fragments using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA piece or the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is linearly related to the fragment length. The distribution of DNA fragment sizes forms a characterization of the DNA piece for use in forensic and research applications.

Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

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

Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.

1989-01-01

Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector /lambda/pSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB/sup +/ clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA/sup +/ transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmidsmore » consistently transduced the markers thrB/sup +/ and lysA/sup +/. The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes.« less

Luciferase assay to study the activity of a cloned promoter DNA fragment.

PubMed

Solberg, Nina; Krauss, Stefan

2013-01-01

Luciferase based assays have become an invaluable tool for the analysis of cloned promoter DNA fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. Here, we describe the use of the Dual-Luciferase(®) Reporter Assay System created by Promega (Promega Corporation, Wisconsin, USA) to study the cloned 6.7 kilobases (kb) mouse (m) Tcf3 promoter DNA fragment in mouse embryonic derived neural stem cells (NSC). In this system, the expression of the firefly luciferase driven by the cloned mTcf3 promoter DNA fragment (including transcription initiation sites) is correlated with a co-transfected control reporter expressing Renilla luciferase from the herpes simplex virus (HSV) thymidine kinase promoter. Using an internal control reporter allows to normalize the activity of the experimental reporter to the internal control, which minimizes experimental variability.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

PubMed

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

PubMed Central

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples. PMID:28052096

Multiplex analysis of DNA

DOEpatents

Church, George M.; Kieffer-Higgins, Stephen

1992-01-01

This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

Homo-trimerization is essential for the transcription factor function of Myrf for oligodendrocyte differentiation.

PubMed

Kim, Dongkyeong; Choi, Jin-Ok; Fan, Chuandong; Shearer, Randall S; Sharif, Mohamed; Busch, Patrick; Park, Yungki

2017-05-19

Myrf is a key transcription factor for oligodendrocyte differentiation and central nervous system myelination. We and others have previously shown that Myrf is generated as a membrane protein in the endoplasmic reticulum (ER), and that it undergoes auto-processing to release its N-terminal fragment from the ER, which enters the nucleus to work as a transcription factor. These previous studies allow a glimpse into the unusual complexity behind the biogenesis and function of the transcription factor domain of Myrf. Here, we report that Myrf N-terminal fragments assemble into stable homo-trimers before ER release. Consequently, Myrf N-terminal fragments are released from the ER only as homo-trimers. Our re-analysis of a previous genetic screening result in Caenorhabditis elegans shows that homo-trimerization is essential for the biological functions of Myrf N-terminal fragment, and that the region adjacent to the DNA-binding domain is pivotal to its homo-trimerization. Further, our computational analysis uncovered a novel homo-trimeric DNA motif that mediates the homo-trimeric DNA binding of Myrf N-terminal fragments. Importantly, we found that homo-trimerization defines the DNA binding specificity of Myrf N-terminal fragments. In sum, our study elucidates the molecular mechanism governing the biogenesis and function of Myrf N-terminal fragments and its physiological significance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

PubMed

Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

2008-04-01

The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

A transmission imaging spectrograph and microfabricated channel system for DNA analysis.

PubMed

Simpson, J W; Ruiz-Martinez, M C; Mulhern, G T; Berka, J; Latimer, D R; Ball, J A; Rothberg, J M; Went, G T

2000-01-01

In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading and contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual laser excitation source and a highly sensitive charge-coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single-stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single-stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.

Molecular and FISH analyses of a 53-kbp intact DNA fragment inserted by biolistics in wheat (Triticum aestivum L.) genome.

PubMed

Partier, A; Gay, G; Tassy, C; Beckert, M; Feuillet, C; Barret, P

2017-10-01

A large, 53-kbp, intact DNA fragment was inserted into the wheat ( Triticum aestivum L.) genome. FISH analyses of individual transgenic events revealed multiple insertions of intact fragments. Transferring large intact DNA fragments containing clusters of resistance genes or complete metabolic pathways into the wheat genome remains a challenge. In a previous work, we showed that the use of dephosphorylated cassettes for wheat transformation enabled the production of simple integration patterns. Here, we used the same technology to produce a cassette containing a 44-kb Arabidopsis thaliana BAC, flanked by one selection gene and one reporter gene. This 53-kb linear cassette was integrated in the bread wheat (Triticum aestivum L.) genome by biolistic transformation. Our results showed that transgenic plants harboring the entire cassette were generated. The inheritability of the cassette was demonstrated in the T1 and T2 generation. Surprisingly, FISH analysis performed on T1 progeny of independent events identified double genomic insertions of intact fragments in non-homoeologous positions. Inheritability of these double insertions was demonstrated by FISH analysis of the T1 generation. Relative conclusions that can be drawn from molecular or FISH analysis are discussed along with future prospects of the engineering of large fragments for wheat transformation or genome editing.

DNA fragmentation of normal spermatozoa negatively impacts embryo quality and intracytoplasmic sperm injection outcome.

PubMed

Avendaño, Conrado; Franchi, Anahí; Duran, Hakan; Oehninger, Sergio

2010-07-01

To evaluate DNA fragmentation in morphologically normal sperm recovered from the same sample used for intracytoplasmic sperm injection (ICSI) and to correlate DNA damage with embryo quality and pregnancy outcome. Prospective study. Academic center. 36 infertile men participating in the ICSI program. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick end labeling (TUNEL) assay and morphologic assessment by phase contrast. Simultaneous assessment of sperm morphology and DNA fragmentation by TUNEL assay was performed in the same cell, then the percentage of normal sperm with fragmented DNA (normal SFD) was correlated with embryo quality and pregnancy outcomes. A highly statistically significant negative correlation was found between the percentage of normal SFD and embryo quality. This association was confirmed for the transferred embryos and for the total embryo cohort. The receiver operating characteristics curve analysis demonstrated that the percentage of normal SFD and embryo quality were statistically significant predictors of pregnancy. When the percentage of normal SFD was

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis.

PubMed

Glavac, Damjan; Potocnik, Uros; Podpecnik, Darja; Zizek, Teofil; Smerkolj, Sava; Ravnik-Glavac, Metka

2002-04-01

We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments. Copyright 2002 Wiley-Liss, Inc.

Identification and nucleotide sequence analysis of the repetitive DNA element in the genome of fish lymphocystis disease virus.

PubMed

Schnitzler, P; Delius, H; Scholz, J; Touray, M; Orth, E; Darai, G

1987-12-01

The genome of the fish lymphocystis disease virus (FLDV) was screened for the existence of repetitive DNA sequences using a defined and complete gene library of the viral genome (98 kbp) by DNA-DNA hybridization, heteroduplex analysis, and restriction fine mapping. A repetitive DNA sequence was detected at the coordinates 0.034 to 0.057 and 0.718 to 0.736 map units (m.u.) of the FLDV genome. The first region (0.034 to 0.057 m.u.) corresponds to the 5' terminus of the EcoRI FLDV DNA fragment B (0.034 to 0.165 m.u.) and the second region (0.718 to 0.736 m.u.) is identical to the EcoRI DNA fragment M of the viral genome. The DNA nucleotide sequence of the EcoRI FLDV DNA fragment M was determined. This analysis revealed the presence of many short direct and inverted repetitions, e.g., a 18-mer direct repetition (TTTAAAATTTAATTAA) that started at nucleotide positions 812 and 942 and a 14-mer inverted repeat (TTAAATTTAAATTT) at nucleotide positions 820 and 959. Only short open reading frames were detected within this region. The DNA repetitions are discussed as sequences that play a possible regulatory role for virus replication. Furthermore, hybridization experiments revealed that the repetitive DNA sequences are conserved in the genome of different strains of fish lymphocystis disease virus isolated from two species of Pleuronectidae (flounder and dab).

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize.

PubMed

Sánchez, Laura; González, Ramón; Crego, Antonio L; Cifuentes, Alejandro

2007-03-01

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

PubMed

Shi, Liang; Khandurina, Julia; Ronai, Zsolt; Li, Bi-Yu; Kwan, Wai King; Wang, Xun; Guttman, András

2003-01-01

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

PubMed

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence. This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest. ClinicalTrials.gov Identifier: NCT01397136.

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

PubMed

van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

1998-06-01

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

PubMed

Amarger, V; Mercier, L

1995-01-01

We have applied the recently developed technique of random amplified polymorphic DNA (RAPD) for the discrimination between two jojoba clones at the genomic level. Among a set of 30 primers tested, a simple reproducible pattern with three distinct fragments for clone D and two distinct fragments for clone E was obtained with primer OPB08. Since RAPD products are the results of arbitrarily priming events and because a given primer can amplify a number of non-homologous sequences, we wondered whether or not RAPD bands, even those of similar size, were derived from different loci in the two clones. To answer this question, two complementary approaches were used: i) cloning and sequencing of the amplification products from clone E; and ii) complementary Southern analysis of RAPD gels using cloned or amplified fragments (directly recovered from agarose gels) as RFLP probes. The data reported here show that the RAPD reaction generates multiple amplified fragments. Some fragments, although resolved as a single band on agarose gels, contain different DNA species of the same size. Furthermore, it appears that the cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals.

The anti-human immunodeficiency virus agent 3'-fluorothymidine induces DNA damage and apoptosis in human lymphoblastoid cells.

PubMed Central

Sundseth, R; Joyner, S S; Moore, J T; Dornsife, R E; Dev, I K

1996-01-01

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo. PMID:8834875

Sperm DNA fragmentation: mechanisms of origin, impact on reproductive outcome, and analysis.

PubMed

Sakkas, Denny; Alvarez, Juan G

2010-03-01

To review the mechanisms responsible for DNA fragmentation in human sperm, including those occurring during spermatogenesis and transport through the reproductive tract. The mechanisms examined include: apoptosis in the seminiferous tubule epithelium, defects in chromatin remodeling during the process of spermiogenesis, oxygen radical-induced DNA damage during sperm migration from the seminiferous tubules to the epididymis, the activation of sperm caspases and endonucleases, damage induced by chemotherapy and radiotherapy, and the effect of environmental toxicants. The different tests currently used for sperm DNA fragmentation analysis and the factors that determine the predictive value of sperm DNA fragmentation testing and their implications in the diagnosis and treatment of infertility are also discussed. Finally, we also scrutinize how the presence in the embryonic genome of DNA strand breaks or modifications of DNA nucleotides inherited from the paternal genome could impact the embryo and offspring. In particular we discuss how abnormal sperm could be dealt with by the oocyte and how sperm DNA abnormalities, which have not been satisfactorily repaired by the oocyte after fertilization, may interfere with normal embryo and fetal development. Sperm DNA can be modified through various mechanisms. The integrity of the paternal genome is therefore of paramount importance in the initiation and maintenance of a viable pregnancy both in a natural conception and in assisted reproduction. The need to diagnose sperm at a nuclear level is an area that needs further understanding so that we can improve treatment of the infertile couple. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Nucleotide Sequence Analysis of RNA Synthesized from Rabbit Globin Complementary DNA

PubMed Central

Poon, Raymond; Paddock, Gary V.; Heindell, Howard; Whitcome, Philip; Salser, Winston; Kacian, Dan; Bank, Arthur; Gambino, Roberto; Ramirez, Francesco

1974-01-01

Rabbit globin complementary DNA made with RNA-dependent DNA polymerase (reverse transcriptase) was used as template for in vitro synthesis of 32P-labeled RNA. The sequences of the nucleotides in most of the fragments resulting from combined ribonuclease T1 and alkaline phosphatase digestion have been determined. Several fragments were long enough to fit uniquely with the α or β globin amino-acid sequences. These data demonstrate that the cDNA was copied from globin mRNA and contained no detectable contaminants. Images PMID:4139714

Identification of genes associated with low furanocoumarin content in grapefruit.

PubMed

Chen, Chunxian; Yu, Qibin; Wei, Xu; Cancalon, Paul F; Gmitter, Fred G

2014-10-01

Some furanocoumarins in grapefruit (Citrus paradisi) are associated with the so-called grapefruit juice effect. Previous phytochemical quantification and genetic analysis suggested that the synthesis of these furanocoumarins may be controlled by a single gene in the pathway. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of fruit tissues was performed to identify the candidate gene(s) likely associated with low furanocoumarin content in grapefruit. Fifteen tentative differentially expressed fragments were cloned through the cDNA-AFLP analysis of the grapefruit variety Foster and its spontaneous low-furanocoumarin mutant Low Acid Foster. Sequence analysis revealed a cDNA-AFLP fragment, Contig 6, was homologous to a substrate-proved psoralen synthase gene, CYP71A22, and was part of citrus unigenes Cit.3003 and Csi.1332, and predicted genes Ciclev10004717m in mandarin and orange1.1g041507m in sweet orange. The two predicted genes contained the highly conserved motifs at one of the substrate recognition sites of CYP71A22. Digital gene expression profile showed the unigenes were expressed only in fruit and seed. Quantitative real-time PCR also proved Contig 6 was down-regulated in Low Acid Foster. These results showed the differentially expressed Contig 6 was related to the reduced furanocoumarin levels in the mutant. The identified fragment, homologs, unigenes, and genes may facilitate further furanocoumarin genetic study and grapefruit variety improvement.

Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact

PubMed Central

Zagoskin, Maxim V.; Lazareva, Valentina I.; Grishanin, Andrey K.; Mukha, Dmitry V.

2014-01-01

The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals. PMID:25215300

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells.

PubMed

Elmroth, Kecke; Stenerlöw, Bo

2005-04-01

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to 125IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

Fragman: an R package for fragment analysis

USDA-ARS?s Scientific Manuscript database

Determination of microsatellite lengths or other DNA fragment types is an important initial component of many genetic studies such as mutation detection, linkage and QTL mapping, genetic diversity, pedigree analysis, and detection of heterozygosity. A handful of commercial and freely available softw...

Analysis of mitochondrial DNA: taxonomic and phylogenetic relationships in two fish taxa (Pisces: Mugilidae and Cyprinidae).

PubMed

Semina, A V; Polyakova, N E; Brykov, Vl A

2007-12-01

To solve some systematic questions as well as to study genetic variability and evolutionary relationships in two groups of fish belonging to the Mugilid (Mugilidae) and Cyprinid (Cyprinidae) families, we have used restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction. The analysis of three mtDNA fragments of 7220 bp total length of six Mugilid species has shown that Mediterranean Liza aurata, L. ramada, L. saliens, and Chelon labrosus form a common cluster, L. aurata and C. labrosus being the closest relatives, whereas L. haematocheilus (syn. C. haematocheilus) of the Sea of Japan forms a sister group to the Mediterranean cluster. It was found that Chelon and Liza genera are paraphyletic, and therefore their division into two genera is unnatural and they should be synonymized. According to priority, Liza species should be ascribed to Chelon genus. Mugil cephalus is the most distant compared to the rest of the species studied. The level of genetic divergence between allopatric samples of M. cephalus from the Sea of Japan and the Mediterranean Sea has proved to be very high--4.5% of nucleotide substitutions. The analysis of four mtDNA fragments of 9340 bp total length of six Cyprinid species has shown that L. waleckii is the most genetically distant. Pseudaspius leptocephalus is a sister group to Tribolodon species. All Tribolodon species form a common cluster with T. sachalinensis as a root. The remaining species form two branches, one of which includes T. nakamurai and T. brandtii, another one combines T. hakonensis and a new form of Tribolodon revealed that is close to T. hakonensis by its mtDNA (2.4% of nucleotide substitutions). This new form might be an independent species.

Isolation of CYP3A5P cDNA from human liver: a reflection of a novel cytochrome P-450 pseudogene.

PubMed

Schuetz, J D; Guzelian, P S

1995-03-14

We have isolated, from a human liver cDNA library, a 1627 bp CYP3A5 cDNA variant (CYP3A5P) that contains several large insertions, deletions, and in-frame termination codons. By comparison with the genomic structure of other CYP3A genes, the major insertions in CYP3A5P cDNA demarcate the inferred sites of several CYP3A5 exons. The segments inserted in CYP3A5P have no homology with splice donor acceptor sites. It is unlikely that CYP3A5P cDNA represents an artifact of the cloning procedures since Southern blot analysis of human genomic DNA disclosed that CYP3A5P cDNA hybridized with a DNA fragment distinct from fragments that hybridized with either CYP3A5, CYP3A3 or CYP3A4. Moreover, analysis of adult human liver RNA on Northern blots hybridized with a CYP3A5P cDNA fragment revealed the presence of an mRNA with the predicted size of CYP3A5P. We conclude that CYP3A5P cDNA was derived from a separate gene, CYP3A5P, most likely a pseudogene evolved from CYP3A5.

Applicability of SCAR markers to food genomics: olive oil traceability.

PubMed

Pafundo, Simona; Agrimonti, Caterina; Maestri, Elena; Marmiroli, Nelson

2007-07-25

DNA analysis with molecular markers has opened a shortcut toward a genomic comprehension of complex organisms. The availability of micro-DNA extraction methods, coupled with selective amplification of the smallest extracted fragments with molecular markers, could equally bring a breakthrough in food genomics: the identification of original components in food. Amplified fragment length polymorphisms (AFLPs) have been instrumental in plant genomics because they may allow rapid and reliable analysis of multiple and potentially polymorphic sites. Nevertheless, their direct application to the analysis of DNA extracted from food matrixes is complicated by the low quality of DNA extracted: its high degradation and the presence of inhibitors of enzymatic reactions. The conversion of an AFLP fragment to a robust and specific single-locus PCR-based marker, therefore, could extend the use of molecular markers to large-scale analysis of complex agro-food matrixes. In the present study is reported the development of sequence characterized amplified regions (SCARs) starting from AFLP profiles of monovarietal olive oils analyzed on agarose gel; one of these was used to identify differences among 56 olive cultivars. All the developed markers were purposefully amplified in olive oils to apply them to olive oil traceability.

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

PubMed

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

1993-09-01

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

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization.

PubMed

Jenkins, Frank J; Kerr, Charles M; Fouquerel, Elise; Bovbjerg, Dana H; Opresko, Patricia L

2017-07-10

There are several different techniques for measuring telomere length, each with their own advantages and disadvantages. The traditional approach, Telomere Restriction Fragment (TRF) analysis, utilizes a DNA hybridization technique whereby genomic DNA samples are digested with restriction enzymes, leaving behind telomere DNA repeats and some sub-telomeric DNA. These are separated by agarose gel electrophoresis, transferred to a filter membrane and hybridized to oligonucleotide probes tagged with either chemiluminescence or radioactivity to visualize telomere restriction fragments. This approach, while requiring a larger quantity of DNA than other techniques such as PCR, can measure the telomere length distribution of a population of cells and allows measurement expressed in absolute kilobases. This manuscript demonstrates a modified DNA hybridization procedure for determining telomere length. Genomic DNA is first digested with restriction enzymes (that do not cut telomeres) and separated by agarose gel electrophoresis. The gel is then dried and the DNA is denatured and hybridized in situ to a radiolabeled oligonucleotide probe. This in situ hybridization avoids loss of telomere DNA and improves signal intensity. Following hybridization, the gels are imaged utilizing phosphor screens and the telomere length is quantified using a graphing program. This procedure was developed by the laboratories of Drs. Woodring Wright and Jerry Shay at the University of Texas Southwestern 1 , 2 . Here, we present a detailed description of this procedure, with some modifications.

Gene expression analysis by cDNA-AFLP highlights a set of new signaling networks and translational control during seed dormancy breaking in Nicotiana plumbaginifolia.

PubMed

Bove, Jérôme; Lucas, Philippe; Godin, Béatrice; Ogé, Laurent; Jullien, Marc; Grappin, Philippe

2005-03-01

Seed dormancy in Nicotiana plumbaginifolia is characterized by an abscisic acid accumulation linked to a pronounced germination delay. Dormancy can be released by 1 year after-ripening treatment. Using a cDNA-amplified fragment length polymorphism (cDNA-AFLP) approach we compared the gene expression patterns of dormant and after-ripened seeds, air-dry or during one day imbibition and analyzed 15,000 cDNA fragments. Among them 1020 were found to be differentially regulated by dormancy. Of 412 sequenced cDNA fragments, 83 were assigned to a known function by search similarities to public databases. The functional categories of the identified dormancy maintenance and breaking responsive genes, give evidence that after-ripening turns in the air-dry seed to a new developmental program that modulates, at the RNA level, components of translational control, signaling networks, transcriptional control and regulated proteolysis.

Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila

PubMed Central

Saveliev, Sergei V.; Cox, Michael M.

2001-01-01

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15–16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form. PMID:11406601

Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila.

PubMed

Saveliev, S V; Cox, M M

2001-06-15

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15-16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form.

A selective plasmin inhibitor, trans-aminomethylcyclohexanecarbonyl-L-(O-picolyl)tyrosine-octylamide (YO-2), induces thymocyte apoptosis.

PubMed

Lee, Eibai; Enomoto, Riyo; Takemura, Kazu; Tsuda, Yuko; Okada, Yoshio

2002-04-01

The treatment of rat thymocytes with YO-2, a novel inhibitor of plasmin, resulted in an increase in DNA fragmentation. DNA fragmentation was also induced by another YO compounds such as YO-0, -3, -4 and -5. These YO compounds are the inhibitor of plasmin activity. On the other hand, YO-1, -6 and -8 that hardly inhibit plasmin activity had no effect on DNA fragmentation. Analysis of fragmented DNA from thymocytes treated with YO-2 by agarose gel electrophoresis revealed that the compound caused internucleosomal DNA fragmentation. In addition, judging from a laser scanning microscopy, annexin V-positive and propidium iodide-negative cells were increased by the treatment of the cells with the compound. Moreover, chromatin condensation was observed in thymocytes treated with the compound. These results demonstrated that YO-2 induces thymocyte apoptosis. There seemed to be some correlation between the apoptosis induced by YO compounds and their plasmin inhibitory effect. However, because the other protease inhibitors including pepstatin A, leupeptin, AEBSF, DFP and E-64-d did not affect DNA fragmentation, YO compounds are likely to have unique mechanism on plasmin or to show the effect on the other plasmin-like proteases. The plasmin inhibitory activity may have an important role in YO-2-induced apoptosis. Furthermore, the stimulations of caspase-8, -9 and -3-like activities were observed in thymocytes treated with YO-2. These results suggest that YO-2 induces thymocyte apoptosis via activation of caspase cascade.

Scar-less multi-part DNA assembly design automation

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

Hillson, Nathan J.

The present invention provides a method of a method of designing an implementation of a DNA assembly. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding flanking homology sequences to each of the DNA oligos. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which tomore » assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding optimized overhang sequences to each of the DNA oligos.« less

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

PubMed Central

Palzkill, T G; Oliver, S G; Newlon, C S

1986-01-01

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity. PMID:3529036

Mitochondrial DNA diagnosis for taeniasis and cysticercosis.

PubMed

Yamasaki, Hiroshi; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Sato, Marcello Otake; Ito, Akira

2006-01-01

Molecular diagnosis for taeniasis and cysticercosis in humans on the basis of mitochondrial DNA analysis was reviewed. Development and application of three different methods, including restriction fragment length polymorphism analysis, base excision sequence scanning thymine-base analysis and multiplex PCR, were described. Moreover, molecular diagnosis of cysticerci found in specimens submitted for histopathology and the molecular detection of taeniasis using copro-DNA were discussed.

Abstracts of papers presented at the 8th workshop of the Virology Section of the Deutsche Gesellschaft für Hygiene und Mikro-biologie, Würzburg, March 17-19, 1983.

PubMed

1983-09-01

17 adenovirus strains were found to be antigenically related to prototype Ad 15 by neutralization. No relationship to Ad 15, but to Ad 9 could be detected by hemagglutination-inhibition; we therefore named them Ad 15/H9 intermediate strains. After analysis of the genome by five different restriction enzymes, the fragment patterns obtained deviated widely from the prototype Ad 15, but only slightly from Ad 9. Differences could also be observed among the variants. After digestion by five restriction enzymes, altogether six genome types could be established among the 17 intermediate strains. To map the variations on the genome of the 15/H9 strains, two methods were employed: the double digestion of the DNA and DNA fragments together with the determination of the terminal fragments made it possible to construct a physical map. The second method depends on a particularity of adenoviruses: the DNA is covalently linked with a 55 kD protein at the 5' terminus. After digestion of the DNA, which does contain this protein, the terminal DNA fragments do not migrate into the agarose gel; after an additional digestion with pronase B, they do migrate into the gel. Thus the terminal fragments were determined by comparing the fragment patterns with and without previous pronase B treatment.

Molecular Characterization and Analysis of 16S Ribosomal DNA in Some Isolates of Demodex folicullorum

PubMed Central

DANESHPARVAR, Afrooz; MOWLAVI, Gholamreza; MIRJALALI, Hamed; HAJJARAN, Homa; MOBEDI, Iraj; NADDAF, Saeed Reza; SHIDFAR, Mohammadreza; SADAT MAKKI, Mahsa

2017-01-01

Background: Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct and transmits through close contact with an infested host. Methods: This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, already obtained from four different patients and identified morphologically though clearing with 10% Potassium hydroxide (KOH) and microscopical examination. Amplified fragments from the isolates were compared with GeneBank database and phylogenetic analysis was carried out using MEGA6 software. Results: A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs) in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Conclusion: Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites. PMID:28761482

Molecular Characterization and Analysis of 16S Ribosomal DNA in Some Isolates of Demodex folicullorum.

PubMed

Daneshparvar, Afrooz; Mowlavi, Gholamreza; Mirjalali, Hamed; Hajjaran, Homa; Mobedi, Iraj; Naddaf, Saeed Reza; Shidfar, Mohammadreza; Sadat Makki, Mahsa

2017-01-01

Demodicosis is one of the most prevalent skin diseases resulting from infestation by Demodex mites. This parasite usually inhabits in follicular infundibulum or sebaceous duct and transmits through close contact with an infested host. This study was carried from September 2014 to January 2016 at Tehran University of Medical Sciences, Tehran, Iran. DNA extraction and amplification of 16S ribosomal RNA was performed on four isolates, already obtained from four different patients and identified morphologically though clearing with 10% Potassium hydroxide (KOH) and microscopical examination. Amplified fragments from the isolates were compared with GeneBank database and phylogenetic analysis was carried out using MEGA6 software. A 390 bp fragment of 16S rDNA was obtained in all isolates and analysis of generated sequences showed high similarity with those submitted to GenBank, previously. Intra-species similarity and distance also showed 99.983% and 0.017, respectively, for the studied isolates. Multiple alignments of the isolates showed Single Nucleotide Polymorphisms (SNPs) in 16S rRNA fragment. Phylogenetic analysis revealed that all 4 isolates clustered with other D. folliculorum, recovered from GenBank database. Our accession numbers KF875587 and KF875589 showed more similarity together in comparison with two other studied isolates. Mitochondrial 16S rDNA is one of the most suitable molecular barcodes for identification D. folliculorum and this fragment can use for intra-species characterization of the most human-infected mites.

20 years since the introduction of DNA barcoding: from theory to application.

PubMed

Fišer Pečnikar, Živa; Buzan, Elena V

2014-02-01

Traditionally, taxonomic identification has relied upon morphological characters. In the last two decades, molecular tools based on DNA sequences of short standardised gene fragments, termed DNA barcodes, have been developed for species discrimination. The most common DNA barcode used in animals is a fragment of the cytochrome c oxidase (COI) mitochondrial gene, while for plants, two chloroplast gene fragments from the RuBisCo large subunit (rbcL) and maturase K (matK) genes are widely used. Information gathered from DNA barcodes can be used beyond taxonomic studies and will have far-reaching implications across many fields of biology, including ecology (rapid biodiversity assessment and food chain analysis), conservation biology (monitoring of protected species), biosecurity (early identification of invasive pest species), medicine (identification of medically important pathogens and their vectors) and pharmacology (identification of active compounds). However, it is important that the limitations of DNA barcoding are understood and techniques continually adapted and improved as this young science matures.

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

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

Tully, D.B.; Cidlowski, J.A.

1989-03-07

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins priormore » to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.« less

High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

PubMed

Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

2015-06-17

High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.

PubMed Central

Sotiropoulos, C; Coloe, P J; Smith, S C

1994-01-01

Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae. Images PMID:7914209

DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica.

PubMed

Milgroom, M G; Lipari, S E; Powell, W A

1992-06-01

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).

DNA analysis in perpetrator identification of terrorism-related disaster: suicide bombing of the Australian Embassy in Jakarta 2004.

PubMed

Sudoyo, Herawati; Widodo, Putut T; Suryadi, Helena; Lie, Yuliana S; Safari, Dodi; Widjajanto, Agung; Kadarmo, D Aji; Hidayat, Soegeng; Marzuki, Sangkot

2008-06-01

We report the strategy that we employed to identify the perpetrator of a suicide car bombing in front of the Australian Embassy in Jakarta, Indonesia, on 9 September 2004. The bomb was so massive that only small tissue pieces of the perpetrator could be recovered, preventing conventional approach to the identification of the bomber, necessitating the introduction of DNA analysis as the primary means for perpetrator identification. Crime scene investigation revealed the trajectory of the bomb blast, which was used to guide the collection of charred tissue fragments of the perpetrator. Mitochondrial DNA analysis was first conducted on 17 tissue fragments, recovered over large areas of the trajectory to, (a) confirm that they are of a common source, i.e. the perpetrator, and thus (b) establish the mtDNA HV1 sequence profile of the perpetrator. The mtDNA of the perpetrator matches that of a maternally related family member of one of four suspects. Standard autosomal STR analysis confirmed the identification. This case is of interest as an illustration of a successful application of DNA analysis as the primary means of disaster perpetrator identification.

Accumulation of linear mitochondrial DNA fragments in the nucleus shortens the chronological life span of yeast.

PubMed

Cheng, Xin; Ivessa, Andreas S

2012-10-01

Translocation of mitochondrial DNA (mtDNA) fragments to the nucleus and insertion of those fragments into nuclear DNA has been observed in several organisms ranging from yeast to plants and mammals. Disruption of specific nuclear genes by de novo insertions of mtDNA fragments has even been linked to the initiation of several human diseases. Recently, we demonstrated that baker's yeast strains with high rates of mtDNA fragments migrating to the nucleus (yme1-1 mutant) exhibit short chronological life spans (CLS). The yeast CLS is determined by the survival of non-dividing cell populations. Here, we show that lack of the non-homologous-end-joining enzyme DNA ligase IV (DNL4) can rescue the short CLS of the yme1-1 mutant. In fission yeast, DNA ligase IV has been shown to be required for the capture of mtDNA fragments during the repair of double-stranded DNA breaks in nuclear DNA. In further analyses using pulse field gel and 2D gel electrophoresis we demonstrate that linear mtDNA fragments with likely nuclear localization accumulate in the yme1-1 mutant. The accumulation of the linear mtDNA fragments in the yme1-1 mutant is suppressed when Dnl4 is absent. We propose that the linear nuclear mtDNA fragments accelerate the aging process in the yme1-1 mutant cells by possibly affecting nuclear processes including DNA replication, recombination, and repair as well as transcription of nuclear genes. We speculate further that Dnl4 protein has besides its function as a ligase also a role in DNA protection. Dnl4 protein may stabilize the linear mtDNA fragments in the nucleus by binding to their physical ends. In the absence of Dnl4 protein the linear fragments are therefore unprotected and possibly degraded by nuclear nucleases. Copyright © 2012 Elsevier GmbH. All rights reserved.

Modulation-frequency encoded multi-color fluorescent DNA analysis in an optofluidic chip.

PubMed

Dongre, Chaitanya; van Weerd, Jasper; Besselink, Geert A J; Vazquez, Rebeca Martinez; Osellame, Roberto; Cerullo, Giulio; van Weeghel, Rob; van den Vlekkert, Hans H; Hoekstra, Hugo J W M; Pollnau, Markus

2011-02-21

We introduce a principle of parallel optical processing to an optofluidic lab-on-a-chip. During electrophoretic separation, the ultra-low limit of detection achieved with our set-up allows us to record fluorescence from covalently end-labeled DNA molecules. Different sets of exclusively color-labeled DNA fragments-otherwise rendered indistinguishable by spatio-temporal coincidence-are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed.

Fragment Length of Circulating Tumor DNA.

PubMed

Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay

2016-07-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

Markers of Developmentally Regulated Programmed Cell Death and Their Analysis in Cereal Seeds.

PubMed

Domínguez, Fernando; Cejudo, Francisco Javier

2018-01-01

Programmed cell death (PCD) is a key process for the development and differentiation of multicellular organisms, which is characterized by well-defined morphological and biochemical features. These include chromatin condensation, DNA degradation and nuclear fragmentation, with nucleases and proteases playing a relevant function in these processes. In this chapter we describe methods routinely used for the analysis of hallmarks of developmentally regulated PCD in cereal seed tissues, which are based on agarose and polyacrylamide gel electrophoresis, in situ staining of DNA fragmentation, and cell-free assays of relevant enzymatic activities.

Freshwater toxic cyanobacteria induced DNA damage in apple (Malus pumila), rape (Brassica napus) and rice (Oryza sativa).

PubMed

Chen, J Z; Ye, J Y; Zhang, H Y; Jiang, X J; Zhang, Y X; Liu, Z L

2011-06-15

Cyanobacteria in freshwater ecosystems can present a harmful effect on growth and development of plants through irrigation with contaminated water. In this study, the effects of microcystins (MCs)-containing cyanobacteria extract (CE) on DNA damage of apple, rape and rice were investigated to explore the phytotoxic mechanism of MCs through DNA fragmentation and RAPD analysis. Determination of DNA fragmentation by fluorescent dye DAPI showed that significant DNA damage was observed in rice seedlings after exposure to CE while DNA fragmentation in rape seedlings and apple cultures did not differ significantly between treatment and control groups. Qualitative characterization of genomic DNA fragmentation by agarose gel electrophoresis supported the quantitative determination using DAPI. The main changes in RAPD profiles of rape seedlings following exposure of lower doses of CE were variation in band intensity for the primers F03 and S01, while higher doses of CE caused loss of normal bands and appearance of new bands except band intensity changes. The data presented here demonstrate that DNA damage in plants occurs following exposure of microcystins, and the polymorphic RAPDs may be used as an investigation tool for environmental toxicology and as a useful biomarker for the detection of genotoxic effects of microcystins on plants. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Anisotropic Brownian motion in ordered phases of DNA fragments.

PubMed

Dobrindt, J; Rodrigo Teixeira da Silva, E; Alves, C; Oliveira, C L P; Nallet, F; Andreoli de Oliveira, E; Navailles, L

2012-01-01

Using Fluorescence Recovery After Photobleaching, we investigate the Brownian motion of DNA rod-like fragments in two distinct anisotropic phases with a local nematic symmetry. The height of the measurement volume ensures the averaging of the anisotropy of the in-plane diffusive motion parallel or perpendicular to the local nematic director in aligned domains. Still, as shown in using a model specifically designed to handle such a situation and predicting a non-Gaussian shape for the bleached spot as fluorescence recovery proceeds, the two distinct diffusion coefficients of the DNA particles can be retrieved from data analysis. In the first system investigated (a ternary DNA-lipid lamellar complex), the magnitude and anisotropy of the diffusion coefficient of the DNA fragments confined by the lipid bilayers are obtained for the first time. In the second, binary DNA-solvent system, the magnitude of the diffusion coefficient is found to decrease markedly as DNA concentration is increased from isotropic to cholesteric phase. In addition, the diffusion coefficient anisotropy measured within cholesteric domains in the phase coexistence region increases with concentration, and eventually reaches a high value in the cholesteric phase.

Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus.

PubMed

Darai, G; Delius, H; Clarke, J; Apfel, H; Schnitzler, P; Flügel, R M

1985-10-30

A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular.

Fragment length polymorphisms among independent isolates of Epstein-Barr virus from immunocompromised and normal hosts.

PubMed

Katz, B Z; Niederman, J C; Olson, B A; Miller, G

1988-02-01

DNA restriction fragment length polymorphisms of Epstein-Barr virus (EBV) DNA were used as a molecular epidemiological tool to study multiple isolates of virus from the same and different individuals. We studied 35 EBV isolates: 19 from seven immunocompromised children and 16 from seven college students with mononucleosis. Analysis of the fragment length polymorphisms in this collection of isolates permitted several conclusions. Sites of polymorphism were most often encountered in regions with repetitive DNA. Epidemiologically unrelated patients harbored viruses that could be readily distinguished; by contrast, two infants and their mothers harbored similar viruses. Isolates from different sites in the same patient were similar. Variations between different clinical isolates of EBV mimic those found between different laboratory strains of the virus. Fragment length polymorphisms thus provide a useful marker for studying transmission and pathogenesis of EBV infections.

The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

PubMed Central

2012-01-01

Background Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics). Results Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05). Conclusions These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics. PMID:23217215

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

NASA Technical Reports Server (NTRS)

Lobrich, M.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

1995-01-01

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

PubMed Central

Thierry, Alain R.; Mouliere, Florent; Gongora, Celine; Ollier, Jeremy; Robert, Bruno; Ychou, Marc; Del Rio, Maguy; Molina, Franck

2010-01-01

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations. PMID:20494973

Fragment Length of Circulating Tumor DNA

PubMed Central

Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

2016-01-01

Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

Palaeoproteomics for human evolution studies

NASA Astrophysics Data System (ADS)

Welker, Frido

2018-06-01

The commonplace sequencing of Neanderthal, Denisovan and ancient modern human DNA continues to revolutionize our understanding of hominin phylogeny and interaction(s). The challenge with older fossils is that the progressive fragmentation of DNA even under optimal conditions, a function of time and temperature, results in ever shorter fragments of DNA. This process continues until no DNA can be sequenced or reliably aligned. Ancient proteins ultimately suffer a similar fate, but are a potential alternative source of biomolecular sequence data to investigate hominin phylogeny given their slower rate of fragmentation. In addition, ancient proteins have been proposed to potentially provide insights into in vivo biological processes and can be used to provide additional ecological information through large scale ZooMS (Zooarchaeology by Mass Spectrometry) screening of unidentifiable bone fragments. However, as initially with ancient DNA, most ancient protein research has focused on Late Pleistocene or Holocene samples from Europe. In addition, only a limited number of studies on hominin remains have been published. Here, an updated review on ancient protein analysis in human evolutionary contexts is given, including the identification of specific knowledge gaps and existing analytical limits, as well as potential avenues to overcome these.

Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism.

PubMed Central

Woods, D E; Edge, M D; Colten, H R

1984-01-01

Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases. Images PMID:6086718

Ancient DNA Reveals Late Pleistocene Existence of Ostriches in Indian Sub-Continent.

PubMed

Jain, Sonal; Rai, Niraj; Kumar, Giriraj; Pruthi, Parul Aggarwal; Thangaraj, Kumarasamy; Bajpai, Sunil; Pruthi, Vikas

2017-01-01

Ancient DNA (aDNA) analysis of extinct ratite species is of considerable interest as it provides important insights into their origin, evolution, paleogeographical distribution and vicariant speciation in congruence with continental drift theory. In this study, DNA hotspots were detected in fossilized eggshell fragments of ratites (dated ≥25000 years B.P. by radiocarbon dating) using confocal laser scanning microscopy (CLSM). DNA was isolated from five eggshell fragments and a 43 base pair (bp) sequence of a 16S rRNA mitochondrial-conserved region was successfully amplified and sequenced from one of the samples. Phylogenetic analysis of the DNA sequence revealed a 92% identity of the fossil eggshells to Struthio camelus and their position basal to other palaeognaths, consistent with the vicariant speciation model. Our study provides the first molecular evidence for the presence of ostriches in India, complementing the continental drift theory of biogeographical movement of ostriches in India, and opening up a new window into the evolutionary history of ratites.

MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

EPA Science Inventory

Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

In situ end labeling of fragmented DNA in induced ovarian atresia.

PubMed

D'Herde, K; De Pestel, G; Roels, F

1994-01-01

Apoptosis is studied in a model of induced follicular atresia in the ovary of Japanese quail (Coturnix coturnix japonica) by in situ end labeling of DNA fragments in granulosa cells using two different techniques (incorporation of labeled nucleotides by DNA polymerase I or terminal deoxynucleotidyl transferase). The most remarkable observation related to apoptosis in this model is the predominant cytoplasmic localization of labeled DNA fragments, while DNA fragmentation appears to be absent from compacted chromatin masses of apoptotic nuclei and apoptotic nuclear fragments. Unstained apoptotic bodies are present adjacent to stained ones, so that their detection rate on hematoxylin + eosin stained sections is better than on the in situ end-labeled sections. This suggests that DNA fragmentation is a late even or not obligatory in apoptotic granulosa cell death. In contrast to similar studies on atretic granulosa in mammalian models, the process of apoptosis is asynchronous in the granulosal epithelium, with a majority of nuclei with normal chromatin configuration remaining negative for DNA fragmentation. Finally it is shown that the techniques used are not specific for apoptosis, as DNA fragmentation in necrotic granulosa cells is detected as well.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

PubMed

Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

2015-03-15

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. Published by Elsevier Inc.

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

USGS Publications Warehouse

Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

2015-01-01

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

PubMed

Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

2014-01-01

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Relationships between human sperm protamines, DNA damage and assisted reproduction outcomes.

PubMed

Simon, Luke; Castillo, Judit; Oliva, Rafael; Lewis, Sheena E M

2011-12-01

The exchange of histones with protamines in sperm DNA results in sperm chromatin compaction and protection. Variations in sperm protamine expression are associated with male infertility. The aim of this study was to investigate relationships between DNA fragmentation, sperm protamines and assisted reproduction treatment. Semen and spermatozoa prepared by density-gradient centrifugation (DGC) from 73 men undergoing IVF and 24 men undergoing intracytoplasmic sperm injection (ICSI) were included in the study. Nuclear DNA fragmentation was assessed using the alkaline Comet assay and protamines were separated by acid-urea polyacrylamide gels. Sperm DNA fragmentation and protamine content (P1-DNA, P2-DNA, P1+P2-DNA) decreased in spermatozoa after DGC. Abnormally high and low P1/P2 ratios were associated with increased sperm DNA fragmentation. Couples with idiopathic infertility had abnormally high P1/P2 ratios. Fertilization rates and embryo quality decreased as sperm DNA fragmentation or protamines increased. Sperm DNA fragmentation was lower in couples achieving pregnancies after IVF, but not after ICSI. There was no correlation between protamine content (P1-DNA, P2-DNA, P1+P2-DNA) or P1/P2 ratios and IVF or ICSI pregnancies. Increased sperm DNA fragmentation was associated with abnormal protamination and resulted in lower fertilization rates, poorer embryo quality and reduced pregnancy rates. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

PubMed Central

Della Valle, G; Fenton, R G; Basilico, C

1981-01-01

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments. Images PMID:6100965

Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

DOEpatents

Wong, Kwong-Kwok

2000-01-01

The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

PubMed Central

Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

2016-01-01

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

Extending the spectrum of DNA sequences retrieved from ancient bones and teeth

PubMed Central

Glocke, Isabelle; Meyer, Matthias

2017-01-01

The number of DNA fragments surviving in ancient bones and teeth is known to decrease with fragment length. Recent genetic analyses of Middle Pleistocene remains have shown that the recovery of extremely short fragments can prove critical for successful retrieval of sequence information from particularly degraded ancient biological material. Current sample preparation techniques, however, are not optimized to recover DNA sequences from fragments shorter than ∼35 base pairs (bp). Here, we show that much shorter DNA fragments are present in ancient skeletal remains but lost during DNA extraction. We present a refined silica-based DNA extraction method that not only enables efficient recovery of molecules as short as 25 bp but also doubles the yield of sequences from longer fragments due to improved recovery of molecules with single-strand breaks. Furthermore, we present strategies for monitoring inefficiencies in library preparation that may result from co-extraction of inhibitory substances during DNA extraction. The combination of DNA extraction and library preparation techniques described here substantially increases the yield of DNA sequences from ancient remains and provides access to a yet unexploited source of highly degraded DNA fragments. Our work may thus open the door for genetic analyses on even older material. PMID:28408382

Rapid estimation of microbial populations in fish samples by using terminal restriction fragment length polymorphism analysis of 16S rDNA.

PubMed

Tanaka, Yuichiro; Takahashi, Hajime; Kitazawa, Nao; Kimura, Bon

2010-01-01

A rapid system using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting 16S rDNA is described for microbial population analysis in edible fish samples. The defined terminal restriction fragment database was constructed by collecting 102 strains of bacteria representing 53 genera that are associated with fish. Digestion of these 102 strains with two restriction enzymes, HhaI and MspI, formed 54 pattern groups with discrimination to the genus level. This T-RFLP system produced results comparable to those from a culture-based method in six natural fish samples with a qualitative correspondence of 71.4 to 92.3%. Using the T-RFLP system allowed an estimation of the microbial population within 7 h. Rapid assay of the microbial population is advantageous for food manufacturers and testing laboratories; moreover, the strategy presented here allows adaptation to specific testing applications.

Method for identifying mutagenic agents which induce large, multilocus deletions in DNA

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

Bradley, W.E.C.; Belouchi, A.; Dewyse, P.

1993-07-13

A method of identifying a mutagenic agent is described which includes a large, multilocus deletions in DNA in mammalian cells comprising: (i) exposing a class III heterozygous CHO cell line to a potential mutagenic agent under investigation, and allowing any mutation of the cell line to proceed, said cell line being characterized in that a restriction fragment length variation exists in on mutation it becomes resistant to 2,6-diaminopurine and in that the DNA sequence adjacent to the two alleles of the APRT gene such that the DNA sequence adjacent to one of the two alleles can be digested with themore » enzyme BclI but the DNA sequence variation adjacent to the other of the two alleles cannot be digested with BclI, (ii) isolating induced mutations of the cell line deficient in APRT function, (iii) isolating DNA from the induced mutants, (iv) digesting the isolated DNA with BclI enzyme to produce digested fragments including a 19 kb fragment and any 2 kb fragment, which fragments hybridize with the labeled probe derived from DNA fragment PDI, (v) separating any digested fragments, (vi) transferring the separated fragments of (v) to a solid support, (vii) hybridizing the supported separated fragments with a labeled probe derived from the clone DNA fragment PD 1, (viii) determining fragments having undergone loss of the 2 kb band identified by the probe, as an identification of parent mutants in which the loss occurred, and (ix) evaluating the mutating ability of the potential mutagenic agent.« less

Novel strategies to construct complex synthetic vectors to produce DNA molecular weight standards.

PubMed

Chen, Zhe; Wu, Jianbing; Li, Xiaojuan; Ye, Chunjiang; Wenxing, He

2009-05-01

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies-sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and "small fragment accumulation"-for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field.

Molecular cloning of a gene encoding translation initiation factor (TIF) from Candida albicans.

PubMed

Mirbod, F; Nakashima, S; Kitajima, Y; Ghannoum, M A; Cannon, R D; Nozawa, Y

1996-01-01

The differential display technique was applied to compare mRNAs from two clinical isolates of Candida albicans with different virulence; high (potent strain, 16240) and low (weak strain, 18084) extracellular phospholipase activities. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. A complementary DNA fragment seen distinctly in the potent phospholipase producing strain was highly homologous to the yeast translation initiation factor (TIF). The selected DNA fragment was then used as a probe to isolate its corresponding complementary DNA clone from a library of C. albicans genomic DNA. The sequence of isolated gene revealed an open reading frame of 1194 nucleotides with the potential to encode a protein of 397 amino acids with a predicted molecular weight of 43 kDa. Over its entire length, the amino acid sequence showed strong homology (78-89%) to Saccharomyces cerevisiae TIF and (63-80%) to mouse eIF-4A proteins. Therefore, our C. albicans gene was identified to be TIF (Ca TIF). Northern blot analysis in the two strains of C. albicans revealed that Ca TIF expression is 1.5-fold higher in the potent phospholipase producing strain. The restriction endonuclease digestion of genomic DNA from this potent strain revealed at least two hybridized bands in Southern blot analysis, suggesting two or more closely related sequences in the C. albicans genome.

Hypervariability of ribosomal DNA at multiple chromosomal sites in lake trout (Salvelinus namaycush).

PubMed

Zhuo, L; Reed, K M; Phillips, R B

1995-06-01

Variation in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) of lake trout (Salvelinus namaycush) was examined. Digestion of genomic DNA with restriction enzymes showed that almost every individual had a unique combination of length variants with most of this variation occurring within rather than between populations. Sequence analysis of a 2.3 kilobase (kb) EcoRI-DraI fragment spanning the 3' end of the 28S coding region and approximately 1.8 kb of the IGS revealed two blocks of repetitive DNA. Putative transcriptional termination sites were found approximately 220 bases (b) downstream from the end of the 28S coding region. Comparison of the 2.3-kb fragments with two longer (3.1 kb) fragments showed that the major difference in length resulted from variation in the number of short (89 b) repeats located 3' to the putative terminator. Repeat units within a single nucleolus organizer region (NOR) appeared relatively homogeneous and genetic analysis found variants to be stably inherited. A comparison of the number of spacer-length variants with the number of NORs found that the number of length variants per individual was always less than the number of NORs. Examination of spacer variants in five populations showed that populations with more NORs had more spacer variants, indicating that variants are present at different rDNA sites on nonhomologous chromosomes.

Sizing of single fluorescently stained DNA fragments by scanning microscopy

PubMed Central

Laib, Stephan; Rankl, Michael; Ruckstuhl, Thomas; Seeger, Stefan

2003-01-01

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates. PMID:14602931

Characterization of transformation related genes in oral cancer cells.

PubMed

Chang, D D; Park, N H; Denny, C T; Nelson, S F; Pe, M

1998-04-16

A cDNA representational difference analysis (cDNA-RDA) and an arrayed filter technique were used to characterize transformation-related genes in oral cancer. From an initial comparison of normal oral epithelial cells and a human papilloma virus (HPV)-immortalized oral epithelial cell line, we obtained 384 differentially expressed gene fragments and arrayed them on a filter. Two hundred and twelve redundant clones were identified by three rounds of back hybridization. Sequence analysis of the remaining clones revealed 99 unique clones corresponding to 69 genes. The expression of these transformation related gene fragments in three nontumorigenic HPV-immortalized oral epithelial cell lines and three oral cancer cell lines were simultaneously monitored using a cDNA array hybridization. Although there was a considerable cell line-to-cell line variability in the expression of these clones, a reliable prediction of their expression could be made from the cDNA array hybridization. Our study demonstrates the utility of combining cDNA-RDA and arrayed filters in high-throughput gene expression difference analysis. The differentially expressed genes identified in this study should be informative in studying oral epithelial cell carcinogenesis.

Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

NASA Astrophysics Data System (ADS)

Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

2015-07-01

Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

DNA fragmentation status in patients with necrozoospermia.

PubMed

Brahem, Sonia; Jellad, Sonia; Ibala, Samira; Saad, Ali; Mehdi, Meriem

2012-12-01

The aim of this study was to determine if a relationship exists between the levels of sperm DNA fragmentation and necrospermia in infertile men. Semen samples obtained from 70 men consulting for infertility evaluation were analyzed according to World Health Organization (WHO) guidelines. Patients were subdivided into three groups according to the percentage of necrotic spermatozoa: normozoospermia (<30%; n = 20), moderate necrozoospermia (50-80%; n = 30), and severe necrozoospermia (>80%; n = 20). DNA fragmentation was detected by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) assay. The sperm DNA fragmentation index (DFI) was 9.28 ± 2.98% in patients with a normal level of necrotic spermatozoa, 20.25 ± 3.21% in patients with moderate necrozoospermia, and 35.31 ± 5.25% in patients with severe necrozoospermia. There was a statistically significant increase of DNA fragmentation in the necrozoospermic group (P < 0.01). A strong correlation was found between the degree of necrozoospermia and sperm DNA fragmentation. We concluded that patients with necrozoospermia showed a high level of DNA fragmentation compared to normozoospermic men. Severe necrozoospermia (>80%) is a predictive factor for increased sperm DNA damage.

Molecular Identification of Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) Markers.

PubMed

Al-Khalifah, Nasser S; Shanavaskhan, A E

2017-01-01

Ambiguity in the total number of date palm cultivars across the world is pointing toward the necessity for an enumerative study using standard morphological and molecular markers. Among molecular markers, DNA markers are more suitable and ubiquitous to most applications. They are highly polymorphic in nature, frequently occurring in genomes, easy to access, and highly reproducible. Various molecular markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) markers have been successfully used as efficient tools for analysis of genetic variation in date palm. This chapter explains a stepwise protocol for extracting total genomic DNA from date palm leaves. A user-friendly protocol for RAPD analysis and a table showing the primers used in different molecular techniques that produce polymorphisms in date palm are also provided.

Detection of restriction fragment length polymorphisms in clinical isolates and serially passaged Pseudomonas aeruginosa strains.

PubMed Central

Hjelm, L N; Branstrom, A A; Warren, R L

1990-01-01

An 800-base-pair HindIII-PstI fragment that flanks a hot spot for Tn7 insertion was isolated from the chromosome of Pseudomonas aeruginosa and cloned into pUC12. The fragment was used to probe XhoI digests of genomic DNA from 18 P. aeruginosa isolates collected from sputum samples of seven cystic fibrosis patients. Only two XhoI restriction fragment length polymorphisms (RFLPs), of 3.7 and 7.7 kilobases (kb), were detected. Isolate WSU3531-1 (3.7-kb XhoI fragment) and WSU3860 (7.7-kb XhoI fragment), while isolated from the same patient, showed different RFLPs. Serial passages of isolate WSU3531-1 demonstrated that this strain was phenotypically stable. In contrast, colony and pigment variants were readily isolated at a frequency of 1% from serial passages of isolate WSU3860. When XhoI-digested genomic DNA from phenotypic variants of serially passaged WSU3860 were probed with the 800-base-pair HindIII-PstI fragment, the probe hybridized to a 10.4-kb XhoI fragment from three isolates. Restriction analysis of the genomic DNA digested with a variety of restriction enzymes showed that a 2.7-kb insertion occurred in the same region for all three isolates. There appeared to be no correlation between changes in the RFLP and changes in colony morphology. Images PMID:1977762

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

PubMed Central

Malc, Ewa P.; Jayakody, Chatura N.; Tsuruta, James K.; Mieczkowski, Piotr A.; Janzen, William P.; Dayton, Paul A.

2015-01-01

A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation. PMID:26186461

Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean.

PubMed

Zhang, Haibo; Yang, Litao; Guo, Jinchao; Li, Xiang; Jiang, Lingxi; Zhang, Dabing

2008-07-23

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.

Evidence for glucocorticoid receptor binding to a site(s) in a remote region of the 5' flanking sequences of the human proopiomelanocortin gene

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

Tully, D.B.; Hillman, D.; Herbert, E.

1986-05-01

Glucocorticoids negatively regulate expression of the human proopiomelanocortin (POMC) gene. It has been postulated that this effect may be modulated by a direct interaction of the glucocorticoid receptor (GR) with DNA in the vicinity of the POMC promoter. In order to investigate interactions of GR with POMC DNA, DNA-cellulose competitive binding assays have been performed using isolated fragments of cloned POMC DNA to compete with calf thymus DNA-cellulose for binding of triamcinolone acetonide affinity-labelled GR prepared from HeLa S/sub 3/ cells. In these assays, two fragments isolated from the 5' flanking sequences of POMC DNA (Fragment 3,-1765 to -677 andmore » Fragment 4, -676 to +125 with respect to the mRNA cap site) have competed favorably, with Fragment 3 consistently competing more strongly than Fragment 4. Additional studies have been conducted utilizing a newly developed South-western Blot procedure in which specific /sup 32/P-labelled DNA fragments are allowed to bind to dexamethasone mesylate labelled GR immobilized on nitrocellulose filters. Results from these studies have also shown preferential binding by POMC DNA fragments 3 and 4. DNA footprinting and gene transfer experiments are now being conducted to further characterize the nature of GR interaction with POMC DNA.« less

A new model for ancient DNA decay based on paleogenomic meta-analysis

PubMed Central

Ware, Roselyn; Smith, Oliver; Collins, Matthew

2017-01-01

Abstract The persistence of DNA over archaeological and paleontological timescales in diverse environments has led to a revolutionary body of paleogenomic research, yet the dynamics of DNA degradation are still poorly understood. We analyzed 185 paleogenomic datasets and compared DNA survival with environmental variables and sample ages. We find cytosine deamination follows a conventional thermal age model, but we find no correlation between DNA fragmentation and sample age over the timespans analyzed, even when controlling for environmental variables. We propose a model for ancient DNA decay wherein fragmentation rapidly reaches a threshold, then subsequently slows. The observed loss of DNA over time may be due to a bulk diffusion process in many cases, highlighting the importance of tissues and environments creating effectively closed systems for DNA preservation. This model of DNA degradation is largely based on mammal bone samples due to published genomic dataset availability. Continued refinement to the model to reflect diverse biological systems and tissue types will further improve our understanding of ancient DNA breakdown dynamics. PMID:28486705

Mass Spectrometry Based Ultrasensitive DNA Methylation Profiling Using Target Fragmentation Assay.

PubMed

Lin, Xiang-Cheng; Zhang, Ting; Liu, Lan; Tang, Hao; Yu, Ru-Qin; Jiang, Jian-Hui

2016-01-19

Efficient tools for profiling DNA methylation in specific genes are essential for epigenetics and clinical diagnostics. Current DNA methylation profiling techniques have been limited by inconvenient implementation, requirements of specific reagents, and inferior accuracy in quantifying methylation degree. We develop a novel mass spectrometry method, target fragmentation assay (TFA), which enable to profile methylation in specific sequences. This method combines selective capture of DNA target from restricted cleavage of genomic DNA using magnetic separation with MS detection of the nonenzymatic hydrolysates of target DNA. This method is shown to be highly sensitive with a detection limit as low as 0.056 amol, allowing direct profiling of methylation using genome DNA without preamplification. Moreover, this method offers a unique advantage in accurately determining DNA methylation level. The clinical applicability was demonstrated by DNA methylation analysis using prostate tissue samples, implying the potential of this method as a useful tool for DNA methylation profiling in early detection of related diseases.

Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

PubMed Central

Jarausch, W; Saillard, C; Dosba, F; Bové, J M

1994-01-01

A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle. Images PMID:7916180

Non-random distribution of DNA double-strand breaks induced by particle irradiation

NASA Technical Reports Server (NTRS)

Lobrich, M.; Cooper, P. K.; Rydberg, B.; Chatterjee, A. (Principal Investigator)

1996-01-01

Induction of DNA double-strand breaks (dsbs) in mammalian cells is dependent on the spatial distribution of energy deposition from the ionizing radiation. For high LET particle radiations the primary ionization sites occur in a correlated manner along the track of the particles, while for X-rays these sites are much more randomly distributed throughout the volume of the cell. It can therefore be expected that the distribution of dsbs linearly along the DNA molecule also varies with the type of radiation and the ionization density. Using pulsed-field gel and conventional gel techniques, we measured the size distribution of DNA molecules from irradiated human fibroblasts in the total range of 0.1 kbp-10 Mbp for X-rays and high LET particles (N ions, 97 keV/microns and Fe ions, 150 keV/microns). On a mega base pair scale we applied conventional pulsed-field gel electrophoresis techniques such as measurement of the fraction of DNA released from the well (FAR) and measurement of breakage within a specific NotI restriction fragment (hybridization assay). The induction rate for widely spaced breaks was found to decrease with LET. However, when the entire distribution of radiation-induced fragments was analysed, we detected an excess of fragments with sizes below about 200 kbp for the particles compared with X-irradiation. X-rays are thus more effective than high LET radiations in producing large DNA fragments but less effective in the production of smaller fragments. We determined the total induction rate of dsbs for the three radiations based on a quantitative analysis of all the measured radiation-induced fragments and found that the high LET particles were more efficient than X-rays at inducing dsbs, indicating an increasing total efficiency with LET. Conventional assays that are based only on the measurement of large fragments are therefore misleading when determining total dsb induction rates of high LET particles. The possible biological significance of this non-randomness for dsb induction is discussed.

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of swine-specific DNA markers for biosensor-based halal authentication.

PubMed

Ali, M E; Hashim, U; Kashif, M; Mustafa, S; Che Man, Y B; Abd Hamid, S B

2012-06-29

The pig (Sus scrofa) mitochondrial genome was targeted to design short (15-30 nucleotides) DNA markers that would be suitable for biosensor-based hybridization detection of target DNA. Short DNA markers are reported to survive harsh conditions in which longer ones are degraded into smaller fragments. The whole swine mitochondrial-genome was in silico digested with AluI restriction enzyme. Among 66 AluI fragments, five were selected as potential markers because of their convenient lengths, high degree of interspecies polymorphism and intraspecies conservatism. These were confirmed by NCBI blast analysis and ClustalW alignment analysis with 11 different meat-providing animal and fish species. Finally, we integrated a tetramethyl rhodamine-labeled 18-nucleotide AluI fragment into a 3-nm diameter citrate-tannate coated gold nanoparticle to develop a swine-specific hybrid nanobioprobe for the determination of pork adulteration in 2.5-h autoclaved pork-beef binary mixtures. This hybrid probe detected as low as 1% pork in deliberately contaminated autoclaved pork-beef binary mixtures and no cross-species detection was recorded, demonstrating the feasibility of this type of probe for biosensor-based detection of pork adulteration of halal and kosher foods.

Cloning of developmentally regulated flagellin genes from Caulobacter crescentus via immunoprecipitation of polyribosomes.

PubMed Central

Milhausen, M; Gill, P R; Parker, G; Agabian, N

1982-01-01

Immunoprecipitation of Caulobacter crescentus polyribosomes with antiflagellin antibody provided RNA for the synthesis of cDNA probes that were used to identify three specific EcoRI restriction fragments (6.8, 10, and 22 kilobases) in genomic digests of Caulobacter DNA. The RNA was present only in polyribosomes isolated from a time interval in the Caulobacter cell cycle that was coincident with flagellin polypeptide synthesis. The structural gene for Mr 27,500 flagellin polypeptide was assigned to a region of the 10-kilobase EcoRI restriction fragment by DNA sequence analysis. Analysis of mutants defective in motility further established a correlation between the Mr 27,500 flagellin gene and the flaE gene locus [Johnson, R. C. & Ely, B. (1979) J. Bacteriol. 137, 627-634]. The other EcoRI fragments that hybridize with the immunoprecipitated polyribosome-derived cDNA probe are also temporally regulated and have features that suggest they encode other polypeptides associated with the flagellum. Modifications were required to adapt the procedure of immunoprecipitation of polyribosomes for use with Caulobacter and should be applicable to the production of specific structural gene probes from other prokaryotic systems. Images PMID:6294658

Isolation and bioinformatics analysis of differentially methylated genomic fragments in human gastric cancer

PubMed Central

Liao, Ai-Jun; Su, Qi; Wang, Xun; Zeng, Bin; Shi, Wei

2008-01-01

AIM: To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa. METHODS: The differentially methylated DNA sequences between gastric cancer and normal gastric mucosa were isolated by methylation-sensitive representational difference analysis (MS-RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with Basic Local Alignment Search Tool (BLAST). RESULTS: Three differentially methylated DNA sequences were obtained, two of which have been accepted by GenBank. The accession numbers are AY887106 and AY887107. AY887107 was highly similar to the 11th exon of LOC440683 (98%), 3’ end of LOC440887 (99%), and promoter and exon regions of DRD5 (94%). AY887106 was consistent (98%) with a CpG island in ribosomal RNA isolated from colorectal cancer by Minoru Toyota in 1999. CONCLUSION: The methylation degree is different between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences can be isolated effectively by MS-RDA. PMID:18322944

Prophagic DNA Fragments in Streptococcus agalactiae Strains and Association with Neonatal Meningitis

PubMed Central

van der Mee-Marquet, Nathalie; Domelier, Anne-Sophie; Mereghetti, Laurent; Lanotte, Philippe; Rosenau, Agnès; van Leeuwen, Willem; Quentin, Roland

2006-01-01

We identified—by randomly amplified polymorphic DNA (RAPD) analysis at the population level followed by DNA differential display, cloning, and sequencing—three prophage DNA fragments (F5, F7, and F10) in Streptococcus agalactiae that displayed significant sequence similarity to the DNA of S. agalactiae and Streptococcus pyogenes. The F5 sequence aligned with a prophagic gene encoding the large subunit of a terminase, F7 aligned with a phage-associated cell wall hydrolase and a phage-associated lysin, and F10 aligned with a transcriptional regulator (ArpU family) and a phage-associated endonuclease. We first determined the prevalence of F5, F7, and F10 by PCR in a collection of 109 strains isolated in the 1980s and divided into two populations: one with a high risk of causing meningitis (HR group) and the other with a lower risk of causing meningitis (LR group). These fragments were significantly more prevalent in the HR group than in the LR group (P < 0.001). Our findings suggest that lysogeny has increased the ability of some S. agalactiae strains to invade the neonatal brain endothelium. We then determined the prevalence of F5, F7, and F10 by PCR in a collection of 40 strains recently isolated from neonatal meningitis cases for comparison with the cerebrospinal fluid (CSF) strains isolated in the 1980s. The prevalence of the three prophage DNA fragments was similar in these two populations isolated 15 years apart. We suggest that the prophage DNA fragments identified have remained stable in many CSF S. agalactiae strains, possibly due to their importance in virulence or fitness. PMID:16517893

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

PubMed

Jorgez, Carolina J; Bischoff, Farideh Z

2009-01-01

Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

Improved Method for Direct Detection of Environmental Microorganisms Using an Amplification of 16S rDNA Region

NASA Astrophysics Data System (ADS)

Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.

2004-12-01

The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection of microorganisms with important genes from more other environments.

An accurate algorithm for the detection of DNA fragments from dilution pool sequencing experiments.

PubMed

Bansal, Vikas

2018-01-01

The short read lengths of current high-throughput sequencing technologies limit the ability to recover long-range haplotype information. Dilution pool methods for preparing DNA sequencing libraries from high molecular weight DNA fragments enable the recovery of long DNA fragments from short sequence reads. These approaches require computational methods for identifying the DNA fragments using aligned sequence reads and assembling the fragments into long haplotypes. Although a number of computational methods have been developed for haplotype assembly, the problem of identifying DNA fragments from dilution pool sequence data has not received much attention. We formulate the problem of detecting DNA fragments from dilution pool sequencing experiments as a genome segmentation problem and develop an algorithm that uses dynamic programming to optimize a likelihood function derived from a generative model for the sequence reads. This algorithm uses an iterative approach to automatically infer the mean background read depth and the number of fragments in each pool. Using simulated data, we demonstrate that our method, FragmentCut, has 25-30% greater sensitivity compared with an HMM based method for fragment detection and can also detect overlapping fragments. On a whole-genome human fosmid pool dataset, the haplotypes assembled using the fragments identified by FragmentCut had greater N50 length, 16.2% lower switch error rate and 35.8% lower mismatch error rate compared with two existing methods. We further demonstrate the greater accuracy of our method using two additional dilution pool datasets. FragmentCut is available from https://bansal-lab.github.io/software/FragmentCut. vibansal@ucsd.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives.

PubMed

Fregene, M A; Vargas, J; Ikea, J; Angel, F; Tohme, J; Asiedu, R A; Akoroda, M O; Roca, W M

1994-11-01

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.

Apoptosis Process in Mouse Leydig Cells during Postnatal Development

NASA Astrophysics Data System (ADS)

Salles Faria, Maria José; Simões, Zilá Paulino; Luz; Orive Lunardi, Laurelucia; Hartfelder, Klaus

2003-02-01

The development of Leydig cells in mammals has been widely described as a biphasic pattern with two temporally mature Leydig cell populations, fetal stage followed by the adult generation beginning at puberty. In the present study, mouse Leydig cells were examined for apoptosis during postnatal testis development using electron microscopy and in situ DNA fragmentation by terminal deoxynucleotidyl transferase staining (TdT). Both the morphological study and the DNA fragmentation analysis showed that cellular death by apoptosis did not occur in Leydig cells during the neonatal, prepubertal, puberty, and adult periods. From these results, we suggest that the remaining fetal Leydig cells in the neonatal testis are associated with the involution or degeneration processes. In contrast, in the prepubertal and puberty stages, fragmentation of apoptotic DNA was detected in germ cells present in some seminiferous tubules.

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

PubMed

Abe, T; Takano, H; Sasaki, N; Mori, K; Kawano, S

2000-02-01

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Cytogenetic and molecular aspects of absolute teratozoospermia: comparison between polymorphic and monomorphic forms.

PubMed

Brahem, Sonia; Elghezal, Hatem; Ghédir, Houda; Landolsi, Hanène; Amara, Abdelbacett; Ibala, Samira; Gribaa, Moez; Saad, Ali; Mehdi, Meriem

2011-12-01

To compare the results of cytogenetic and molecular analysis between absolute polymorphic and monomorphic teratozoospermia. The semen samples from patients with polymorphic teratozoospermia (n = 20), globozoospermia (n = 8), or macrocephalic sperm head syndrome (n = 12), and healthy fertile men (n = 20) were analyzed according to the World Health Organization criteria. The constitutional blood karyotype of the patients was performed on cultured lymphocytes, according to standard techniques. Microdeletion analysis of the Y chromosomes used a sequence tagged site-polymerase chain reaction technique. Triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18 were used to analyze the meiotic segregation. DNA fragmentation was detected using the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling assay. Whatever the type of teratozoospermia, a normal karyotype and an absence of Y chromosome microdeletion were shown for all patients. A significant increase in the sperm aneuploidy rate and DNA fragmentation were shown, regardless of the type of teratozoospermia. Spermatozoa of the patients with globozoospermia carry an abnormal chromosomal constitution and DNA damage rate with the same frequency as that found in the sperm of patients with absolute polymorphic teratozoospermia. However, a greater sperm aneuploidy rate and DNA fragmentation were found in patients whose teratozoospermia was mainly characterized by increased rates of spermatozoa with macrocephalic head and multiple flagella. Our data have demonstrated that DNA fragmentation and sperm aneuploidy are critical tests in teratozoospermic men, because the results could negatively affect the intracytoplasmic sperm injection outcomes and might play an important role in the counseling of couples considering intracytoplasmic sperm injection. Copyright © 2011 Elsevier Inc. All rights reserved.

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy

PubMed Central

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog

2016-01-01

Abstract Aims: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. Results: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. Innovation: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. Conclusion: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072–1083. PMID:26935406

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

PubMed

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog; Jurkunas, Ula V

2016-06-20

Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein–Barr virus ZEBRA protein

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

Morand, Patrice; Laboratoire de Virologie Moléculaire et Structurale, EA 2939, Université Joseph Fourier, Grenoble; Budayova-Spano, Monika

A C-terminal fragment of the Epstein–Barr virus lytic switch protein ZEBRA has been crystallized in complex with DNA. A C-terminal fragment of the Epstein–Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 Å resolution using synchrotron radiationmore » (λ = 0.976 Å). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 Å, β = 103.9°.« less

Multiplex pyrosequencing of InDel markers for forensic DNA analysis.

PubMed

Bus, Magdalena M; Karas, Ognjen; Allen, Marie

2016-12-01

The capillary electrophoresis (CE) technology is commonly used for fragment length separation of markers in forensic DNA analysis. In this study, pyrosequencing technology was used as an alternative and rapid tool for the analysis of biallelic InDel (insertion/deletion) markers for individual identification. The DNA typing is based on a subset of the InDel markers that are included in the Investigator ® DIPplex Kit, which are sequenced in a multiplex pyrosequencing analysis. To facilitate the analysis of degraded DNA, the polymerase chain reaction (PCR) fragments were kept short in the primer design. Samples from individuals of Swedish origin were genotyped using the pyrosequencing strategy and analysis of the Investigator ® DIPplex markers with CE. A comparison between the pyrosequencing and CE data revealed concordant results demonstrating a robust and correct genotyping by pyrosequencing. Using optimal marker combination and a directed dispensation strategy, five markers could be multiplexed and analyzed simultaneously. In this proof-of-principle study, we demonstrate that multiplex InDel pyrosequencing analysis is possible. However, further studies on degraded samples, lower DNA quantities, and mixtures will be required to fully optimize InDel analysis by pyrosequencing for forensic applications. Overall, although CE analysis is implemented in most forensic laboratories, multiplex InDel pyrosequencing offers a cost-effective alternative for some applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

PubMed

Mazzanti, Morena; Alessandrini, Federica; Tagliabracci, Adriano; Wells, Jeffrey D; Campobasso, Carlo P

2010-02-25

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments. 2009 Elsevier Ireland Ltd. All rights reserved.

Linkage map of the fragments of herpesvirus papio DNA.

PubMed Central

Lee, Y S; Tanaka, A; Lau, R Y; Nonoyama, M; Rabin, H

1981-01-01

Herpesvirus papio (HVP), an Epstein-Barr-like virus, causes lymphoblastoid disease in baboons. The physical map of HVP DNA was constructed for the fragments produced by cleavage of HVP DNA with restriction endonucleases EcoRI, HindIII, SalI, and PvuI, which produced 12, 12, 10, and 4 fragments, respectively. The total molecular size of HVP DNA was calculated as close to 110 megadaltons. The following methods were used for construction of the map; (i) fragments near the ends of HVP DNA were identified by treating viral DNA with lambda exonuclease before restriction enzyme digestion; (ii) fragments containing nucleotide sequences in common with fragments from the second enzyme digest of HVP DNA were examined by Southern blot hybridization; and (iii) the location of some fragments was determined by isolating individual fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. Terminal heterogeneity and internal repeats were found to be unique features of HVP DNA molecule. One to five repeats of 0.8 megadaltons were found at both terminal ends. Although the repeats of both ends shared a certain degree of homology, it was not determined whether they were identical repeats. The internal repeat sequence of HVP DNA was found in the EcoRI-C region, which extended from 8.4 to 23 megadaltons from the left end of the molecule. The average number of the repeats was calculated to be seven, and the molecular size was determined to be 1.8 megadaltons. Similar unique features have been reported in EBV DNA (D. Given and E. Kieff, J. Virol. 28:524-542, 1978). Images PMID:6261015

Supramolecular gel electrophoresis of large DNA fragments.

PubMed

Tazawa, Shohei; Kobayashi, Kazuhiro; Oyoshi, Takanori; Yamanaka, Masamichi

2017-10-01

Pulsed-field gel electrophoresis is a frequent technique used to separate exceptionally large DNA fragments. In a typical continuous field electrophoresis, it is challenging to separate DNA fragments larger than 20 kbp because they migrate at a comparable rate. To overcome this challenge, it is necessary to develop a novel matrix for the electrophoresis. Here, we describe the electrophoresis of large DNA fragments up to 166 kbp using a supramolecular gel matrix and a typical continuous field electrophoresis system. C 3 -symmetric tris-urea self-assembled into a supramolecular hydrogel in tris-boric acid-EDTA buffer, a typical buffer for DNA electrophoresis, and the supramolecular hydrogel was used as a matrix for electrophoresis to separate large DNA fragments. Three types of DNA marker, the λ-Hind III digest (2 to 23 kbp), Lambda DNA-Mono Cut Mix (10 to 49 kbp), and Marker 7 GT (10 to 165 kbp), were analyzed in this study. Large DNA fragments of greater than 100 kbp showed distinct mobility using a typical continuous field electrophoresis system. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A fluorescent aptasensor for sensitive analysis oxytetracycline based on silver nanoclusters.

PubMed

Hosseini, Morteza; Mehrabi, Fatemeh; Ganjali, Mohammad Reza; Norouzi, Parviz

2016-11-01

A fluorescent aptasensor for detection of oxytetracycline (OTC) was presented based on fluorescence quenching of DNA aptamer-templated silver nanoclusters (AgNCs). The specific DNA scaffolds with two different nucleotides fragments were used: one was enriched with a cytosine sequence fragment (C12) that could produce DNA-AgNCs via a chemical reduction method, and another was the OTC aptamer fragment that could selectively bind to the OTC antibiotic. Thus, the as-prepared AgNCs could exhibit quenched fluorescence after binding to the target OTC. The fluorescence ratio of the DNA-AgNCs was quenched in a linearly proportional manner to the concentration of the target in the range of 0.5 nM to 100 nM with a detection limit of 0.1 nM. This proposed nanobiosensor was demonstrated to be sensitive, selective, and simple, introducing a viable alternative for rapid determination of toxin OTC in honey and water samples. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

PubMed

Zeng, Fanli; Zang, Jinping; Zhang, Suhua; Hao, Zhimin; Dong, Jingao; Lin, Yibin

2017-11-14

Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

PubMed Central

Takita, Eiji; Kohda, Katsunori; Tomatsu, Hajime; Hanano, Shigeru; Moriya, Kanami; Hosouchi, Tsutomu; Sakurai, Nozomu; Suzuki, Hideyuki; Shinmyo, Atsuhiko; Shibata, Daisuke

2013-01-01

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. PMID:23897972

Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal

PubMed Central

Skoglund, Pontus; Northoff, Bernd H.; Shunkov, Michael V.; Derevianko, Anatoli P.; Pääbo, Svante; Krause, Johannes; Jakobsson, Mattias

2014-01-01

One of the main impediments for obtaining DNA sequences from ancient human skeletons is the presence of contaminating modern human DNA molecules in many fossil samples and laboratory reagents. However, DNA fragments isolated from ancient specimens show a characteristic DNA damage pattern caused by miscoding lesions that differs from present day DNA sequences. Here, we develop a framework for evaluating the likelihood of a sequence originating from a model with postmortem degradation—summarized in a postmortem degradation score—which allows the identification of DNA fragments that are unlikely to originate from present day sources. We apply this approach to a contaminated Neandertal specimen from Okladnikov Cave in Siberia to isolate its endogenous DNA from modern human contaminants and show that the reconstructed mitochondrial genome sequence is more closely related to the variation of Western Neandertals than what was discernible from previous analyses. Our method opens up the potential for genomic analysis of contaminated fossil material. PMID:24469802

Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations.

PubMed

Korolev, Nikolay; Yu, Hang; Lyubartsev, Alexander P; Nordenskiöld, Lars

2014-10-01

The positively charged N-terminal histone tails play a crucial role in chromatin compaction and are important modulators of DNA transcription, recombination, and repair. The detailed mechanism of the interaction of histone tails with DNA remains elusive. To model the unspecific interaction of histone tails with DNA, all-atom molecular dynamics (MD) simulations were carried out for systems of four DNA 22-mers in the presence of 20 or 16 short fragments of the H4 histone tail (variations of the 16-23 a. a. KRHRKVLR sequence, as well as the unmodified fragment a. a.13-20, GGAKRHRK). This setup with high DNA concentration, explicit presence of DNA-DNA contacts, presence of unstructured cationic peptides (histone tails) and K(+) mimics the conditions of eukaryotic chromatin. A detailed account of the DNA interactions with the histone tail fragments, K(+) and water is presented. Furthermore, DNA structure and dynamics and its interplay with the histone tail fragments binding are analysed. The charged side chains of the lysines and arginines play major roles in the tail-mediated DNA-DNA attraction by forming bridges and by coordinating to the phosphate groups and to the electronegative sites in the minor groove. Binding of all species to DNA is dynamic. The structure of the unmodified fully-charged H4 16-23 a.a. fragment KRHRKVLR is dominated by a stretched conformation. The H4 tail a. a. fragment GGAKRHRK as well as the H4 Lys16 acetylated fragment are highly flexible. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction and dynamics. © 2014 Wiley Periodicals, Inc.

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.

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.

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.

DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

PubMed

Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

2015-03-01

DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

Crystal structure of an Okazaki fragment at 2-A resolution

NASA Technical Reports Server (NTRS)

Egli, M.; Usman, N.; Zhang, S. G.; Rich, A.

1992-01-01

In DNA replication, Okazaki fragments are formed as double-stranded intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type conformation at the junction between RNA.DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.

Apoptosis in human unfertilized oocytes after intracytoplasmic sperm injection.

PubMed

Bosco, Liana; Ruvolo, Giovanni; Morici, Giovanni; Manno, Maurizio; Cittadini, Ettore; Roccheri, Maria C

2005-11-01

To investigate the presence of programmed cell death in unfertilized oocytes after intracytoplasmic sperm injection (ICSI), assuming that previous apoptotic events could be correlated with the fertilization failure. Comparison of the rate of DNA fragmentation in human oocytes at different stages of maturation soon after pick-up (control) and in unfertilized oocytes after ICSI treatment. In vitro fertilization (IVF) laboratory with extensive ICSI experience. Sixty-three patients undergoing assisted fertilization by ICSI. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay and anticaspase-3 cleaved immunoassay to detect apoptosis in control and ICSI-treated oocytes. Differences in the percentage of oocytes demonstrating DNA fragmentation between control oocytes and unfertilized ICSI treated oocytes at different stages of maturation. The DNA fragmentation, by TUNEL assay, appeared in all the immature control oocytes, but only 37% of mature oocytes showed DNA fragmentation. This DNA fragmentation was observed in 88.8% of the oocytes unfertilized after ICSI; furthermore, DNA fragmentation appeared as well in the sperm injected into the cytoplasm. The study has shown DNA fragmentation in human oocytes unfertilized after ICSI. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. Caspase-3 immunoassay suggests the presence of apoptosis. The high percentage of oocytes demonstrating DNA fragmentation in the unfertilized oocytes could be correlated with fertilization failure.

Bacterial DNA in water and dialysate: detection and significance for patient outcomes.

PubMed

Handelman, Garry J; Megdal, Peter A; Handelman, Samuel K

2009-01-01

The fluid used for hemodialysis may contain DNA fragments from bacteria, which could be harmful for patient outcomes. DNA fragments from bacteria, containing the nonmethylated CpG motif, can trigger inflammation through the monocyte and lymphocyte Toll-like receptor 9, and these DNA fragments have been observed in dialysate. The fragments may transfer across the dialyzer into the patient's bloodstream during hemodialysis treatment. During hemodiafiltration, the fragments would be introduced directly into the bloodstream. The DNA fragments may arise from biofilm in the pipes of the water system, from growth of bacteria in the water, or as contaminants in the bicarbonate and salt mixture used for preparation of dialysate. Current filtration methods, such as Diasafe filters, are not able to remove these fragments. It would be prudent to seek to reduce or eliminate these contaminants. However, the cost and effort of decreasing bacterial DNA content may ultimately require substantial facility improvements; we therefore need to fund research studies to determine if modifications to reduce bacterial DNA content are clinically warranted. This research will require methods to accurately determine the species of bacteria that contribute the DNA, since this information will allow the source to be established as biofilm, bicarbonate mixtures, or other problems in the dialysis system such as bacterial growth or leakage during water preparation. In this review, the evidence for bacterial DNA fragments will be examined and suggestions for further studies will be described.

Separation efficiency of free-solution conjugated electrophoresis with drag-tags incorporating a synthetic amino acid.

PubMed

Seo, Kyung-Ho; Chu, Hun-Su; Yoo, Tae Hyeon; Lee, Sun-Gu; Won, Jong-In

2016-03-01

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge-to-friction ratio in free solution, DNA fragments cannot be separated by size. However, size-based separation of DNA is possible in free-solution conjugate electrophoresis (FSCE) if a "drag-tag" is attached to DNA fragments because the tag breaks the linear charge-to-friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free-solution conjugated electrophoresis, generation of a monodisperse drag-tag and identification of a strong, site-specific conjugation method between a DNA fragment and a drag-tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin-like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site-specific conjugation, a methionine residue in drag-tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

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

Evenson, Donald P.; Wixon, Regina

Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to {approx}1-2 x 106 sperm/ml,more » treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for elevated sperm DNA fragmentation. Extensive DNA fragmentation probably cannot be repaired by the egg and the spontaneous abortion rate is {approx}2x higher if a man has more than 30% of sperm showing DNA fragmentation. DNA fragmentation is an excellent marker for exposure to potential reproductive toxicants and a diagnostic/prognostic tool for potential male infertility.« less

Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization.

PubMed

Mills, D; Russell, B W; Hanus, J W

1997-08-01

ABSTRACT Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

Sperm DNA oxidative damage and DNA adducts

PubMed Central

Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

2015-01-01

The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps = 0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps = 0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps = 0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm. PMID:26653986

RAPD-SCAR marker and genetic relationship analysis of three Demodex species (Acari: Demodicidae).

PubMed

Zhao, Ya-E; Wu, Li-Ping

2012-06-01

For a long time, classification of Demodex mites has been mainly based on their hosts and phenotype characteristics. The study was the first to conduct molecular identification and genetic relationship analysis for six isolates of three Demodex species by random amplified polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) marker. Totally, 239 DNA fragments were amplified from six Demodex isolates with 10 random primers in RAPD, of which 165 were polymorphic. Using a single primer, at least five fragments and at most 40 in the six isolates were amplified, whereas within a single isolate, a range of 35-49 fragments were amplified. DNA fingerprints of primers CZ 1-9 revealed intra- and interspecies difference in six Demodex isolates, whereas primer CZ 10 only revealed interspecies difference. The genetic distance and dendrogram showed the intraspecific genetic distances were closer than the interspecific genetic distances. The interspecific genetic distances of Demodex folliculorum and Demodex canis (0.7931-0.8140) were shorter than that of Demodex brevis and D. canis (0.8182-0.8987). The RAPD-SCAR marker displayed primer CZ 10 could be applied to identify the three Demodex species. The 479-bp fragment was specific for D. brevis, and the 261-bp fragment was specific for D. canis. The conclusion was that the RAPD-SCAR multi-marker was effective in molecular identification of three Demodex species. The genetic relationship between D. folliculorum and D. canis was nearer than that between D. folliculorum and D. brevis.

Microfabricated plastic chips by hot embossing methods and their applications for DNA separation and detection

NASA Astrophysics Data System (ADS)

Lee, Gwo-Bin; Chen, Shu-Hui; Huang, Guan-Ruey; Lin, Yen-Heng; Sung, Wang-Chou

2000-08-01

Design and fabrication of microfluidic devices on polymethylmethacrylate (PMMA) substrates using novel microfabrication methods are described. The image of microfluidic devices is transferred from quartz master templates possessing inverse image of the devices to plastic plates by using hot embossing method. The micro channels on master templates are formed by the combination of metal etch mask and wet chemical etching. The micromachined quartz templates can be used repeatedly to fabricate cheap and disposable plastic devices. The reproducibility of the hot embossing method is evaluated after using 10 channels on different plastics. The relative standard deviation of the plastic channel profile from ones on quartz templates is less than 1%. In this study, the PMMA chips have been demonstrated as a micro capillary electrophoresis ((mu) -CE) device for DNA separation and detection. The capability of the fabricated chip for electrophoretic injection and separation is characterized via the analysis of DNA fragments (phi) X174. Results indicate that all of the 11 DNA fragments of the size marker could be identified in less than 3 minutes with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. Moreover, with the use of near IR dye, fluorescence signals of the higher molecular weight fragments ($GTR 603 bp in length) could be detected at total DNA concentrations as low as 0.1 (mu) g/mL. In addition to DNA fragments (phi) X174, DNA sizing of hepatitis C viral (HCV) amplicon is also achieved using microchip electrophoresis fabricated on PMMA substrate.

DNA sequencing using fluorescence background electroblotting membrane

DOEpatents

Caldwell, Karin D.; Chu, Tun-Jen; Pitt, William G.

1992-01-01

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through said smino groups contained on the surface thereof. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to said target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membrances may be reprobed numerous times.

DNA sequencing using fluorescence background electroblotting membrane

DOEpatents

Caldwell, K.D.; Chu, T.J.; Pitt, W.G.

1992-05-12

A method for the multiplex sequencing on DNA is disclosed which comprises the electroblotting or specific base terminated DNA fragments, which have been resolved by gel electrophoresis, onto the surface of a neutral non-aromatic polymeric microporous membrane exhibiting low background fluorescence which has been surface modified to contain amino groups. Polypropylene membranes are preferably and the introduction of amino groups is accomplished by subjecting the membrane to radio or microwave frequency plasma discharge in the presence of an aminating agent, preferably ammonia. The membrane, containing physically adsorbed DNA fragments on its surface after the electroblotting, is then treated with crosslinking means such as UV radiation or a glutaraldehyde spray to chemically bind the DNA fragments to the membrane through amino groups contained on the surface. The DNA fragments chemically bound to the membrane are subjected to hybridization probing with a tagged probe specific to the sequence of the DNA fragments. The tagging may be by either fluorophores or radioisotopes. The tagged probes hybridized to the target DNA fragments are detected and read by laser induced fluorescence detection or autoradiograms. The use of aminated low fluorescent background membranes allows the use of fluorescent detection and reading even when the available amount of DNA to be sequenced is small. The DNA bound to the membranes may be reprobed numerous times. No Drawings

Sperm DNA damage or progressive motility: which one is the better predictor of fertilization in vitro?

PubMed

Simon, Luke; Lewis, Sheena E M

2011-06-01

Sperm progressive motility has been reported to be one of the key factors influencing in vitro fertilization rates. However, recent studies have shown that sperm DNA fragmentation is a more robust predictor of assisted reproductive outcomes including reduced fertilization rates, embryo quality, and pregnancy rates. This study aimed to compare the usefulness of sperm progressive motility and DNA damage as predictive tools of in vitro fertilization rates. Here, 136 couples provided 1,767 eggs with an overall fertilization rate of 64.2%. The fertilization rate in vitro correlated with both sperm progressive motility (r² = 0.236; P = 0.002) and DNA fragmentation (r² = -0.318; P < 0.001). The relative risk of a poor fertilization rate was 9.5 times higher in sperm of men with high DNA fragmentation (>40%) compared with 2.6 times in sperm with poor motility (<40%). Further, sperm DNA fragmentation gave a higher specificity (93.3%) in predicting the fertilization rate than progressive motility (77.8%). Finally, the odds ratio to determine fertilization rate (>70%) was 4.81 (1.89-12.65) using progressive motility compared with 24.18 (5.21-154.51) using DNA fragmentation. This study shows that fertilization rates are directly dependent upon both sperm progressive motility and DNA fragmentation, but sperm DNA fragmentation is a much stronger test.

A new model for ancient DNA decay based on paleogenomic meta-analysis.

PubMed

Kistler, Logan; Ware, Roselyn; Smith, Oliver; Collins, Matthew; Allaby, Robin G

2017-06-20

The persistence of DNA over archaeological and paleontological timescales in diverse environments has led to a revolutionary body of paleogenomic research, yet the dynamics of DNA degradation are still poorly understood. We analyzed 185 paleogenomic datasets and compared DNA survival with environmental variables and sample ages. We find cytosine deamination follows a conventional thermal age model, but we find no correlation between DNA fragmentation and sample age over the timespans analyzed, even when controlling for environmental variables. We propose a model for ancient DNA decay wherein fragmentation rapidly reaches a threshold, then subsequently slows. The observed loss of DNA over time may be due to a bulk diffusion process in many cases, highlighting the importance of tissues and environments creating effectively closed systems for DNA preservation. This model of DNA degradation is largely based on mammal bone samples due to published genomic dataset availability. Continued refinement to the model to reflect diverse biological systems and tissue types will further improve our understanding of ancient DNA breakdown dynamics. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

FSH treatment in infertile males candidate to assisted reproduction improved sperm DNA fragmentation and pregnancy rate.

PubMed

Garolla, Andrea; Ghezzi, Marco; Cosci, Ilaria; Sartini, Barbara; Bottacin, Alberto; Engl, Bruno; Di Nisio, Andrea; Foresta, Carlo

2017-05-01

The purpose of this study is to evaluate whether follicle-stimulating hormone treatment improves sperm DNA parameters and pregnancy outcome in infertile male candidates to in-vitro fertilization.Observational study in 166 infertile male partners of couples undergoing in-vitro fertilization. Eighty-four patients were receiving follicle-stimulating hormone treatment (cases) and 82 refused treatment (controls). Semen parameters, sexual hormones, and sperm nucleus (fluorescence in-situ hybridization, acridine orange, TUNEL, and γH2AX) were evaluated at baseline (T0) and after 3 months (T1), when all subjects underwent assisted reproduction techniques. Statistical analysis was performed by analysis of variance.Compared to baseline, cases showed significant improvements in seminal parameters and DNA fragmentation indexes after follicle-stimulating hormone therapy (all P < 0.05), whereas no changes were observed in controls. Within cases, follicle-stimulating hormone treatment allowed to perform intrauterine insemination in 35 patients with a pregnancy rate of 23.2 %. Intracytoplasmic sperm injection was performed in all controls and in 49 patients from cases, with pregnancy rates of 23.2 and 40.8 %, respectively (P < 0.05). After 3 months (T0 vs. T1) of follicle-stimulating hormone therapy, cases with positive outcome had reduced DNA fragmentation index and lower double strand breaks (P < 0.05 and P < 0.001 vs. negative outcome, respectively).In this observational study, we showed that follicle-stimulating hormone treatment improves sperm DNA fragmentation, which in turn leads to increased pregnancy rates in infertile males undergoing in-vitro fertilization. In particular, double strand breaks (measured with γH2AX test) emerged as the most sensible parameter to follicle-stimulating hormone treatment in predicting reproductive outcome.

Subacute Low Dose Nerve Agent Exposure Causes DNA Fragmentation in Guinea Pig Leukocytes

DTIC Science & Technology

2005-10-01

1 SUBACUTE LOW DOSE NERVE AGENT EXPOSURE CAUSES DNA FRAGMENTATION IN GUINEA PIG LEUKOCYTES. Jitendra R. Dave1, John R. Moffett1, Sally M...DNA fragmentation in blood leukocytes from guinea pigs by ‘Comet’ assay after exposure to soman at doses ranging from 0.1LD50 to 0.4 LD50, once per...computer. Data obtained for exposure to soman demonstrated significant increases in DNA fragmentation in circulating leukocytes in CWNA treated guinea pigs as

High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System

PubMed Central

Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C. T.; Shui, Lingling

2017-01-01

DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1–10 Kbp fragment lengths with a yield of 75.30–91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future. PMID:28098208

High Efficiency Hydrodynamic DNA Fragmentation in a Bubbling System.

PubMed

Li, Lanhui; Jin, Mingliang; Sun, Chenglong; Wang, Xiaoxue; Xie, Shuting; Zhou, Guofu; van den Berg, Albert; Eijkel, Jan C T; Shui, Lingling

2017-01-18

DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1-10 Kbp fragment lengths with a yield of 75.30-91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.

Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

PubMed

Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

2016-01-01

Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample types that can be processed and minimizes the time between sample collection, sample processing and analysis, and generation of actionable intelligence. The fully integrated Expert System is capable of interpreting a wide range or sample types and input DNA quantities, allowing samples to be processed and interpreted without a technical operator.

Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation.

PubMed

Hao, Yu-Jin; You, Chun-Xiang; Deng, Xui-Xin

2002-01-01

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.

Sliding Window Analyses for Optimal Selection of Mini-Barcodes, and Application to 454-Pyrosequencing for Specimen Identification from Degraded DNA

PubMed Central

Boyer, Stephane; Brown, Samuel D. J.; Collins, Rupert A.; Cruickshank, Robert H.; Lefort, Marie-Caroline; Malumbres-Olarte, Jagoba; Wratten, Stephen D.

2012-01-01

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential ‘mini-barcodes’ for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation. PMID:22666489

RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

PubMed Central

Spiering, Michelle M.; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J.

2017-01-01

The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior—that is, the signaling mechanism—have not been definitively identified. We examined the role of RNA primer–primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer–primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer–primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer–primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes. PMID:28507156

RNA primer-primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication.

PubMed

Spiering, Michelle M; Hanoian, Philip; Gannavaram, Swathi; Benkovic, Stephen J

2017-05-30

The opposite strand polarity of duplex DNA necessitates that the leading strand is replicated continuously whereas the lagging strand is replicated in discrete segments known as Okazaki fragments. The lagging-strand polymerase sometimes recycles to begin the synthesis of a new Okazaki fragment before finishing the previous fragment, creating a gap between the Okazaki fragments. The mechanism and signal that initiate this behavior-that is, the signaling mechanism-have not been definitively identified. We examined the role of RNA primer-primase complexes left on the lagging ssDNA from primer synthesis in initiating early lagging-strand polymerase recycling. We show for the T4 bacteriophage DNA replication system that primer-primase complexes have a residence time similar to the timescale of Okazaki fragment synthesis and the ability to block a holoenzyme synthesizing DNA and stimulate the dissociation of the holoenzyme to trigger polymerase recycling. The collision with primer-primase complexes triggering the early termination of Okazaki fragment synthesis has distinct advantages over those previously proposed because this signal requires no transmission to the lagging-strand polymerase through protein or DNA interactions, the mechanism for rapid dissociation of the holoenzyme is always collision, and no unique characteristics need to be assigned to either identical polymerase in the replisome. We have modeled repeated cycles of Okazaki fragment initiation using a collision with a completed Okazaki fragment or primer-primase complexes as the recycling mechanism. The results reproduce experimental data, providing insights into events related to Okazaki fragment initiation and the overall functioning of DNA replisomes.

DNA Length Modulates the Affinity of Fragments of Genomic DNA for the Nuclear Matrix In Vitro.

PubMed

García-Vilchis, David; Aranda-Anzaldo, Armando

2017-12-01

Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Effects of semen storage and separation techniques on sperm DNA fragmentation.

PubMed

Jackson, Robert E; Bormann, Charles L; Hassun, Pericles A; Rocha, André M; Motta, Eduardo L A; Serafini, Paulo C; Smith, Gary D

2010-12-01

To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Controlled clinical study. An assisted reproductive technology laboratory. Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. DNA fragmentation as measured by SCD. There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

PWHATSHAP: efficient haplotyping for future generation sequencing.

PubMed

Bracciali, Andrea; Aldinucci, Marco; Patterson, Murray; Marschall, Tobias; Pisanti, Nadia; Merelli, Ivan; Torquati, Massimo

2016-09-22

Haplotype phasing is an important problem in the analysis of genomics information. Given a set of DNA fragments of an individual, it consists of determining which one of the possible alleles (alternative forms of a gene) each fragment comes from. Haplotype information is relevant to gene regulation, epigenetics, genome-wide association studies, evolutionary and population studies, and the study of mutations. Haplotyping is currently addressed as an optimisation problem aiming at solutions that minimise, for instance, error correction costs, where costs are a measure of the confidence in the accuracy of the information acquired from DNA sequencing. Solutions have typically an exponential computational complexity. WHATSHAP is a recent optimal approach which moves computational complexity from DNA fragment length to fragment overlap, i.e., coverage, and is hence of particular interest when considering sequencing technology's current trends that are producing longer fragments. Given the potential relevance of efficient haplotyping in several analysis pipelines, we have designed and engineered PWHATSHAP, a parallel, high-performance version of WHATSHAP. PWHATSHAP is embedded in a toolkit developed in Python and supports genomics datasets in standard file formats. Building on WHATSHAP, PWHATSHAP exhibits the same complexity exploring a number of possible solutions which is exponential in the coverage of the dataset. The parallel implementation on multi-core architectures allows for a relevant reduction of the execution time for haplotyping, while the provided results enjoy the same high accuracy as that provided by WHATSHAP, which increases with coverage. Due to its structure and management of the large datasets, the parallelisation of WHATSHAP posed demanding technical challenges, which have been addressed exploiting a high-level parallel programming framework. The result, PWHATSHAP, is a freely available toolkit that improves the efficiency of the analysis of genomics information.

The characterization of four gene expression analysis in circulating tumor cells made by Multiplex-PCR from the AdnaTest kit on the lab-on-a-chip Agilent DNA 1000 platform.

PubMed

Škereňová, Markéta; Mikulová, Veronika; Čapoun, Otakar; Zima, Tomáš

2016-01-01

Nowadays, on-a-chip capillary electrophoresis is a routine method for the detection of PCR fragments. The Agilent 2100 Bioanalyzer was one of the first commercial devices in this field. Our project was designed to study the characteristics of Agilent DNA 1000 kit in PCR fragment analysis as a part of circulating tumour cell (CTC) detection technique. Despite the common use of this kit a complex analysis of the results from a long-term project is still missing. A commercially available Agilent DNA 1000 kit was used as a final step in the CTC detection (AdnaTest) for the determination of the presence of PCR fragments generated by Multiplex PCR. Data from 30 prostate cancer patients obtained during two years of research were analyzed to determine the trueness and precision of the PCR fragment size determination. Additional experiments were performed to demonstrate the precision (repeatability, reproducibility) and robustness of PCR fragment concentration determination. The trueness and precision of the size determination was below 3% and 2% respectively. The repeatability of the concentration determination was below 15%. The difference in concentration determination increases when Multiplex-PCR/storage step is added between the two measurements of one sample. The characteristics established in our study are in concordance with the manufacturer's specifications established for a ladder as a sample. However, the concentration determination may vary depending on chip preparation, sample storage and concentration. The 15% variation of concentration determination repeatability was shown to be partly proportional and can be suppressed by proper normalization.

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites.

PubMed

Balani, Valério Américo; de Lima Neto, Quirino Alves; Takeda, Karen Izumi; Gimenes, Fabrícia; Fiorini, Adriana; Debatisse, Michelle; Fernandez, Maria Aparecida

2010-11-01

The aim of this work was to determine whether intrinsically bent DNA sites are present at, or close to, the mammalian replication origins oriGNAI3 and oriB in the Chinese hamster AMPD2 locus. Using an electrophoretic mobility shift assay and in silico analysis, we located four intrinsically bent DNA sites (b1 to b4) in a fragment that contains the oriGNAI3 and one site (b5) proximal to oriB. The helical parameters show that each bent DNA site is curved in a left-handed superhelical writhe. A 2D projection of 3D fragment trajectories revealed that oriGNAI3 is located in a relatively straight segment flanked by bent sites b1 and b2, which map in previously identified Scaffold/Matrix Attachment Region. Sites b3 and b4 are located approximately 2 kb downstream and force the fragment into a strong closed loop structure. The b5 site is also located in an S/MAR that is found just downstream of oriB.

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

PubMed Central

2012-01-01

Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants. PMID:22883984

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia.

PubMed

Zuiter, Afnan Saeid; Sawwan, Jammal; Al Abdallat, Ayed

2012-08-10

Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.

Essentials of Conservation Biotechnology: A mini review

NASA Astrophysics Data System (ADS)

Merlyn Keziah, S.; Subathra Devi, C.

2017-11-01

Equilibrium of biodiversity is essential for the maintenance of the ecosystem as they are interdependent on each other. The decline in biodiversity is a global problem and an inevitable threat to the mankind. Major threats include unsustainable exploitation, habitat destruction, fragmentation, transformation, genetic pollution, invasive exotic species and degradation. This review covers the management strategies of biotechnology which include sin situ, ex situ conservation, computerized taxonomic analysis through construction of phylogenetic trees, calculating genetic distance, prioritizing the group for conservation, digital preservation of biodiversities within the coding and decoding keys, molecular approaches to asses biodiversity like polymerase chain reaction, real time, randomly amplified polymorphic DNA, restriction fragment length polymorphism, amplified fragment length polymorphism, single sequence repeats, DNA finger printing, single nucleotide polymorphism, cryopreservation and vitrification.

Morphometric comparison by the ISAS® CASA-DNAf system of two techniques for the evaluation of DNA fragmentation in human spermatozoa

PubMed Central

Sadeghi, Sara; García-Molina, Almudena; Celma, Ferran; Valverde, Anthony; Fereidounfar, Sogol; Soler, Carles

2016-01-01

DNA fragmentation has been shown to be one of the causes of male infertility, particularly related to repeated abortions, and different methods have been developed to analyze it. In the present study, two commercial kits based on the SCD technique (Halosperm® and SDFA) were evaluated by the use of the DNA fragmentation module of the ISAS® v1 CASA system. Seven semen samples from volunteers were analyzed. To compare the results between techniques, the Kruskal–Wallis test was used. Data were used for calculation of Principal Components (two PCs were obtained), and subsequent subpopulations were identified using the Halo, Halo/Core Ratio, and PC data. Results from both kits were significantly different (P < 0.001). In each case, four subpopulations were obtained, independently of the classification method used. The distribution of subpopulations differed depending on the kit used. From the PC data, a discriminant analysis matrix was obtained and a good a posteriori classification was obtained (97.1% for Halosperm and 96.6% for SDFA). The present results are the first approach on morphometric evaluation of DNA fragmentation from the SCD technique. This approach could be used for the future definition of a classification matrix surpassing the current subjective evaluation of this important sperm factor. PMID:27678463

Morphometric comparison by the ISAS® CASA-DNAf system of two techniques for the evaluation of DNA fragmentation in human spermatozoa.

PubMed

Sadeghi, Sara; García-Molina, Almudena; Celma, Ferran; Valverde, Anthony; Fereidounfar, Sogol; Soler, Carles

2016-01-01

DNA fragmentation has been shown to be one of the causes of male infertility, particularly related to repeated abortions, and different methods have been developed to analyze it. In the present study, two commercial kits based on the SCD technique (Halosperm ® and SDFA) were evaluated by the use of the DNA fragmentation module of the ISAS ® v1 CASA system. Seven semen samples from volunteers were analyzed. To compare the results between techniques, the Kruskal-Wallis test was used. Data were used for calculation of Principal Components (two PCs were obtained), and subsequent subpopulations were identified using the Halo, Halo/Core Ratio, and PC data. Results from both kits were significantly different (P < 0.001). In each case, four subpopulations were obtained, independently of the classification method used. The distribution of subpopulations differed depending on the kit used. From the PC data, a discriminant analysis matrix was obtained and a good a posteriori classification was obtained (97.1% for Halosperm and 96.6% for SDFA). The present results are the first approach on morphometric evaluation of DNA fragmentation from the SCD technique. This approach could be used for the future definition of a classification matrix surpassing the current subjective evaluation of this important sperm factor.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): taxonomic implications for the Great Lakes species flock.

PubMed

Reed, K M; Dorschner, M O; Todd, T N; Phillips, R B

1998-09-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens of C. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Norrie disease: linkage analysis using a 4.2-kb RFLP detected by a human ornithine aminotransferase cDNA probe.

PubMed

Ngo, J T; Bateman, J B; Cortessis, V; Sparkes, R S; Mohandas, T; Inana, G; Spence, M A

1989-05-01

Previous study has shown that the usual DNA marker for Norrie disease, the L1.28 probe which identifies the DXS7 locus, can recombine with the disease locus. In this study, we used a human ornithine aminotransferase (OAT) cDNA which detects OAT-related DNA sequences mapped to the same region on the X chromosome as that of the L1.28 probe to investigate the family with Norrie disease who exhibited the recombinational event. When genomic DNA from this family was digested with the PvuII restriction endonuclease, we found a restriction fragment length polymorphism (RFLP) of 4.2 kb in size. This fragment was absent in the affected males and cosegregated with the disease locus; we calculated a lod score of 0.602, at theta = 0.00. No deletion could be detected by chromosomal analysis or on Southern blots with other enzymes. These results suggest that one of the OAT-related sequences on the X chromosome may be in close proximity to the Norrie disease locus and represent the first report which indicates that the OAT cDNA may be useful for the identification of carrier status and/or prenatal diagnosis.

A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

PubMed

Thierry, Alain R

2016-01-01

Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring, and follow-up of cancer patients expanding the scope of personalized cancer medicine.

Freeze-dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays.

PubMed

Olaciregui, M; Luño, V; Martí, J I; Aramayona, J; Gil, L

2016-11-01

During the freeze-drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well-established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff-Quik (DQ) with two established procedures: acridine orange test (AOT) and sperm chromatin dispersion (SCD) on freeze-dried (FD) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze-dried in basic medium supplemented with two different chelating agents: EGTA or EDTA. After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt, AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD. The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation (r = 0.528), whereas a negative correlation was observed between SCD, DQ and AOT (r = -0.134 and r = -0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable. © 2016 Blackwell Verlag GmbH.

Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method.

PubMed

Ding, Wenwen; Weng, Huanjiao; Jin, Peng; Du, Guocheng; Chen, Jian; Kang, Zhen

2017-05-04

Efficient assembly of multiple DNA fragments is a pivotal technology for synthetic biology. A scarless and sequence-independent DNA assembly method (DATEL) using thermal exonucleases has been developed recently. Here, we present a simplified DATEL (sDATEL) for efficient assembly of unphosphorylated DNA fragments with low cost. The sDATEL method is only dependent on Taq DNA polymerase and Taq DNA ligase. After optimizing the committed parameters of the reaction system such as pH and the concentration of Mg 2+ and NAD+, the assembly efficiency was increased by 32-fold. To further improve the assembly capacity, the number of thermal cycles was optimized, resulting in successful assembly 4 unphosphorylated DNA fragments with an accuracy of 75%. sDATEL could be a desirable method for routine manual and automated assembly.

Screening of eye-position related genes with DD-RT-PCR and RDA in the hybrids between Japanese flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus

NASA Astrophysics Data System (ADS)

Chen, Yanjie; Zhang, Quanqi; Qi, Jie; Sun, Yeying; Zhong, Qiwang; Wang, Xubo; Wang, Zhigang; Li, Shuo; Li, Chunmei

2009-02-01

Flatfish or flounder moves one eye to change body proportion into vertebral asymmetry during metamorphosis, during which some become sinistral while others dextral. However, the mechanism behinds the eye-position has not been well understood. In this research, hybrids between Japanese flounder(♀) and stone flounder (♂) show mixed eye-location in both dextral type and sinistral type, and thus become good samples for studying the eye-migration. mRNAs from pro-metamorphosis sinistral and dextral hybrids larvae were screened with classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA (cDNA-RDA); 30 and 47 putative fragments were isolated, respectively. The cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited eye-position expression patterns during metamorphosis. However, none of the fragments was proved to be related to flatfishes’ eye-position specifically. Therefore, further studies and more sensitive gene isolated methods are needed to solve the problems.

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism.

PubMed

Kanchanaketu, T; Sangduen, N; Toojinda, T; Hongtrakul, V

2012-04-13

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.

Cloning of the Pseudomonas aeruginosa outer membrane porin protein P gene: evidence for a linked region of DNA homology.

PubMed Central

Siehnel, R J; Worobec, E A; Hancock, R E

1988-01-01

The gene encoding the outer membrane phosphate-selective porin protein P from Pseudomonas aeruginosa was cloned into Escherichia coli. The protein product was expressed and transported to the outer membrane of an E. coli phoE mutant and assembled into functional trimers. Expression of a product of the correct molecular weight was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, using polyclonal antibodies to protein P monomer and trimer forms. Protein P trimers were partially purified from the E. coli clone and shown to form channels with the same conductance as those formed by protein P from P. aeruginosa. The location and orientation of the protein P-encoding (oprP) gene on the cloned DNA was identified by three methods: (i) mapping the insertion point of transposon Tn501 in a previously isolated P. aeruginosa protein P-deficient mutant; (ii) hybridization of restriction fragments from the cloned DNA to an oligonucleotide pool synthesized on the basis of the amino-terminal protein sequence of protein P; and (iii) fusion of a PstI fragment of the cloned DNA to the amino terminus of the beta-galactosidase gene of pUC8, producing a fusion protein that contained protein P-antigenic epitopes. Structural analysis of the cloned DNA and P. aeruginosa chromosomal DNA revealed the presence of two adjacent PstI fragments which cross-hybridized, suggesting a possible gene duplication. The P-related (PR) region hybridized to the oligonucleotide pool described above. When the PstI fragment which contained the PR region was fused to the beta-galactosidase gene of pUC8, a fusion protein was produced which reacted with a protein P-specific antiserum. However, the restriction endonuclease patterns of the PR region and the oprP gene differed significantly beyond the amino-terminal one-third of the two genes. Images PMID:2834340

Phylogenetic relationships of German heavy draught horse breeds inferred from mitochondrial DNA D-loop variation.

PubMed

Aberle, K S; Hamann, H; Drögemüller, C; Distl, O

2007-04-01

We analysed a 610-bp mitochondrial (mt)DNA D-loop fragment in a sample of German draught horse breeds and compared the polymorphic sites with sequences from Arabian, Hanoverian, Exmoor, Icelandic, Sorraia and Przewalski's Horses as well as with Suffolk, Shire and Belgian horses. In a total of 65 horses, 70 polymorphic sites representing 47 haplotypes were observed. The average percentage of polymorphic sites was 11.5% for the mtDNA fragment analysed. In the nine different draught horse breeds including South German, Mecklenburg, Saxon Thuringa coldblood, Rhenisch German, Schleswig Draught Horse, Black Forest Horse, Shire, Suffolk and Belgian, 61 polymorphic sites and 24 haplotypes were found. The phylogenetic analysis failed to show monophyletic groups for the draught horses. The analysis indicated that the draught horse populations investigated consist of diverse genetic groups with respect to their maternal lineage.

The mechanism of thioacetamide-induced apoptosis in the L37 albumin-SV40 T-antigen transgenic rat hepatocyte-derived cell line occurs without DNA fragmentation.

PubMed

Bulera, S J; Sattler, C A; Gast, W L; Heath, S; Festerling, T A; Pitot, H C

1998-10-01

The hepatotoxicant thioacetamide (TH) has classically been used as a model to study hepatic necrosis; however, recent studies have shown that TH can also induce apoptosis. In this report we demonstrate that 2.68+/-0.54% of the albumin-SV40 T-antigen transgenic rat hepatocytes undergo TH-induced apoptosis, a level comparable to other in vivo models of liver apoptosis. In addition, TH could induce apoptosis and necrosis in the L37 albumin-SV40 T-antigen transgenic rat liver-derived cell line. Examination of dying L37 cells treated with 100 mM TH by electron microscopy revealed distinct morphological characteristics that could be attributed to apoptosis. Quantitation of apoptosis by FACS analysis 24 h after treatment with 100 mM TH revealed that 81.3+/-1.6% of the cells were undergoing apoptosis. In contrast, when L37 cells were treated with 250 mM TH, cells exhibited characteristics consistent with necrotic cell death. DNA fragmentation ladders were produced by growth factor withdrawal-induced apoptosis; however, in 100 mM TH-induced apoptosis, DNA fragmentation ladders were not observed. Analysis of endonuclease activity in L37 cells revealed that the enzymes were not inactivated in the presence of 100 mM TH. The data presented in this report indicate that the L37 cell line could be used to study the mechanism of TH-induced apoptosis that was not mediated through a mechanism requiring DNA fragmentation.

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis

PubMed Central

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval. PMID:28440264

Use of testicular sperm for intracytoplasmic sperm injection in men with high sperm DNA fragmentation: a SWOT analysis.

PubMed

Esteves, Sandro C; Roque, Matheus; Garrido, Nicolás

2018-01-01

Spermatozoa retrieved from the testis of men with high levels of sperm DNA fragmentation (SDF) in the neat semen tend to have better DNA quality. Given the negative impact of SDF on the outcomes of Assisted Reproductive Technology (ART), an increased interest has emerged about the use of testicular sperm for intracytoplasmic sperm injection (Testi-ICSI). In this article, we used a SWOT (strengths, weaknesses, opportunities, and threats) analysis to summarize the advantages and drawbacks of this intervention. The rationale of Testi-ICSI is bypass posttesticular DNA fragmentation caused by oxidative stress during sperm transit through the epididymis. Hence, oocyte fertilization by genomically intact testicular spermatozoa may be optimized, thus increasing the chances of creating a normal embryonic genome and the likelihood of achieving a live birth, as recently demonstrated in men with high SDF. However, there is still limited evidence as regards the clinical efficacy of Testi-ICSI, thus creating opportunities for further confirmatory clinical research as well as investigation of Testi-ICSI in clinical scenarios other than high SDF. Furthermore, Testi-ICSI can be compared to other laboratory preparation methods for deselecting sperm with damaged DNA. At present, the available literature supports the use of testicular sperm when performing ICSI in infertile couples whose male partners have posttesticular SDF. Due to inherent risks of sperm retrieval, Testi-ICSI should be offered when less invasive treatments for alleviating DNA damage have failed. A call for continuous monitoring is nonetheless required concerning the health of generated offspring and the potential complications of sperm retrieval.

Identification of two new polymorphisms in the manganese superoxide dismutase gene (Mn-SOD); Part of the etiology of Parkinson`s disease?

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

Eggers, B.; Kurth, J.H.; Kurth, M.C.

1994-09-01

Epidemiological studies suggest that several different environmental agents interact with a number of genetic elements to cause Parkinson`s disease (PD), a common neurodegenerative disease. Abnormalities of oxidative metabolism may be central to this process. Specifically, the production and degradation of dopamine may lead to toxic by-products and increased oxidative stress. Toxic by-products include hydrogen peroxide, superoxide, and hydroxyl radicals, all of which are implicated in the aging process of the central nervous system. Superoxide dismutase (SOD) catalyzes superoxide to hydrogen peroxide. Genetic predisposition to PD may be at least partially a result of certain SOD alleles. Using the cDNA sequencemore » of Mn-SOD gene, oligonucleotide primers were designed which span several presumptive splice junction sites. An approximatley 2.4kb PCR product was amplified from gDNA samples that span one or more intron near the 3{prime} end of the Mn-SOD cDNA sequence. The resultant product was screened with a panel of 4-cutters to identify fragments appropriate for SSCP analysis. Twenty-two gDNA samples were screened for SSCP and size differences of these PCR products. After digestion with AluI, two polymorphisms were observed. Two alleles with a size difference of 2-4 bp were observed by denaturing PAGE in one of the fragments. SSCP analysis revealed a polymorphism with 2 alleles in another fragment. Sequence analysis of these polymorphisms is in progress. DNA from several DEPH families was used to confirm Mendelian inheritance of these polymorphisms. Genomic DNA samples have been collected from 265 PD patients and 169 control individuals; allelic frequencies will be determined for these populations, compared by {chi}{sup 2} analysis, and relative risk calculated. These results may support a contribution of Mn-SOD in the genetic predisposition to PD.« less

The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation*

PubMed Central

Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

2015-01-01

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

PubMed

Sha, A H; Lin, X H; Huang, J B; Zhang, D P

2005-07-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

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

PubMed Central

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

2009-01-01

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

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

PubMed Central

Kosturko, L D; Daub, E; Murialdo, H

1989-01-01

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure. Images PMID:2521383

Sperm DNA damage has a negative association with live-birth rates after IVF.

PubMed

Simon, L; Proutski, I; Stevenson, M; Jennings, D; McManus, J; Lutton, D; Lewis, S E M

2013-01-01

Sperm DNA damage has a negative impact on pregnancy rates following assisted reproduction treatment (ART). The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage. Following IVF, couples with <25% sperm DNA fragmentation had a live-birth rate of 33%; in contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13%. Following ICSI, no significant differences in sperm DNA damage were found between any groups of patients. Sperm DNA damage was also associated with low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men with idiopathic infertility have high sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Sperm DNA damage has a negative impact on assisted reproduction treatment outcome, in particular, on pregnancy rates. The aim of the present study was to examine the relationship between sperm DNA fragmentation and live-birth rates after IVF and intracytoplasmic sperm injection (ICSI). The alkaline Comet assay was employed to measure sperm DNA fragmentation in native semen and in spermatozoa following density-gradient centrifugation in semen samples from 203 couples undergoing IVF and 136 couples undergoing ICSI. Men were divided into groups according to sperm DNA damage and treatment outcome. Following IVF, couples with <25% sperm DNA fragmentation had a live birth rate of 33%. In contrast, couples with >50% sperm DNA fragmentation had a much lower live-birth rate of 13% following IVF. Following ICSI, there were no significant differences in levels of sperm DNA damage between any groups of patients. Sperm DNA damage was also associated with the very low live-birth rates following IVF in both men and couples with idiopathic infertility: 39% of couples and 41% of men have high level of sperm DNA damage. Sperm DNA damage assessed by the Comet assay has a close inverse relationship with live-birth rates after IVF. Copyright © 2012 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.

PubMed

Balasuriya, A; Serhal, P; Doshi, A; Harper, J C

2014-03-01

Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART. © 2012 Blackwell Verlag GmbH.

High-resolution hydrodynamic chromatographic separation of large DNA using narrow, bare open capillaries: a rapid and economical alternative technology to pulsed-field gel electrophoresis?

PubMed

Liu, Lei; Veerappan, Vijaykumar; Pu, Qiaosheng; Cheng, Chang; Wang, Xiayan; Lu, Liping; Allen, Randy D; Guo, Guangsheng

2014-01-07

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10(5) plates for the longest (48.5 kbp) and 3.7 × 10(5) plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsed-field gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

NASA Technical Reports Server (NTRS)

Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

1995-01-01

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

Telomere Restriction Fragment (TRF) Analysis.

PubMed

Mender, Ilgen; Shay, Jerry W

2015-11-20

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al. , 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al. , 1990; Ouellette et al. , 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Spontaneous apoptotic DNA fragmentation in cultured guinea pig gastric mucosal cells.

PubMed

Tsutsumi, S; Rokutan, K; Tsuchiya, T; Mizushima, T

2000-02-01

The purpose of this study was to elucidate the mechanism of spontaneous and rapid cell death of cultured gastric pit cells. Gastric pit cells have a rapid cell turnover rate in vivo. We here show that guinea pig gastric pit cells in culture undergo spontaneous and rapid apoptotic DNA fragmentation, which may represent the rapid cell turnover cycle of gastric pit cells in vivo. This spontaneous apoptotic DNA fragmentation required the presence of fetal calf serum in the culture media. Furthermore, the spontaneous apoptotic DNA fragmentation was prevented by protein synthesis and caspase inhibitors.

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

PubMed Central

Den Dunnen, J T; Grootscholten, P M; Bakker, E; Blonden, L A; Ginjaar, H B; Wapenaar, M C; van Paassen, H M; van Broeckhoven, C; Pearson, P L; van Ommen, G J

1989-01-01

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron. Images Figure 1 Figure 4 PMID:2573997

Cloning of hydrogenase genes and fine structure analysis of an operon essential for H/sub 2/ metabolism in Escherichia coli

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

Sankar, P.; Lee, J.H.; Shanmugam, K.T.

1985-04-01

Escherichia coli has two unlinked genes that code for hydrogenase synthesis and activity. The DNA fragments containing the two genes (hydA and hydB) were cloned into a plasmid vector, pBR322. The plasmids containing the hyd genes (pSE-290 and pSE-111 carrying the hydA and hydB genes, respectively) were used to genetically map a total of 51 mutant strains with defects in hydrogenase activity. A total of 37 mutants carried a mutation in the hydB gene, whereas the remaining 14 hyd were hydA. This complementation analysis also established the presence of two new genes, so far unidentified, one coding for formate dehydrogenase-2more » (fdv) and another producing an electron transport protein (fhl) coupling formate dehydrogenase-2 to hydrogenase. Three of the four genes, hydB, fhl, and fdv, may constitute a single operon, and all three genes are carried by a 5.6-kilobase-pair chromosomal DNA insert in plasmid pSE-128. Plasmids carrying a part of this 5.6-kilobase-pair DNA (pSE-130) or fragments derived from this DNA in different orientations (pSE-126 and pSE-129) inhibited the production of active formate hydrogenlyase. This inhibition occurred even in a prototrophic E. coli, strain K-10, but only during an early induction period. These results, based on complementation analysis with cloned DNA fragments, show that both hydA and hydB genes are essential for the production of active hydrogenase. For the expression of active formate hydrogenlyase, two other gene products, fhl and fdv are also needed. All four genes map between 58 and 59 min in the E. coli chromosome.« less

[Molecular-genetic analysis of wheat (T. aestivum L.) genome with introgression of Ae. cylindrica Host genetic elements].

PubMed

Galaev, A V; Sivolap, Iu M

2005-01-01

Wheat-aegilops hybrid plants Triticum aestivum L. (2n = 42) x Aegilops cylindrica Host (2n = 28) were investigated with using microsatellite markers. In two BC1F9 lines some genome modifications connected with losing DNA fragments of initial variety or appearing of Aegilops genome elements were detected. In some investigated hybrids new amplicons lacking in parental plants were found. Substitution of wheat chromosomes for aegilops chromosomes was not revealed. Analysis of microsatellite loci in BC2F5 plants showed stable introgression of aegilops genetic elements into wheat; elimination of some transferred aegilops DNA fragments in the course of backcrossing; decreasing size of introgressive elements after backcrossing. Introgressive lines were classified according to genome changes.

The role of heat shock protein 70 (Hsp 70) in male infertility: is it a line of defense against sperm DNA fragmentation?

PubMed

Erata, Gül Ozdemirler; Koçak Toker, Necla; Durlanik, Ozgür; Kadioğlu, Ateş; Aktan, Gülşen; Aykaç Toker, Gülçin

2008-08-01

To clarify the role of heat shock protein 70 (Hsp 70) and its relation with DNA damage in male infertility. Prospective study. Andrology laboratory of Istanbul Medical Faculty. Semen samples from 37 infertile men and 13 fertile men (as controls). The percentage of DNA fragmentation was assayed with the use of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Sperm Hsp 70 expression was determined by using Western blot analysis. Both the percentages of sperm DNA fragmentation and Hsp 70 expression were correlated with semen analysis parameters. TUNEL-positive spermatozoa in the infertile group (18.7% for asthenospermics and 13.0% for oligoasthenospermics) were higher than the fertile group (4.9%). Significant inverse correlations were detected between percentage of TUNEL-positive cells and both concentration (r = -0.487) and motility (r = -0.377) of spermatozoa. No expression of Hsp 70 was observed in azospermic group, whereas Hsp 70 levels were found increased significantly in infertile group (U = 62 for asthenospermics and U = 38 for oligoasthenospermics) compared to fertile group as analyzed by using Mann-Whitney U Wilcoxon rank sum test. Furthermore, significant positive correlation was found between percentage of TUNEL-positive cells and Hsp 70 expression (r = 0.357). Hsp 70 expression may have been increased as a protective mechanism against apoptosis in spermatozoa of infertile men.

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps).

PubMed

Schmidt, Volker; Klasen, Linus; Schneider, Juliane; Hübel, Jens; Pees, Michael

2017-03-01

Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons ( Pogona vitticeps ), six veiled chameleons ( Chamaeleo calyptratus ), and six panther chameleons ( Furcifer pardalis ) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity. Copyright © 2017 American Society for Microbiology.

Characterization of Metarhizium viride Mycosis in Veiled Chameleons (Chamaeleo calyptratus), Panther Chameleons (Furcifer pardalis), and Inland Bearded Dragons (Pogona vitticeps)

PubMed Central

Klasen, Linus; Schneider, Juliane; Hübel, Jens; Pees, Michael

2016-01-01

ABSTRACT Metarhizium viride has been associated with fatal systemic mycoses in chameleons, but subsequent data on mycoses caused by this fungus in reptiles are lacking. The aim of this investigation was therefore to obtain information on the presence of M. viride in reptiles kept as pets in captivity and its association with clinical signs and pathological findings as well as improvement of diagnostic procedures. Beside 18S ribosomal DNA (rDNA) (small subunit [SSU]) and internal transcribed spacer region 1 (ITS-1), a fragment of the large subunit (LSU) of 28S rDNA, including domain 1 (D1) and D2, was sequenced for the identification of the fungus and phylogenetic analysis. Cultural isolation and histopathological examinations as well as the pattern of antifungal drug resistance, determined by using agar diffusion testing, were additionally used for comparison of the isolates. In total, 20 isolates from eight inland bearded dragons (Pogona vitticeps), six veiled chameleons (Chamaeleo calyptratus), and six panther chameleons (Furcifer pardalis) were examined. Most of the lizards suffered from fungal glossitis, stomatitis, and pharyngitis or died due to visceral mycosis. Treatment with different antifungal drugs according to resistance patterns in all three different lizard species was unsuccessful. Sequence analysis resulted in four different genotypes of M. viride based on differences in the LSU fragment, whereas the SSU and ITS-1 were identical in all isolates. Sequence analysis of the SSU fragment revealed the first presentation of a valid large fragment of the SSU of M. viride. According to statistical analysis, genotypes did not correlate with differences in pathogenicity, antifungal susceptibility, or species specificity. PMID:28003420

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

NASA Astrophysics Data System (ADS)

Wang, Tiegu; Huang, Qunce; Feng, Weisen

2007-10-01

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

Sequence analysis of the mitochondrial DNA control region of ciscoes (genus Coregonus): Taxonomic implications for the Great Lakes species flock

USGS Publications Warehouse

Reed, Kent M.; Dorschner, Michael O.; Todd, Thomas N.; Phillips, Ruth B.

1998-01-01

Sequence variation in the control region (D-loop) of the mitochondrial DNA (mtDNA) was examined to assess the genetic distinctiveness of the shortjaw cisco (Coregonus zenithicus). Individuals from within the Great Lakes Basin as well as inland lakes outside the basin were sampled. DNA fragments containing the entire D-loop were amplified by PCR from specimens ofC. zenithicus and the related species C. artedi, C. hoyi, C. kiyi, and C. clupeaformis. DNA sequence analysis revealed high similarity within and among species and shared polymorphism for length variants. Based on this analysis, the shortjaw cisco is not genetically distinct from other cisco species.

Production of transgenic chickens using an avian retroviral vector

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

Kopchick, J.; Mills, E.; Rosenblum C.

1987-05-01

The authors efforts to insert genes into the chicken germ line are dependent upon the ability of exogenous avian retroviruses to infect chicken germ cells. They have used a transformation defective Schmidt Ruppin A strain of Rous Sarcoma Virus (RSV-SRA) in their initial experiments. The general protocol involved generating RSV-SRA viremic female chickens (Go), which shed exogenous virus via the oviduct. As the fertilized egg passes through the oviduct, embryonic cells are exposed to the virus. If the germ cell precursors are infected by the virus, offspring (G1) should be generated which are capable of passing the viral DNA tomore » the next generation (G2). Fifteen viremic G1 males were selected for breeding and progeny testing. Since male chickens do not congenitally pass retroviruses through semen, production of viremic G2 offspring indicates germ line DNA transmission. This is confirmed by DNA analysis of the experimental chickens. Using a specific probe for exogenous retrovirus, they have detected the presence of RSV-SRA DNA in viremic chickens. Southern DNA analysis revealed junction fragments for RSV-SRA DNA in viremic G2 chickens, but not in non-viremic siblings. Furthermore, DNA isolated from various tissues of a viremic G2 chicken showed an identical DNA junction fragment pattern, indicating all tissues were derived from the same embryonic cell which contained integrated provirus. To date they have generated 50 transgenic chickens.« less

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

PubMed

Santos, E M; Paula, J F R; Motta, P M C; Heinemann, M B; Leite, R C; Haddad, J P A; Del Puerto, H L; Reis, J K P

2010-08-17

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

Sperm quality and DNA integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons.

PubMed

Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Chiu, Chien-Chih; Zhou, Guodong; Chou, Chon-Kit; Lin, Wen-Yi

2016-11-18

The objective of this study was to assess sperm quality and deoxyribonucleic acid (DNA) integrity of coke oven workers exposed to polycyclic aromatic hydrocarbons (PAHs) as compared to control subjects. The coke oven workers (N = 52) and administrative staff (N = 35) of a steel plant served as the exposed and control groups, respectively. Exposure to PAHs was assessed by measuring 1-hydroxypyren. Analysis of sperm quality (concentration, motility, vitality, and morphology) was performed simultaneously with sperm DNA integrity analysis, including DNA fragmentation, denaturation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). A questionnaire was conducted to collect demographic and potential confounding data. The coke oven workers had lower percentages of sperm motility, vitality and normal morphology than the control group, but the difference was not significant. For DNA integrity, the coke oven workers had significantly higher concentrations of bulky DNA adducts and 8-oxo-dGuo than the control subjects (p = 0.009 and p = 0.048, respectively). However, DNA fragmentation percentages did not significantly increase as compared to those in the subjects from the control group (p = 0.232). There was no correlation between sperm quality parameters and DNA integrity indicators. Occupational exposure of the coke oven workers to PAHs was associated with decreased sperm DNA integrity. Int J Occup Med Environ Health 2016;29(6):915-926. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

The roles of family B and D DNA polymerases in Thermococcus species 9°N Okazaki fragment maturation.

PubMed

Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F

2015-05-15

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Synchronization of DNA array replication kinetics

NASA Astrophysics Data System (ADS)

Manturov, Alexey O.; Grigoryev, Anton V.

2016-04-01

In the present work we discuss the features of the DNA replication kinetics at the case of multiplicity of simultaneously elongated DNA fragments. The interaction between replicated DNA fragments is carried out by free protons that appears at the every nucleotide attachment at the free end of elongated DNA fragment. So there is feedback between free protons concentration and DNA-polymerase activity that appears as elongation rate dependence. We develop the numerical model based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) for DNA elongation process with conditions pointed above and we study the possibility of the DNA polymerases movement synchronization. The results obtained numerically can be useful for DNA polymerase movement detection and visualization of the elongation process in the case of massive DNA replication, eg, under PCR condition or for DNA "sequencing by synthesis" sequencing devices evaluation.

Structure and DNA-binding of meiosis-specific protein Hop2

NASA Astrophysics Data System (ADS)

Zhou, Donghua; Moktan, Hem; Pezza, Roberto

2014-03-01

Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

Epidemiologic and Molecular Relationship Between Vaccine Manufacture and Autism Spectrum Disorder Prevalence.

PubMed

Deisher, Theresa A; Doan, Ngoc V; Koyama, Kumiko; Bwabye, Sarah

2015-01-01

To assess the public health consequences of fetal cell line manufactured vaccines that contain residual human fetal DNA fragments utilizing laboratory and ecological approaches including statistics, molecular biology and genomics. MMR coverage and autism disorder or autism spectrum disorder prevalence data for Norway, Sweden and the UK were obtained from public and government websites as well as peer reviewed published articles. Biologically, the size and quantity of the contaminating fetal DNA in Meruvax II and Havrix as well as the propensity of various cell lines for cellular and nuclear uptake of primitive human DNA fragments were measured and quantified using gel electrophoresis, fluorescence microscopy and fluorometry. Lastly, genomic analysis identified the specific sites where fetal DNA fragment integration into a child's genome is most likely to occur. The average MMR coverage for the three countries fell below 90% after Dr. Wakefield's infamous 1998 publication but started to recover slowly after 2001 until reaching over 90% coverage again by 2004. During the same time period, the average autism spectrum disorder prevalence in the United Kingdom, Norway and Sweden dropped substantially after birth year 1998 and gradually increased again after birth year 2000. Average single stranded DNA and double stranded DNA in Meruvax II were 142.05 ng/vial and 35.00 ng/vial, respectively, and 276.00 ng/vial and 35.74 ng/vial in Havrix respectively. The size of the fetal DNA fragments in Meruvax II was approximately 215 base pairs. There was spontaneous cellular and nuclear DNA uptake in HFF1 and NCCIT cells. Genes that have been linked to autism (autism associated genes; AAGs) have a more concentrated susceptibility for insults to genomic stability in comparison to the group of all genes contained within the human genome. Of the X chromosome AAGs, 15 of 19 have double strand break motifs less than 100 kilobases away from the center of a meiotic recombination hotspot located within an exon. Vaccines manufactured in human fetal cell lines contain unacceptably high levels of fetal DNA fragment contaminants. The human genome naturally contains regions that are susceptible to double strand break formation and DNA insertional mutagenesis. The "Wakefield Scare" created a natural experiment that may demonstrate a causal relationship between fetal cell-line manufactured vaccines and ASD prevalence.

Trapping and breaking of in vivo nicked DNA during pulsed-field gel electrophoresis

PubMed Central

Khan, Sharik R.; Kuzminov, Andrei

2013-01-01

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percent of chromosomal DNA entering the gel. The degree of separation in PFG depends upon the size of DNA, as well as various conditions of electrophoresis, such as electric field strength (FS), time of electrophoresis, switch time and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that sub-chromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single strand interruptions results in artefactual decrease in molecular weight of linear DNA making accurate determination of the number of double strand breaks difficult. While breakage of nicked sub-chromosomal fragments is FS-independent, some high molecular weight sub-chromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage. PMID:23770235

Scanning the human genome at kilobase resolution.

PubMed

Chen, Jun; Kim, Yeong C; Jung, Yong-Chul; Xuan, Zhenyu; Dworkin, Geoff; Zhang, Yanming; Zhang, Michael Q; Wang, San Ming

2008-05-01

Normal genome variation and pathogenic genome alteration frequently affect small regions in the genome. Identifying those genomic changes remains a technical challenge. We report here the development of the DGS (Ditag Genome Scanning) technique for high-resolution analysis of genome structure. The basic features of DGS include (1) use of high-frequent restriction enzymes to fractionate the genome into small fragments; (2) collection of two tags from two ends of a given DNA fragment to form a ditag to represent the fragment; (3) application of the 454 sequencing system to reach a comprehensive ditag sequence collection; (4) determination of the genome origin of ditags by mapping to reference ditags from known genome sequences; (5) use of ditag sequences directly as the sense and antisense PCR primers to amplify the original DNA fragment. To study the relationship between ditags and genome structure, we performed a computational study by using the human genome reference sequences as a model, and analyzed the ditags experimentally collected from the well-characterized normal human DNA GM15510 and the leukemic human DNA of Kasumi-1 cells. Our studies show that DGS provides a kilobase resolution for studying genome structure with high specificity and high genome coverage. DGS can be applied to validate genome assembly, to compare genome similarity and variation in normal populations, and to identify genomic abnormality including insertion, inversion, deletion, translocation, and amplification in pathological genomes such as cancer genomes.

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

Screening of Israeli Holstein-Friesian cattle for restriction fragment length polymorphisms using homologous and heterologous deoxyribonucleic acid probes.

PubMed

Hallerman, E M; Nave, A; Soller, M; Beckmann, J S

1988-12-01

Genomic DNA of Israeli Holstein-Friesian dairy cattle were screened with a battery of 17 cloned or subcloned DNA probes in an attempt to document restriction fragment length polymorphisms at a number of genetic loci. Restriction fragment length polymorphisms were observed at the chymosin, oxytocin-neurophysin I, lutropin beta, keratin III, keratin VI, keratin VII, prolactin, and dihydrofolate reductase loci. Use of certain genomic DNA fragments as probes produced hybridization patterns indicative of satellite DNA at the respective loci. Means for distinguishing hybridizations to coding sequences for unique genes from those to satellite DNA were developed. Results of this study are discussed in terms of strategy for the systematic development of large numbers of bovine genomic polymorphisms.

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

PubMed

Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

2017-08-15

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.

PubMed

Musharova, Olga; Klimuk, Evgeny; Datsenko, Kirill A; Metlitskaya, Anastasia; Logacheva, Maria; Semenova, Ekaterina; Severinov, Konstantin; Savitskaya, Ekaterina

2017-04-07

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF PCR-AMPLIFIED NIFH SEQUENCES FROM WETLAND PLANT RHIZOSPHERE COMMUNITIES

EPA Science Inventory

We describe a method to assess the community structure of N2-fixing bacteria in the rhizosphere. Total DNA was extracted from Spartina alterniflora and Sesbania macrocarpa root zones by bead-beating and purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 ...

Molecular Characterization of a Chromosomal Determinant Conferring Resistance to Zinc and Cobalt Ions in Staphylococcus aureus

PubMed Central

Xiong, Anming; Jayaswal, Radheshyam K.

1998-01-01

A DNA fragment conferring resistance to zinc and cobalt ions was isolated from a genomic DNA library of Staphylococcus aureus RN450. The DNA sequence analysis revealed two consecutive open reading frames, designated zntR and zntA. The predicted ZntR and ZntA showed significant homology to members of ArsR and cation diffusion families, respectively. A mutant strain containing the null allele of zntA was more sensitive to zinc and cobalt ions than was the parent strain. The metal-sensitive phenotype of the mutant was complemented by a 2.9-kb DNA fragment containing zntR and zntA. An S. aureus strain harboring multiple copies of zntR and zntA showed an increased resistance to zinc. The resistance to zinc in the wild-type strain was inducible. Transcriptional analysis indicated that zntR and zntA genes were cotranscribed. The zinc uptake studies suggested that the zntA product was involved in the export of zinc ions out of cells. PMID:9696746

Safety assessment of sodium acetate, sodium diacetate and potassium sorbate food additives.

PubMed

Mohammadzadeh-Aghdash, Hossein; Sohrabi, Yousef; Mohammadi, Ali; Shanehbandi, Dariush; Dehghan, Parvin; Ezzati Nazhad Dolatabadi, Jafar

2018-08-15

Cytotoxicity and genotoxicity of sodium acetate (SA), sodium diacetate (SDA), and potassium sorbate (PS) was tested on Human Umbilical Vein Endothelial Cells (HUVEC). Cytotoxicity was investigated by MTT assay and flow cytometry analysis, while genotoxicity was evaluated using DNA fragmentation and DAPI staining assays. The growth of treated HUVECs with various concentrations of SA, SDA and PS decreased in a dose-and time-dependent manner. The IC50 of 487.71, 485.82 and 659.96 µM after 24 h and IC50 of 232.05, 190.19 and 123.95 µM after 48 h of treatment were attained for SA, SDA and PS, respectively. Flow cytometry analysis showed that early and late apoptosis percentage in treated cells was not considerable. Also neither considerable DNA fragmentation nor DNA smear was observed using DAPI staining and DNA ladder assays. Overall, it can be concluded that the aforementioned food additives can be used as safe additives at low concentration in food industry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

PubMed

Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

2014-03-01

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

PubMed Central

Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

2014-01-01

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

Temporal patterns of damage and decay kinetics of DNA retrieved from plant herbarium specimens.

PubMed

Weiß, Clemens L; Schuenemann, Verena J; Devos, Jane; Shirsekar, Gautam; Reiter, Ella; Gould, Billie A; Stinchcombe, John R; Krause, Johannes; Burbano, Hernán A

2016-06-01

Herbaria archive a record of changes of worldwide plant biodiversity harbouring millions of specimens that contain DNA suitable for genome sequencing. To profit from this resource, it is fundamental to understand in detail the process of DNA degradation in herbarium specimens. We investigated patterns of DNA fragmentation and nucleotide misincorporation by analysing 86 herbarium samples spanning the last 300 years using Illumina shotgun sequencing. We found an exponential decay relationship between DNA fragmentation and time, and estimated a per nucleotide fragmentation rate of 1.66 × 10(-4) per year, which is six times faster than the rate estimated for ancient bones. Additionally, we found that strand breaks occur specially before purines, and that depurination-driven DNA breakage occurs constantly through time and can to a great extent explain decreasing fragment length over time. Similar to what has been found analysing ancient DNA from bones, we found a strong correlation between the deamination-driven accumulation of cytosine to thymine substitutions and time, which reinforces the importance of substitution patterns to authenticate the ancient/historical nature of DNA fragments. Accurate estimations of DNA degradation through time will allow informed decisions about laboratory and computational procedures to take advantage of the vast collection of worldwide herbarium specimens.

Individual specific DNA fingerprints from a hypervariable region probe: alpha-globin 3'HVR.

PubMed

Fowler, S J; Gill, P; Werrett, D J; Higgs, D R

1988-06-01

A probe detecting a hypervariable region (HVR) 3' to the alpha globin locus on chromosome 16 has been used to produce DNA fingerprints. Segregation analysis has revealed multiple, randomly dispersed DNA fragments inherited in a Mendelian fashion with minimal allelism and linkage. The fingerprints are highly polymorphic (probability of chance association between random individuals much less than 10(-14]. The probe is, therefore, a powerful discriminating tool: it is envisaged that this probe will have forensic applications, including paternity cases, and will be informative in linkage analysis.

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.

PubMed

Zhang, Zijie; Liu, Juewen

2016-03-01

Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.

An Authentic RFLP Lab for High School or College Biology Students.

ERIC Educational Resources Information Center

Guilfoile, Patrick G.; Plum, Stephen

1998-01-01

Explains how students can perform an alternative authentic DNA fingerprinting analysis. Presents restriction fragment length polymorphism (RFLP) analysis and can serve as a simulated molecular epidemiology laboratory or as a simulated forensic laboratory exercise. (DDR)

Differential gene expression for Curvularia eragrostidis pathogenic incidence in crabgrass (Digitaria sanguinalis) revealed by cDNA-AFLP analysis.

PubMed

Wang, Jianshu; Wang, Xuemin; Yuan, Bohua; Qiang, Sheng

2013-01-01

Gene expression profiles of Digitaria sanguinalis infected by Curvularia eragrostidis strain QZ-2000 at two concentrations of conidia and two dew durations were analyzed by cDNA amplified fragment length polymorphisms (cDNA-AFLP). Inoculum strength was more determinant of gene expression than dew duration. A total of 256 primer combinations were used for selective amplification and 1214 transcript-derived fragments (TDFs) were selected for their differential expression. Of these, 518 up-regulated differentially expressed TDFs were identified. Forty-six differential cDNA fragments were chosen to be cloned and 35 of them were successfully cloned and sequenced, of which 25 were homologous to genes of known function according to the GenBank database. Only 6 genes were up-regulated in Curvularia eragrostidis-inoculated D. sanguinalis, with functions involved in signal transduction, energy metabolism, cell growth and development, stress responses, abscisic acid biosynthesis and response. It appears that a few pathways may be important parts of the pathogenic strategy of C. eragrostidis strain QZ-2000 on D. sanguinalis. Our study provides the fundamentals to further study the pathogenic mechanism, screen for optimal C. eragrostidis strains as potential mycoherbicide and apply this product to control D. sanguinalis.

Longitudinal study of sperm DNA fragmentation as measured by terminal uridine nick end-labelling assay.

PubMed

Sergerie, M; Laforest, G; Boulanger, K; Bissonnette, F; Bleau, G

2005-07-01

One major limitation in the use of sperm DNA fragmentation as measured by the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL) assay is the paucity of solid data on the stability of this parameter. The objective of our study was to evaluate variations in the degree of sperm DNA fragmentation, as measured by the TUNEL assay, over a 6 month period. Five donors provided semen samples (total 107) on the average three times per month, and 10 infertility patients provided semen samples every 4 weeks (total 58). The mean percentage of sperm DNA fragmentation for donors was 13.18%, the within-donor standard deviation (SD(W) = 3.79%) was small compared to between-donor (SD(B) = 17.56%). For the group of patients, the mean percentage of sperm DNA fragmentation was 22.44%, with SD(W) of 4.43% within patients and SD(B) of 29.48% between patients. No seasonal rhythm was observed during the study. The intra-class correlation coefficient for all subjects combined was 0.83. Compared to sperm concentration, individual coefficients of variation for sperm DNA fragmentation indicated less variability in four subjects, but were similar in the others. This longitudinal study shows that sperm DNA fragmentation is a parameter with good stability (repeatability) over time; it can be taken as a baseline both in healthy fertile men and in patients from infertility couples.

Asian Elephants in China: Estimating Population Size and Evaluating Habitat Suitability

PubMed Central

Zhang, Li; Dong, Lu; Lin, Liu; Feng, Limin; Yan, Fan; Wang, Lanxin; Guo, Xianming; Luo, Aidong

2015-01-01

We monitored the last remaining Asian elephant populations in China over the past decade. Using DNA tools and repeat genotyping, we estimated the population sizes from 654 dung samples collected from various areas. Combined with morphological individual identifications from over 6,300 elephant photographs taken in the wild, we estimated that the total Asian elephant population size in China is between 221 and 245. Population genetic structure and diversity were examined using a 556-bp fragment of mitochondrial DNA, and 24 unique haplotypes were detected from DNA analysis of 178 individuals. A phylogenetic analysis revealed two highly divergent clades of Asian elephants, α and β, present in Chinese populations. Four populations (Mengla, Shangyong, Mengyang, and Pu’Er) carried mtDNA from the α clade, and only one population (Nangunhe) carried mtDNA belonging to the β clade. Moreover, high genetic divergence was observed between the Nangunhe population and the other four populations; however, genetic diversity among the five populations was low, possibly due to limited gene flow because of habitat fragmentation. The expansion of rubber plantations, crop cultivation, and villages along rivers and roads had caused extensive degradation of natural forest in these areas. This had resulted in the loss and fragmentation of elephant habitats and had formed artificial barriers that inhibited elephant migration. Using Geographic Information System, Global Positioning System, and Remote Sensing technology, we found that the area occupied by rubber plantations, tea farms, and urban settlements had dramatically increased over the past 40 years, resulting in the loss and fragmentation of elephant habitats and forming artificial barriers that inhibit elephant migration. The restoration of ecological corridors to facilitate gene exchange among isolated elephant populations and the establishment of cross-boundary protected areas between China and Laos to secure their natural habitats are critical for the survival of Asian elephants in this region. PMID:25992617

Asian elephants in China: estimating population size and evaluating habitat suitability.

PubMed

Zhang, Li; Dong, Lu; Lin, Liu; Feng, Limin; Yan, Fan; Wang, Lanxin; Guo, Xianming; Luo, Aidong

2015-01-01

We monitored the last remaining Asian elephant populations in China over the past decade. Using DNA tools and repeat genotyping, we estimated the population sizes from 654 dung samples collected from various areas. Combined with morphological individual identifications from over 6,300 elephant photographs taken in the wild, we estimated that the total Asian elephant population size in China is between 221 and 245. Population genetic structure and diversity were examined using a 556-bp fragment of mitochondrial DNA, and 24 unique haplotypes were detected from DNA analysis of 178 individuals. A phylogenetic analysis revealed two highly divergent clades of Asian elephants, α and β, present in Chinese populations. Four populations (Mengla, Shangyong, Mengyang, and Pu'Er) carried mtDNA from the α clade, and only one population (Nangunhe) carried mtDNA belonging to the β clade. Moreover, high genetic divergence was observed between the Nangunhe population and the other four populations; however, genetic diversity among the five populations was low, possibly due to limited gene flow because of habitat fragmentation. The expansion of rubber plantations, crop cultivation, and villages along rivers and roads had caused extensive degradation of natural forest in these areas. This had resulted in the loss and fragmentation of elephant habitats and had formed artificial barriers that inhibited elephant migration. Using Geographic Information System, Global Positioning System, and Remote Sensing technology, we found that the area occupied by rubber plantations, tea farms, and urban settlements had dramatically increased over the past 40 years, resulting in the loss and fragmentation of elephant habitats and forming artificial barriers that inhibit elephant migration. The restoration of ecological corridors to facilitate gene exchange among isolated elephant populations and the establishment of cross-boundary protected areas between China and Laos to secure their natural habitats are critical for the survival of Asian elephants in this region.

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

PubMed

Samadpour, M; Grimm, L M; Desai, B; Alfi, D; Ongerth, J E; Tarr, P I

1993-12-01

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.

Identification of victims of the 1998 Taoyuan Airbus crash accident using DNA analysis.

PubMed

Hsu, C M; Huang, N E; Tsai, L C; Kao, L G; Chao, C H; Linacre, A; Lee, J C

1999-01-01

In February 1998 a civilian aeroplane carrying 196 individuals crashed in Taiwan and killed another 6 people on the ground. Although there were dental and medical records, fingerprints, photographic evidence and personal effects to identify some of the victims, DNA analysis was required to further identify severely damaged remains. From the 202 people known to have perished in the plane crash, a total of 685 fragments of human remains were subjected to DNA analysis. The analysis was carried out using nine microsatellite loci, plus amelogenin to cluster the 685 fragments into 202 groups, accounting for all the victims. To establish genetic relatedness of the victims to other victims and living relatives, additional DNA loci were used. In this case the paternity index was increased by using HLA DQA1 plus Polymarker. The same 16 DNA loci were used to test blood samples from 201 relatives to establish parent/child and sibling relationships. With the exception of 19 victims identified by non-genetic evidence, 183 victims were successfully identified by DNA typing with relatively high values of paternity index by the direct or indirect comparison of relatives. The 202 victims were from 37 different families, ranging in size from 2 to 13 members and 74 individuals known to be unrelated to any other victim. The DNA from living relatives was used to identify one member of a family group, from which other victims of the family could be identified. ABO blood group information was further used to confirm genetic relatedness within families. A comparison of the DNA profiling results to the ABO blood group of the victims showed no discrepancies with the exception of two mutations in the FGA locus. In cases of severely damaged victims from a plane crash, DNA analysis proved to be the best choice to identify victims.

Genome Fragmentation Is Not Confined to the Peridinin Plastid in Dinoflagellates

PubMed Central

Espelund, Mari; Minge, Marianne A.; Gabrielsen, Tove M.; Nederbragt, Alexander J.; Shalchian-Tabrizi, Kamran; Otis, Christian; Turmel, Monique; Lemieux, Claude; Jakobsen, Kjetill S.

2012-01-01

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3′-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates. PMID:22719952

[Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2016-01-01

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses.

PubMed

Schnitzler, P; Darai, G

1989-09-01

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.

Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal-organic framework.

PubMed

Qiu, Gui-Hua; Weng, Zi-Hua; Hu, Pei-Pei; Duan, Wen-Jun; Xie, Bao-Ping; Sun, Bin; Tang, Xiao-Yan; Chen, Jin-Xiang

2018-04-01

From a three-dimensional (3D) metal-organic framework (MOF) of {[Cu(Cmdcp)(phen)(H 2 O)] 2 ·9H 2 O} n (1, H 3 CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π … π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction.

PubMed

Kurien, B T; Kaufman, K M; Harley, J B; Scofield, R H

2001-09-15

A simple method for extracting DNA from agarose gel slices is described. The extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying from 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and higher (70-90%). This range of efficiency was maintained when the starting material was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit. Copyright 2001 Academic Press.

Sperm DNA fragmentation in the total and vital fractions before and after density gradient centrifugation: Significance in male fertility diagnosis.

PubMed

Punjabi, U; Van Mulders, H; Goovaerts, I; Peeters, K; Clasen, K; Janssens, P; Zemtsova, O; De Neubourg, D

2018-05-21

Sperm DNA fragmentation measured by different techniques make comparisons impossible due to lack of standardization. Induction of DNA damage after sperm preparation in the entire fraction has been observed on independent occasions but findings are not consistent. Men presenting at a University hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. Sperm motility in neat semen inversely correlated with sperm DNA fragmentation in the total fraction, but, total count, leukocytes and immature germ cells significantly affected the vital fraction. Sperm DNA fragmentation was observed both in normal and subnormal semen samples, but was significantly different in the total fraction of astheno-, asthenoterato- and oligoteratozoospermic men. After density gradient centrifugation, sperm DNA fragmentation increased significantly in the total but decreased in the vital fraction. Advancing male age significantly influenced damage in the total but not in the vital population. These findings provide opportunities to investigate the significance of the total and the vital fractions both in natural conception and after different assisted reproductive technologies. Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

DNA damage in spermatozoa from infertile men with varicocele evaluated by sperm chromatin dispersion and DBD-FISH.

PubMed

Cortés-Gutiérrez, Elva I; Dávila-Rodríguez, Martha I; Fernández, José Luis; López-Fernández, Carmen; Aragón-Tovar, Anel R; Urbina-Bernal, Luis C; Gosálvez, Jaime

2016-01-01

Evaluation of DNA integrity is an important test, possessing greater diagnostic and prognostic significance for couples requiring assisted reproduction. In this study, we evaluate the levels of DNA damage in infertile patients with varicocele with respect to fertile males by the sperm chromatin dispersion (SCD) test. The presence of DNA breaks in spermatozoa was confirmed by DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). In this study, the frequency of sperm cells with fragmented DNA was studied in a group of 20 infertile patients with varicocele and compared with 20 fertile males. The spermatozoa were processed to classify different levels of DNA fragmentation using the Halosperm(®) kit, an improved SCD test, and DBD-FISH. Patients with varicocele showed 25.54 ± 28.17 % of spermatozoa with fragmented DNA, significantly higher than those of the group of fertile subjects (11.54 ± 3.88 %). The proportion of degraded cells in total sperm cells with fragmented DNA was sixfold higher in the case of patients with varicocele. The presence of DNA breaks in spermatozoa was confirmed by DBD-FISH. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions. Our finding preliminary demonstrated an increase of DNA fragmentation associated to severe sperm damage, in infertile patients with varicocele with respect to fertile males. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions.

Identification of differentially expressed genes in brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae) responding to host plant resistance.

PubMed

Yang, Zhifan; Zhang, Futie; Zhu, Lili; He, Guangcun

2006-02-01

The brown planthopper Nilaparvata lugens Stål is one of the major insect pests of rice Oryza sativa L. The host resistance exhibits profound effects on growth, development and propagation of N. lugens. To investigate the molecular response of N. lugens to host resistance, a cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique was employed to identify the differentially expressed genes in the nymphs feeding on three rice varieties. Of the 2,800 cDNA bands analysed, 54 were up-regulated and seven down-regulated qualitatively in N. lugens when the ingestion sources were changed from susceptible rice plants to resistant ones. Sequence analysis of the differential transcript-derived fragments showed that the genes involved in signalling, stress response, gene expression regulation, detoxification and metabolism were regulated by host resistance. Four of the transcript-derived fragments corresponding to genes encoding for a putative B subunit of phosphatase PP2A, a nemo kinase, a cytochrome P450 monooxygenase and a prolyl endopeptidase were further characterized in detail. Northern blot analysis confirmed that the expression of the four genes was enhanced in N. lugens feeding on resistant rice plants. The roles of these genes in the defensive response of N. lugens to host plant resistance were discussed.

An innovative platform for quick and flexible joining of assorted DNA fragments

DOE PAGES

De Paoli, Henrique Cestari; Tuskan, Gerald A.; Yang, Xiaohan

2016-01-13

Successful synthetic biology efforts rely on conceptual and experimental designs in combination with testing of multi-gene constructs. Despite recent progresses, several limitations still hinder the ability to flexibly assemble and collectively share different types of DNA segments. We describe an advanced system for joining DNA fragments from a universal library that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation (domestication) of fragments in order to work properly. Moreover, we find that this system, which is enhanced by a unique buffer formulation, provides unforeseen capabilities for testing, and sharing, complex multi-gene circuitrymore » assembled from different DNA fragments.« less

Linkage and homology analysis divides the eight genes for the small subunit of petunia ribulose 1,5-bisphosphate carboxylase into three gene families

PubMed Central

Dean, Caroline; van den Elzen, Peter; Tamaki, Stanley; Dunsmuir, Pamela; Bedbrook, John

1985-01-01

Twenty-six λ phage clones with homology to coding sequences of the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase have been isolated from an EMBL3 λ phage bank of Petunia (Mitchell) DNA. Restriction mapping of the phage inserts shows that the clones were obtained from five nonoverlapping regions of petunia DNA that carry seven SSU genes. Comparison of the HindIII genomic fragments of petunia DNA with the HindIII restriction fragments of the isolated phage indicates that petunia nuclear DNA encodes eight SSU genes, seven of which are present in the phage clones. Two incomplete genes, which contain only the 3′ end of an SSU gene, were also found in the phage clones. We demonstrate that the eight SSU genes of petunia can be divided into three gene families based on homology to three petunia cDNA clones. Two gene families contain single SSU genes and the third contains six genes, four of which are closely linked within petunia nuclear DNA. Images PMID:16593584

Presence of a consensus DNA motif at nearby DNA sequence of the mutation susceptible CG nucleotides.

PubMed

Chowdhury, Kaushik; Kumar, Suresh; Sharma, Tanu; Sharma, Ankit; Bhagat, Meenakshi; Kamai, Asangla; Ford, Bridget M; Asthana, Shailendra; Mandal, Chandi C

2018-01-10

Complexity in tissues affected by cancer arises from somatic mutations and epigenetic modifications in the genome. The mutation susceptible hotspots present within the genome indicate a non-random nature and/or a position specific selection of mutation. An association exists between the occurrence of mutations and epigenetic DNA methylation. This study is primarily aimed at determining mutation status, and identifying a signature for predicting mutation prone zones of tumor suppressor (TS) genes. Nearby sequences from the top five positions having a higher mutation frequency in each gene of 42 TS genes were selected from a cosmic database and were considered as mutation prone zones. The conserved motifs present in the mutation prone DNA fragments were identified. Molecular docking studies were done to determine putative interactions between the identified conserved motifs and enzyme methyltransferase DNMT1. Collective analysis of 42 TS genes found GC as the most commonly replaced and AT as the most commonly formed residues after mutation. Analysis of the top 5 mutated positions of each gene (210 DNA segments for 42 TS genes) identified that CG nucleotides of the amino acid codons (e.g., Arginine) are most susceptible to mutation, and found a consensus DNA "T/AGC/GAGGA/TG" sequence present in these mutation prone DNA segments. Similar to TS genes, analysis of 54 oncogenes not only found CG nucleotides of the amino acid Arg as the most susceptible to mutation, but also identified the presence of similar consensus DNA motifs in the mutation prone DNA fragments (270 DNA segments for 54 oncogenes) of oncogenes. Docking studies depicted that, upon binding of DNMT1 methylates to this consensus DNA motif (C residues of CpG islands), mutation was likely to occur. Thus, this study proposes that DNMT1 mediated methylation in chromosomal DNA may decrease if a foreign DNA segment containing this consensus sequence along with CG nucleotides is exogenously introduced to dividing cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Soil pretreatment and fast cell lysis for direct polymerase chain reaction from forest soils for terminal restriction fragment length polymorphism analysis of fungal communities

Treesearch

Fei Cheng; Lin Hou; Keith Woeste; Zhengchun Shang; Xiaobang Peng; Peng Zhao; Shuoxin Zhang

2016-01-01

Humic substances in soil DNA samples can influence the assessment of microbial diversity and community composition. Using multiple steps during or after cell lysis adds expenses, is time-consuming, and causes DNA loss. A pretreatment of soil samples and a single step DNA extraction may improve experimental results. In order to optimize a protocol for obtaining high...

Aqueous trifluorethanol solutions simulate the environment of DNA in the crystalline state.

PubMed

Kypr, J; Chládková, J; Zimulová, M; Vorlícková, M

1999-09-01

We took 28 fragments of DNA whose crystal structures were known and used CD spectroscopy to search for conditions stabilising the crystal structures in solution. All 28 fragments switched into their crystal structures in 60-80% aqueous trifluorethanol (TFE) to indicate that the crystals affected the conformation of DNA like the concentrated TFE. The fragments crystallising in the B-form also underwent cooperative TFE-induced changes that took place within the wide family of B-form structures, suggesting that the aqueous and crystal B-forms differed as well. Spermine and magnesium or calcium cations, which were contained in the crystallisation buffers, promoted or suppressed the TFE-induced changes of several fragments to indicate that the crystallisation agents can decide which of the possible structures is adopted by the DNA fragment in the crystal.

Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis

EPA Science Inventory

A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

DNA methylation and genome rearrangement characteristics of phase change in cultured shoots of Sequoia sempervirens.

PubMed

Huang, Li-Chun; Hsiao, Lin-June; Pu, Szu-Yuan; Kuo, Ching-I; Huang, Bau-Lian; Tseng, Tsung-Che; Huang, Hao-Jen; Chen, Yu-Ting

2012-06-01

Epigenetic machinery regulates the expression of individual genes and plays a crucial role in globally shaping and maintaining developmental patterning. We studied the extent of DNA methylation in the nucleus, mitochondrion and chloroplast in cultured Sequoia sempervirens (coast redwood) adult, juvenile and rejuvenated shoots by measuring the ratio of methylcytosine to total cytosine using high-performance liquid chromatography (HPLC). We also analyzed nuclear DNA (nuDNA) polymorphisms of different shoot types by methylation-sensitive amplified fragment length polymorphism (MSAP) and Southern blot analysis. The extent of nuDNA methylation was greater in the adult vegetative than juvenile and rejuvenated shoots (8% vs 6.5-7.5%). In contrast, the proportion of methylcytosine was higher in mitochondrial DNA (mDNA) of juvenile and rejuvenated shoots than adult shoots (6.6% vs 7.8-8.2%). MSAP and Southern blot analyses identified three MSAP fragments which could be applied as phase-specific molecular markers. We also found nuclear genome and mtDNA rearrangement may be as important as DNA methylation status during the phase change. Our findings strongly suggest that DNA methylation and genome rearrangement may affect the dynamic tissue- and cell type-specific changes that determine the developmental phase of S. sempervirens shoots. Copyright © Physiologia Plantarum 2012.

Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis

PubMed Central

1985-01-01

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full- length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain- specific expression of V kappa Ser-associated phenotypic markers are discussed. PMID:3926938

Charging YOYO-1 on Capillary Wall for Online DNA Intercalation and Integrating This Approach with Multiplex PCR and Bare Narrow Capillary–Hydrodynamic Chromatography for Online DNA Analysis

PubMed Central

2016-01-01

Multiplex polymerase chain reaction (PCR) has been widely utilized for high-throughput pathogen identification. Often, a dye is used to intercalate the amplified DNA fragments, and identifications of the pathogens are carried out by DNA melting curve analysis or gel electrophoresis. Integrating DNA amplification and identification is a logic path toward maximizing the benefit of multiplex PCR. Although PCR and gel electrophoresis have been integrated, replenishing the gels after each run is tedious and time-consuming. In this technical note, we develop an approach to address this issue. We perform multiplex PCR inside a capillary, transfer the amplified fragments to a bare narrow capillary, and measure their lengths online using bare narrow capillary–hydrodynamic chromatography (BaNC-HDC), a new technique recently developed in our laboratory for free-solution DNA separation. To intercalate the DNA with YOYO-1 (a fluorescent dye) for BaNC-HDC, we flush the capillary column with a YOYO-1 solution; positively charged YOYO-1 is adsorbed (or charged) onto the negatively charged capillary wall. As DNA molecules are driven down the column for separation, they react with the YOYO-1 stored on the capillary wall and are online-intercalated with the dye. With a single YOYO-1 charging, the column can be used for more than 40 runs, although the fluorescence signal intensities of the DNA peaks decrease gradually. Although the dye-DNA intercalation occurs during the separation, it does not affect the retention times, separation efficiencies, or resolutions. PMID:25555111

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina

PubMed Central

Marjanović, Damir; Hadžić Metjahić, Negra; Čakar, Jasmina; Džehverović, Mirela; Dogan, Serkan; Ferić, Elma; Džijan, Snježana; Škaro, Vedrana; Projić, Petar; Madžar, Tomislav; Rod, Eduard; Primorac, Dragan

2015-01-01

Aim To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Methods Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. Results A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. Conclusion DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them. PMID:26088850

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

PubMed

Marjanović, Damir; Hadžić Metjahić, Negra; Čakar, Jasmina; Džehverović, Mirela; Dogan, Serkan; Ferić, Elma; Džijan, Snježana; Škaro, Vedrana; Projić, Petar; Madžar, Tomislav; Rod, Eduard; Primorac, Dragan

2015-06-01

To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons' relatives and collect referent samples from them.

FragIdent--automatic identification and characterisation of cDNA-fragments.

PubMed

Seelow, Dominik; Goehler, Heike; Hoffmann, Katrin

2009-03-02

Many genetic studies and functional assays are based on cDNA fragments. After the generation of cDNA fragments from an mRNA sample, their content is at first unknown and must be assigned by sequencing reactions or hybridisation experiments. Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available. Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs) within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones. We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at http://compbio.charite.de/genetik/FragIdent/.

Agarose gel electrophoresis for the separation of DNA fragments.

PubMed

Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

2012-04-20

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight(3). The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along(4). The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation(5); 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate concentration for their needs Prepare an agarose gel for electrophoresis of DNA samples Set up the gel electrophoresis apparatus and power supply Select an appropriate voltage for the separation of DNA fragments Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands Determine the sizes of separated DNA fragments.

Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

NASA Astrophysics Data System (ADS)

Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

2016-03-01

We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

Raman-based system for DNA sequencing-mapping and other separations

DOEpatents

Vo-Dinh, Tuan

1994-01-01

DNA sequencing and mapping are performed by using a Raman spectrometer with a surface enhanced Raman scattering (SERS) substrate to enhance the Raman signal. A SERS label is attached to a DNA fragment and then analyzed with the Raman spectrometer to identify the DNA fragment according to characteristics of the Raman spectrum generated.

Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage.

PubMed

Xavier, Miguel J; Nixon, Brett; Roman, Shaun D; Aitken, Robert John

2018-01-01

Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.

Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

PubMed Central

Pena, S D; Barreto, G; Vago, A R; De Marco, L; Reinach, F C; Dias Neto, E; Simpson, A J

1994-01-01

Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA. Images PMID:8127912

Current genetic methodologies in the identification of disaster victims and in forensic analysis.

PubMed

Ziętkiewicz, Ewa; Witt, Magdalena; Daca, Patrycja; Zebracka-Gala, Jadwiga; Goniewicz, Mariusz; Jarząb, Barbara; Witt, Michał

2012-02-01

This review presents the basic problems and currently available molecular techniques used for genetic profiling in disaster victim identification (DVI). The environmental conditions of a mass disaster often result in severe fragmentation, decomposition and intermixing of the remains of victims. In such cases, traditional identification based on the anthropological and physical characteristics of the victims is frequently inconclusive. This is the reason why DNA profiling became the gold standard for victim identification in mass-casualty incidents (MCIs) or any forensic cases where human remains are highly fragmented and/or degraded beyond recognition. The review provides general information about the sources of genetic material for DNA profiling, the genetic markers routinely used during genetic profiling (STR markers, mtDNA and single-nucleotide polymorphisms [SNP]) and the basic statistical approaches used in DNA-based disaster victim identification. Automated technological platforms that allow the simultaneous analysis of a multitude of genetic markers used in genetic identification (oligonucleotide microarray techniques and next-generation sequencing) are also presented. Forensic and population databases containing information on human variability, routinely used for statistical analyses, are discussed. The final part of this review is focused on recent developments, which offer particularly promising tools for forensic applications (mRNA analysis, transcriptome variation in individuals/populations and genetic profiling of specific cells separated from mixtures).

Sequences in the intergenic spacer influence RNA Pol I transcription from the human rRNA promoter

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

Li, W.M.; Sylvester, J.E.

1994-09-01

In most eucaryotic species, ribosomal genes are tandemly repeated about 100-5000 times per haploid genome. The 43 Kb human rDNA repeat consists of a 13 Kb coding region for the 18S, 5.8S, 28S ribosomal RNAs (rRNAs) and transcribed spacers separated by a 30 Kb intergenic spacer. For species such as frog, mouse and rat, sequences in the intergenic spacer other than the gene promoter have been shown to modulate transcription of the ribosomal gene. These sequences are spacer promoters, enhancers and the terminator for spacer transcription. We are addressing whether the human ribosomal gene promoter is similarly influenced. In-vitro transcriptionmore » run-off assays have revealed that the 4.5 kb region (CBE), directly upstream of the gene promoter, has cis-stimulation and trans-competition properties. This suggests that the CBE fragment contains an enhancer(s) for ribosomal gene transcription. Further experiments have shown that a fragment ({approximately}1.6 kb) within the CBE fragment also has trans-competition function. Deletion subclones of this region are being tested to delineate the exact sequences responsible for these modulating activities. Previous sequence analysis and functional studies have revealed that CBE contains regions of DNA capable of adopting alternative structures such as bent DNA, Z-DNA, and triple-stranded DNA. Whether these structures are required for modulating transcription remains to be determined as does the specific DNA-protein interaction involved.« less

Evaluation of methods for the extraction of DNA from drinking water distribution system biofilms.

PubMed

Hwang, Chiachi; Ling, Fangqiong; Andersen, Gary L; LeChevallier, Mark W; Liu, Wen-Tso

2012-01-01

While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories.

Cloning of human prourokinase cDNA without the signal peptide and expression in Escherichia coli.

PubMed

Hu, B; Li, J; Yu, W; Fang, J

1993-01-01

Human prourokinase (pro-UK) cDNA without the signal peptide was obtained using synthetic oligonucleotide and DNA recombination techniques and was successfully expressed in E. coli. The plasmid pMMUK which contained pro-UK cDNA (including both the entire coding sequence and the sequence for signal peptide) was digested with Hind III and PstI, so that the N-terminal 371-bp fragment could be recovered. A 304-bp fragment was collected from the 371-bp fragment after partial digestion with Fnu4HI in order to remove the signal peptide sequence. An intermediate plasmid was formed after this 304-bp fragment and the synthetic oligonucleotide was ligated with pUC18. Correctness of the ligation was confirmed by enzyme digestion and sequencing. By joining the PstI-PstI fragment of pro-UK to the plasmid we obtained the final plasmid which contained the entire coding sequence of pro-UK without the signal peptide. The coding sequence with correct orientation was inserted into pBV220 under the control of the temperature-induced promoter PRPL, and mature pro-UK was expressed in E. coli at 42 degrees C. Both sonicated supernatant and inclusion bodies of the bacterial host JM101 showed positive results by ELISA and FAPA assays. After renaturation, the biological activity of the expressed product was increased from 500-1000IU/L to about 60,000IU/L. The bacterial pro-UK showed a molecular weight of about 47,000 daltons by Western blot analysis. It can be completely inhibited by UK antiserum but not by t-PA antiserum nor by normal rabbit serum.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

PubMed

Liu, Chao; Wang, Mingbo; Wang, Lingli; Guo, Qigao; Liang, Guolu

2018-04-24

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

PubMed Central

Winnacker, E L

1975-01-01

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA. PMID:1117487

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks*

PubMed Central

Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

2013-01-01

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments.

PubMed Central

Gray, A J; Beecher, D E; Olson, M V

1984-01-01

A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments. Images PMID:6320097

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways.

PubMed

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T; Gasparutto, Didier; Geacintov, Nicholas E; Saparbaev, Murat

2015-06-05

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways*

PubMed Central

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T.; Gasparutto, Didier; Geacintov, Nicholas E.; Saparbaev, Murat

2015-01-01

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506–2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. PMID:25903131

Analysis of DNA from post-blast pipe bomb fragments for identification and determination of ancestry.

PubMed

Tasker, Esiri; LaRue, Bobby; Beherec, Charity; Gangitano, David; Hughes-Stamm, Sheree

2017-05-01

Improvised explosive devices (IEDs) such as pipe bombs are weapons used to detrimentally affect people and communities. A readily accessible brand of exploding targets called Tannerite® has been identified as a potential material for abuse as an explosive in pipe bombs. The ability to recover and genotype DNA from such weapons may be vital in the effort to identify suspects associated with these devices. While it is possible to recover DNA from post-blast fragments using short tandem repeat markers (STRs), genotyping success can be negatively affected by low quantities of DNA, degradation, and/or PCR inhibitors. Alternative markers such as insertion/null (INNULs) and single nucleotide polymorphisms (SNPs) are bi-allelic genetic markers that are shorter genomic targets than STRs for amplification, which are more likely to resist degradation. In this study, we constructed pipe bombs that were spiked with known amounts of biological material to: 1) recover "touch" DNA from the surface of the device, and 2) recover traces of blood from the ends of wires (simulated finger prick). The bombs were detonated with the binary explosive Tannerite® using double-base smokeless powder to initiate the reaction. DNA extracted from the post-blast fragments was quantified with the Quantifiler® Trio DNA Quantification Kit. STR analysis was conducted using the GlobalFiler® Amplification Kit, INNULs were amplified using an early-access version of the InnoTyper™ 21 Kit, and SNP analysis via massively parallel sequencing (MPS) was performed using the HID-Ion Ampliseq™ Identity and Ancestry panels using the Ion Chef and Ion PGM sequencing system. The results of this study showed that INNUL markers resulted in the most complete genetic profiles when compared to STR and SNP profiles. The random match probabilities calculated for samples using INNULs were lower than with STRs when less than 14 STR alleles were reported. These results suggest that INNUL analysis may be well suited for low-template and/or degraded DNA samples, and may be used to supplement incomplete or failed STR analysis. Human identification using SNP analysis via MPS showed variable success with low-level post-blast samples in this study (<150pg). While neat DNA samples (6μL input as recommended) resulted in <50% of SNP calls, samples that were concentrated from 15μL to 6μL (15μL was added for STR and INNUL typing) resulted in more complete SNP profiles. Five out of six blood samples recovered from the wires attached to the pipe-bombs resulted in the correct ancestry predictions. Copyright © 2017 Elsevier B.V. All rights reserved.

Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA.

PubMed

Sha, Y; Melcher, U; Davis, R E; Fletcher, J

1995-11-01

Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by SVTS2.

Target capture enrichment of nuclear SNP markers for massively parallel sequencing of degraded and mixed samples.

PubMed

Bose, Nikhil; Carlberg, Katie; Sensabaugh, George; Erlich, Henry; Calloway, Cassandra

2018-05-01

DNA from biological forensic samples can be highly fragmented and present in limited quantity. When DNA is highly fragmented, conventional PCR based Short Tandem Repeat (STR) analysis may fail as primer binding sites may not be present on a single template molecule. Single Nucleotide Polymorphisms (SNPs) can serve as an alternative type of genetic marker for analysis of degraded samples because the targeted variation is a single base. However, conventional PCR based SNP analysis methods still require intact primer binding sites for target amplification. Recently, probe capture methods for targeted enrichment have shown success in recovering degraded DNA as well as DNA from ancient bone samples using next-generation sequencing (NGS) technologies. The goal of this study was to design and test a probe capture assay targeting forensically relevant nuclear SNP markers for clonal and massively parallel sequencing (MPS) of degraded and limited DNA samples as well as mixtures. A set of 411 polymorphic markers totaling 451 nuclear SNPs (375 SNPs and 36 microhaplotype markers) was selected for the custom probe capture panel. The SNP markers were selected for a broad range of forensic applications including human individual identification, kinship, and lineage analysis as well as for mixture analysis. Performance of the custom SNP probe capture NGS assay was characterized by analyzing read depth and heterozygote allele balance across 15 samples at 25 ng input DNA. Performance thresholds were established based on read depth ≥500X and heterozygote allele balance within ±10% deviation from 50:50, which was observed for 426 out of 451 SNPs. These 426 SNPs were analyzed in size selected samples (at ≤75 bp, ≤100 bp, ≤150 bp, ≤200 bp, and ≤250 bp) as well as mock degraded samples fragmented to an average of 150 bp. Samples selected for ≤75 bp exhibited 99-100% reportable SNPs across varied DNA amounts and as low as 0.5 ng. Mock degraded samples at 1 ng and 10 ng exhibited >90% reportable SNPs. Finally, two-person male-male mixtures were tested at 10 ng in contributor varying ratios. Overall, 85-100% of alleles unique to the minor contributor were observed at all mixture ratios. Results from these studies using the SNP probe capture NGS system demonstrates proof of concept for application to forensically relevant degraded and mixed DNA samples. Copyright © 2018 Elsevier B.V. All rights reserved.

A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.

PubMed

Shinozuka, Hiroshi; Cogan, Noel O I; Shinozuka, Maiko; Marshall, Alexis; Kay, Pippa; Lin, Yi-Han; Spangenberg, German C; Forster, John W

2015-04-11

Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.

Genome-wide analysis of DNA methylation in five tissues of sika deer (Cervus nippon).

PubMed

Yang, Chun; Zhang, Yan; Liu, Wenyuan; Lu, Xiao; Li, Chunyi

2018-03-01

DNA methylation plays an important role in regulating gene expression during tissue development and differentiation in eukaryotes. In contrast to domestic animals, epigenetic studies have been seldom conducted in wild animals. In the present study, we conducted the genome-wide profiling of DNA methylation for five tissues of sika deer using the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique. Overall, a total of 104,131 fragments were amplified including 41,951 methylated fragments using 32 pairs of selected primers. The average incidence of DNA methylation was approximately 38.18% in muscle, 40.32% in heart, 41.86% in liver, 41.20% in lung, and 41.68% in kidney, respectively. Also, the significant differences of the DNA methylation levels were found between the different tissue types (P<0.05), which indicates that the differences of genome-wide DNA methylation levels may be related to gene expression during tissue development and differentiation. In addition, 37 tissue-specific differentially methylated regions (T-DMRs) were identified and recovered by MSAP in five tissues, and were further confirmed by Southern blot analysis. Our study presents the first look at the T-DMRs in sika deer and represents an initial step towards understanding of epigenetic regulatory mechanism underlying tissue development and differentiation in sika deer. Copyright © 2017. Published by Elsevier B.V.

Effects of Spirulina platensis on DNA damage and chromosomal aberration against cadmium chloride-induced genotoxicity in rats.

PubMed

Aly, Fayza M; Kotb, Ahmed M; Hammad, Seddik

2018-04-01

Todays, bioactive compounds extracted from Spirulina platensis have been intensively studied for their therapeutical values. Therefore, in the present study, we aimed to evaluate the effects of S. platensis extract on DNA damage and chromosomal aberrations induced by cadmium in rats. Four groups of male albino rats (n = 7 rats) were used. The first group served as a control group and received distilled water. The second group was exposed intraperitoneally to cadmium chloride (CdCl 2 ) (3.5 mg/kg body weight dissolved in 2 ml distilled water). The third group included the rats that were orally treated with S. platensis extract (1 g/kg dissolved in 5 ml distilled water, every other day for 30 days). The fourth group included the rats that were intraperitoneally and orally exposed to cadmium chloride and S. platensis, respectively. The experiment in all groups was extended for 60 days. The results of cadmium-mediated toxicity revealed significant genetic effects (DNA fragmentation, deletion or disappearance of some base pairs of DNA, and appearance of few base pairs according to ISSR-PCR analysis). Moreover, chromosomes showed structural aberrations such as reduction of chromosomal number, chromosomal ring, chromatid deletions, chromosomal fragmentations, and dicentric chromosomes. Surprisingly, S. platensis extract plus CdCl 2 -treated group showed less genetic effects compared with CdCl 2 alone. Further, S. platensis extract upon CdCl 2 toxicity was associated with less chromosomal aberration number and nearly normal appearance of DNA fragments as indicated by the bone marrow and ISSR-PCR analysis, respectively. In conclusion, the present novel study showed that co-treatment with S. platensis extract could reduce the genotoxic effects of CdCl 2 in rats.

Molecular identification and phylogenetic study of Demodex caprae.

PubMed

Zhao, Ya-E; Cheng, Juan; Hu, Li; Ma, Jun-Xian

2014-10-01

The DNA barcode has been widely used in species identification and phylogenetic analysis since 2003, but there have been no reports in Demodex. In this study, to obtain an appropriate DNA barcode for Demodex, molecular identification of Demodex caprae based on mitochondrial cox1 was conducted. Firstly, individual adults and eggs of D. caprae were obtained for genomic DNA (gDNA) extraction; Secondly, mitochondrial cox1 fragment was amplified, cloned, and sequenced; Thirdly, cox1 fragments of D. caprae were aligned with those of other Demodex retrieved from GenBank; Finally, the intra- and inter-specific divergences were computed and the phylogenetic trees were reconstructed to analyze phylogenetic relationship in Demodex. Results obtained from seven 429-bp fragments of D. caprae showed that sequence identities were above 99.1% among three adults and four eggs. The intraspecific divergences in D. caprae, Demodex folliculorum, Demodex brevis, and Demodex canis were 0.0-0.9, 0.5-0.9, 0.0-0.2, and 0.0-0.5%, respectively, while the interspecific divergences between D. caprae and D. folliculorum, D. canis, and D. brevis were 20.3-20.9, 21.8-23.0, and 25.0-25.3, respectively. The interspecific divergences were 10 times higher than intraspecific ones, indicating considerable barcoding gap. Furthermore, the phylogenetic trees showed that four Demodex species gathered separately, representing independent species; and Demodex folliculorum gathered with canine Demodex, D. caprae, and D. brevis in sequence. In conclusion, the selected 429-bp mitochondrial cox1 gene is an appropriate DNA barcode for molecular classification, identification, and phylogenetic analysis of Demodex. D. caprae is an independent species and D. folliculorum is closer to D. canis than to D. caprae or D. brevis.

Band-broadening suppressed effect in long turned geometry channel and high-sensitive analysis of DNA sample by using floating electrokinetic supercharging on a microchip.

PubMed

Xu, Zhongqi; Murata, Kenji; Arai, Akihiro; Hirokawa, Takeshi

2010-03-12

A featured microchip owning three big reservoirs and long turned geometry channel was designed to improve the detection limit of DNA fragments by using floating electrokinetic supercharging (FEKS) method. The novel design matches the FEKS preconcentration needs of a large sample volume introduction with electrokinetic injection (EKI), as well as long duration of isotachophoresis (ITP) process to enrich low concentration sample. In the curved channel [ approximately 45.6 mm long between port 1 (P1) and the intersection point of two channels], EKI and ITP were performed while the side port 3 (P3) was electrically floated. The turn-induced band broadening with or without ITP process was investigated by a computer simulation (using CFD-ACE+ software) when the analytes traveling through the U-shaped geometry. It was found that the channel curvature determined the extent of band broadening, however, which could be effectively eliminated by the way of ITP. After the ITP-stacked zones passed the intersection point from P1, they were rapidly destacked for separation and detection from ITP to zone electrophoresis by using leading ions from P3. The FEKS carried on the novel chip successfully contributed to higher sensitivities of DNA fragments in comparison with our previous results realized on either a single channel or a cross microchip. The analysis of low concentration 50 bp DNA step ladders (0.23 mugml after 1500-fold diluted) was achieved with normal UV detection at 260 nm. The obtained limit of detections (LODs) were on average 100 times better than using conventional pinched injection, down to several ngml for individual DNA fragment.

[Microbial community structure in bio-ceramics and biological activated carbon analyzed by PCR-SSCP technique].

PubMed

Liu, Xiao-Lin; Liu, Wen-Jun

2007-04-01

Analyses of microbial community structure in bio-ceramics (BC) and biological activated carbon (BAC), which widely used in drinking water treatment were performed by polymerase-chain-reaction-single-strand-conformation-polymorphism (PCR-SSCP) targeted eubacterial 16S ribosomal RNA gene. Microorganisms on bio-ceramics and biological activated carbon were detached by ultrasonic, culturing on R2A and LB agar, respectively, followed by genome DNA extracting. Results show that larger than 10 kb genome DNA could be extracted from all the samples except the BAC samples processed by ultrasonic. However, quantities of the extracted DNA were different. 408 bp gene fragments were observed after PCR using the extracted genome DNA as templates. These gene fragments were digested with lambda exonuclease followed by SSCP electrophoresis. Same SSCP profiles were observed between ultrasonic eluting, R2A and LB agar culturing. The identity of the segment from bio-ceramics with uncultured Pseudomonas sp. Clone FTL201 16S rDNA (GenBank, AF509293.1) fragment was 92%, and identities of the two segments from BAC with Bacillus sp. JH19 16S rDNA (GenBank , DQ232748.1) fragment and Bacterium VA-S-11 16S rDNA (GenBank, AY395279.1) fragment were 100% and 99%, respectively.

Raman-based system for DNA sequencing-mapping and other separations

DOEpatents

Vo-Dinh, T.

1994-04-26

DNA sequencing and mapping are performed by using a Raman spectrometer with a surface enhanced Raman scattering (SERS) substrate to enhance the Raman signal. A SERS label is attached to a DNA fragment and then analyzed with the Raman spectrometer to identify the DNA fragment according to characteristics of the Raman spectrum generated. 11 figures.

High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

NASA Astrophysics Data System (ADS)

Miller, Cynthia K.; Temin, Howard M.

1983-05-01

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

PubMed

Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

2015-12-01

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Laser mass spectrometry for DNA fingerprinting for forensic applications

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

Chen, C.H.; Tang, K.; Taranenko, N.I.

The application of DNA fingerprinting has become very broad in forensic analysis, patient identification, diagnostic medicine, and wildlife poaching, since every individual`s DNA structure is identical within all tissues of their body. DNA fingerprinting was initiated by the use of restriction fragment length polymorphisms (RFLP). In 1987, Nakamura et al. found that a variable number of tandem repeats (VNTR) often occurred in the alleles. The probability of different individuals having the same number of tandem repeats in several different alleles is very low. Thus, the identification of VNTR from genomic DNA became a very reliable method for identification of individuals.more » DNA fingerprinting is a reliable tool for forensic analysis. In DNA fingerprinting, knowledge of the sequence of tandem repeats and restriction endonuclease sites can provide the basis for identification. The major steps for conventional DNA fingerprinting include (1) specimen processing (2) amplification of selected DNA segments by PCR, and (3) gel electrophoresis to do the final DNA analysis. In this work we propose to use laser desorption mass spectrometry for fast DNA fingerprinting. The process and advantages are discussed.« less

Evaluation of DNA extraction methods for the analysis of microbial community in biological activated carbon.

PubMed

Zheng, Lu; Gao, Naiyun; Deng, Yang

2012-01-01

It is difficult to isolate DNA from biological activated carbon (BAC) samples used in water treatment plants, owing to the scarcity of microorganisms in BAC samples. The aim of this study was to identify DNA extraction methods suitable for a long-term, comprehensive ecological analysis of BAC microbial communities. To identify a procedure that can produce high molecular weight DNA, maximizes detectable diversity and is relatively free from contaminants, the microwave extraction method, the cetyltrimethylammonium bromide (CTAB) extraction method, a commercial DNA extraction kit, and the ultrasonic extraction method were used for the extraction of DNA from BAC samples. Spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) analysis were conducted to compare the yield and quality of DNA obtained using these methods. The results showed that the CTAB method produce the highest yield and genetic diversity of DNA from BAC samples, but DNA purity was slightly less than that obtained with the DNA extraction-kit method. This study provides a theoretical basis for establishing and selecting DNA extraction methods for BAC samples.

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

PubMed

Razvi, F; Gargiulo, G; Worcel, A

1983-08-01

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

An anti-DNA antibody prefers damaged dsDNA over native.

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2017-01-01

DNA-protein interactions, including DNA-antibody complexes, have both fundamental and practical significance. In particular, antibodies against double-stranded DNA play an important role in the pathogenesis of autoimmune diseases. Elucidation of structural mechanisms of an antigen recognition and interaction of anti-DNA antibodies provides a basis for understanding the role of DNA-containing immune complexes in human pathologies and for new treatments. Here we used Molecular Dynamic simulations of bimolecular complexes of a segment of dsDNA with a monoclonal anti-DNA antibody's Fab-fragment to obtain detailed structural and physical characteristics of the dynamic intermolecular interactions. Using a computationally modified crystal structure of a Fab-DNA complex (PDB: 3VW3), we studied in silico equilibrium Molecular Dynamics of the Fab-fragment associated with two homologous dsDNA fragments, containing or not containing dimerized thymine, a product of DNA photodamage. The Fab-fragment interactions with the thymine dimer-containing DNA was thermodynamically more stable than with the native DNA. The amino acid residues constituting a paratope and the complementary nucleotide epitopes for both Fab-DNA constructs were identified. Stacking and electrostatic interactions were shown to play the main role in the antibody-dsDNA contacts, while hydrogen bonds were less significant. The aggregate of data show that the chemically modified dsDNA (containing a covalent thymine dimer) has a higher affinity toward the antibody and forms a stronger immune complex. These findings provide a mechanistic insight into formation and properties of the pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus, associated with skin photosensibilization and DNA photodamage.

Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.

PubMed

Berger, Cordula; Parson, Walther

2009-06-01

The degradation state of some biological traces recovered from the crime scene requires the amplification of very short fragments to attain a useful mitochondrial (mt)DNA sequence. We have previously introduced two mini-multiplex assays that amplify 10 overlapping control region (CR) fragments in two separate multiplex PCRs, which brought successful CR consensus sequences from even highly degraded DNA extracts. This procedure requires a total of 20 sequencing reactions per sample, which is laborious and cost intensive. For only moderately degraded samples that we encounter more frequently with typical mtDNA casework material, we developed two new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded CR consensus sequence. We used a preceding mtDNA quantitation step by real-time PCR with two different target fragments (143 and 283 bp) that roughly correspond to the average fragment sizes of the different multiplex approaches to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes with respect to quality of the results and required costs.

Determining Phylogenetic Relationships Among Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers.

PubMed

Haider, Nadia

2017-01-01

Investigation of genetic variation and phylogenetic relationships among date palm (Phoenix dactylifera L.) cultivars is useful for their conservation and genetic improvement. Various molecular markers such as restriction fragment length polymorphisms (RFLPs), simple sequence repeat (SSR), representational difference analysis (RDA), and amplified fragment length polymorphism (AFLP) have been developed to molecularly characterize date palm cultivars. PCR-based markers random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) are powerful tools to determine the relatedness of date palm cultivars that are difficult to distinguish morphologically. In this chapter, the principles, materials, and methods of RAPD and ISSR techniques are presented. Analysis of data generated from these two techniques and the use of these data to reveal phylogenetic relationships among date palm cultivars are also discussed.

Characterization of Betula platyphylla gene transcripts associated with early development of male inflorescence.

PubMed

Xing, Lei; Liu, Xue-Mei

2012-02-01

Birch (Betula platyphylla), an eminent tree species in Northeast and Inner Mongolia of China, has been widely used in architecture, furniture, and paper making in recent years. In order to retrieve genes involved in early development of B. platyphylla male inflorescence, RNA populations extracted from early and late developmental stage were analyzed by cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) technique. Following amplification of 256 pairs of primer combinations, ~7000 fragments were generated, of which 350 transcripts expressing more in early stage than late. Of 350 specific transcripts, 198 clear and reproducible electrophoresis bands were retrieved and sequenced successfully, 74 of them (37%) showing significant homologies to known genes after GO annotation. Majority of the predicted gene products were involved in metabolism (24.56%), cellular process (27.19%), response to stimulus (11.4%) and cell growth (8.7%). Transcripts ME56, ME108, ME206 and ME310, representing metabolism, cellular process, response to stimulus and cell growth, respectively, were selected for further study to validate cDNA-AFLP expression patterns via RT-PCR and qRT-PCR analysis. RT-PCR and qRT-PCR expression pattern results were consistent with cDNA-AFLP analysis results.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

PubMed Central

Soares, Vítor Yamashiro Rocha; da Silva, Jailthon Carlos; da Silva, Kleverton Ribeiro; Cruz, Maria do Socorro Pires e; Santos, Marcos Pérsio Dantas; Ribolla, Paulo Eduardo Martins; Alonso, Diego Peres; Coelho, Luiz Felipe Leomil; Costa, Dorcas Lamounier; Costa, Carlos Henrique Nery

2014-01-01

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA. PMID:24821056

In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

PubMed Central

Shafiu Kamba, Abdullahi; Ismail, Maznah; Tengku Ibrahim, Tengku Azmi; Zakaria, Zuki Abu Bakar; Hassan Gusau, Lawal

2014-01-01

Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system. PMID:25028650

Process to Selectively Distinguish Viable from Non-Viable Bacterial Cells

NASA Technical Reports Server (NTRS)

LaDuc, Myron T.; Bernardini, Jame N.; Stam, Christina N.

2010-01-01

The combination of ethidium monoazide (EMA) and post-fragmentation, randomly primed DNA amplification technologies will enhance the analytical capability to discern viable from non-viable bacterial cells in spacecraft-related samples. Intercalating agents have been widely used since the inception of molecular biology to stain and visualize nucleic acids. Only recently, intercalating agents such as EMA have been exploited to selectively distinguish viable from dead bacterial cells. Intercalating dyes can only penetrate the membranes of dead cells. Once through the membrane and actually inside the cell, they intercalate DNA and, upon photolysis with visible light, produce stable DNA monoadducts. Once the DNA is crosslinked, it becomes insoluble and unable to be fragmented for post-fragmentation, randomly primed DNA library formation. Viable organisms DNA remains unaffected by the intercalating agents, allowing for amplification via post-fragmentation, randomly primed technologies. This results in the ability to carry out downstream nucleic acid-based analyses on viable microbes to the exclusion of all non-viable cells.

Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes

PubMed Central

O’Farrell, Katrina A.; Janssen, Peter H.

1999-01-01

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil. PMID:10473454

A low-cost efficient multiplex PCR for prenatal sex determination in bovine fetus using free fetal DNA in maternal plasma.

PubMed

Davoudi, Arash; Seighalani, Ramin; Aleyasin, Seyed Ahmad; Tarang, Alireza; Salehi, Abdolreza Salehi; Tahmoressi, Farideh

2012-04-01

In order to establish a reliable non-invasive method for sex determination in a bovine fetus in a routine setting, the possibility of identifying specific sequence in the fetal X and Y-chromosomes has been evaluated in maternal plasma using conventional multiplex polymerase chain reaction (PCR) analysis. The aim of this study was to provide a rapid and reliable method for sexing bovine fetuses. In this experimental study, peripheral blood samples were taken from 38 pregnant heifers with 8 to 38 weeks of gestation. DNA template was extracted by phenol-chloroform method from 350 µl maternal plasma. Two primer pairs for bovine amelogenin gene (bAML) and BC1.2 were used to amplify fragments from X and Y chromosomes. A multiplex PCR reaction has been optimized for amplification of 467 bp and 341 bp fragments from X and Y bAML gene and a 190 bp fragment from BC1.2 related to Y chromosome. The 467 bp fragment was observed in all 38 samples. Both 341 and 190 bp fragments were detected only in 24 plasma samples from male calves. The sensitivity and specificity of test were 100% with no false negative or false positive results. The results showed that phenol-chloroform method is a simple and suitable method for isolation of fetal DNA in maternal plasma. The multiplex PCR method is an available non-invasive approach which is cost efficient and reliable for sexing bovine fetuses.

Typing single polymorphic nucleotides in mitochondrial DNA as a way to access Middle Pleistocene DNA

PubMed Central

Valdiosera, Cristina; García, Nuria; Dalén, Love; Smith, Colin; Kahlke, Ralf-Dietrich; Lidén, Kerstin; Angerbjörn, Anders; Arsuaga, Juan Luis; Götherström, Anders

2006-01-01

In this study, we have used a technique designed to target short fragments containing informative mitochondrial substitutions to extend the temporal limits of DNA recovery and study the molecular phylogeny of Ursus deningeri. We present a cladistic analysis using DNA recovered from 400 kyr old U. deningeri remains, which demonstrates U. deningeri's relation to Ursus spelaeus. This study extends the limits of recovery from skeletal remains by almost 300 kyr. Plant material from permafrost environments has yielded DNA of this age in earlier studies, and our data suggest that DNA in teeth from cave environments may be equally well preserved. PMID:17148299

Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility.

PubMed

Boe-Hansen, G B; Christensen, P; Vibjerg, D; Nielsen, M B F; Hedeboe, A M

2008-04-01

Extended semen doses from some boars used for AI have been shown to develop high levels of sperm DNA fragmentation during storage. Studies in other animals and humans have shown that if DNA damage is present in a certain percentage of the sperm cells the fertility potential of the semen sample is reduced. The objectives of the present study was to determine the relationship between sperm DNA fragmentation measured using the sperm chromatin structure assay (SCSA) in extended stored semen and field fertility in the boar. Three ejaculates from each of 145 boars were collected. Preparation of the semen doses included dilution with an EDTA extender and storage for up to 72 h post collection. The semen doses were assessed using flow cytometric methods for the percentage of viable sperm (PI/SYBR-14) and sperm DNA fragmentation (SCSA) at 0, 24, 48, and 72 h. A total of 3276 experimental inseminations in Danish breeding herds were conducted. The results showed that for 11 (7.6%) of the boars at least one of the three samples showed a value of DNA fragmentation index (DFI) above 20% within the storage period. Total number of piglets born (litter size) for Hampshire, Landrace and Danish Large White boars was, respectively, 0.5, 0.7 and 0.9 piglets smaller per litter when DFI values were above 2.1% as opposed to below this value. In conclusion the SCSA technique appears to be able to identify individuals with lower fertility with respect to litter size, and could in the future be implemented by the pig industry after a cost-benefit analysis.

Differential Nuclear and Mitochondrial DNA Preservation in Post-Mortem Teeth with Implications for Forensic and Ancient DNA Studies

PubMed Central

Higgins, Denice; Rohrlach, Adam B.; Kaidonis, John; Townsend, Grant; Austin, Jeremy J.

2015-01-01

Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard extraction methods, without the need for specialised equipment or large-volume demineralisation steps. PMID:25992635

Chromatin conformation in living cells: support for a zig-zag model of the 30 nm chromatin fiber

NASA Technical Reports Server (NTRS)

Rydberg, B.; Holley, W. R.; Mian, I. S.; Chatterjee, A.

1998-01-01

A new method was used to probe the conformation of chromatin in living mammalian cells. The method employs ionizing radiation and is based on the concept that such radiation induces correlated breaks in DNA strands that are in spatial proximity. Human dermal fibroblasts in G0 phase of the cell cycle and Chinese hamster ovary cells in mitosis were irradiated by X-rays or accelerated ions. Following lysis of the cells, DNA fragments induced by correlated breaks were end-labeled and separated according to size on denaturing polyacrylamide gels. A characteristic peak was obtained for a fragment size of 78 bases, which is the size that corresponds to one turn of DNA around the nucleosome. Additional peaks between 175 and 450 bases reflect the relative position of nearest-neighbor nucleosomes. Theoretical calculations that simulate the indirect and direct effect of radiation on DNA demonstrate that the fragment size distributions are closely related to the chromatin structure model used. Comparison of the experimental data with theoretical results support a zig-zag model of the chromatin fiber rather than a simple helical model. Thus, radiation-induced damage analysis can provide information on chromatin structure in the living cell. Copyright 1998 Academic Press.

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

PubMed

Warwicker, J; Ollis, D; Richards, F M; Steitz, T A

1985-12-05

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: A Design of Experiments approach

PubMed Central

Sun, Mingyun; Lin, Jennifer S.

2012-01-01

Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses (HECs) with average molar masses of ~27 kDa and ~1 MDa were blended with a second class of polymer, high-molar mass (~7 MDa) linear polyacrylamide (LPA). Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1 Kb DNA extension ladder (200 bp to 40,000 bp) was completed in 2 minutes. An orthogonal Design of Experiments (DOE) was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 kbp and 10 kbp, and large dsDNA fragments above 10 kbp. PMID:22009451

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders.

PubMed

Pérez-Llano, Begoña; Enciso, María; García-Casado, Pedro; Sala, Rubén; Gosálvez, Jaime

2006-12-01

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis.

PubMed

Nezis, Ioannis P; Shravage, Bhupendra V; Sagona, Antonia P; Lamark, Trond; Bjørkøy, Geir; Johansen, Terje; Rusten, Tor Erik; Brech, Andreas; Baehrecke, Eric H; Stenmark, Harald

2010-08-23

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.

Microtubule antagonists activate programmed cell death (apoptosis) in cultured rat hepatocytes.

PubMed Central

Tsukidate, K.; Yamamoto, K.; Snyder, J. W.; Farber, J. L.

1993-01-01

We investigated the mechanism of lethal injury following the disruption of microtubules in cultured hepatocytes treated with vinblastine (VBL) or colchicine (COL). These agents kill hepatocytes by a process readily distinguished from two well-known pathways that lead to a loss of viability, namely, oxidative stress and inhibition of mitochondrial electron transport. Cell killing with VBL and COL was accompanied by fragmentation of DNA. Both the loss of viability and the fragmentation of DNA were prevented by the inhibition of protein synthesis within 6 hours following exposure to VBL or COL. Cell death and the fragmentation of DNA were also prevented when Ca2+ was removed from the culture medium. By contrast, the inhibition of protein kinase C prevented cell killing by VBL or COL, but did not alter the extent of DNA fragmentation. The requirements here for protein synthesis, extracellular Ca2+, and protein kinase C activity define a model of apoptosis, or programmed cell death, that seems to involve mechanisms that can be dissociated from the fragmentation of DNA. Images Figure 2 PMID:8362985

Universal DNA-based methods for assessing the diet of grazing livestock and wildlife from feces.

PubMed

Pegard, Anthony; Miquel, Christian; Valentini, Alice; Coissac, Eric; Bouvier, Frédéric; François, Dominique; Taberlet, Pierre; Engel, Erwan; Pompanon, François

2009-07-08

Because of the demand for controlling livestock diets, two methods that characterize the DNA of plants present in feces were developed. After DNA extraction from fecal samples, a short fragment of the chloroplastic trnL intron was amplified by PCR using a universal primer pair for plants. The first method generates a signature that is the electrophoretic migration pattern of the PCR product. The second method consists of sequencing several hundred DNA fragments from the PCR product through pyrosequencing. These methods were validated with a blind analysis of feces from concentrate- and pasture-fed lambs. The signature method allowed differentiation of the two diets and confirmed the presence of concentrate in one of them. The pyrosequencing method allowed the identification of up to 25 taxa in a diet. These methods are complementary to the chemical methods already used. They could be applied to the control of diets and the study of food preferences.

The thiostrepton-resistance-encoding gene in Streptomyces laurentii is located within a cluster of ribosomal protein operons.

PubMed

Smith, T M; Jiang, Y F; Shipley, P; Floss, H G

1995-10-16

A common approach to identify and clone biosynthetic gene from an antibiotic-producing streptomycete is to clone the resistance gene for the antibiotic of interest and then use that gene to clone DNA that is linked to it. As a first step toward cloning the genes responsible for the biosynthesis of thiostrepton (Th) in Streptomyces laurentii (Sl), the Th resistance-encoding gene (tsnR) was cloned as a 1.5-kb BamHI-PvuII fragment in Escherichia coli (Ec), and shown to confer Th resistance when introduced into S. lividans TK24. The tsnR-containing DNA fragment was used as a probe to isolate clones from cosmid libraries of DNA in the Ec cosmid vector SuperCos, and pOJ446 (an Ec/streptomycete) cosmid vector. Sequence and genetic analysis of the DNA flanking the tsnR indicates that the Sl tsnR is not closely linked to biosynthetic genes. Instead it is located within a cluster of ribosomal protein operons.

Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.

PubMed Central

Higgins, M J; Turmel, C; Noolandi, J; Neumann, P E; Lalande, M

1990-01-01

Pulsed-field gel electrophoresis (PFGE) and deletion mapping are being used to construct a physical map of the long arm of human chromosome 13. The present study reports a 2700-kilobase (kb) Not I long-range restriction map encompassing the 13q14-specific loci D13S10, D13S21, and D13S22, which are detected by the cloned DNA markers p7D2, pG24E2.4, and pG14E1.9, respectively. Analysis of a panel of seven cell lines that showed differential methylation at a Not I site between D13S10 and D13S21 proved physical linkage of the two loci to the same 875-kb Not I fragment. D13S22 mapped to a different Not I fragment, precluding the possibility that D13S22 is located between D13S10 and D13S21. PFGE analysis of Not I partial digests placed the 1850-kb Not I fragment containing D13S22 immediately adjacent to the 875-kb fragment containing the other two loci. The proximal rearrangement breakpoint in a cell line carrying a del13(q14.1q21.2) was detected by D13S21 but not by D13S10, demonstrating that D13S21 lies proximal to D13S10. Quantitative analysis of hybridization signals of the three DNA probes to DNA from the same cell line indicated that only D13S10 was deleted, establishing the order of these loci to be cen-D13S22-D13S21-D13S10-tel. Surprisingly, this order was estimated to be 35,000 times less likely than that favored by genetic linkage analysis. Images PMID:1970636

Multisegment nanowire sensors for the detection of DNA molecules.

PubMed

Wang, Xu; Ozkan, Cengiz S

2008-02-01

We describe a novel application for detecting specific single strand DNA sequences using multisegment nanowires via a straightforward surface functionalization method. Nanowires comprising CdTe-Au-CdTe segments are fabricated using electrochemical deposition, and electrical characterization indicates a p-type behavior for the multisegment nanostructures, in a back-to-back Schottky diode configuration. Such nanostructures modified with thiol-terminated probe DNA fragments could function as high fidelity sensors for biomolecules at very low concentration. The gold segment is utilized for functionalization and binding of single strand DNA (ssDNA) fragments while the CdTe segments at both ends serve to modulate the equilibrium Fermi level of the heterojunction device upon hybridization of the complementary DNA fragments (cDNA) to the ssDNA over the Au segment. Employing such multisegment nanowires could lead to the fabrication more sophisticated and high multispecificity biosensors via selective functionalization of individual segments for biowarfare sensing and medical diagnostics applications.

Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

PubMed

Fiscus, R R; Leung, C P; Yuen, J P; Chan, H C

2001-01-01

Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines. Copyright 2001 Academic Press.

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

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

Pröpper, Kevin; Instituto de Biologia Molecular de Barcelona; Meindl, Kathrin

2014-06-01

The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite themore » fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.« less

Biophysical modeling of fragment length distributions of DNA plasmids after X and heavy-ion irradiation analyzed by atomic force microscopy.

PubMed

Elsässer, Thilo; Brons, Stephan; Psonka, Katarzyna; Scholz, Michael; Gudowska-Nowak, Ewa; Taucher-Scholz, Gisela

2008-06-01

The investigation of fragment length distributions of plasmid DNA gives insight into the influence of localized energy distribution on the induction of DNA damage, particularly the clustering of double-strand breaks. We present an approach that determines the fragment length distributions of plasmid DNA after heavy-ion irradiation by using the Local Effect Model. We find a good agreement of our simulations with experimental fragment distributions derived from atomic force microscopy (AFM) studies by including experimental constraints typical for AFM. Our calculations reveal that by comparing the fragmentation in terms of fluence, we can uniquely distinguish the effect of different radiation qualities. For very high-LET irradiation using nickel or uranium ions, no difference between their fragment distributions can be expected for the same dose level. However, for carbon ions with an intermediate LET, the fragmentation pattern differs from the distribution for very high-LET particles. The results of the model calculations can be used to determine the optimal experimental parameters for a demonstration of the influence of track structure on primary radiation damage. Additionally, we compare the results of our model for two different plasmid geometries.

Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease

PubMed Central

Nicholls, Thomas J.; Zsurka, Gábor; Peeva, Viktoriya; Schöler, Susanne; Szczesny, Roman J.; Cysewski, Dominik; Reyes, Aurelio; Kornblum, Cornelia; Sciacco, Monica; Moggio, Maurizio; Dziembowski, Andrzej; Kunz, Wolfram S.; Minczuk, Michal

2014-01-01

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA. PMID:24986917

The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size

PubMed Central

Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

2016-01-01

Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of −0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process. PMID:27104527

The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.

PubMed

Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

2016-04-20

Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process.

Development of SCAR Markers for the DNA-Based Detection of the Asian Long-Horned Beetle; Anoplophora glabripennis (Motschulsky)

Treesearch

Damodar R. Kethidi; David B. Roden; Tim R. Ladd; Peter J. Krell; Arthur Ratnakaran; Qili Feng

2003-01-01

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence charaterized amplified regions (SCARS) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after...

Visualization of DNA in highly processed botanical materials.

PubMed

Lu, Zhengfei; Rubinsky, Maria; Babajanian, Silva; Zhang, Yanjun; Chang, Peter; Swanson, Gary

2018-04-15

DNA-based methods have been gaining recognition as a tool for botanical authentication in herbal medicine; however, their application in processed botanical materials is challenging due to the low quality and quantity of DNA left after extensive manufacturing processes. The low amount of DNA recovered from processed materials, especially extracts, is "invisible" by current technology, which has casted doubt on the presence of amplifiable botanical DNA. A method using adapter-ligation and PCR amplification was successfully applied to visualize the "invisible" DNA in botanical extracts. The size of the "invisible" DNA fragments in botanical extracts was around 20-220 bp compared to fragments of around 600 bp for the more easily visualized DNA in botanical powders. This technique is the first to allow characterization and visualization of small fragments of DNA in processed botanical materials and will provide key information to guide the development of appropriate DNA-based botanical authentication methods in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint.

PubMed

Budd, Martin E; Antoshechkin, Igor A; Reis, Clara; Wold, Barbara J; Campbell, Judith L

2011-05-15

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27 (scFEN1) , encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27 (ScFEN1) processes most of the Okazaki fragments, while Dna2 processes only a subset.

A Genetic Linkage Map of Longleaf Pine (Pinus palustris Mill.) Based on Random Amplified Polymorphic DNAs

Treesearch

C.D. Nelson; Thomas L. Kubisiak; M. Stine; W.L. Nance

1994-01-01

Eight megagametophyte DNA samples from a single longleaf pine (Pinus palustris Mill.) tree were used to screen 576 oligonucleotide primers for random amplified polymorphic DNA (RAPD) fragments. Primers amplifying repeatable polymorphic fragments were further characterized within a sample of 72 megagametophytes from the same tree. Fragments...

An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

PubMed

Gonçalves, A M; Nehme, N S; Morel, C M

1990-01-01

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.

Detection of damage on single- or double-stranded DNA in a population exposed to arsenic in drinking water.

PubMed

Jiménez-Villarreal, J; Rivas-Armendariz, D I; Pineda-Belmontes, C P; Betancourt-Martínez, N D; Macías-Corral, M A; Guerra-Alanis, A J; Niño-Castañeda, M S; Morán-Martínez, J

2017-05-18

Different studies have suggested an association between arsenic (As) exposure and damage to single-stranded DNA by reactive oxygen species derived from the biotransformation of arsenic. The single strand damages are converted to double strand damage upon interaction with ultraviolet radiation. Analysis of genomic integrity is important for assessing the genotoxicity caused by environmental pollutants. In this study, we compared the concentration of As in drinking water, nutritional status, lifestyle variables, and the level of genotoxicity in an exposed population and a control group. Arsenic content of water was determined using a portable Arsenator ® kit. DNA fragmentation was determined using the two-tailed comet assay. Our results show that the exposed population had low nutritional consumption compared to the control group (P < 0.05). Furthermore, the water consumed by the exposed group had As concentration of 14.3 ± 8.4 mg/L, whereas the As level in the water consumed by the control group was 7.7 ± 3.5 mg/L. Analysis shows that the frequency of double strand break (DSB) fragmentation was higher in the population exposed to higher levels of As compared to that of the control group. These results suggest a possible association between the concentration of As in drinking water and lifestyle variables, with increasing fragmentation of DSBs in the exposed population.

Crystallization of DNA fragments from water-salt solutions, containing 2-methylpentane-2,3-diol.

PubMed

Osica, V D; Sukharevsky, B Y; Vasilchenko, V N; Verkin, B I; Polyvtsev, O F

1976-09-01

Fragments of calf thymus DNA have been crystallized by precipitation from water-salt solutions, containing 2-methylpentane-2,3-diol (MPD). DNA crystals usually take the form either of spherulites up to 100 mu in diameter or of needles with the length up to 50 mu. No irreversible denaturation of DNA occurs during the crystallization process. X-ray diffraction from dense slurries of DNA crystals yields crystalline powder patterns.

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

PubMed Central

Eduardoff, Mayra; Xavier, Catarina; Strobl, Christina; Casas-Vargas, Andrea; Parson, Walther

2017-01-01

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories. PMID:28934125

High content analysis of differentiation and cell death in human adipocytes.

PubMed

Doan-Xuan, Quang Minh; Sarvari, Anitta K; Fischer-Posovszky, Pamela; Wabitsch, Martin; Balajthy, Zoltan; Fesus, Laszlo; Bacso, Zsolt

2013-10-01

Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFα)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

PubMed Central

Tsui, Nancy B. Y.; Jiang, Peiyong; Chow, Katherine C. K.; Su, Xiaoxi; Leung, Tak Y.; Sun, Hao; Chan, K. C. Allen; Chiu, Rossa W. K.; Lo, Y. M. Dennis

2012-01-01

Background Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine. Methodology We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine. Principal Findings Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length. Conclusions With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded. PMID:23118982

Review: Diagnosis and impact of sperm DNA alterations in assisted reproduction.

PubMed

Simon, Luke; Emery, Benjamin R; Carrell, Douglas T

2017-10-01

Sperm nuclear and chromatin abnormalities are common among infertile men and are known to influence natural reproduction. These abnormalities are also considered detrimental to normal fertilization, embryo development, and successful implantation and pregnancies following assisted reproductive treatment (ART). Abnormalities in the sperm nucleus can be broadly classified into sperm chromosomal abnormalities (aneuploidies) and sperm DNA abnormalities such as abnormal packing, DNA integrity, or DNA fragmentation. For the past 30 years, numerous tests have been developed to quantify these abnormalities in sperm. In this chapter, we review the causes of sperm DNA and chromosomal abnormalities, describe the commonly used tests to evaluate these abnormalities, and finally review the impact of these abnormalities on male fertility and ART outcomes. We also performed a comprehensive meta-analysis and systematic review from the existing literature to summarize the effect of sperm DNA fragmentation on ART outcomes such as fertilization rate, embryo quality, and clinical pregnancies. A review of the literature presented in this chapter suggests that sperm nuclear and chromatin abnormalities are associated with male infertility, and they reduce the probability of a successful pregnancy following ART. Copyright © 2017. Published by Elsevier Ltd.

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

PubMed

Wang, Yongming; Lin, Xiuyun; Dong, Bo; Wang, Yingdian; Liu, Bao

2004-01-01

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

PubMed

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-02-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

PubMed Central

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-01-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. Images PMID:3003377

Co-precipitation of protein and polyester as a method to isolate high molecular weight DNA.

PubMed

Dixson, Jamie D

2005-02-01

DNA isolation is often the limiting step in genetic analysis using PCR and automated fragment analysis due to low quality or purity of DNA, the need to determine and adjust DNA concentrations after isolation etc. Several protocols have been developed which are either safe and provide good quality DNA or hazardous and provide excellent quality DNA. In this brief communication I describe a new and rapid method of DNA isolation which employs the co-precipitation of protein and polyester, in the presence of acetone, to remove contaminating proteins from a lysed-tissue sample, thus leaving high quality pure DNA. The advantages of this method are increased safety over the phenol:chloroform and the chaotrophic salt methods and increased purity over the salting-out method. Since the concentrations of DNA isolated using this method are relatively consistent regardless of the amount of starting tissue (within limits), adjustments of the DNA concentrations before use as templates in PCR's are not necessary.

Comprehensive Study On The Metastable Negative Ion Fragmentation Of Individual Dna Components And Larger Oligonucleotides

NASA Astrophysics Data System (ADS)

Ingolfsson, O.; Flosadottir, H. D.; Omarsson, B.; Ilko, B.

2010-07-01

Here we present a systematic study on the unimolecular decay pathways of the deprotonated building blocks of DNA and RNA to address the following questions: 1. Are the negative ion fragmentation patterns observed in the metastable decay of individual DNA components still evident when these are combined to larger oligonucleotides? 2. What is the significance of the charge location in determining the fragmentation pathways in the metastable decay process? 3. Are those metastable decay channels relevant in dissociative electron attachment to DNA components? To address these questions we have studied the fragmentation patterns of the deprotonated ribose and ribose 5'-monophosphate, the fragmentation patterns of the individual bases, all nucleosides and all 2'-deoxynucleosides as well as the individual nucleotides and several combinations of hexameric oligonucleotides. Furthermore, to understand the significance of the charge location in determining the fragmentation path in the metastable decay process of these deprotonated ions we have also studied modified uridine and guanosine. These have been modified to block different deprotonation sites and thus to control the initial step in the in the fragmentation process i.e. the site of deprotonation. In addition to our experimental approach we have also simulated the metastable fragmentation of the deprotonated uridine and 2'-deoxyguanosine to clarify the mechanisms and fragmentation patterns observed. Where data is available, the results are compared to dissociative electron attachment to DNA components and discussed in context to the underlying mechanism. Experiments on modified nucleosides where selected deprotonation sites have been blocked are used to verify the predicted reaction paths and imulations on uridine and 2'-deoxyguanosine are compared to the experimental results and used to shed light on the mechanisms involved.

Detection and size analysis of proteins with switchable DNA layers.

PubMed

Rant, Ulrich; Pringsheim, Erika; Kaiser, Wolfgang; Arinaga, Kenji; Knezevic, Jelena; Tornow, Marc; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2009-04-01

We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform.

A novel gene: sawD related to the differentiation of streptomyces ansochromogenes.

PubMed

Gang, L; Wei, C; Yuqing, T; Huarong, T; Chater, K F; Buttner, M J

1999-01-01

A 1.3 kb DNA fragment was cloned from a total DNA library of Streptomyces ansochromogenes using Southern hybridization. Nucleotide sequencing analysis indicated that the 1320 bp DNA fragment contained a complete open reading frame (ORF). In search of databases, the deduced product of ORF containing 213 amino acids is homologous to the serine protease of Caulobacter cresceatus, and a conserved serine-catalytic active site (GPSAG) exists. The gene was designated as sawD. The function of this gene was studied with the strategy of gene disruption, and the result showed that the sawD may be related to sporulation and especially to the spore septation in Streptomyces ansochromogenes. The preliminary result indicated that sawD mutant could produce abundant pigment in contrast with the wild type, it seems that sawD gene may be involved in pigment biosynthesis, and this gene is also dispensable for biosynthesis of nikkomycin in Streptomyces ansochromogenes.

An efficient approach for cloning the dNDP-glucose synthase gene from actinomycetes and its application in Streptomyces spectabilis, a spectinomycin producer.

PubMed

Hyun, C; Kim, S S; Sohng, J K; Hahn, J; Kim, J; Suh, J

2000-02-01

Specifically designed PCR primers were applied to amplify a segment of dTDP-glucose synthase gene from six actinomycete strains. About 300-bp or 580-bp DNA fragments were obtained from all the organisms tested. By DNA sequence analysis, seven amplified fragments showed high homology with dTDP-glucose synthase genes that participate in the biosynthesis of secondary metabolites or in deoxy-sugar moieties in lipopolysaccharides. In addition, we have cloned a 45-kb region of DNA from Streptomyces spectabilis ATCC27741, a spectinomycin producer which contained the dTDP-glucose synthase and dTDP-glucose 4,6-dehydratase genes named spcD and spcE, respectively. The spcE gene was expressed in Escherichia coli and the activity was assayed in cell extracts. The enzyme showed substrate specificity only to dTDP-glucose.

Ultra-low background DNA cloning system.

PubMed

Goto, Kenta; Nagano, Yukio

2013-01-01

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an "ultra-low background DNA cloning system" on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp(r)). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp(r) 5' UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp(r) 3' UTR. This cassette allowed conversion of the Amp(r)-containing vector into the yeast/E. coli shuttle vector through use of the Amp(r) sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific "origins of replication" to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

A glass fiber/diethylaminoethyl double filter binding assay that measures apoptotic internucleosomal DNA fragmentation.

PubMed

Erusalimsky, J D; John, J; Hong, Y; Moore, M

1996-11-15

A filter binding assay that measures internucleosomal DNA fragmentation associated with apoptosis is described. The assay is based on a novel principle that consists of using simultaneously two kinds of glass fiber filters to harvest [3H]thymidine-prelabeled cells following their incubation with inducers of apoptosis. One filter, which is neutral, traps intact chromatin and high-molecular-weight DNA. The other filter, which is positively charged with DEAE active groups, traps low-molecular-weight DNA fragments. DNA fragmentation is quantified by measuring the radioactivity retained by each of the filters. The assay was evaluated with the histiocytic lymphoma cell line U937 and the topoisomerase inhibitors camptothecin, etoposide, and doxorubicin. These agents caused a dose-dependent decrease of radioactivity in the neutral filter and a parallel increase of radioactivity in the DEAE filter. Irradiation-induced single strand breaks and topoisomerase-mediated primary DNA damage were not detected by this method. Consistent with the detection of internucleosomal DNA fragmentation, the effects measured by this assay were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using this assay were validated by observation of DNA ladders on agarose gels and by morphologic examination of apoptotic features. Evaluation of the assay in a mock screen demonstrated that the introduction of the DEAE filter increases the assay sensitivity and eliminates false positives. Thus, this assay may be used in high-throughput screening approaches to discover novel modulators of apoptosis.

Post-Fragmentation Whole Genome Amplification-Based Method

NASA Technical Reports Server (NTRS)

Benardini, James; LaDuc, Myron T.; Langmore, John

2011-01-01

This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice

PubMed Central

Alyamkina, Ekaterina A; Dolgova, Evgenia V; Likhacheva, Anastasia S; Rogachev, Vladimir A; Sebeleva, Tamara E; Nikolin, Valeriy P; Popova, Nelly A; Orishchenko, Konstantin E; Strunkin, Dmitriy N; Chernykh, Elena R; Zagrebelniy, Stanislav N; Bogachev, Sergei S; Shurdov, Mikhail A

2009-01-01

Background When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment. Methods Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls. Results An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection. Conclusion Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are grafted to mice after this combined treatment. PMID:19682353

Histological analysis and ancient DNA amplification of human bone remains found in caius iulius polybius house in pompeii.

PubMed

Cipollaro, M; Di Bernado, G; Forte, A; Galano, G; De Masi, L; Galderisi, U; Guarino, F M; Angelini, F; Cascino, A

1999-09-01

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein.

PubMed

Kabeya, Hidenori; Maruyama, Soichi; Hirano, Kouji; Mikami, Takeshi

2003-01-01

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.

High-sensitivity stable-isotope probing by a quantitative terminal restriction fragment length polymorphism protocol.

PubMed

Andeer, Peter; Strand, Stuart E; Stahl, David A

2012-01-01

Stable-isotope probing (SIP) has proved a valuable cultivation-independent tool for linking specific microbial populations to selected functions in various natural and engineered systems. However, application of SIP to microbial populations with relatively minor buoyant density increases, such as populations that utilize compounds as a nitrogen source, results in reduced resolution of labeled populations. We therefore developed a tandem quantitative PCR (qPCR)-TRFLP (terminal restriction fragment length polymorphism) protocol that improves resolution of detection by quantifying specific taxonomic groups in gradient fractions. This method combines well-controlled amplification with TRFLP analysis to quantify relative taxon abundance in amplicon pools of FAM-labeled PCR products, using the intercalating dye EvaGreen to monitor amplification. Method accuracy was evaluated using mixtures of cloned 16S rRNA genes, DNA extracted from low- and high-G+C bacterial isolates (Escherichia coli, Rhodococcus, Variovorax, and Microbacterium), and DNA from soil microcosms amended with known amounts of genomic DNA from bacterial isolates. Improved resolution of minor shifts in buoyant density relative to TRFLP analysis alone was confirmed using well-controlled SIP analyses.

Identification and phylogenetic analysis of novel cytochrome P450 1A genes from ungulate species.

PubMed

Darwish, Wageh Sobhy; Kawai, Yusuke; Ikenaka, Yoshinori; Yamamoto, Hideaki; Muroya, Tarou; Ishizuka, Mayumi

2010-09-01

As part of an ongoing effort to understand the biological response of wild and domestic ungulates to different environmental pollutants such as dioxin-like compounds, cDNAs encoding for CYP1A1 and CYP1A2 were cloned and characterized. Four novel CYP1A cDNA fragments from the livers of four wild ungulates (elephant, hippopotamus, tapir and deer) were identified. Three fragments from hippopotamus, tapir and deer were classified as CYP1A2, and the other fragment from elephant was designated as CYP1A1/2. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 76 to 97% with other animal CYP1As. The phylogenetic analysis of these fragments showed that both elephant and hippopotamus CYP1As made separate branches, while tapir and deer CYP1As were located beside that of horse and cattle respectively in the phylogenetic tree. Analysis of dN/dS ratio among the identified CYP1As indicated that odd toed ungulate CYP1A2s were exposed to different selection pressure.

DNA fragmentation and nuclear phenotype in tendons exposed to low-intensity infrared laser

NASA Astrophysics Data System (ADS)

de Paoli, Flavia; Ramos Cerqueira, Larissa; Martins Ramos, Mayara; Campos, Vera M.; Ferreira-Machado, Samara C.; Geller, Mauro; de Souza da Fonseca, Adenilson

2015-03-01

Clinical protocols are recommended in device guidelines outlined for treating many diseases on empirical basis. However, effects of low-intensity infrared lasers at fluences used in clinical protocols on DNA are controversial. Excitation of endogenous chromophores in tissues and free radicals generation could be described as a consequence of laser used. DNA lesions induced by free radicals cause changes in DNA structure, chromatin organization, ploidy degrees and cell death. In this work, we investigated whether low-intensity infrared laser therapy could alter the fibroblasts nuclei characteristics and induce DNA fragmentation. Tendons of Wistar rats were exposed to low-intensity infrared laser (830 nm), at different fluences (1, 5 and 10 J/cm2), in continuous wave (power output of 10mW, power density of 79.6 mW/cm2). Different frequencies were analyzed for the higher fluence (10 J/cm2), at pulsed emission mode (2.5, 250 and 2500 Hz), with the laser source at surface of skin. Geometric, densitometric and textural parameters obtained for Feulgen-stained nuclei by image analysis were used to define nuclear phenotypes. Significant differences were observed on the nuclear phenotype of tendons after exposure to laser, as well as, high cell death percentages was observed for all fluences and frequencies analyzed here, exception 1 J/cm2 fluence. Our results indicate that low-intensity infrared laser can alter geometric, densitometric and textural parameters in tendon fibroblasts nuclei. Laser can also induce DNA fragmentation, chromatin lost and consequently cell death, using fluences, frequencies and emission modes took out from clinical protocols.

Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

PubMed

Bounartzi, Theofania; Dafopoulos, Konstantinos; Anifandis, George; Messini, Christina I; Koutsonikou, Chrysoula; Kouris, Spyros; Satra, Maria; Sotiriou, Sotirios; Vamvakopoulos, Nicholas; Messinis, Ioannis E

2016-04-01

The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p < 0.05) than f-spDNA (AUC = 0.6, p > 0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success.

2000 Year-old ancient equids: an ancient-DNA lesson from pompeii remains.

PubMed

Di Bernardo, Giovanni; Del Gaudio, Stefania; Galderisi, Umberto; Cipollaro, Marilena

2004-11-15

Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern horses to assess their genetic relationship. The 16S rRNA mitochondrial gene was also examined to unravel the post-mortem base modification feature and to test the status of Pompeian equids taxon on the basis of a Mae III restriction site polymorphism. Copyright 2004 Wiley-Liss, Inc.

A DNA logic gate based on strand displacement reaction and rolling circle amplification, responding to multiple low-abundance DNA fragment input signals, and its application in detecting miRNAs.

PubMed

Chen, Yuqi; Song, Yanyan; Wu, Fan; Liu, Wenting; Fu, Boshi; Feng, Bingkun; Zhou, Xiang

2015-04-25

A conveniently amplified DNA AND logic gate platform was designed for the highly sensitive detection of low-abundance DNA fragment inputs based on strand displacement reaction and rolling circle amplification strategy. Compared with others, this system can detect miRNAs in biological samples. The success of this strategy demonstrates the potential of DNA logic gates in disease diagnosis.

Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

PubMed Central

Sun, Fei; Huang, Li

2013-01-01

Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667

Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis genome.

PubMed

Ponce, M R; Quesada, V; Micol, J L

1998-05-01

An improvement to previous methods for recovering Arabidopsis thaliana genomic DNA flanking T-DNA insertions is presented that allows for the avoidance of some of the cloning difficulties caused by the concatameric nature of T-DNA inserts. The principle of the procedure is to categorize by size restriction fragments of mutant DNA, produced in separate digestions with NdeI and Bst1107I. Given that the sites for these two enzymes are contiguous within the pGV3850:1003 T-DNA construct, the restriction fragments obtained fall into two categories: those showing identical size in both digestions, which correspond to sequences internal to T-DNA concatamers; and those of different sizes, that contain the junctions between plant DNA and the T-DNA insert. Such a criterion makes it possible to easily distinguish the digestion products corresponding to internal T-DNA parts, which do not deserve further attention, and those which presumably include a segment of the locus of interest. Discrimination between restriction fragments of genomic mutant DNA can be made on rescued plasmids, inverse PCR amplification products or bands in a genomic blot.

Apoptotic cell death and the proliferative capacity of human breast cancers.

PubMed

Losa, G A; Graber, R

1998-01-01

The proliferative capacity (%S-phase fraction), DNA ploidy, apoptosis frequency (DNA fragmentation) and steroid hormone receptor status (estrogen receptor, ER; progesterone receptor, PR) of 110 samples of human breast tissues with ductal invasive carcinoma were measured using biochemical and cytofluorimetric procedures. The DNA fragmentation had a left-skewed frequency distribution and an overall median value of 1.64%, whilst the median %S-phase fraction was 8%. The median %DNA fragmentation and %S-phase fraction were 1.96% and 16% in hyperdiploid tumours (n = 29; DNA index >1.1) higher than in hypodiploid tumors (n = 10; DNA index <0.96), 0.38% and 7.5%. DNA diploid tumours (n = 71) had median %DNA fragmentation and %S-phase values of 1.68% and 6%, consistently lower than the median values of DNA hyperdiploid tumours. The ER content of hypodiploid tumours was about one half (median: 5.9 fmol/mg) the median values in hyperdiploid (10.6 fmol/mg) and diploid tumours (14.6 fmol/mg). This may correlate with the lowest frequency of apoptosis in hypodiploid tumours, at least when measured by biochemical methods which only detect cells in the late phases of apoptosis. In contrast, the median PR was lowest in hyperdiploid tumours than in hypo and/or diploid tumours. The %S-phase/%fragmented DNA ratio for the hypodiploid tumours was 19.7, significantly higher than the ratios for hyperdiploid (8.2) and diploid tumours (3.6). These findings indicated that there is an imbalance between proliferative capacity and cell death or growth arrest in human breast tumours. This imbalance may well be linked to a loss of steroid hormone control.

Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA.

PubMed

Miklenić, Marina; Štafa, Anamarija; Bajić, Ana; Žunar, Bojan; Lisnić, Berislav; Svetec, Ivan-Krešimir

2013-05-01

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/ PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/microg DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality.

PubMed

Dejmek, Annika; Zendehrokh, Nooreldin; Tomaszewska, Malgorzata; Edsjö, Anders

2013-07-01

Personalized oncology requires molecular analysis of tumor cells. Several studies have demonstrated that cytological material is suitable for DNA analysis, but to the authors' knowledge there are no systematic studies comparing how the yield and quality of extracted DNA is affected by the various techniques used for the preparation of cytological material. DNA yield and quality were compared using cultured human lung cancer cells subjected to different preparation techniques used in routine cytology, including fixation, mounting medium, and staining. The results were compared with the outcome of epidermal growth factor receptor (EGFR) genotyping of 66 clinical cytological samples using the same DNA preparation protocol. All tested protocol combinations resulted in fragment lengths of at least 388 base pairs. The mounting agent EcoMount resulted in higher yields than traditional xylene-based medium. Spray and ethanol fixation resulted in both a higher yield and better DNA quality than air drying. In liquid-based cytology (LBC) methods, CytoLyt solution resulted in a 5-fold higher yield than CytoRich Red. Papanicolaou staining provided twice the yield of hematoxylin and eosin staining in both liquid-based preparations. Genotyping outcome and quality control values from the clinical EGFR genotyping demonstrated a sufficient amount and amplifiability of DNA in both spray-fixed and air-dried cytological samples. Reliable clinical genotyping can be performed using all tested methods. However, in the cell line experiments, spray- or ethanol-fixed, Papanicolaou-stained slides provided the best results in terms of yield and fragment length. In LBC, the DNA recovery efficiency of the preserving medium may differ considerably, which should be taken into consideration when introducing LBC. Cancer (Cancer Cytopathol) 2013;121:344-353. © 2013 American Cancer Society. © 2013 American Cancer Society.

Integrated microfluidic chip for rapid DNA digestion and time-resolved capillary electrophoresis analysis

PubMed Central

Lin, Che-Hsin; Wang, Yao-Nan; Fu, Lung-Ming

2012-01-01

An integrated microfluidic chip is proposed for rapid DNA digestion and time-resolved capillary electrophoresis (CE) analysis. The chip comprises two gel-filled chambers for DNA enrichment and purification, respectively, a T-form micromixer for DNA/restriction enzyme mixing, a serpentine channel for DNA digestion reaction, and a CE channel for on-line capillary electrophoresis analysis. The DNA and restriction enzyme are mixed electroomostically using a pinched-switching DC field. The experimental and numerical results show that a mixing performance of 97% is achieved within a distance of 1 mm from the T-junction when a driving voltage of 90 V/cm and a switching frequency of 4 Hz are applied. Successive mixing digestion and capillary electrophoresis operation clearly present the changes on digesting φx-174 DNA in different CE runs. The time-resolved electropherograms show that the proposed device enables a φx-174 DNA sample comprising 11 fragments to be concentrated and analyzed within 24 min. Overall, the results presented in this study show that the proposed microfluidic chip provides a rapid and effective tool for DNA digestion and CE analysis applications. PMID:22662085

Evaluation of Methods for the Extraction of DNA from Drinking Water Distribution System Biofilms

PubMed Central

Hwang, Chiachi; Ling, Fangqiong; Andersen, Gary L.; LeChevallier, Mark W.; Liu, Wen-Tso

2012-01-01

While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories. PMID:22075624

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

PubMed

Freemont, P S; Ollis, D L; Steitz, T A; Joyce, C M

1986-09-01

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis

PubMed Central

Marsh, Terence L.; Saxman, Paul; Cole, James; Tiedje, James

2000-01-01

Rapid analysis of microbial communities has proven to be a difficult task. This is due, in part, to both the tremendous diversity of the microbial world and the high complexity of many microbial communities. Several techniques for community analysis have emerged over the past decade, and most take advantage of the molecular phylogeny derived from 16S rRNA comparative sequence analysis. We describe a web-based research tool located at the Ribosomal Database Project web site (http://www.cme.msu.edu/RDP/html/analyses.html) that facilitates microbial community analysis using terminal restriction fragment length polymorphism of 16S ribosomal DNA. The analysis function (designated TAP T-RFLP) permits the user to perform in silico restriction digestions of the entire 16S sequence database and derive terminal restriction fragment sizes, measured in base pairs, from the 5′ terminus of the user-specified primer to the 3′ terminus of the restriction endonuclease target site. The output can be sorted and viewed either phylogenetically or by size. It is anticipated that the site will guide experimental design as well as provide insight into interpreting results of community analysis with terminal restriction fragment length polymorphisms. PMID:10919828

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

PubMed

Gryson, Nicolas

2010-03-01

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

The Neandertal type site revisited: Interdisciplinary investigations of skeletal remains from the Neander Valley, Germany

PubMed Central

Schmitz, Ralf W.; Serre, David; Bonani, Georges; Feine, Susanne; Hillgruber, Felix; Krainitzki, Heike; Pääbo, Svante; Smith, Fred H.

2002-01-01

The 1856 discovery of the Neandertal type specimen (Neandertal 1) in western Germany marked the beginning of human paleontology and initiated the longest-standing debate in the discipline: the role of Neandertals in human evolutionary history. We report excavations of cave sediments that were removed from the Feldhofer caves in 1856. These deposits have yielded over 60 human skeletal fragments, along with a large series of Paleolithic artifacts and faunal material. Our analysis of this material represents the first interdisciplinary analysis of Neandertal remains incorporating genetic, direct dating, and morphological dimensions simultaneously. Three of these skeletal fragments fit directly on Neandertal 1, whereas several others have distinctively Neandertal features. At least three individuals are represented in the skeletal sample. Radiocarbon dates for Neandertal 1, from which a mtDNA sequence was determined in 1997, and a second individual indicate an age of ≈40,000 yr for both. mtDNA analysis on the same second individual yields a sequence that clusters with other published Neandertal sequences. PMID:12232049

Comparative gene expression in sexual and apomictic ovaries of Pennisetum ciliare (L.) Link.

PubMed

Vielle-Calzada, J P; Nuccio, M L; Budiman, M A; Thomas, T L; Burson, B L; Hussey, M A; Wing, R A

1996-12-01

Limited emphasis has been given to the molecular study of apomixis, an asexual method of reproduction where seeds are produced without fertilization. Most buffelgrass (Pennisetum ciliare (L.) Link syn = Cenchrus ciliaris L.) genotypes reproduce by obligate apomixis (apospory); however, rare sexual plants have been recovered. A modified differential display procedure was used to compare gene expression in unpollinated ovaries containing ovules with either sexual or apomictic female gametophytes. The modification incorporated end-labeled poly(A)+ anchored primers as the only isotopic source, and was a reliable and consistent approach for detecting differentially displayed transcripts. Using 20 different decamers and two anchor primers, 2268 cDNA fragments between 200 and 600 bp were displayed. From these, eight reproducible differentially displayed cDNAs were identified and cloned. Based on northern analysis, one cDNA was detected in only the sexual ovaries, two cDNAs in only apomictic ovaries and one cDNA was present in both types of ovaries. Three fragments could not be detected and one fragment was detected in ovaries, stems, and leaves. Comparison of gene expression during sexual and apomictic development in buffelgrass represents a new model system and a strategy for investigating female reproductive development in the angiosperms.

DNA barcoding insect–host plant associations

PubMed Central

Jurado-Rivera, José A.; Vogler, Alfried P.; Reid, Chris A.M.; Petitpierre, Eduard; Gómez-Zurita, Jesús

2008-01-01

Short-sequence fragments (‘DNA barcodes’) used widely for plant identification and inventorying remain to be applied to complex biological problems. Host–herbivore interactions are fundamental to coevolutionary relationships of a large proportion of species on the Earth, but their study is frequently hampered by limited or unreliable host records. Here we demonstrate that DNA barcodes can greatly improve this situation as they (i) provide a secure identification of host plant species and (ii) establish the authenticity of the trophic association. Host plants of leaf beetles (subfamily Chrysomelinae) from Australia were identified using the chloroplast trnL(UAA) intron as barcode amplified from beetle DNA extracts. Sequence similarity and phylogenetic analyses provided precise identifications of each host species at tribal, generic and specific levels, depending on the available database coverage in various plant lineages. The 76 species of Chrysomelinae included—more than 10 per cent of the known Australian fauna—feed on 13 plant families, with preference for Australian radiations of Myrtaceae (eucalypts) and Fabaceae (acacias). Phylogenetic analysis of beetles shows general conservation of host association but with rare host shifts between distant plant lineages, including a few cases where barcodes supported two phylogenetically distant host plants. The study demonstrates that plant barcoding is already feasible with the current publicly available data. By sequencing plant barcodes directly from DNA extractions made from herbivorous beetles, strong physical evidence for the host association is provided. Thus, molecular identification using short DNA fragments brings together the detection of species and the analysis of their interactions. PMID:19004756

Regulation of apoptosis of interleukin 2-dependent mouse T-cell line by protein tyrosine phosphorylation and polyamines.

PubMed

Min, A; Hasuma, T; Yano, Y; Matsui-Yuasa, I; Otani, S

1995-12-01

We examined the effect of inhibitors of tyrosine kinase and tyrosine phosphatase on DNA fragmentation, protein tyrosine phosphorylation, and polyamine metabolism in the murine T-cell line CTLL-2. When cells were exposed to herbimycin A, a specific inhibitor of tyrosine kinase (Uehara et al., 1989, Biochem. Biophys. Res. Commun., 163:803-809), in the presence of interleukin 2 (IL-2), DNA was degraded into oligonucleosomal fragments in a dose-dependent fashion. Genistein, another inhibitor of tyrosine kinase (Akiyama et al., 1987, J. Biol. Chem., 262:5592-5596), had similar effects. Exposure of CTLL-2 cells to vanadate, a tyrosine phosphatase inhibitor, blocked with the DNA fragmentation induced by herbimycin A. Tyrosine phosphorylation of 55 Kd protein was inhibited by herbimycin A, and the inhibition was reduced by vanadate. Ornithine decarboxylase (ODC) activity decreased rapidly after herbimycin A was added to CTLL-2 cell cultures, while vanadate increased ODC activity. The exogenous addition of putrescine or spermine, but not that of spermidine, attenuated herbimycin A-induced DNA fragmentation. These findings suggest that phosphorylation of tyrosine residues of 55 Kd protein prevents DNA fragmentation and that polyamines are involved in regulation of apoptosis.

Cell-free DNA fragment-size distribution analysis for non-invasive prenatal CNV prediction.

PubMed

Arbabi, Aryan; Rampášek, Ladislav; Brudno, Michael

2016-06-01

Non-invasive detection of aneuploidies in a fetal genome through analysis of cell-free DNA circulating in the maternal plasma is becoming a routine clinical test. Such tests, which rely on analyzing the read coverage or the allelic ratios at single-nucleotide polymorphism (SNP) loci, are not sensitive enough for smaller sub-chromosomal abnormalities due to sequencing biases and paucity of SNPs in a genome. We have developed an alternative framework for identifying sub-chromosomal copy number variations in a fetal genome. This framework relies on the size distribution of fragments in a sample, as fetal-origin fragments tend to be smaller than those of maternal origin. By analyzing the local distribution of the cell-free DNA fragment sizes in each region, our method allows for the identification of sub-megabase CNVs, even in the absence of SNP positions. To evaluate the accuracy of our method, we used a plasma sample with the fetal fraction of 13%, down-sampled it to samples with coverage of 10X-40X and simulated samples with CNVs based on it. Our method had a perfect accuracy (both specificity and sensitivity) for detecting 5 Mb CNVs, and after reducing the fetal fraction (to 11%, 9% and 7%), it could correctly identify 98.82-100% of the 5 Mb CNVs and had a true-negative rate of 95.29-99.76%. Our source code is available on GitHub at https://github.com/compbio-UofT/FSDA CONTACT: : brudno@cs.toronto.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Whole Genome Sequence Analysis of Mutations Accumulated in rad27Δ Yeast Strains with Defects in the Processing of Okazaki Fragments Indicates Template-Switching Events

PubMed Central

Omer, Sumita; Lavi, Bar; Mieczkowski, Piotr A.; Covo, Shay; Hazkani-Covo, Einat

2017-01-01

Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a S. cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing. Out of the 455 changes observed in 10 colonies isolated the two most common types of events were insertions or deletions (INDELs) in simple sequence repeats (SSRs) and INDELs mediated by short direct repeats. Surprisingly, we also detected a previously neglected class of 21 template-switching events. These events were presumably generated by quasi-palindrome to palindrome correction, as well as palindrome elongation. The formation of these events is best explained by folding back of the stalled nascent strand and resumption of DNA synthesis using the same nascent strand as a template. Evidence of quasi-palindrome to palindrome correction that could be generated by template switching appears also in yeast genome evolution. Out of the 455 events, 55 events appeared in multiple isolates; further analysis indicates that these loci are mutational hotspots. Since Rad27 acts on the lagging strand when the leading strand should not contain any gaps, we propose a mechanism favoring intramolecular strand switching over an intermolecular mechanism. We note that our results open new ways of understanding template switching that occurs during genome instability and evolution. PMID:28974572

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

PubMed Central

Margalit, Y; Yarus, S; Shapira, E; Gruenbaum, Y; Fainsod, A

1993-01-01

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture. Images PMID:7909943

What Hinders Electron Transfer Dissociation (ETD) of DNA Cations?

NASA Astrophysics Data System (ADS)

Hari, Yvonne; Leumann, Christian J.; Schürch, Stefan

2017-12-01

Radical activation methods, such as electron transfer dissociation (ETD), produce structural information complementary to collision-induced dissociation. Herein, electron transfer dissociation of 3-fold protonated DNA hexamers was studied to gain insight into the fragmentation mechanism. The fragmentation patterns of a large set of DNA hexamers confirm cytosine as the primary target of electron transfer. The reported data reveal backbone cleavage by internal electron transfer from the nucleobase to the phosphate linker leading either to a•/ w or d/ z• ion pairs. This reaction pathway contrasts with previous findings on the dissociation processes after electron capture by DNA cations, suggesting multiple, parallel dissociation channels. However, all these channels merely result in partial fragmentation of the precursor ion because the charge-reduced DNA radical cations are quite stable. Two hypotheses are put forward to explain the low dissociation yield of DNA radical cations: it is either attributed to non-covalent interactions between complementary fragments or to the stabilization of the unpaired electron in stacked nucleobases. MS3 experiments suggest that the charge-reduced species is the intact oligonucleotide. Moreover, introducing abasic sites significantly increases the dissociation yield of DNA cations. Consequently, the stabilization of the unpaired electron by π-π-stacking provides an appropriate rationale for the high intensity of DNA radical cations after electron transfer. [Figure not available: see fulltext.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

PubMed

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

A Single Molecular Beacon Probe Is Sufficient for the Analysis of Multiple Nucleic Acid Sequences

PubMed Central

Gerasimova, Yulia V.; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M.

2010-01-01

Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping. PMID:20665615

Comparisons of molecular karyotype and RAPD patterns of anuran trypanosome isolates during long-term in vitro cultivation.

PubMed

Lun, Z R; Desser, S S

1996-01-01

The patterns of random amplified fragments and molecular karyotypes of 12 isolates of anuran trypanosomes continuously cultured in vitro were compared by random amplified polymorphic DNA (RAPD) analysis and pulsed field gradient gel electrophoresis (PFGE). The time interval between preparation of two series of samples was one year. Changes were not observed in the number and size of sharp, amplified fragments of DNA samples from both series examined with the ten primers used. Likewise, changes in the molecular karyotypes were not detected between the two samples of these isolates. These results suggest that the molecular karyotype and the RAPD patterns of the anuran trypanosomes remain stable after being cultured continuously in vitro for one year.

Dna Sequencing

DOEpatents

Tabor, Stanley; Richardson, Charles C.

1995-04-25

A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

[Improvement of laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera Vibrio biovar El Tor].

PubMed

Savel'eva, I V; Khatsukov, K X; Savel'eva, E I; Moskvitina, S I; Kovalev, D A; Savel'ev, V N; Kulichenko, A N; Antonenko, A D; Babenyshev, B V

2015-01-01

Improvement of laboratory diagnostics of cholera taking into the account appearance of hybrid variants of cholera vibrio El Tor biovar in the 1990s. Phenotypic and molecular-genetic properties of typical toxigenic (151 strains) and hybrid (102 strains) variants of El Tor biovar cholera vibrios, isolated in the Caucuses in 1970-1990 and 1993-1998, respectively, were studied. Toxigenicity gene DNA fragments, inherent to El Tor biovars or classic, were detected by using a reagent kit "Genes of Vibrio cholerae variant ctxB-rstR-rstC, REF" developed by us. Reagent kit "Genes of V. cholerae variant ctxB-rstR-rstC, REF" is proposed to be used for laboratory diagnostics of cholera during study of material from humans or environmental objects and for identification of V. cholerae 01 on genome level in PCR-analysis as a necessary addition to the classic scheme of bacteriological analysis. Laboratory diagnostics of cholera due to genetically altered (hybrid) variants of cholera vibrio El Tor biovar is based on a complex study of material from humans and environmental objects by routine bacteriologic and PCR-analysis methods with the aim of detection of gene DNA fragments in the studied material, that determine biovar (classic or El Tor), identification of V. cholerae O1 strains with differentiation of El Tor vibrios into typical and altered, as well as determination of enterotoxin, produced by the specific cholera vibrio strain (by the presence ctxB(El) or ctxB(Cl) gene DNA fragment, coding biosynthesis of CT-2 or CT-1, respectively).

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

PubMed

Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

2013-09-24

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

PubMed Central

Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

2013-01-01

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp. PMID:24019490

Multiplex screening for RB1 germline mutations in 106 patients with hereditary retinoblastoma

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

Lohmann, D.R.; Brandt, B.; Passarge, E.

1994-09-01

The identification of germline mutations in the retinoblastoma susceptibility gene (RB1) is important for genetic counseling in hereditary retinoblastoma. Due to the complex genomic organization of this gene and the heterogeneity of mutations, efficient screening procedures are important for rapid mutation detection. We have developed methods based on simultaneous analysis of multiple regions of this gene in an ABI automated DNA fragment analyzer to examine 106 patients with hereditary retinoblastoma in which no alteration was identified by Southern blot hybridization. Primers for the amplification of all 27 exons of the RB1 gene as well as the promoter and poly(A) signalmore » sequences were labelled with distinct fluorescent dyes (FAM, HEX, TAMRA) to enable simultaneous electrophoretic analysis of PCR products with similar mobility. PCR fragments distinguishable by size or color were co-amplified by multiplex PCR and analyzed for length by GENESCAN analysis. Using this approach, small deletions ranging from 1 bp to 22 bp were identified in 24 patients (23%). Short sequence repeats or polypyrimidine runs were present in the vicinity of most of these deletions. In 4 patients (4%), insertions from 1 bp to 4 bp were found. The majority of length mutations resulted in a truncated gene product due to frameshift and premature termination. No mutation was identified in exons 25 to 27 possibly indicating that the encoded protein domains have minor functional importance. In order to screen for base substitutions that are not detectable by fragment length analysis, we adapted heteroduplex analysis for the use in the DNA fragment analyzer. During the optimization of this method we detected 10 single base substitutions most of which generated stop codons. Intriguingly, two identical missense mutations were identified in two unrelated families with a low-penetrance phenotype.« less

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

Atomic Force Microscopy Studies on DNA Structural Changes Induced by Vincristine Sulfate and Aspirin

NASA Astrophysics Data System (ADS)

Zhu, Yi; Zeng, Hu; Xie, Jianming; Ba, Long; Gao, Xiang; Lu, Zuhong

2004-04-01

We report that atomic force microscopy (AFM) studies on structural variations of a linear plasmid DNA interact with various concentrations of vincristine sulfate and aspirin. The different binding images show that vincrinstine sulfate binding DNA chains caused some loops and cleavages of the DNA fragments, whereas aspirin interaction caused the width changes and conformational transition of the DNA fragments. Two different DNA structural alternations could be explained by the different mechanisms of the interactions with these two components. Our work indicates that the AFM is a powerful tool in studying the interaction between DNA and small molecules.

Randomized DNA libraries construction tool: a new 3-bp 'frequent cutter' TthHB27I/sinefungin endonuclease with chemically-induced specificity.

PubMed

Krefft, Daria; Papkov, Aliaksei; Prusinowski, Maciej; Zylicz-Stachula, Agnieszka; Skowron, Piotr M

2018-05-11

Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5'-CAARCA-3' is converted into a combined 3.2-3.0-bp 'site' or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA.

The effect of swim-up and gradient sperm preparation techniques on deoxyribonucleic acid (DNA) fragmentation in subfertile patients.

PubMed

Oguz, Yuksel; Guler, Ismail; Erdem, Ahmet; Mutlu, Mehmet Firat; Gumuslu, Seyhan; Oktem, Mesut; Bozkurt, Nuray; Erdem, Mehmet

2018-03-23

To compare the effect of two different sperm preparation techniques, including swim-up and gradient methods on sperm deoxyribonucleic acid (DNA) fragmentation status of semen samples from unexplained and mild male factor subfertile patients undergoing intrauterine insemination (IUI). A prospective randomized study was conducted in 65 subfertile patients, including 34 unexplained and 31 male factor infertility to compare basal and post-procedure DNA fragmentation rates in swim-up and gradient techniques. Sperm DNA fragmentation rates were evaluated by a sperm chromatin dispersion (SCD) test in two portions of each sample of semen that was prepared with either swim-up or gradient techniques. Sperm motility and morphology were also assessed based on WHO 2010 criteria. Swim-up but not gradient method yielded a statistically significant reduction in the DNA fragmented sperm rate after preparation as compared to basal rates, in the semen samples of both unexplained (41.85 ± 22.04 vs. 28.58 ± 21.93, p < 0.001 for swim-up; and 41.85 ± 22.04 vs. 38.79 ± 22.30, p = 0.160 for gradient) and mild male factor (46.61 ± 19.38 vs. 30.32 ± 18.20, p < 0.001 for swim-up and 46.61 ± 19.38 vs. 44.03 ± 20.87, p = 0.470 for gradient) subgroups. Swim-up method significantly reduces sperm DNA fragmentation rates and may have some prognostic value on intrauterine insemination in patients with decreased sperm DNA integrity.

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

PubMed

Schouten, Henk J; Vande Geest, Henri; Papadimitriou, Sofia; Bemer, Marian; Schaart, Jan G; Smulders, Marinus J M; Perez, Gabino Sanchez; Schijlen, Elio

2017-03-01

Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

The Effect of Glyphosate on Human Sperm Motility and Sperm DNA Fragmentation.

PubMed

Anifandis, George; Katsanaki, Katerina; Lagodonti, Georgia; Messini, Christina; Simopoulou, Mara; Dafopoulos, Konstantinos; Daponte, Alexandros

2018-05-30

Glyphosate is the active ingredient of Roundup ® , which is one of the most popular herbicides worldwide. Although many studies have focused on the reproductive toxicity of glyphosate or glyphosate-based herbicides, the majority of them have concluded that the effect of the specific herbicide is negligible, while only a few studies indicate the male reproductive toxicity of glyphosate alone. The aim of the present study was to investigate the effect of 0.36 mg/L glyphosate on sperm motility and sperm DNA fragmentation (SDF). Thirty healthy men volunteered to undergo semen analysis for the purpose of the study. Sperm motility was calculated according to WHO 2010 guidelines at collection time (zero time) and 1 h post-treatment with glyphosate. Sperm DNA fragmentation was evaluated with Halosperm ® G2 kit for both the control and glyphosate-treated sperm samples. Sperm progressive motility of glyphosate-treated samples was significantly reduced after 1 h post-treatment in comparison to the respective controls, in contrast to the SDF of glyphosate-treated samples, which was comparable to the respective controls. Conclusively, under these in vitro conditions, at high concentrations that greatly exceed environmental exposures, glyphosate exerts toxic effects on sperm progressive motility but not on sperm DNA integrity, meaning that the toxic effect is limited only to motility, at least in the first hour.

[Construction of large fragment metagenome library of natural mangrove soil].

PubMed

Jiang, Yun-Xia; Zheng, Tian-Ling

2007-11-01

Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significant increased. The large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. All of the clones had recombinant Cosmids and each differed in their fragment profiles when Cosmid DNA was extracted from 12 randomly picked colonies and digested with BamHI. The average insert size for this library was larger than 35 kbp. This culturing-independent library at least encompassed 335 Mbp valuable genetic information of mangrove soil microbes. It allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.

Oxidative stress negatively affects human sperm mitochondrial respiration.

PubMed

Ferramosca, Alessandra; Pinto Provenzano, Sara; Montagna, Daniela Domenica; Coppola, Lamberto; Zara, Vincenzo

2013-07-01

To correlate the level of oxidative stress in serum and seminal fluid and the level of sperm deoxyribonucleic acid (DNA) fragmentation with sperm mitochondrial respiratory efficiency. Sperm mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. A possible relationship between sperm mitochondrial respiratory efficiency, the level of oxidative stress, and the level of sperm DNA fragmentation was investigated. Sperm motility was positively correlated with mitochondrial respiration but negatively correlated with oxidative stress and DNA fragmentation. Interestingly, sperm mitochondrial respiratory activity was negatively affected by oxidative stress and DNA fragmentation. Our data indicate that sperm mitochondrial respiration is decreased in patients with high levels of reactive oxygen species by an uncoupling between electron transport and adenosine triphosphate synthesis. This reduction in mitochondrial functionality might be 1 of the reasons responsible for the decrease in spermatozoa motility. Copyright © 2013 Elsevier Inc. All rights reserved.

A new mutation in the CFTR gene, composed of two adjacent DNA alterations, is a common cause of cystic fibrosis among Georgian Jews

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

Shoshani, T.; Berkun, Y.; Yahav, Y.

Five Jewish cystic fibrosis (CF) patients from four unrelated families, all of whom emigrated from what was Soviet Georgia were studied. The parents in two of the families are first-degree relatives. The clinical phenotype of the patients seems to be associated with a severe disease, as reflected by early age of diagnosis (before the age of 1 year), high sweat chloride level (105-140 meq/liter), and pancreatic insufficiency. The pulmonary function and nutritional status of these patients are normal. These patients were tested for [Delta]F508 by analysis of heteroduplex DNA (4). None of the CF chromosomes was found to carry themore » [Delta]F508 mutation. Subsequently, PCR-amplified genomic DNA samples from two of these patients were subjected to direct sequencing (5) of regions containing exons 7, 9-12, an 19-21 of the CF gene using the oligonucleotides previously described (3, 6). In exon 7, two DNA alterations 3 bp apart were identified in both patients. The first alteration in a C [yields] A transversion at nucleotide position 1207, changing the glutamine codon to lysine (Q359K). The second DNA alteration is a C [yields] A transversion at nucleotide position 1211 changing the threonine codon to lysine (T360K). The two DNA alterations cause nonconservative amino acid substitutions, changing each of the two uncharged polar amino acids (glutamine and threonine) to a basic amino acid, lysine. The Q359K substitution destroys an Rsal recognition site and can be detected by PCR amplification of exon 7 using 7i-5 and 7i-3 oligonucleotides (6), followed by Rsal digestion and electrophoresis on 10% polyacrylamide gels. Two Rsal sites are found in a normal amplified DNA fragment, resulting in three restriction fragments of 292, 68, and 50 bp. Digestion of the PCR fragment of an individual homozygous for this substitution resulted in only two fragments of 342 and 68 bp. 6 refs., 3 figs.« less

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

PubMed

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

NASA Astrophysics Data System (ADS)

Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

2015-09-01

Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

Nucleic acid isolation

DOEpatents

Longmire, J.L.; Lewis, A.K.; Hildebrand, C.E.

1988-01-21

A method is provided for isolating DNA from eukaryotic cell and flow sorted chromosomes. When DNA is removed from chromosome and cell structure, detergent and proteolytic digestion products remain with the DNA. These products can be removed with organic extraction, but the process steps associated with organic extraction reduces the size of DNA fragments available for experimental use. The present process removes the waste products by dialyzing a solution containing the DNA against a solution containing polyethylene glycol (PEG). The waste products dialyze into the PEG leaving isolated DNA. The remaining DNA has been prepared with fragments containing more than 160 kb. The isolated DNA has been used in conventional protocols without effect on the protocol.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples.

PubMed

Ogunremi, Dele; Nadin-Davis, Susan; Dupras, Andrée Ann; Márquez, Imelda Gálvan; Omidi, Katayoun; Pope, Louise; Devenish, John; Burke, Teresa; Allain, Ray; Leclair, Daniel

2017-02-01

A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella -specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non- Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

[On the use of FTA technology for collection, archieving, and molecular analysis of microsporidia dna from clinical stool samples].

PubMed

Sokolova, O I; Dem'ianov, A V; Bovers, L S; Did'e, E S; Sokolova, Iu Ia

2011-01-01

The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates.

Plant genotoxicity: a molecular cytogenetic approach in plant bioassays.

PubMed

Maluszynska, Jolanta; Juchimiuk, Jolanta

2005-06-01

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).

Ultrasensitive electrochemical detection of DNA based on Zn²⁺ assistant DNA recycling followed with hybridization chain reaction dual amplification.

PubMed

Qian, Yong; Wang, Chunyan; Gao, Fenglei

2015-01-15

A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

First ancient DNA sequences from the Late Pleistocene red deer (Cervus elaphus) in the Crimea, Ukraine

NASA Astrophysics Data System (ADS)

Stanković, Ana; Nadachowski, Adam; Doan, Karolina; Stefaniak, Krzysztof; Baca, Mateusz; Socha, Paweł; Wegleński, Piotr; Ridush, Bogdan

2010-05-01

The Late Pleistocene has been a period of significant population and species turnover and extinctions among the large mammal fauna. Massive climatic and environmental changes during Pleistocene significantly influenced the distribution and also genetic diversity of plants and animals. The model of glacial refugia and habitat contraction to southern peninsulas in Europe as areas for the survival of temperate animal species during unfavourable Pleistocene glaciations is at present widely accepted. However, both molecular data and the fossil record indicate the presence of northern and perhaps north-eastern refugia in Europe. In recent years, much new palaeontological data have been obtained in the Crimean Peninsula, Ukraine, following extensive investigations. The red deer (Cervus elaphus) samples for aDNA studies were collected in Emine-Bair-Khosar Cave, situated on the north edge of Lower Plateau of the Chatyrdag Massif (Crimean Mountains). The cave is a vertical shaft, which functioned as a huge mega-trap over a long period of time (probably most of the Pleistocene). The bone assemblages provided about 5000 bones belonging to more than 40 species. The C. elaphus bones were collected from three different stratigraphical levels, radiocarbon dated by accelerator mass spectrometry (AMS) method. The bone fragments of four specimens of red deer were used for the DNA isolation and analysis. The mtDNA (Cytochome b) was successfully isolated from three bone fragments and the cytochrome b sequences were amplified by multiplex PCR. The sequences obtained so far allowed for the reconstruction of only preliminary phylogenetic trees. A fragment of metatarsus from level dated to ca. 48,500±2,000 years BP, yielded a sequence of 513 bp, allowing to locate the specimen on the phylogenetic tree within modern C. elaphus specimens from southern and middle Europe. The second bone fragment, a fragment of mandible, collected from level dated approximately to ca. 33,500±400 years BP, yielded a sequence (696 bp) locating this specimen much closer to the modern C. elaphus specimens from China and Far East. From the third bone fragment (metatarsus), dated between ca. 12,000 years BP and 30,000 years BP, the sequence of only 346 bp has been obtained. It locates this specimen between European and Asiatic haplogroups. The preliminary results of analysis of the DNA from Crimean C. elaphus fossils reveal the great genetic heterogeneity and a complex phylogeographical pattern of the material studied. The obtained results support the opinion that Crimean Peninsula was the most north-eastern refugium in Europe during Late Pleistocene playing a major role in recolonization and dispersal processes of temperate species during and after the Late Pleistocene in this part of the Euro-Asian continent.

Real-time Tracking of DNA Fragment Separation by Smartphone.

PubMed

Tao, Chunxian; Yang, Bo; Li, Zhenqing; Zhang, Dawei; Yamaguchi, Yoshinori

2017-06-01

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Useful DNA polymorphisms are identified by snapback, a midrepetitive element in Tribolium castaneum.

PubMed

Stuart, J J; De Gortari, M J; Hall, P S; Maxwell, M E; Mocelin, G; Brown, S J; Muir, W M

1996-06-01

The red flour bettle, Tribolium castaneum, is both a pest of stored grain products and an important experimental organism. To improve its facility as a genetic model, we are developing DNA fingerprinting methods for this insect. A Tribolium DNA fragment, snapback-1 (SBI), identified among sequences that reassociate before a Cot of 0.03 mol.s/L, was found to produce a banding pattern in restriction endonuclease digested genomic DNA that is characteristic of a midrepetitive element. DNA fingerprints of individual beetles demonstrated that unvarying inherited DNA polymorphism is revealed, and that polymorphism is inherited in a dominant Mendelian fashion. Linkage between bands was minimal. The sequence of SBI was determined, and hybridization experiments indicated that SBI is a fragment of a larger midrepetitive element. Fingerprinting individuals with known inbreeding coefficients indicated that SBI loci have relatively high mutation rates. The possibility that SBI is a fragment of a transposable element is discussed.

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

PubMed Central

Hykin, Sarah M.; Bi, Ke; McGuire, Jimmy A.

2015-01-01

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis. PMID:26505622

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

PubMed

Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

2015-01-01

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Sperm deoxyribonucleic acid damage in normozoospermic men is related to age and sperm progressive motility.

PubMed

Belloc, Stephanie; Benkhalifa, Moncef; Cohen-Bacrie, Martine; Dalleac, Alain; Amar, Edouard; Zini, Armand

2014-06-01

To evaluate sperm DNA fragmentation in normozoospermic male partners of couples undergoing infertility evaluation. Retrospective cohort study. Clinical andrology laboratory. A total of 1,974 consecutive normozoospermic men selected from a larger cohort of 4,345 consecutive, nonazoospermic men presenting for infertility evaluation. None. Clinical parameters, conventional semen parameters, and sperm DNA fragmentation assessed by flow cytometry-based TUNEL assay and reported as percent sperm DNA fragmentation (%SDF). The mean (± SD) %SDF and the proportion of men with high %SDF (>30%) were significantly lower in the normozoospermic compared with the entire cohort of 4,345 evaluable infertile men (17.6% ± 10.1% vs. 20.7% ± 12.4% and 11% vs. 20%, respectively). In the group of 1,974 normozoospermic men, %SDF was positively correlated with paternal age (r = 0.17) and inversely correlated with progressive motility (r = -0.26). In the subset of normozoospermic men with sperm parameters above the 50th percentile (≥ 73 × 10(6) sperm/mL, ≥ 55% progressive motility, and ≥ 14% normal forms, World Health Organization 2010 guidelines), 5% (4 of 83) had elevated %SDF (>30%). In this large cohort of normozoospermic men presenting for infertility evaluation, DNA fragmentation level is related to sperm motility and paternal age, and 11% of these men have high levels of sperm DNA fragmentation. Furthermore, the data indicate that a nonnegligible proportion (5%) of normozoospermic men with high-normal sperm parameters may also have significant sperm DNA fragmentation. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

Theoretical study of the Hoogsteen-Watson-Crick junctions in DNA.

PubMed

Cubero, Elena; Luque, F Javier; Orozco, Modesto

2006-02-01

A series of d (AT)(n) oligonucleotides containing mixtures of normal B-type Watson-Crick and antiparallel Hoogsteen helices have been studied using molecular dynamics simulation techniques to analyze the structural and thermodynamic impact of the junction between Watson-Crick and antiparallel Hoogsteen structures. Analysis of molecular dynamics simulations strongly suggests that for all oligonucleotides studied the antiparallel Hoogsteen appears as a reasonable conformation, only slightly less stable than the canonical B-type Watson-Crick one. The junctions between the Watson-Crick and Hoogsteen structures introduces a priori a sharp discontinuity in the helix, because the properties of each type of conformation are very well preserved in the corresponding fragments. However, and quite counterintuitively, junctions do not largely distort the duplex in structural, dynamics or energetic terms. Our results strongly support the possibility that small fragments of antiparallel Hoogsteen duplex might be embedded into large fragments of B-type Watson-Crick helices, making possible protein-DNA interactions that are specific of the antiparallel Hoogsteen conformation.

Theoretical Study of the Hoogsteen–Watson-Crick Junctions in DNA

PubMed Central

Cubero, Elena; Luque, F. Javier; Orozco, Modesto

2006-01-01

A series of d (AT)n oligonucleotides containing mixtures of normal B-type Watson-Crick and antiparallel Hoogsteen helices have been studied using molecular dynamics simulation techniques to analyze the structural and thermodynamic impact of the junction between Watson-Crick and antiparallel Hoogsteen structures. Analysis of molecular dynamics simulations strongly suggests that for all oligonucleotides studied the antiparallel Hoogsteen appears as a reasonable conformation, only slightly less stable than the canonical B-type Watson-Crick one. The junctions between the Watson-Crick and Hoogsteen structures introduces a priori a sharp discontinuity in the helix, because the properties of each type of conformation are very well preserved in the corresponding fragments. However, and quite counterintuitively, junctions do not largely distort the duplex in structural, dynamics or energetic terms. Our results strongly support the possibility that small fragments of antiparallel Hoogsteen duplex might be embedded into large fragments of B-type Watson-Crick helices, making possible protein-DNA interactions that are specific of the antiparallel Hoogsteen conformation. PMID:16287814

Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions

NASA Astrophysics Data System (ADS)

Millar, David P.; Carver, Theodore E.

1994-08-01

Time-resolved fluorescence measurements of optical probes incorporated at specific sites in DNA provides a new approach to studies of DNA structure and DNA:protein interactions. This approach can be used to study complex multi-state behavior, such as the folding of DNA into alternative higher order structures or the transfer of DNA between multiple binding sites on a protein. In this study, fluorescence anisotropy decay of an internal dansyl probe attached to 17/27-mer oligonucleotides was used to monitor the distribution of DNA 3' termini bound at either the polymerase of 3' to 5' exonuclease sites of the Klenow fragment of DNA polymerase I. Partitioning of the primer terminus between the two active sites of the enzyme resulted in a heterogeneous probe environment, reflected in the associative behavior of the fluorescence anisotropy decay. Analysis of the anisotropy decay with a two state model of solvent-exposed and protein-associated dansyl probes was used to determine the fraction of DNA bound at each site. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3-fold increase in the equilibrium partitioning of DNA into the exonuclease site, while two or more consecutive G:G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250- fold greater than that of the corresponding matched DNA sequence. Internal single mismatches located up to four bases from the primer terminus produced larger effects than the same mismatch at the primer terminus. These results provide insight into the recognition mechanisms that enable DNA polymerases to proofread misincorporated bases during DNA replication.

Positive and negative ion mode comparison for the determination of DNA/peptide noncovalent binding sites through the formation of "three-body" noncovalent fragment ions.

PubMed

Brahim, Bessem; Tabet, Jean-Claude; Alves, Sandra

2018-02-01

Gas-phase fragmentation of single strand DNA-peptide noncovalent complexes is investigated in positive and negative electrospray ionization modes.Collision-induced dissociation experiments, performed on the positively charged noncovalent complex precursor ions, have confirmed the trend previously observed in negative ion mode, i.e. a high stability of noncovalent complexes containing very basic peptidic residues (i.e. R > K) and acidic nucleotide units (i.e. Thy units), certainly incoming from the existence of salt bridge interactions. Independent of the ion polarity, stable noncovalent complex precursor ions were found to dissociate preferentially through covalent bond cleavages of the partners without disrupting noncovalent interactions. The resulting DNA fragment ions were found to be still noncovalently linked to the peptides. Additionally, the losses of an internal nucleic fragment producing "three-body" noncovalent fragment ions were also observed in both ion polarities, demonstrating the spectacular salt bridge interaction stability. The identical fragmentation patterns (regardless of the relative fragment ion abundances) observed in both polarities have shown a common location of salt bridge interaction certainly preserved from solution. Nonetheless, most abundant noncovalent fragment ions (and particularly three-body ones) are observed from positively charged noncovalent complexes. Therefore, we assume that, independent of the preexisting salt bridge interaction and zwitterion structures, multiple covalent bond cleavages from single-stranded DNA/peptide complexes rely on an excess of positive charges in both electrospray ionization ion polarities.

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

PubMed Central

2011-01-01

Background Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. Results An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). Conclusions The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy. PMID:21864341

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

PubMed

Sobolewski, Ireneusz; Polska, Katarzyna; Zylicz-Stachula, Agnieszka; Jeżewska-Frąckowiak, Joanna; Rak, Janusz; Skowron, Piotr

2011-08-24

Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

Genetic and Epigenetic Alterations of Brassica nigra Introgression Lines from Somatic Hybridization: A Resource for Cauliflower Improvement.

PubMed

Wang, Gui-Xiang; Lv, Jing; Zhang, Jie; Han, Shuo; Zong, Mei; Guo, Ning; Zeng, Xing-Ying; Zhang, Yue-Yun; Wang, You-Ping; Liu, Fan

2016-01-01

Broad phenotypic variations were obtained previously in derivatives from the asymmetric somatic hybridization of cauliflower "Korso" (Brassica oleracea var. botrytis, 2n = 18, CC genome) and black mustard "G1/1" (Brassica nigra, 2n = 16, BB genome). However, the mechanisms underlying these variations were unknown. In this study, 28 putative introgression lines (ILs) were pre-selected according to a series of morphological (leaf shape and color, plant height and branching, curd features, and flower traits) and physiological (black rot/club root resistance) characters. Multi-color fluorescence in situ hybridization revealed that these plants contained 18 chromosomes derived from "Korso." Molecular marker (65 simple sequence repeats and 77 amplified fragment length polymorphisms) analysis identified the presence of "G1/1" DNA segments (average 7.5%). Additionally, DNA profiling revealed many genetic and epigenetic differences among the ILs, including sequence alterations, deletions, and variation in patterns of cytosine methylation. The frequency of fragments lost (5.1%) was higher than presence of novel bands (1.4%), and the presence of fragments specific to Brassica carinata (BBCC 2n = 34) were common (average 15.5%). Methylation-sensitive amplified polymorphism analysis indicated that methylation changes were common and that hypermethylation (12.4%) was more frequent than hypomethylation (4.8%). Our results suggested that asymmetric somatic hybridization and alien DNA introgression induced genetic and epigenetic alterations. Thus, these ILs represent an important, novel germplasm resource for cauliflower improvement that can be mined for diverse traits of interest to breeders and researchers.

Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

PubMed

Fu, Changlin; Donovan, William P; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.

Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

PubMed Central

Fu, Changlin; Donovan, William P.; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H.

2014-01-01

Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%. PMID:25551825

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

PubMed

Yu, Stephanie C Y; Chan, K C Allen; Zheng, Yama W L; Jiang, Peiyong; Liao, Gary J W; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y; Go, Attie T J I; van Vugt, John M G; Minekawa, Ryoko; Oudejans, Cees B M; Nicolaides, Kypros H; Chiu, Rossa W K; Lo, Y M Dennis

2014-06-10

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

PubMed Central

Yu, Stephanie C. Y.; Chan, K. C. Allen; Zheng, Yama W. L.; Jiang, Peiyong; Liao, Gary J. W.; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y.; Go, Attie T. J. I.; van Vugt, John M. G.; Minekawa, Ryoko; Oudejans, Cees B. M.; Nicolaides, Kypros H.; Chiu, Rossa W. K.; Lo, Y. M. Dennis

2014-01-01

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring. PMID:24843150

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

1995-01-01

A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

NASA Technical Reports Server (NTRS)

Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

2000-01-01

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

PubMed

Speck, Janina; Stebel, Sabine C; Arndt, Katja M; Müller, Kristian M

2011-01-01

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.

DNA Extraction from Museum Specimens of Parasitic Hymenoptera

PubMed Central

Andersen, Jeremy C.; Mills, Nicholas J.

2012-01-01

At the same time that molecular researchers are improving techniques to extract DNA from museum specimens, this increased demand for access to museum specimens has created tension between the need to preserve specimens for maintaining collections and morphological research and the desire to conduct molecular analyses. To address these concerns, we examined the suitability of non-invasive DNA extraction techniques on three species of parasitic Hymenoptera (Braconidae), and test the effects of body size (parasitoid species), age (time since collection), and DNA concentration from each extract on the probability of amplifying meaningful fragments of two commonly used genetic loci. We found that age was a significant factor for determining the probability of success for sequencing both 28S and COI fragments. While the size of the braconid parasitoids significantly affected the total amount of extracted DNA, neither size nor DNA concentration were significant factors for the amplification of either gene region. We also tested several primer combinations of various lengths, but were unable to amplify fragments longer than ∼150 base pairs. These short fragments of 28S and COI were however sufficient for species identification, and for the discovery of within species genetic variation. PMID:23077493

Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

PubMed Central

Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

1996-01-01

Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction. PMID:8795234

Southern blotting.

PubMed

Brown, T

2001-05-01

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.

Studies on DNA damage: discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate, obtained for several compounds. Possible explanations of the discrepancies.

PubMed

Parodi, S; Balbi, C; Abelmoschi, M L; Pala, M; Russo, P; Santi, L

1983-12-01

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically , is more strictly correlated with the initiation of DNA unwinding.

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

PubMed Central

Khan, A S; Repaske, R; Garon, C F; Chan, H W; Rowe, W P; Martin, M A

1982-01-01

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA. Images PMID:6281459

Optimisation of DNA extraction from the crustacean Daphnia

PubMed Central

Athanasio, Camila Gonçalves; Chipman, James K.; Viant, Mark R.

2016-01-01

Daphnia are key model organisms for mechanistic studies of phenotypic plasticity, adaptation and microevolution, which have led to an increasing demand for genomics resources. A key step in any genomics analysis, such as high-throughput sequencing, is the availability of sufficient and high quality DNA. Although commercial kits exist to extract genomic DNA from several species, preparation of high quality DNA from Daphnia spp. and other chitinous species can be challenging. Here, we optimise methods for tissue homogenisation, DNA extraction and quantification customised for different downstream analyses (e.g., LC-MS/MS, Hiseq, mate pair sequencing or Nanopore). We demonstrate that if Daphnia magna are homogenised as whole animals (including the carapace), absorbance-based DNA quantification methods significantly over-estimate the amount of DNA, resulting in using insufficient starting material for experiments, such as preparation of sequencing libraries. This is attributed to the high refractive index of chitin in Daphnia’s carapace at 260 nm. Therefore, unless the carapace is removed by overnight proteinase digestion, the extracted DNA should be quantified with fluorescence-based methods. However, overnight proteinase digestion will result in partial fragmentation of DNA therefore the prepared DNA is not suitable for downstream methods that require high molecular weight DNA, such as PacBio, mate pair sequencing and Nanopore. In conclusion, we found that the MasterPure DNA purification kit, coupled with grinding of frozen tissue, is the best method for extraction of high molecular weight DNA as long as the extracted DNA is quantified with fluorescence-based methods. This method generated high yield and high molecular weight DNA (3.10 ± 0.63 ng/µg dry mass, fragments >60 kb), free of organic contaminants (phenol, chloroform) and is suitable for large number of downstream analyses. PMID:27190714

Sperm DNA Fragmentation Index and Hyaluronan Binding Ability in Men from Infertile Couples and Men with Testicular Germ Cell Tumor

PubMed Central

Filipiak, Eliza; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Sobkiewicz, Slawomir; Wojt, Malgorzata; Chmiel, Jacek; Kula, Krzysztof; Slowikowska-Hilczer, Jolanta

2016-01-01

Objective. To investigate sperm DNA fragmentation and sperm functional maturity in men from infertile couples (IC) and men with testicular germ cell tumor (TGCT). Materials and Methods. Semen samples were collected from 312 IC men and 23 men with TGCT before unilateral orchiectomy and oncological treatment. The sperm chromatin dispersion test was performed to determine DNA fragmentation index (DFI) and the ability of sperm to bind with hyaluronan (HA) was assessed. Results. In comparison with the IC men, the men with TGCT had a higher percentage of sperm with fragmented DNA (median 28% versus 21%; p < 0.01) and a lower percentage of HA-bound sperm (24% versus 66%; p < 0.001). Normal results of both analyses were observed in 24% of IC men and 4% of men with TGCT. Negative Spearman's correlations were found between DFI and the percentage of HA-bound sperm in the whole group and in IC subjects and those with TGCT analyzed separately. Conclusions. Approximately 76% of IC men and 96% with TGCT awaiting orchiectomy demonstrated DNA fragmentation and/or sperm immaturity. We therefore recommend sperm banking after unilateral orchiectomy, but before irradiation and chemotherapy; the use of such a deposit appears to be a better strategy to obtain functionally efficient sperms. PMID:27999814

Plasma cell-free DNA quantification is highly correlated to tumor burden in children with neuroblastoma.

PubMed

Wang, Xisi; Wang, Lijun; Su, Yan; Yue, Zhixia; Xing, Tianyu; Zhao, Wen; Zhao, Qian; Duan, Chao; Huang, Cheng; Zhang, Dawei; Jin, Mei; Cheng, Xianfeng; Chen, Shenglan; Liu, Yi; Ma, Xiaoli

2018-06-14

To evaluate plasma cell-free DNA (cfDNA) as a promising biomarker for neuroblastoma (NB) tumor burden. Seventy-nine eligible patients with newly diagnosed NB were recruited from Beijing Children's Hospital between April 2016 and April 2017. Additionally, from September 2011 to June 2017, 79 patients with stable NB were evaluated with a median follow-up time of 21 months. Approximately 2 mL of peripheral blood was drawn upon enrollment, and plasma cfDNA levels were measured via quantitative polymerase chain reaction (qPCR). Total cfDNA analysis was performed using the long interspersed nuclear element 1 (LINE-1) 79 bp fragment, and DNA integrity was calculated by the ratio of the LINE-1 300 bp fragment to the LINE-1 79 bp fragment. A total of 79 NB patients with a median age of 36 months comprised the group of newly diagnosed NB patients. The main primary tumor site was the retroperitoneal and adrenal region (81%). Three or more metastatic sites were found in 17.7% of patients. Stable NB patients older than 18 months comprised 98.7% of the stable NB patients. Neuron-specific enolase (NSE), lactate dehydrogenase (LDH), and cfDNA levels were dramatically increased in the newly diagnosed NB patients and significantly different from those in the stable NB patients. Moreover, the concentration of cfDNA was much higher in patients with larger tumors. By analyzing the area under the receiver operator characteristic (ROC) curve (AUC), the areas of total cfDNA, NSE, and LDH levels were 0.953, 0.929, and 0.906, respectively. The sensitivity and specificity data clarified that the level of circulating cfDNA in plasma can be considered as a reliable biomarker for describing tumor load in NB. The plasma cfDNA concentration was as good as the levels of LDH and NSE to discriminate the tumor burden in children with NB. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Ultra-High-Speed DNA Fragment Separations Using Microfabricated Capillary Array Electrophoresis Chips

NASA Astrophysics Data System (ADS)

Woolley, Adam T.; Mathies, Richard A.

1994-11-01

Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of φX174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 μm) have been explored. By use of channels with an effective length of only 3.5 cm, separations of φX174 Hae III DNA fragments from ≈70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQα alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes.

PubMed Central

Santangelo, J D; Jones, D T; Woods, D R

1991-01-01

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. Images PMID:1991710

Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha.

PubMed

Tikhomirova, L P; Ikonomova, R N; Kuznetsova, E N

1986-01-01

For the transformation of the yeast Hansenula polymorpha we have constructed a set of hybrid plasmids carrying the LEU2 gene of Saccharomyces cerevisiae as a selective marker and fragments of mitochondrial DNA of Candida utilis and H. polymorpha or chromosomal DNA fragments of H. polymorpha as replicator sequences. The replication properties of chimeric plasmids in the yeast H. polymorpha were investigated. We showed that for plasmids propagated autonomously in this yeast the plasmid monomers could be detected in the transformants only during the immediate time after the transformation event. Further growth under selective conditions led to the selection of polymeric forms of plasmid DNA as it was clearly shown for transformants carrying cosmid pL2 with mtDNA fragment of C. utilis. Such transformants carrying polymerized plasmids showed a remarkably increased stability of the transformed phenotype. Cosmid pL2 was able to shuttle between Escherichia coli, S. cerevisiae and H. polymorpha, whereas plasmids with DNA fragments from H. polymorpha did not transform S. cerevisiae effectively.

Nucleic acid isolation process

DOEpatents

Longmire, Jonathan L.; Lewis, Annette K.; Hildebrand, Carl E.

1990-01-01

A method is provided for isolating DNA from eukaryotic cell and flow sorted chromosomes. When DNA is removed from chromosome and cell structure, detergent and proteolytic digestion products remain with the DNA. These products can be removed with organic extraction, but the process steps associated with organic extraction reduce the size of DNA fragments available for experimental use. The present process removes the waste products by dialyzing a solution containing the DNA against a solution containing polyethylene glycol (PEG). The waste products dialyze into the PEG leaving isolated DNA. The remaining DNA has been prepared with fragments containing more than 160 kb. The isolated DNA has been used in conventional protocols without affect on the protocol.

RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

PubMed Central

Halász, László; Karányi, Zsolt; Boros-Oláh, Beáta; Kuik-Rózsa, Tímea; Sipos, Éva; Nagy, Éva; Mosolygó-L, Ágnes; Mázló, Anett; Rajnavölgyi, Éva; Halmos, Gábor; Székvölgyi, Lóránt

2017-01-01

The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection. PMID:28341774

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

PubMed Central

Busti, Elena; Bordoni, Roberta; Castiglioni, Bianca; Monciardini, Paolo; Sosio, Margherita; Donadio, Stefano; Consolandi, Clarissa; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

2002-01-01

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria. PMID:12243651

Crystallization and preliminary X-ray analysis of a complex formed between the antibiotic simocyclinone D8 and the DNA breakage–reunion domain of Escherichia coli DNA gyrase

PubMed Central

Edwards, Marcus J.; Flatman, Ruth H.; Mitchenall, Lesley A.; Stevenson, Clare E. M.; Maxwell, Anthony; Lawson, David M.

2009-01-01

Crystals of a complex formed between the 59 kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage–reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I-centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75 Å from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development. PMID:19652356

E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

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

Turner, Roberta L.; Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu; Ornelles, David A., E-mail: ornelles@wakehealth.edu

2014-05-15

Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose)more » polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.« less

Flexible bent rod model with a saturating induced dipole moment to study the electric linear dichroism of DNA fragments

NASA Astrophysics Data System (ADS)

Bertolotto, Jorge A.; Umazano, Juan P.

2016-06-01

In the present work we make a theoretical study of the steady state electric linear dichroism of DNA fragments in aqueous solution. The here developed theoretical approach considers a flexible bent rod model with a saturating induced dipole moment. The electric polarizability tensor of bent DNA fragments is calculated considering a phenomenological model which theoretical and experimental backgroung is presented here. The model has into account the electric polarizability longitudinal and transversal to the macroion. Molecular flexibility is described using an elastic potential. We consider DNA fragments originally bent with bending fluctuations around an average bending angle. The induced dipole moment is supposed constant once the electric field strength grows up at critical value. To calculate the reduced electric linear dichroism we determine the optical factor considering the basis of the bent DNA perpendicular to the molecular axis. The orientational distribution function has into account the anisotropic electric properties and the molecule flexibility. We applied the present theoretical background to fit electric dichroism experimental data of DNA fragments reported in the bibliography in a wide range of molecular weight and electric field. From these fits, values of DNA physical properties are estimated. We compare and discuss the results here obtained with the theoretical and experimental data presented by other authors. The original contributions of this work are: the inclusion of the transversal electric polarizability saturating with the electric field, the description of the electric properties with an electric polarizability tensor dependant on the bending angle and the use of an arc model originally bent.

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

PubMed Central

Richardson, Ruth E.; Suzuki, Yo

2015-01-01

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work. PMID:26348330

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

PubMed

Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

2008-12-15

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Factor IX[sub Madrid 2]: A deletion/insertion in Facotr IX gene which abolishes the sequence of the donor junction at the exon IV-intron d splice site

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

Solera, J.; Magallon, M.; Martin-Villar, J.

1992-02-01

DNA from a patient with severe hemophilia B was evaluated by RFLP analysis, producing results which suggested the existence of a partial deletion within the factor IX gene. The deletion was further localized and characterized by PCR amplification and sequencing. The altered allele has a 4,442-bp deletion which removes both the donor splice site located at the 5[prime] end of intron d and the two last coding nucleotides located at the 3[prime] end of exon IV in the normal factor IX gene; this fragment has been inserted in inverted orientation. Two homologous sequences have been discovered at the ends ofmore » the deleted DNA fragment.« less

PCR-based Analysis of Microbial Communities in Extreme Environment: Results from EuroGeoMars MDRS campaign

NASA Astrophysics Data System (ADS)

Thiel, C.; Wills, D.; Foing, B.; Wadham, J.; Cullen, D.; van Sluis, C.

2009-04-01

Deoxyribonucleic acid (DNA) is found in almost all living organisms. The main function of DNA molecules is the long-term storage of genetic information.They are passed on from generation to generation as the hereditary material. This molecular structure is often compared to a genetic blueprint, a fingerprint, which is unique for each organism and can therefore be used as a mean of identification. In 1984 a revolutionary technique called polymerase chain reaction (PCR) was established, able to amplify a single or few copies of DNA molecules across several orders of magnitude, generating millions of copies of the original DNA fragment. PCR is nowadays a common technique used in medical and biological research laboratories for a large variety of applications like functional analysis of genes, DNA-based phylogeny, diagnosis of hereditary diseases, detection and diagnosis of infectious diseases, and identification of genetic fingerprints. This powerful tool gives us the opportunity to investigate, if there is or was life on Mars since DNA fragments are highly stable what allows not only amplification from living organisms but also from samples with an age of several thousand years. If we assume that micro-organisms were exchanged between Mars and Earth via meteorites, it is imaginable that Martian life might also be based on DNA as carrier of genetic information. Therefore our goal is to establish a routine for detection of DNA from micro-organisms based on the effective but also robust and simple PCR technique, demonstrated during the EuroGeoMars simulation campaign at Mars Desert Research Station (MDRS). We have already analysed some MDRS soil samples at ESTEC ExoGeoLab facility. During the MDRS simulation we will show that it is possible to establish a minimal molecular biology lab in the habitat for an immediate on site analysis by PCR after sample collection. Samples will be taken from different locations and soil depths. The sample analysis will start immediately after returning to the habitat and will be finished during the following days. DNA will be isolated from micro-organisms by Powersoil DNA isolation kit and serves as template for PCR using oligonucleotides specific for ribosomal DNA to identify representatives of the different groups of micro-organisms: archaea, bacteria and eukaryotes. PCR products will be analysed by agarose gel electrophoresis and documented via UV-trans-illuminator and digital camera.

[Association of phytoplasma with Bermuda grass white-leaf disease].

PubMed

Tan, Weijun; Chen, Yong; Zhang, Wu; Han, Chengchou; Tan, Zhiyuan; Zhang, Juming

2008-10-01

Bermuda grass white leaf is an important disease on Bermuda grass all over the world. The aim of this research is to identify the pathogen which leads to Bermuda grass white leaf occurring on the Chinese mainland. PCR amplification technique, sequence analysis and Southern hybridization were used. A 1.3 kb fragment was amplified by PCR phytoplasma universal primers and total DNA sample extracted from ill Bermuda grass as the amplified template. Sequence analysis of the amplified fragment indicated it clustered into Candidatus Phytoplasm Cynodontis. Southern hybridization analysis showed differential cingulums. The pathogen of Bermuda grass white leaf on the Chinese mainland contains phytoplasma, which provides a scientific basis for further identification, prevention and control of the disease.

Pulsed-field gel electrophoresis typing of Staphylococcus aureus isolates

USDA-ARS?s Scientific Manuscript database

Pulsed-field gel electrophoresis (PFGE) is the most applied and effective genetic typing method for epidemiological studies and investigation of foodborne outbreaks caused by different pathogens, including Staphylococcus aureus. The technique relies on analysis of large DNA fragments generated by th...

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

PubMed

Santillán, Orlando; Ramírez-Romero, Miguel A; Dávila, Guillermo

2017-06-25

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1

PubMed Central

Erdal, Erkin; Haider, Syed; Rehwinkel, Jan; Harris, Adrian L.

2017-01-01

Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3′–5′ exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics. PMID:28279982

Vitrification of neat semen alters sperm parameters and DNA integrity.

PubMed

Khalili, Mohammad Ali; Adib, Maryam; Halvaei, Iman; Nabi, Ali

2014-05-06

Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was significantly higher after vitrification compared to fresh samples in normal (24.76 ± 5.03 and 16.41 ± 4.53, P = .002) and abnormal (34.29 ± 10.02 and 23.5 ± 8.31, P < .0001), respectively. There was negative correlation between sperm motility and sperm DNA integrity in both groups after vitrification. Vitrification of neat ejaculates has negative impact on sperm parameters as well as DNA integrity, particularly among abnormal semen subjects. It is, therefore, recommend to process semen samples and vitrify the sperm pellets.

Generation of marker-free Bt transgenic indica rice and evaluation of its yellow stem borer resistance.

PubMed

Kumar, S; Arul, L; Talwar, D

2010-01-01

We report on generation of marker-free (‘clean DNA’) transgenic rice (Oryza sativa), carrying minimal gene-expression-cassettes of the genes of interest, and evaluation of its resistance to yellow stem borer Scirpophaga incertulas (Lepidoptera: Pyralidae). The transgenic indica rice harbours a translational fusion of 2 different Bacillus thuringiensis (Bt) genes, namely cry1B-1Aa, driven by the green-tissue-specific phosphoenol pyruvate carboxylase (PEPC) promoter. Mature seed-derived calli of an elite indica rice cultivar Pusa Basmati-1 were co-bombarded with gene-expression-cassettes (clean DNA fragments) of the Bt gene and the marker hpt gene, to generate marker-free transgenic rice plants. The clean DNA fragments for bombardment were obtained by restriction digestion and gel extraction. Through biolistic transformation, 67 independent transformants were generated. Transformation frequency reached 3.3%, and 81% of the transgenic plants were co-transformants. Stable integration of the Bt gene was confirmed, and the insert copy number was determined by Southern analysis. Western analysis and ELISA revealed a high level of Bt protein expression in transgenic plants. Progeny analysis confirmed stable inheritance of the Bt gene according to the Mendelian (3:1) ratio. Insect bioassays revealed complete protection of transgenic plants from yellow stem borer infestation. PCR analysis of T2 progeny plants resulted in the recovery of up to 4% marker-free transgenic rice plants.

PCR-based detection of a rare linear DNA in cell culture.

PubMed

Saveliev, Sergei V.

2002-11-11

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 10(7) or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials.

PCR-based detection of a rare linear DNA in cell culture

PubMed Central

2002-01-01

The described method allows for detection of rare linear DNA fragments generated during genomic deletions. The predicted limit of the detection is one DNA molecule per 107 or more cells. The method is based on anchor PCR and involves gel separation of the linear DNA fragment and chromosomal DNA before amplification. The detailed chemical structure of the ends of the linear DNA can be defined with the use of additional PCR-based protocols. The method was applied to study the short-lived linear DNA generated during programmed genomic deletions in a ciliate. It can be useful in studies of spontaneous DNA deletions in cell culture or for tracking intracellular modifications at the ends of transfected DNA during gene therapy trials. PMID:12734566

Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

PubMed

Treulen, F; Uribe, P; Boguen, R; Villegas, J V

2016-02-01

Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study in male infertility. None. The study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT (F.T.). The authors declare no conflict of interest. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

PubMed

Pavco, P A; Van Tuyle, G C

1985-01-01

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.

Evidence for the proteolytic processing of dentin matrix protein 1. Identification and characterization of processed fragments and cleavage sites.

PubMed

Qin, Chunlin; Brunn, Jan C; Cook, Richard G; Orkiszewski, Ralph S; Malone, James P; Veis, Arthur; Butler, William T

2003-09-05

Full-length cDNA coding for dentin matrix protein 1 (DMP1) has been cloned and sequenced, but the corresponding complete protein has not been isolated. In searching for naturally occurring DMP1, we recently discovered that the extracellular matrix of bone contains fragments originating from DMP1. Shortened forms of DMP1, termed 37K and 57K fragments, were treated with alkaline phosphatase and then digested with trypsin. The resultant peptides were purified by a two-dimensional method: size exclusion followed by reversed-phase high performance liquid chromatography. Purified peptides were sequenced by Edman degradation and mass spectrometry, and the sequences compared with the DMP1 sequence predicted from cDNA. Extensive sequencing of tryptic peptides revealed that the 37K fragments originated from the NH2-terminal region, and the 57K fragments were from the COOH-terminal part of DMP1. Phosphate analysis indicated that the 37K fragments contained 12 phosphates, and the 57K fragments had 41. From 37K fragments, two peptides lacked a COOH-terminal lysine or arginine; instead they ended at Phe173 and Ser180 and were thus COOH termini of 37K fragments. Two peptides were from the NH2 termini of 57K fragments, starting at Asp218 and Asp222. These findings indicated that DMP1 is proteolytically cleaved at four bonds, Phe173-Asp174, Ser180-Asp181, Ser217-Asp218, and Gln221-Asp222, forming eight fragments. The uniformity of cleavages at the NH2-terminal peptide bonds of aspartyl residues suggests that a single proteinase is involved. Based on its reported specificity, we hypothesize that these scissions are catalyzed by PHEX protein. We envision that the proteolytic processing of DMP1 plays a crucial role during osteogenesis and dentinogenesis.

Computational model of chromosome aberration yield induced by high- and low-LET radiation exposures.

PubMed

Ponomarev, Artem L; George, Kerry; Cucinotta, Francis A

2012-06-01

We present a computational model for calculating the yield of radiation-induced chromosomal aberrations in human cells based on a stochastic Monte Carlo approach and calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. A previously developed DNA-fragmentation model for high- and low-LET radiation called the NASARadiationTrackImage model was enhanced to simulate a stochastic process of the formation of chromosomal aberrations from DNA fragments. The current version of the model gives predictions of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G(0)/G(1) cell cycle phase during the first cell division after irradiation. As the model can predict smaller-sized deletions and rings (<3 Mbp) that are below the resolution limits of current cytogenetic analysis techniques, we present predictions of hypothesized small deletions that may be produced as a byproduct of properly repaired DNA double-strand breaks (DSB) by nonhomologous end-joining. Additionally, the model was used to scale chromosomal exchanges in two or three chromosomes that were obtained from whole-chromosome FISH painting analysis techniques to whole-genome equivalent values.

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits.

PubMed

Himuro, Yasuyo; Tanaka, Hidenori; Hashiguchi, Masatsugu; Ichikawa, Takanari; Nakazawa, Miki; Seki, Motoaki; Fujita, Miki; Shinozaki, Kazuo; Matsui, Minami; Akashi, Ryo; Hoffmann, Franz

2011-01-15

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant. Copyright © 2010 Elsevier GmbH. All rights reserved.

An analysis on DNA fingerprints of thirty papaya cultivars (Carica papaya L.), grown in Thailand with the use of amplified fragment length polymorphisms technique.

PubMed

Ratchadaporn, Janthasri; Sureeporn, Katengam; Khumcha, U

2007-09-15

The experiment was carried out at the Department of Horticulture, Ubon Ratchathani University, Ubon Ratchathani province, Northeast Thailand during June 2002 to May 2003 aims to identify DNA fingerprints of thirty papaya cultivars with the use of Amplified Fragment Length Polymorphisms (AFLP) technique. Papaya cultivars were collected from six different research centers in Thailand. Papaya plants of each cultivar were grown under field conditions up to four months then leaf numbers 2 and 3 of each cultivar (counted from top) were chosen for DNA extraction and the samples were used for AFLP analysis. Out of 64 random primers being used, 55 pairs gave an increase in DNA bands but only 12 pairs of random primers were randomly chosen for the final analysis of the experiment. The results showed that AFLP markers gave Polymorphic Information Contents (PIC) of three ranges i.e., AFLP markers of 235 lied on a PIC range of 0.003-0.05, 47 for a PIC range of 0.15-0.20 and 12 for a PIC range of 0.35-0.40. The results on dendrogram cluster analysis revealed that the thirty papaya cultivars were classified into six groups i.e., (1) Kaeg Dum and Malador (2) Kaeg Nuan (3) Pakchong and Solo (4) Taiwan (5) Co Coa Hai Nan and (6) Sitong. Nevertheless, in spite of the six papaya groups all papaya cultivars were genetically related to each other where diversity among the cultivars was not significantly found.

Mapped DNA probes from Ioblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

Treesearch

M.R. Ahuja; M.E. Devey; A.T. Groover; K.D. Jermstad; D.B Neale

1994-01-01

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

Multiple tag labeling method for DNA sequencing

DOEpatents

Mathies, R.A.; Huang, X.C.; Quesada, M.A.

1995-07-25

A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster.

PubMed Central

Prieto, R; Yousibova, G L; Woloshuk, C P

1996-01-01

Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A. flavus genomic DNA carried by these clones. Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively.These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9. Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid. Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid. The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid. PMID:8967772

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

PubMed

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

2017-12-01

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

Method for in vitro recombination

DOEpatents

Gibson, Daniel Glenn; Smith, Hamilton O

2013-05-07

The present invention relates to an in vitro method, using isolated protein reagents, for joining two double-stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest.

Extracellular self-DNA as a damage-associated molecular pattern (DAMP) that triggers self-specific immunity induction in plants.

PubMed

Duran-Flores, Dalia; Heil, Martin

2017-10-16

Mammals sense self or non-self extracellular or extranuclear DNA fragments (hereinafter collectively termed eDNA) as indicators of injury or infection and respond with immunity. We hypothesised that eDNA acts as a damage-associated molecular pattern (DAMP) also in plants and that it contributes to self versus non-self discrimination. Treating plants and suspension-cultured cells of common bean (Phaseolus vulgaris) with fragmented self eDNA (obtained from other plants of the same species) induced early, immunity-related signalling responses such as H 2 O 2 generation and MAPK activation, decreased the infection by a bacterial pathogen (Pseudomonas syringae) and increased an indirect defence to herbivores (extrafloral nectar secretion). By contrast, non-self DNA (obtained from lima bean, Phaseolus lunatus, and Acacia farnesiana) had significantly lower or no detectable effects. Only fragments below a size of 700 bp were active, and treating the eDNA preparation DNAse abolished its inducing effects, whereas treatment with RNAse or proteinase had no detectable effect. These findings indicate that DNA fragments, rather than small RNAs, single nucleotides or proteins, accounted for the observed effects. We suggest that eDNA functions a DAMP in plants and that plants discriminate self from non-self at a species-specific level. The immune systems of plants and mammals share multiple central elements, but further work will be required to understand the mechanisms and the selective benefits of an immunity response that is triggered by eDNA in a species-specific manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Mannose Induces an Endonuclease Responsible for DNA Laddering in Plant Cells

PubMed Central

Stein, Joshua C.; Hansen, Geneviève

1999-01-01

The effect of d-mannose (Man) on plant cells was studied in two different systems: Arabidopsis roots and maize (Zea mays) suspension-cultured cells. In both systems, exposure to d-Man was associated with a subset of features characteristic of apoptosis, as assessed by oligonucleosomal fragmentation and microscopy analysis. Furthermore, d-Man induced the release of cytochrome c from mitochondria. The specificity of d-Man was evaluated by comparing the effects of diastereomers such as l-Man, d-glucose, and d-galactose. Of these treatments, only d-Man caused a reduction in final fresh weight with concomitant oligonucleosomal fragmentation. Man-induced DNA laddering coincided with the activation of a DNase in maize cytosolic extracts and with the appearance of single 35-kD band detected using an in-gel DNase assay. The DNase activity was further confirmed by using covalently closed circular plasmid DNA as a substrate. It appears that d-Man, a safe and readily accessible compound, offers remarkable features for the study of apoptosis in plant cells. PMID:10482662

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

PubMed

Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

2015-08-01

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

Information-theoretic signatures of biodiversity in the barcoding gene.

PubMed

Barbosa, Valmir C

2018-08-14

Analyzing the information content of DNA, though holding the promise to help quantify how the processes of evolution have led to information gain throughout the ages, has remained an elusive goal. Paradoxically, one of the main reasons for this has been precisely the great diversity of life on the planet: if on the one hand this diversity is a rich source of data for information-content analysis, on the other hand there is so much variation as to make the task unmanageable. During the past decade or so, however, succinct fragments of the COI mitochondrial gene, which is present in all animal phyla and in a few others, have been shown to be useful for species identification through DNA barcoding. A few million such fragments are now publicly available through the BOLD systems initiative, thus providing an unprecedented opportunity for relatively comprehensive information-theoretic analyses of DNA to be attempted. Here we show how a generalized form of total correlation can yield distinctive information-theoretic descriptors of the phyla represented in those fragments. In order to illustrate the potential of this analysis to provide new insight into the evolution of species, we performed principal component analysis on standardized versions of the said descriptors for 23 phyla. Surprisingly, we found that, though based solely on the species represented in the data, the first principal component correlates strongly with the natural logarithm of the number of all known living species for those phyla. The new descriptors thus constitute clear information-theoretic signatures of the processes whereby evolution has given rise to current biodiversity, which suggests their potential usefulness in further related studies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Use of restriction fragment length polymorphisms to investigate strain variation within Neisseria meningitidis

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

Williams, S.D.

1989-01-01

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collectionmore » of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty-six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P{sup 32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analyzed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population.« less

The effects of metal ions on the DNA damage induced by hydrogen peroxide.

PubMed

Kobayashi, S; Ueda, K; Komano, T

1990-01-01

The effects of metal ions on DNA damage induced by hydrogen peroxide were investigated using two methods, agarose-gel electrophoretic analysis of supercoiled DNA and sequencing-gel analysis of single end-labeled DNA fragments of defined sequences. Hydrogen peroxide induced DNA damage when iron or copper ion was present. At least two classes of DNA damage were induced, one being direct DNA-strand cleavage, and the other being base modification labile to hot piperidine. The investigation of the damaged sites and the inhibitory effects of radical scavengers revealed that hydroxyl radical was the species which attacked DNA in the reaction of H2O2/Fe(II). On the other hand, two types of DNA damage were induced by H2O2/Cu(II). Type I damage was predominant and inhibited by potassium iodide, but type II was not. The sites of the base-modification induced by type I damage were similar to those by lipid peroxidation products and by ascorbate in the presence of Cu(II), suggesting the involvement of radical species other than free hydroxyl radical in the damaging reactions.

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

PubMed Central

Hayford, Alice E.; Petersen, Anne; Vogensen, Finn K.; Jakobsen, Mogens

1999-01-01

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates. PMID:10388723

Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD.

PubMed

Di Bernardo, G; Galderisi, U; Del Gaudio, S; D'Aniello, A; Lanave, C; De Robertis, M T; Cascino, Antonino; Cipollaro, M

2004-05-01

DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals-donkeys, horses, or crossbreeds-were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth.

PCR-based analysis of microbial communities during the EuroGeoMars campaign at Mars Desert Research Station, Utah

NASA Astrophysics Data System (ADS)

Thiel, Cora S.; Ehrenfreund, Pascale; Foing, Bernard; Pletser, Vladimir; Ullrich, Oliver

2011-07-01

The search for evidence of past or present life on Mars will require the detection of markers that indicate the presence of life. Because deoxyribonucleic acid (DNA) is found in all known living organisms, it is considered to be a ‘biosignature’ of life. The main function of DNA is the long-term storage of genetic information, which is passed on from generation to generation as hereditary material. The Polymerase Chain Reaction (PCR) is a revolutionary technique which allows a single fragment or a small number of fragments of a DNA molecule to be amplified millions of times, making it possible to detect minimal traces of DNA. The compactness of the contemporary PCR instruments makes routine sample analysis possible with a minimum amount of laboratory space. Furthermore the technique is effective, robust and straightforward. Our goal was to establish a routine for the detection of DNA from micro-organisms using the PCR technique during the EuroGeoMars simulation campaign. This took place at the Mars Society's Mars Desert Research Station (MDRS) in Utah in February 2009 (organized with the support of the International Lunar Exploration Working Group (ILEWG), NASA Ames and the European Space Research and Technology Centre (ESTEC)). During the MDRS simulation, we showed that it is possible to establish a minimal molecular biology lab in the habitat for the immediate on-site analysis of samples by PCR after sample collection. Soil and water samples were taken at different locations and soil depths. The sample analysis was started immediately after the crew returned to the habitat laboratory. DNA was isolated from micro-organisms and used as a template for PCR analysis of the highly conserved ribosomal DNA to identify representatives of the different groups of micro-organisms (bacteria, archaea and eukarya). The PCR products were visualized by agarose gel electrophoresis and documented by transillumination and digital imaging. The microbial diversity in the collected samples was analysed with respect to sampling depth and the presence or absence of vegetation. For the first time, we have demonstrated that it is possible to perform direct on-site DNA analysis by PCR at MDRS, a simulated planetary habitat in an extreme environment that serves as a model for preparation and optimization of techniques to be used for future Mars exploration.

Use of Restriction Fragment Length Polymorphisms to Investigate Strain Variation Within Neisseria Meningitidis.

NASA Astrophysics Data System (ADS)

Williams, Shelley Diane

Similarity within bacterial populations is difficult to assess due to the limited number of characters available for evaluation and the heterogeneity of bacterial species. Currently, the preferred method used to evaluate the structure of bacterial populations is multilocus enzyme electrophoresis. However, this method is extremely cumbersome and only offers an indirect measure of genetic similarities. The development of a more direct and less cumbersome method for this purpose is warranted. Restriction fragment length polymorphism analysis was evaluated as a tool for use in the study of bacterial population structures and in the epidemiology and surveillance of infectious disease. A collection of Neisseria meningitidis was available for use in the investigation of this technique. Neisseria meningitidis is the causative agent of epidemic cerebrospinal meningitis and septicemia as well as a variety of other clinical manifestations. Each isolate in the collection was defined in terms of serogroup specificity, clinical history, geographic source, and date of isolation. Forty -six strains were chosen for this study. The DNA from each strain was restricted with Pst1 and EcoR1 and electrophoresed on agarose gels. The DNA was transferred to nylon filters and hybridized with P ^{32} labeled DNA probes. Two randomly generated probes and a gene-specific probe were used to estimate the genetic similarities between and among the strains in the study population. A total of 28 different restriction fragment migration types were detected by the probes used. Data obtained from the RFLP analysis was analysed by cluster analysis and multivariate statistical methods. A total of 7 clones groups were detected. Two of these appear to be major clones that comprise 35% of the population. This analysis demonstrates the lack of structure within Neisseria meningitidis due primarily to a heterogenous population and the lack of geographic segregation. The potential utility of this technique as a tool in epidemiologic surveillance is addressed. Further work is needed in the evaluation of RFLP analysis in the taxonomy bacteria.

A Modified Gibson Assembly Method for Cloning Large DNA Fragments with High GC Contents.

PubMed

Li, Lei; Jiang, Weihong; Lu, Yinhua

2018-01-01

Gibson one-step, isothermal assembly method (Gibson assembly) can be used to efficiently assemble large DNA molecules by in vitro recombination involving a 5'-exonuclease, a DNA polymerase and a DNA ligase. In the past few years, this robust DNA assembly method has been widely applied to seamlessly construct genes, genetic pathways and even entire genomes. Here, we expand this method to clone large DNA fragments with high GC contents, such as antibiotic biosynthetic gene clusters from Streptomyces . Due to the low isothermal condition (50 °C) in the Gibson reaction system, the complementary overlaps with high GC contents are proposed to easily form mismatched linker pairings, which leads to low assembly efficiencies mainly due to vector self-ligation. So, we modified this classic method by the following two steps. First, a pair of universal terminal single-stranded DNA overhangs with high AT contents are added to the ends of the BAC vector. Second, two restriction enzyme sites are introduced into the respective sides of the designed overlaps to achieve the hierarchical assembly of large DNA molecules. The optimized Gibson assembly method facilitates fast acquisition of large DNA fragments with high GC contents from Streptomyces.

Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

PubMed Central

Nassar, A; Darrasse, A; Lemattre, M; Kotoujansky, A; Dervin, C; Vedel, R; Bertheau, Y

1996-01-01

Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes. PMID:8779560

Flow cytometry application in the assessment of sperm DNA integrity of men with asthenozoospermia.

PubMed

Piasecka, M; Gaczarzewicz, D; Laszczyńska, M; Starczewski, A; Brodowska, A

2007-01-01

Sperm genomic integrity and ultrastructural features of ejaculated spermatozoa contributing to the assessment of gamete fertility potential in patients with asthenozoospermia are discussed. The proportion of TUNEL-positive cells was significantly higher in the semen of patients with low sperm motility (n=40; p<0.01) as compared to men with normal sperm motility (n=54). Sperm DNA fragmentation negatively correlated (n=94) with sperm motility, sperm concentration, and integrity of the sperm cellular membrane (HOS-test). Two categories of patients were distinguished: (1) patients (23 out of 94 subjects) with < or = 4% of TUNEL-positive cells and (2) patients (71 subjects) with 4% of TUNEL-positive cells. A significant difference was noted in the sperm motility and HOS-test results between patients from both groups. Large numbers of immature spermatozoa with extensive cytoplasmic retention, ultrastructural chromatin and midpiece abnormalities, and conglomerates containing sperm fragments were present more frequently in the semen of asthenozoospermic subjects with >4% of TUNEL-positive sperm cells. Low sperm motility seems to be accompanied by serious defects of gamete chromatin expressed as diminished sperm genomic integrity and abnormal DNA condensation and by defects of sperm midpiece. These abnormalities may reflect developmental failure during the spermatogenic remodeling process. The DNA fragmentation test may be considered as an additional assay for the evaluation of spermatozoa beside standard analysis and taken together with electron microscopy may help to determine the actual number of "healthy" spermatozoa thereby playing an important role during diagnosis and treatment of male infertility.

Genomic fragmentation and extrachromosomal telomeric repeats impact assessment of telomere length in human spermatozoa: quantitative experiments and systematic review.

PubMed

Kurjanowicz, P; Moskovtsev, S; Librach, C

2017-11-01

Can differences in DNA isolation alter assessment of sperm telomere length (spTL) and do they account for conflicting results in the literature on spTL and male fertility? DNA isolation methods preferentially include or exclude short, extrachromosomal (EC) telomere-specific sequences that alter spTL measurements, and are responsible for a proportion of the disparity observed between investigations. The relationship between spTL and male fertility has become an active area of research. The results across investigations, however, have been discordant, generating a need to critically evaluate the existing body of knowledge to guide future investigations. Quantitative experiments determined the effect of DNA isolation on the integrity of sperm DNA and measures of spTL, while a systematic analysis of the current literature evaluated the effect of DNA isolation and study design on experimental outcomes. Two DNA isolation methods were compared: Genomic Tips which isolate 'High Molecular Weight' (HMW) DNA exclusively, and QIAamp® DNA Mini which isolates 'Total' genomic DNA irrespective of size. DNA quality was assessed via field inversion gel electrophoresis (FIGE) and spTL was measured via terminal restriction fragment analysis. In addition, major databases in medicine, health and the life sciences were subject to a targeted search, and results were independently screened according to defined exclusion/inclusion criterion. Findings from primary articles were analyzed for concordance and study designs were compared across six moderator variables (sample size, participant age, fertility status, semen fraction, telomere population and type of analysis). HMW DNA spTL was significantly longer than spTL measured from total DNA (P < 0.01), indicating that Total DNA contained short, EC telomeric repeats that shifted downstream assessment towards shorter spTL. HMW DNA spTL reflected the length of intact, chromosomal telomeres. Major findings on spTL showed the greatest concordance amongst studies that implemented HMW DNA isolation prior to spTL assessment. Studies that utilized Total DNA varied in concordance, but outcomes were similar if (i) a comparative analysis was applied or (ii) a sample size threshold of 81 was achieved for correlative analysis. Chromosomal and EC telomeric DNA were distinguished based on outcomes of HMW DNA isolation and size. Further experiments are required to determine the nature and function of these two types of telomeric sequences. This study reveals a dramatic impact of upstream DNA processing and study design on measurements of spTL, which accounts for conflicting results in the literature. Future assessments of spTL should incorporate independent detection of chromosomal and EC telomeric DNA and specific experimental planning. This study was funded by CReATe Fertility Centre, Toronto, Ontario, Canada. The authors have declared no conflict of interest. N/A. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

[Construction of thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene].

PubMed

Wei, Qing; Liu, Yi-Min; Wang, Hui; Zhao, Xiao-Lin; Ren, Tie-ling; Xiao, Yong-mei

2006-09-01

To construct Thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene and to provide scientific base for deeply researching on the function of cytochrome 1A1 gene (CYP1A1) and the mechanism of carcinogenesis. According to cDNA sequence of human CYP1A1 gene, universal primers (Pm3/Pm4) and mutant primers (Pt15/Pt16 and Pt17/Pt18) containing restriction enzyme site and mutation site were designed. The first set of primers involving Pm3/Pt16 and Pm3/Pt18 amplified a forward 1.5kb fragment from pGEM-T-CYP1A1 plasmid. The second set of primers involving Pt15/Pm4 and Pt17/Pm4 amplified a reverse 177-bp fragment from 10ng pGEM-T-CYP1A1 plasmid. The third set of primers involving Pm3/Pm4 amplified a 1.5kb fragment from the fomer PCR amplifications. The third PCR products were separated, purified and recovered from 1% agarose gel, then inserted into pMD-T vector. Subsequently the conjunct products were transformed into E. coil strain DH-5alpha., then the single clone was screened out and plasmids were extracted from such clone finally verified by restriction endonuclease analysis and sequencing. A 1.5kb fragment of tricycle PCR amplifications were digested by restriction endonucleases (BamHI and SailI) and sequenced bidirectionally by universal primers(T7p and SP6). The results verified that the cloned fragment including Asn461 and Val462 mutant site had 99.9% homology with the human cDNA of CYP1A1 gene in Genebank. The objective fragment containing Asn461 and Va462 mutant site with cDNA of the CYP1A1 gene has been successfully constructed in this experiment.

Surfactin production by strains of Bacillus mojavensis

USDA-ARS?s Scientific Manuscript database

Bacillus mojavensis, RRC101 is an endophytic bacterium patented for control of fungal diseases in maize and other plants. DNA fingerprint analysis of the rep-PCR fragments of 35 B. mojavensis and 4 B. subtilis strains using the Diversilab genotyping system revealed genotypic distinctive strains alon...

Lyme disease with facial nerve palsy: rapid diagnosis using a nested polymerase chain reaction-restriction fragment length polymorphism analysis.

PubMed

Hashimoto, Y; Takahashi, H; Kishiyama, K; Sato, Y; Nakao, M; Miyamoto, K; Iizuka, H

1998-02-01

A 64-year-old woman with Lyme disease and manifesting facial nerve palsy had been bitten by a tick on the left frontal scalp 4 weeks previously. Erythema migrans appeared on the left forehead, accompanied by left facial paralysis. Nested polymerase chain reaction-restriction fragment length polymorphism analysis (nested PCR-RFLP) was performed on DNA extracted from a skin biopsy of the erythema on the left forehead. Borrelia flagellin gene DNA was detected and its RFLP pattern indicated that the organism was B. garinii, Five weeks later, B. garinii was isolated by conventional culture from the erythematous skin lesion, but not from the cerebrospinal fluid. After treatment with ceftriaxone intravenously for 10 days and oral administration of minocycline for 7 days, both the erythema and facial nerve palsy improved significantly. Nested PCR and culture taken after the lesion subsided, using skin samples obtained from a site adjacent to the original biopsy, were both negative. We suggest that nested PCR-RFLP analysis might be useful for the rapid diagnosis of Lyme disease and for evaluating therapy.

The death of Adolf Hitler--forensic aspects.

PubMed

Marchetti, Daniela; Boschi, Ilaria; Polacco, Matteo; Rainio, Juha

2005-09-01

The death of Adolf Hitler is one of the unsolved mysteries of the twentieth century. Numerous historians and journalists have attempted to piece together the details, but despite the interest in the forensic literature regarding the identification of the body, there has not been much scientific debate about the alleged cause of death--cyanide poisoning, gunshot injury, or both. The available literature concerning Hitler's cause of death is incomplete because the toxicological analysis has not been performed and because the skull bone fragment with a gunshot wound possibly from Hitler's corpse has not been properly examined. This has given basis for various theories, which are reviewed. We believe that mtDNA analysis of the skull fragments and of Hitler's jaw, now filed in Moscow, and samples from maternal relatives of Hitler are crucial linking the skull fragment with the gunshot wound to Hitler.

Isolation and characterization of cDNA clones for human erythrocyte beta-spectrin.

PubMed Central

Prchal, J T; Morley, B J; Yoon, S H; Coetzer, T L; Palek, J; Conboy, J G; Kan, Y W

1987-01-01

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical alpha (Mr 240,000) and beta (Mr 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. We report here the isolation and characterization of a human erythroid-specific beta-spectrin cDNA clone that encodes parts of the beta-9 through beta-12 repeat segments. This cDNA was used as a hybridization probe to assign the beta-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte beta-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the beta-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities. Images PMID:3478706

Genetic diversity and differentiation in Prunus species (Rosaceae) using chloroplast and mitochondrial DNA CAPS markers.

PubMed

Ben Mustapha, S; Ben Tamarzizt, H; Baraket, G; Abdallah, D; Salhi Hannachi, A

2015-04-27

Chloroplast (cpDNA) and mitochondrial DNA (mtDNA) were analyzed to establish genetic relationships among Tunisian plum cultivars using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Two mtDNA regions (nad 1 b/c and nad 4 1/2) and a cpDNA region (trnL-trnF) were amplified and digested using restriction enzymes. Seventy and six polymorphic sites were revealed in cpDNA and mtDNA, respectively. As a consequence, cpDNA appears to be more polymorphic than mtDNA. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed that accessions were distributed independently of their geographical origin, and introduced and local cultivars appear to be closely related. Both UPGMA and principal component analysis grouped Tunisian plum accessions into similar clusters. The analysis of the pooled sequences allowed the detection of 17 chlorotypes and 12 mitotypes. The unique haplotypes detected for cultivars are valuable for management and preservation of the plum local resources. From this study, PCR-RFLP analysis appears to be a useful approach to detect and identify cytoplasmic variation in plum trees. Our results also provide useful information for the management of genetic resources and to establish a program to improve the genetic resources available for plums.

Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe

PubMed Central

Lenglez, Sandrine; Hermand, Damien; Decottignies, Anabelle

2010-01-01

Chromosomal double-strand breaks (DSBs) threaten genome integrity and repair of these lesions is often mutagenic. How and where DSBs are formed is a major question conveniently addressed in simple model organisms like yeast. NUMTs, nuclear DNA sequences of mitochondrial origin, are present in most eukaryotic genomes and probably result from the capture of mitochondrial DNA (mtDNA) fragments into chromosomal breaks. NUMT formation is ongoing and was reported to cause de novo human genetic diseases. Study of NUMTs is likely to contribute to the understanding of naturally occurring chromosomal breaks. We show that Schizosaccharomyces pombe NUMTs are exclusively located in noncoding regions with no preference for gene promoters and, when located into promoters, do not affect gene transcription level. Strikingly, most noncoding regions comprising NUMTs are also associated with a DNA replication origin (ORI). Chromatin immunoprecipitation experiments revealed that chromosomal NUMTs are probably not acting as ORI on their own but that mtDNA insertions occurred directly next to ORIs, suggesting that these loci may be prone to DSB formation. Accordingly, induction of excessive DNA replication origin firing, a phenomenon often associated with human tumor formation, resulted in frequent nucleotide deletion events within ORI3001 subtelomeric chromosomal locus, illustrating a novel aspect of DNA replication-driven genomic instability. How mtDNA is fragmented is another important issue that we addressed by sequencing experimentally induced NUMTs. This highlighted regions of S. pombe mtDNA prone to breaking. Together with an analysis of human NUMTs, we propose that these fragile sites in mtDNA may correspond to replication pause sites. PMID:20688779

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

NASA Astrophysics Data System (ADS)

Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

2016-07-01

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

Cloning and heterologous expression of blasticidin S biosynthetic genes from Streptomyces griseochromogenes.

PubMed

Cone, M C; Petrich, A K; Gould, S J; Zabriskie, T M

1998-06-01

Two small chromosomal DNA fragments (2.6 and 4.8 kb) from the blasticidin S producer Streptomyces griseochromogenes were cloned in the high copy number vector pIJ702 and shown to confer increased resistance to blasticidin S upon S. lividans TK24. These fragments were used to screen a library of S. griseochromogenes DNA prepared in the cosmid shuttle vector pOJ446. Cosmids containing DNA inserts of at least 23 kb were identified which hybridized to one or the other resistance fragment, but not to both. Transformation of S. lividans TK24 with several cosmids hybridizing with the 4.8 kb resistance fragment resulted in clones that produced cytosylglucuronic acid, the first intermediate of the blasticidin S biosynthetic pathway, and other blasticidin-related metabolites. A strain of S. lividans TK24 harboring both the 4.8 kb-hybridizing cosmid and the 2.6 kb resistance fragment cloned in pIJ702 produced 12.5 times as much demethylblasticidin S as the transformant harboring the cosmid alone.

Detection of pork adulteration in processed meat by species-specific PCR-QIAxcel procedure based on D-loop and cytb genes.

PubMed

Barakat, Hassan; El-Garhy, Hoda A S; Moustafa, Mahmoud M A

2014-12-01

Detection of pork meat adulteration in "halal" meat products is a crucial issue in the fields of modern food inspection according to implementation of very strict procedures for halal food labelling. Present study aims at detecting and quantifying pork adulteration in both raw and cooked manufactured sausages. This is by applying an optimized species-specific PCR procedure followed by QIAxcel capillary electrophoresis system. Manufacturing experiment was designed by incorporating pork with beef meat at 0.01 to 10 % substitution levels beside beef and pork sausages as negative and positive controls, respectively. Subsequently, sausages were divided into raw and cooked sausages then subjected to DNA extraction. Results indicated that PCR amplifications of mitochondrial D-loop and cytochrome b (cytb) genes by porcine-specific primers produced 185 and 117 bp pork-specific DNA fragments in sausages, respectively. No DNA fragments were detected when PCR was applied on beef sausage DNA confirming primers specificity. For internal control, a 141-bp DNA fragment of eukaryotic 18S ribosomal RNA (rRNA) gene was amplified from pork and beef DNA templates. Although PCR followed by either QIAxcel or agarose techniques were efficient for targeted DNA fragments differentiation even as low as 0.01 % (pork/meat: w/w). For proficiency, adequacy, and performance, PCR-QIA procedure is highly sensitive, a time-saver, electronically documented, mutagenic-reagent free, of little manual errors, accurate in measuring PCR fragments length, and quantitative data supplier. In conclusion, it can be suggested that optimized PCR-QAI is considered as a rapid and sensitive method for routine pork detection and quantification in raw or processed meat.

Cloning and expression analysis of a new anther-specific gene CaMF4 in Capsicum annuum.

PubMed

Hao, Xuefeng; Chen, Changming; Chen, Guoju; Cao, Bihao; Lei, Jianjun

2017-03-01

Our previous study on the genic male sterile-fertile line 114AB of Capsicum annuum indicated a diversity of differentially expressed cDNA fragments in fertile and sterile lines. In this study, a transcript-derived fragment (TDF), male fertile 4 (CaMF4) was chosen for further investigation to observe that this specific fragment accumulates in the flower buds of the fertile line. The full genomic DNA sequence of CaMF4 was 894 bp in length, containing two exons and one intron, and the complete coding sequence encoded a putative 11.53 kDa protein of 109 amino acids. The derived protein of CaMF4 shared similarity with the members of PGPS/D3 protein family. The expression of CaMF4 was detected in both the flower buds at stage 8 and open flowers of the male fertile line. In contrast to this observation, expression of CaMF4 was not detected in any organs of the male sterile line. Further analysis revealed that CaMF4 was expressed particularly in anthers of the fertile line. Our results suggest that CaMF4 is an anther-specific gene and might be indispensable for anther or pollen development in C. annuum.

From milk to diet: feed recognition for milk authenticity.

PubMed

Ponzoni, E; Gianì, S; Mastromauro, F; Breviario, D

2009-11-01

The presence of plastidial DNA fragments of plant origin in animal milk samples has been confirmed. An experimental plan was arranged with 4 groups of goats, each provided with a different monophytic diet: 3 fresh forages (oats, ryegrass, and X-triticosecale) and one 2-wk-old silage (X-triticosecale). Feed-derived rubisco (ribulose bisphosphate carboxylase, rbcL) DNA fragments were detected in 100% of the analyzed goat milk samples, and the nucleotide sequence of the PCR-amplified fragments was found to be 100% identical to the corresponding fragments amplified from the plant species consumed in the diet. Two additional chloroplast-based molecular markers were used to set up an assay for distinctiveness, conveniently based on a simple PCR. In one case, differences in single nucleotides occurring within the gene encoding for plant maturase K (matK) were exploited. In the other, plant species recognition was based on the difference in the length of the intron present within the transfer RNA leucine (trnL) gene. The presence of plastidial plant DNA, ascertained by the PCR-based amplification of the rbcL fragment, was also assessed in raw cow milk samples collected directly from stock farms or taken from milk sold on the commercial market. In this case, the nucleotide sequence of the amplified DNA fragments reflected the multiple forages present in the diet fed to the animals.

Impact of the Z potential technique on reducing the sperm DNA fragmentation index, fertilization rate and embryo development.

PubMed

Duarte, Carlos; Núñez, Víctor; Wong, Yat; Vivar, Carlos; Benites, Elder; Rodriguez, Urso; Vergara, Carlos; Ponce, Jorge

2017-12-01

In assisted reproduction procedures, we need to develop and enhance new protocols to optimize sperm selection. The aim of this study is to evaluate the ability of the Z potential technique to select sperm with intact DNA in non-normospermic patients and evaluate the impact of this selection on embryonic development. We analyzed a total of 174 human seminal samples with at least one altered parameter. We measured basal, post density gradients, and post density gradients + Z potential DNA fragmentation index. To evaluate the impact of this technique on embryo development, 54 cases were selected. The embryo development parameters evaluated were fertilization rate, cleavage rate, top quality embryos at the third day and blastocysts rate. We found significant differences in the study groups when we compared the sperm fragmentation index by adding the Z potential technique to density gradient selection vs. density gradients alone. Furthermore, there was no significant difference in the embryo development parameters between the low sperm fragmentation index group vs. the moderate and high sperm fragmentation index groups, when selecting sperms with this new technique. The Z potential technique is a very useful tool for sperm selection; it significantly reduces the DNA fragmentation index and improves the parameters of embryo development. This technique could be considered routine for its simplicity and low cost.

Evaluation of Microbial Diversity in Wetland through Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP)

DTIC Science & Technology

2006-06-01

51 Appendix C. Promega Restriction Digest Protocol ....................................................53...Rsa1 Restriction Digest Results............................................................................180 9. DNA Base Pair Comparison...particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme (Edwards

Genetic and Epigenetic Alterations of Brassica nigra Introgression Lines from Somatic Hybridization: A Resource for Cauliflower Improvement

PubMed Central

Wang, Gui-xiang; Lv, Jing; Zhang, Jie; Han, Shuo; Zong, Mei; Guo, Ning; Zeng, Xing-ying; Zhang, Yue-yun; Wang, You-ping; Liu, Fan

2016-01-01

Broad phenotypic variations were obtained previously in derivatives from the asymmetric somatic hybridization of cauliflower “Korso” (Brassica oleracea var. botrytis, 2n = 18, CC genome) and black mustard “G1/1” (Brassica nigra, 2n = 16, BB genome). However, the mechanisms underlying these variations were unknown. In this study, 28 putative introgression lines (ILs) were pre-selected according to a series of morphological (leaf shape and color, plant height and branching, curd features, and flower traits) and physiological (black rot/club root resistance) characters. Multi-color fluorescence in situ hybridization revealed that these plants contained 18 chromosomes derived from “Korso.” Molecular marker (65 simple sequence repeats and 77 amplified fragment length polymorphisms) analysis identified the presence of “G1/1” DNA segments (average 7.5%). Additionally, DNA profiling revealed many genetic and epigenetic differences among the ILs, including sequence alterations, deletions, and variation in patterns of cytosine methylation. The frequency of fragments lost (5.1%) was higher than presence of novel bands (1.4%), and the presence of fragments specific to Brassica carinata (BBCC 2n = 34) were common (average 15.5%). Methylation-sensitive amplified polymorphism analysis indicated that methylation changes were common and that hypermethylation (12.4%) was more frequent than hypomethylation (4.8%). Our results suggested that asymmetric somatic hybridization and alien DNA introgression induced genetic and epigenetic alterations. Thus, these ILs represent an important, novel germplasm resource for cauliflower improvement that can be mined for diverse traits of interest to breeders and researchers. PMID:27625659

Restriction fragment length polymorphism of the major histocompatibility complex of the dog.

PubMed

Sarmiento, U M; Storb, R F

1988-01-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3 + Dw4 + D1, Dw8 + D10, D7 + D16 + D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.

Comparative analysis by polymerase chain reaction amplified minicircles of kinetoplast DNA of a stable strain of Trypanosoma cruzi from São Felipe, Bahia, its clones and subclones: possibility of predominance of a principal clone in this area.

PubMed

Campos, R F; Gonçalves, M S; dos Reis, E A; dos Reis, M G; Andrade, S G

1999-01-01

Molecular characterization of one stable strain of Trypanosoma cruzi, the 21 SF, representative of the pattern of strains isolated from the endemic area of São Felipe, State of Bahia, Brazil, maintained for 15 years in laboratory by serial passages in mice and classified as biodeme Type II and zymodeme 2 has been investigated. The kinetoplast DNA (kDNA) of parental strain, 5 clones and 14 subclones were analyzed. Schizodeme was established by comparative study of the fragments obtained from digestion of the 330-bp fragments amplified by polymerase chain reaction (PCR) from the variable regions of the minicircles, and digested by restriction endonucleases Rsa I and Hinf I. Our results show a high percentual of similarity between the restriction fragment length polymorphism (RFLP) for the parental strain and its clones and among these individual clones and their subclones at a level of 80 to 100%. This homology indicates a predominance of the same "principal clone" in the 21SF strain and confirms the homogeneity previously observed at biological and isozymic analysis. These results suggest the possibility that the T. cruzi strains with similar biological and isoenzymic patterns, circulating in this endemic area, are representative of one dominant clone. The presence of "principal clones" could be responsible for a predominant tropism of the parasites for specific organs and tissues and this could contribute to the pattern of clinico-pathological manifestations of Chagas's disease in one geographical area.

Population Genetic Structure and Phylogeography of Camellia flavida (Theaceae) Based on Chloroplast and Nuclear DNA Sequences

PubMed Central

Wei, Su-Juan; Lu, Yong-Bin; Ye, Quan-Qing; Tang, Shao-Qing

2017-01-01

Camellia flavida is an endangered species of yellow camellia growing in limestone mountains in southwest China. The current classification of C. flavida into two varieties, var. flavida and var. patens, is controversial. We conducted a genetic analysis of C. flavida to determine its taxonomic structure. A total of 188 individual plants from 20 populations across the entire distribution range in southwest China were analyzed using two DNA fragments: a chloroplast DNA fragment from the small single copy region and a single-copy nuclear gene called phenylalanine ammonia-lyase (PAL). Sequences from both chloroplast and nuclear DNA were highly diverse; with high levels of genetic differentiation and restricted gene flow. This result can be attributed to the high habitat heterogeneity in limestone karst, which isolates C. flavida populations from each other. Our nuclear DNA results demonstrate that there are three differentiated groups within C. flavida: var. flavida 1, var. flavida 2, and var. patens. These genetic groupings are consistent with the morphological characteristics of the plants. We suggest that the samples included in this study constitute three taxa and the var. flavida 2 group is the genuine C. flavida. The three groups should be recognized as three management units for conservation concerns. PMID:28579991

Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

PubMed

Lam, Kathy N; Charles, Trevor C

2015-01-01

Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite widespread use of E. coli to propagate foreign DNA in metagenomic libraries, the effects of in vivo transcriptional activity on clone stability are not well understood. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

DNA methylation profiling using HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)

PubMed Central

Suzuki, Masako; Greally, John M.

2010-01-01

The HELP assay is a technique that allows genome-wide analysis of cytosine methylation. Here we describe the assay, its relative strengths and weaknesses, and the transition of the assay from a microarray to massively-parallel sequencing-based foundation. PMID:20434563

QUALITY ASSURANCE CONSIDERATIONS FOR USE OF THE FLUORIMAGER SI AND FRAGMENT ANALYSIS SOFTWARE

EPA Science Inventory

The Fluorimager SI (FSI) from Molecular Dynamics is one of several scanning instruments available for the detection of fluorescent emissions associated with DNA samples in a variety of matrices (agarose and polyacrylamide gels, membranes and microplates). In our laboratory, we m...

Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

DOE PAGES

Kostylev, Maxim; Otwell, Anne E.; Richardson, Ruth E.; ...

2015-09-08

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processesmore » associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.« less

Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

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

Kostylev, Maxim; Otwell, Anne E.; Richardson, Ruth E.

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processesmore » associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.« less

Studies with infections fragments of phage DNA. Final report, January 1, 1970--June 30, 1976

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

Schachtele, C. F.

The minute, virulent and structurally intricate Bacillus subtilis bacteriophage phi 29 was utilized to study in vivo viral development. Purified strands of phi 29 DNA were used to analyze transcription of the viral genome. Early mRNA hybridizes to the light DNA strand which controls DNA replication and other early functions. Late mRNA hybridizes to the heavy DNA strand which codes for phage structural proteins. The temporal sequence of specific viral protein synthesis was analyzed by gel electrophoresis and was shown to directly correlate with the RNA transcription pattern. The genes carried by phi 29 have been marked with ts andmore » sus mutations and mapped by appropriate crosses yielding a linear map of 17 cistrons. Fragments of the phi 29 DNA were shown to retain their biological activity and marker rescue studies indicated that gene transfer could be performed with pieces having a molecular weight of less than 1 million daltons. Mutant infection under nonpermissive conditions and the analysis of precursor structures has allowed the formation of a tentative morphogenetic pathway leading to the formation of infectious particles. Work with phi 29 has established this virus as an advantageous model system for studying a variety of problems in molecular biology and approximately a dozen laboratories in the country and abroad are working with this phage.« less

The combi-targeting concept: in vitro and in vivo fragmentation of a stable combi-nitrosourea engineered to interact with the epidermal growth factor receptor while remaining DNA reactive.

PubMed

Qiu, Qiyu; Domarkas, Juozas; Banerjee, Ranjita; Merayo, Nuria; Brahimi, Fouad; McNamee, James P; Gibbs, Bernard F; Jean-Claude, Bertrand J

2007-01-01

JDA58 (NSC 741282), a "combi-molecule" optimized in the context of the "combi-targeting concept," is a nitrosourea moiety tethered to an anilinoquinazoline. Here, we sought to show its binary epidermal growth factor receptor (EGFR)/DNA targeting property and to study its fragmentation in vitro and in vivo. The fragmentation of JDA58 was detected in cells in vitro and in vivo by fluorescence microscopy and tandem mass spectrometry. EGFR phosphorylation and DNA damage were determined by Western blotting and comet assay, respectively. Tumor data were examined for statistical significance using the Student's t test. JDA58 inhibited EGFR tyrosine kinase (IC(50), 0.2 micromol/L) and blocked EGFR phosphorylation in human DU145 prostate cancer cells. It induced significant levels of DNA damage in DU145 cells in vitro or in vivo and showed potent antiproliferative activity both in vitro and in a DU145 xenograft model. In cell-free medium, JDA58 was hydrolyzed to JDA35, a fluorescent amine that could be observed in tumor cells both in vitro and in vivo. In tumor cells in vitro or in vivo, or in plasma collected from mice, the denitrosated species JDA41 was the predominant metabolite. However, mass spectrometric analysis revealed detectable levels of the hydrolytic product JDA35 in tumor cells both in vitro and in vivo. The results in toto suggest that growth inhibition in vitro and in vivo may be sustained by the intact combi-molecule plus JDA35 plus JDA41, three inhibitors of EGFR, and the concomitantly released DNA-damaging species. This leads to a model wherein a single molecule carries a complex multitargeted-multidrug combination.

Extraction of genomic DNA from yeasts for PCR-based applications.

PubMed

Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

2011-05-01

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Live Cell Characterization of DNA Aggregation Delivered through Lipofection

PubMed Central

Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A.; Gratton, Enrico; Jones, Mark R

2015-01-01

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation. PMID:26013547

Study of the family of a patient with male-limited precocious puberty (MPP) due to T1193C transition in exon 11 of LH receptor gene.

PubMed

Ignacak, M; Starzyk, J; Dziatkowiak, H; Trzeciak, W H

2002-03-01

Molecular diagnostics of the LHR gene was conducted in a 5-year-old boy with clinical symptoms and hormonal profile typical of precocious puberty. His parents and 4 sisters were also diagnosed. Single-strand conformation polymorphism analysis under temperature gradient conditions (Multitemperature SSCP) of 3 overlapping fragments of exon 11 of LHR gene revealed a mutation in the fragment spanning nucleotides 1072 to 1804. This mutation was found in the patient, in his mother and in his 4 sisters, and was confirmed by digestion with the use of restriction enzyme Bbr Cl. Direct sequencing revealed a heterozygous T1193C transition in the DNA fragment of the patient and in one of the alleles of his mother's and sister's DNA. This mutation causes Met398Thr substitution in the second transmembrane helix and results in a constitutive activation of LH receptor. This is the second identical mutation detected in Poland and one of the 7 identified so far in the world population.

The Evolutionary Relationships between Endosymbiotic Green Algae of Paramecium bursaria Syngens Originating from Different Geographical Locations.

PubMed

Zagata, Patrycja; Greczek-Stachura, Magdalena; Tarcz, Sebastian; Rautian, Maria

2016-01-01

Paramecium bursaria (Ehrenberg 1831), a freshwater ciliate, typically harbors hundreds of green algal symbionts inside the cell. The aim of present study was the molecular identification of newly analyzed P. bursaria symbionts. The second aspect of the present survey was testing a hypothesis whether endosymbionts prefer the specified syngen of the host, and the specified geographical distribution. Ten strains of endosymbionts isolated from strains of P. bursaria originating from different geographical locations were studied. We analyzed for the first time, both the fragment of plastid genome containing 3'rpl36-5' infA genes and a fragment of a nuclear gene encoding large subunit ribosomal RNA (LSU rDNA). The analysis of the LSU rDNA sequences showed the existence of 3 haplotypes and the haplotype diversity of 0.733, and 8 haplotypes for the 3'rpl36-5' infA gene fragment and haplotype diversity of 0.956. The endosymbionts isolated from P. bursaria strains were identified as Chlorella vulgaris, Ch. variabilis and Micractinium conductrix. There was no correlation between the syngen of P. bursaria and the species of endosymbiont.

Annual time-series analysis of aqueous eDNA reveals ecologically relevant dynamics of lake ecosystem biodiversity

PubMed Central

Bista, Iliana; Carvalho, Gary R.; Walsh, Kerry; Seymour, Mathew; Hajibabaei, Mehrdad; Lallias, Delphine; Christmas, Martin; Creer, Simon

2017-01-01

The use of environmental DNA (eDNA) in biodiversity assessments offers a step-change in sensitivity, throughput and simultaneous measures of ecosystem diversity and function. There remains, however, a need to examine eDNA persistence in the wild through simultaneous temporal measures of eDNA and biota. Here, we use metabarcoding of two markers of different lengths, derived from an annual time series of aqueous lake eDNA to examine temporal shifts in ecosystem biodiversity and in an ecologically important group of macroinvertebrates (Diptera: Chironomidae). The analyses allow different levels of detection and validation of taxon richness and community composition (β-diversity) through time, with shorter eDNA fragments dominating the eDNA community. Comparisons between eDNA, community DNA, taxonomy and UK species abundance data further show significant relationships between diversity estimates derived across the disparate methodologies. Our results reveal the temporal dynamics of eDNA and validate the utility of eDNA metabarcoding for tracking seasonal diversity at the ecosystem scale. PMID:28098255

Annual time-series analysis of aqueous eDNA reveals ecologically relevant dynamics of lake ecosystem biodiversity

NASA Astrophysics Data System (ADS)

Bista, Iliana; Carvalho, Gary R.; Walsh, Kerry; Seymour, Mathew; Hajibabaei, Mehrdad; Lallias, Delphine; Christmas, Martin; Creer, Simon

2017-01-01

The use of environmental DNA (eDNA) in biodiversity assessments offers a step-change in sensitivity, throughput and simultaneous measures of ecosystem diversity and function. There remains, however, a need to examine eDNA persistence in the wild through simultaneous temporal measures of eDNA and biota. Here, we use metabarcoding of two markers of different lengths, derived from an annual time series of aqueous lake eDNA to examine temporal shifts in ecosystem biodiversity and in an ecologically important group of macroinvertebrates (Diptera: Chironomidae). The analyses allow different levels of detection and validation of taxon richness and community composition (β-diversity) through time, with shorter eDNA fragments dominating the eDNA community. Comparisons between eDNA, community DNA, taxonomy and UK species abundance data further show significant relationships between diversity estimates derived across the disparate methodologies. Our results reveal the temporal dynamics of eDNA and validate the utility of eDNA metabarcoding for tracking seasonal diversity at the ecosystem scale.

Effect of pyrimido[1,6-a]benzimidazoles, quinolones, and Ca2+ on the DNA gyrase-mediated cleavage reaction.

PubMed Central

Gmünder, H; Kuratli, K; Keck, W

1995-01-01

The quinolones inhibit the A subunit of DNA gyrase in the presence of Mg2+ by interrupting the DNA breakage and resealing steps, and the latter step is also retarded without quinolones if Mg2+ is replaced by Ca2+. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca2+, we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca2+ was in addition dependent on the length of the DNA fragments. PMID:7695300

Development of procedures for the identification of human papilloma virus DNA fragments in laser plume

NASA Astrophysics Data System (ADS)

Woellmer, Wolfgang; Meder, Tom; Jappe, Uta; Gross, Gerd; Riethdorf, Sabine; Riethdorf, Lutz; Kuhler-Obbarius, Christina; Loening, Thomas

1996-01-01

For the investigation of laser plume for the existence of HPV DNA fragments, which possibly occur during laser treatment of virus infected tissue, human papillomas and condylomas were treated in vitro with the CO2-laser. For the sampling of the laser plume a new method for the trapping of the material was developed by use of water-soluble gelatine filters. These samples were analyzed with the polymerase chain reaction (PCR) technique, which was optimized in regard of the gelatine filters and the specific primers. Positive PCR results for HPV DNA fragments up to the size of a complete oncogene were obtained and are discussed regarding infectiousity.

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA.

PubMed

Ludgate, Jackie L; Wright, James; Stockwell, Peter A; Morison, Ian M; Eccles, Michael R; Chatterjee, Aniruddha

2017-08-31

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing. We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

Nucleotide Sequence of the Protective Antigen Gene of Bacillus Anthracis

DTIC Science & Technology

1988-02-02

the bands excised, and the DNA extracted with phenol for cloning in M13 . 6 Nuclotida sequence analysis. The two fragments were each cloned into phages ...DNA; and strain JM103 (29) was used to propagate M13 ph&ge derivatives. -1 Subcloning and detection of PA-producing rsccmbinants. The isolation of...method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 19. Lauben, J. 0., and J. 2. K. Nielsen. 1982. Penicillinase and

The Snail-Induced Sulfonation Pathway in Breast Cancer Metastasis

DTIC Science & Technology

2014-09-01

of the SNAIL protein with DNA The model of SNAIL, containing 4 Zn fingers bound to DNA, was created using PDB structures 1tf3 (TFIIIA protein, for...AutoDOCK (17) analysis of fragmented LIMD2 structure against that of the pdb struc- ture 3kmw (ILK/a-Parvin), rethreading the LIMD2 structure through the top...Fig. 5E). We assessed the structural similarity between LIMD2 and other reported LIM structures present in the PDB . The superposition of LIMD2 onto the