Detection of Non-Amplified Genomic DNA

CERN Document Server

Corradini, Roberto

2012-01-01

This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

Genomic DNA extraction from sapwood of Pinus roxburghii for ...

African Journals Online (AJOL)

Ashish

2013-02-22

Feb 22, 2013 ... A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, .... DNA as a template. PCR was performed on a thermal cycler. (Biorad, Mycycler) incorporating 10 ng genomic DNA to a 25 µl reaction mix containing 1X Taq buffer, 3 mM MgCl2, 0.2 mM each of dNTPs ...

Sequencing intractable DNA to close microbial genomes.

Directory of Open Access Journals (Sweden)

Richard A Hurt

Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

Sequencing Intractable DNA to Close Microbial Genomes

Energy Technology Data Exchange (ETDEWEB)

Hurt, Jr., Richard Ashley [ORNL; Brown, Steven D [ORNL; Podar, Mircea [ORNL; Palumbo, Anthony Vito [ORNL; Elias, Dwayne A [ORNL

2012-01-01

Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled intractable resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such difficult regions in the non-contiguous finished Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. These developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

Directory of Open Access Journals (Sweden)

Zdepski Anna

2011-05-01

Full Text Available Abstract Background High throughput sequencing (HTS technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination. Results We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR. We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment. Conclusions Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.

Recurrence time statistics: versatile tools for genomic DNA sequence analysis.

Science.gov (United States)

Cao, Yinhe; Tung, Wen-Wen; Gao, J B

2004-01-01

With the completion of the human and a few model organisms' genomes, and the genomes of many other organisms waiting to be sequenced, it has become increasingly important to develop faster computational tools which are capable of easily identifying the structures and extracting features from DNA sequences. One of the more important structures in a DNA sequence is repeat-related. Often they have to be masked before protein coding regions along a DNA sequence are to be identified or redundant expressed sequence tags (ESTs) are to be sequenced. Here we report a novel recurrence time based method for sequence analysis. The method can conveniently study all kinds of periodicity and exhaustively find all repeat-related features from a genomic DNA sequence. An efficient codon index is also derived from the recurrence time statistics, which has the salient features of being largely species-independent and working well on very short sequences. Efficient codon indices are key elements of successful gene finding algorithms, and are particularly useful for determining whether a suspected EST belongs to a coding or non-coding region. We illustrate the power of the method by studying the genomes of E. coli, the yeast S. cervisivae, the nematode worm C. elegans, and the human, Homo sapiens. Computationally, our method is very efficient. It allows us to carry out analysis of genomes on the whole genomic scale by a PC.

Quantification of trace-level DNA by real-time whole genome amplification.

Science.gov (United States)

Kang, Min-Jung; Yu, Hannah; Kim, Sook-Kyung; Park, Sang-Ryoul; Yang, Inchul

2011-01-01

Quantification of trace amounts of DNA is a challenge in analytical applications where the concentration of a target DNA is very low or only limited amounts of samples are available for analysis. PCR-based methods including real-time PCR are highly sensitive and widely used for quantification of low-level DNA samples. However, ordinary PCR methods require at least one copy of a specific gene sequence for amplification and may not work for a sub-genomic amount of DNA. We suggest a real-time whole genome amplification method adopting the degenerate oligonucleotide primed PCR (DOP-PCR) for quantification of sub-genomic amounts of DNA. This approach enabled quantification of sub-picogram amounts of DNA independently of their sequences. When the method was applied to the human placental DNA of which amount was accurately determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES), an accurate and stable quantification capability for DNA samples ranging from 80 fg to 8 ng was obtained. In blind tests of laboratory-prepared DNA samples, measurement accuracies of 7.4%, -2.1%, and -13.9% with analytical precisions around 15% were achieved for 400-pg, 4-pg, and 400-fg DNA samples, respectively. A similar quantification capability was also observed for other DNA species from calf, E. coli, and lambda phage. Therefore, when provided with an appropriate standard DNA, the suggested real-time DOP-PCR method can be used as a universal method for quantification of trace amounts of DNA.

Partial digestion with restriction enzymes of ultraviolet-irradiated human genomic DNA: a method for identifying restriction site polymorphisms

International Nuclear Information System (INIS)

Nobile, C.; Romeo, G.

1988-01-01

A method for partial digestion of total human DNA with restriction enzymes has been developed on the basis of a principle already utilized by P.A. Whittaker and E. Southern for the analysis of phage lambda recombinants. Total human DNA irradiated with uv light of 254 nm is partially digested by restriction enzymes that recognize sequences containing adjacent thymidines because of TT dimer formation. The products resulting from partial digestion of specific genomic regions are detected in Southern blots by genomic-unique DNA probes with high reproducibility. This procedure is rapid and simple to perform because the same conditions of uv irradiation are used for different enzymes and probes. It is shown that restriction site polymorphisms occurring in the genomic regions analyzed are recognized by the allelic partial digest patterns they determine

Restriction site extension PCR: a novel method for high-throughput characterization of tagged DNA fragments and genome walking.

Directory of Open Access Journals (Sweden)

Jiabing Ji

Full Text Available BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR, touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well.

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing.

Science.gov (United States)

Feehan, Joanna M; Scheibel, Katherine E; Bourras, Salim; Underwood, William; Keller, Beat; Somerville, Shauna C

2017-03-31

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 µg DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., > 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

Directory of Open Access Journals (Sweden)

Yang Moon-Sik

2004-09-01

Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

Functional interrogation of non-coding DNA through CRISPR genome editing.

Science.gov (United States)

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

Simplified extraction of good quality genomic DNA from a variety of ...

African Journals Online (AJOL)

Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques. Here, we optimized and employed a simple genomic DNA isolation protocol suitable for a variety of plant materials ...

A tailing genome walking method suitable for genomes with high local GC content.

Science.gov (United States)

Liu, Taian; Fang, Yongxiang; Yao, Wenjuan; Guan, Qisai; Bai, Gang; Jing, Zhizhong

2013-10-15

The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5' polycytosine and 3' polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content. Copyright © 2013 Elsevier Inc. All rights reserved.

A protocol for large scale genomic DNA isolation for cacao genetics ...

African Journals Online (AJOL)

Advances in DNA technology, such as marker assisted selection, detection of quantitative trait loci and genomic selection also require the isolation of DNA from a large number of samples and the preservation of tissue samples for future use in cacao genome studies. The present study proposes a method for the ...

Genomic DNA extraction from sapwood of Pinus roxburghii for ...

African Journals Online (AJOL)

A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted DNA ranged ...

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

Science.gov (United States)

Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

2018-05-14

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk

Directory of Open Access Journals (Sweden)

Jose Carlos Ribeiro Junior

2016-10-01

Full Text Available The spore-forming microbiota is mainly responsible for the deterioration of pasteurized milk with long shelf life in the United States. The identification of these microorganisms, using molecular tools, is of particular importance for the maintenance of the quality of milk. However, these molecular techniques are not only costly but also labor-intensive and time-consuming. The aim of this study was to compare the efficiency of boiling in conjunction with four other methods for the genomic DNA extraction of sporulated bacteria with proteolytic and lipolytic potential isolated from raw milk in the states of Paraná and Maranhão, Brazil. Protocols based on cellular lysis by enzymatic digestion, phenolic extraction, microwave-heating, as well as the use of guanidine isothiocyanate were used. This study proposes a method involving simple boiling for the extraction of genomic DNA from these microorganisms. Variations in the quality and yield of the extracted DNA among these methods were observed. However, both the cell lysis protocol by enzymatic digestion (commercial kit and the simple boiling method proposed in this study yielded sufficient DNA for successfully carrying out the Polymerase Chain Reaction (PCR of the rpoB and 16S rRNA genes for all 11 strains of microorganisms tested. Other protocols failed to yield sufficient quantity and quality of DNA from all microorganisms tested, since only a few strains have showed positive results by PCR, thereby hindering the search for new microorganisms. Thus, the simple boiling method for DNA extraction from sporulated bacteria in spoiled milk showed the same efficacy as that of the commercial kit. Moreover, the method is inexpensive, easy to perform, and much less time-consuming.

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model

Directory of Open Access Journals (Sweden)

Elaine C. Maggi

2018-01-01

Full Text Available The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Genome-wide mapping of DNA strand breaks.

Directory of Open Access Journals (Sweden)

Frédéric Leduc

Full Text Available Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP, uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

Sequencing of chloroplast genome using whole cellular DNA and Solexa sequencing technology

Directory of Open Access Journals (Sweden)

Jian eWu

2012-11-01

Full Text Available Sequencing of the chloroplast genome using traditional sequencing methods has been difficult because of its size (>120 kb and the complicated procedures required to prepare templates. To explore the feasibility of sequencing the chloroplast genome using DNA extracted from whole cells and Solexa sequencing technology, we sequenced whole cellular DNA isolated from leaves of three Brassica rapa accessions with one lane per accession. In total, 246 Mb, 362Mb, 361 Mb sequence data were generated for the three accessions Chiifu-401-42, Z16 and FT, respectively. Microreads were assembled by reference-guided assembly using the cpDNA sequences of B. rapa, Arabidopsis thaliana, and Nicotiana tabacum. We achieved coverage of more than 99.96% of the cp genome in the three tested accessions using the B. rapa sequence as the reference. When A. thaliana or N. tabacum sequences were used as references, 99.7–99.8% or 95.5–99.7% of the B. rapa chloroplast genome was covered, respectively. These results demonstrated that sequencing of whole cellular DNA isolated from young leaves using the Illumina Genome Analyzer is an efficient method for high-throughput sequencing of chloroplast genome.

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

Science.gov (United States)

Gouveia, Juceli Gonzalez; Wolf, Ivan Rodrigo; de Moraes-Manécolo, Vivian Patrícia Oliveira; Bardella, Vanessa Belline; Ferracin, Lara Munique; Giuliano-Caetano, Lucia; da Rosa, Renata; Dias, Ana Lúcia

2016-12-01

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C 0 t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C 0 t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.

Genome instabilities arising from ribonucleotides in DNA.

Science.gov (United States)

Klein, Hannah L

2017-08-01

Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of FTA ® paper for storage of oral meta-genomic DNA.

Science.gov (United States)

Foitzik, Magdalena; Stumpp, Sascha N; Grischke, Jasmin; Eberhard, Jörg; Stiesch, Meike

2014-10-01

The purpose of the present study was to evaluate the short-term storage of meta-genomic DNA from native oral biofilms on FTA(®) paper. Thirteen volunteers of both sexes received an acrylic splint for intraoral biofilm formation over a period of 48 hours. The biofilms were collected, resuspended in phosphate-buffered saline, and either stored on FTA(®) paper or directly processed by standard laboratory DNA extraction. The nucleic acid extraction efficiencies were evaluated by 16S rDNA targeted SSCP fingerprinting. The acquired banding pattern of FTA-derived meta-genomic DNA was compared to a standard DNA preparation protocol. Sensitivity and positive predictive values were calculated. The volunteers showed inter-individual differences in their bacterial species composition. A total of 200 bands were found for both methods and 85% of the banding patterns were equal, representing a sensitivity of 0.941 and a false-negative predictive value of 0.059. Meta-genomic DNA sampling, extraction, and adhesion using FTA(®) paper is a reliable method for storage of microbial DNA for a short period of time.

A high throughput DNA extraction method with high yield and quality

Directory of Open Access Journals (Sweden)

Xin Zhanguo

2012-07-01

Full Text Available Abstract Background Preparation of large quantity and high quality genomic DNA from a large number of plant samples is a major bottleneck for most genetic and genomic analyses, such as, genetic mapping, TILLING (Targeting Induced Local Lesion IN Genome, and next-generation sequencing directly from sheared genomic DNA. A variety of DNA preparation methods and commercial kits are available. However, they are either low throughput, low yield, or costly. Here, we describe a method for high throughput genomic DNA isolation from sorghum [Sorghum bicolor (L. Moench] leaves and dry seeds with high yield, high quality, and affordable cost. Results We developed a high throughput DNA isolation method by combining a high yield CTAB extraction method with an improved cleanup procedure based on MagAttract kit. The method yielded large quantity and high quality DNA from both lyophilized sorghum leaves and dry seeds. The DNA yield was improved by nearly 30 fold with 4 times less consumption of MagAttract beads. The method can also be used in other plant species, including cotton leaves and pine needles. Conclusion A high throughput system for DNA extraction from sorghum leaves and seeds was developed and validated. The main advantages of the method are low cost, high yield, high quality, and high throughput. One person can process two 96-well plates in a working day at a cost of $0.10 per sample of magnetic beads plus other consumables that other methods will also need.

An automated annotation tool for genomic DNA sequences using

Indian Academy of Sciences (India)

Genomic sequence data are often available well before the annotated sequence is published. We present a method for analysis of genomic DNA to identify coding sequences using the GeneScan algorithm and characterize these resultant sequences by BLAST. The routines are used to develop a system for automated ...

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Our method uses a single-CpG-resolution, whole-genome methylation ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, ...... methylation is prevalent in embryonic stem cells andmaybe mediated.

Isolation of Retroelement from Plant Genomic DNA

OpenAIRE

sprotocols

2014-01-01

Author: Pat Heslop-Harrison ### Abstract: Retroelements and their derivatives are an ubiquitous and abundant component of plant genomes. From the 1990s, PCR based techniques have been developed to isolate the elements from genomic DNA of different plants, and the methods and primers used are presented here. Major classes of retroelements include the Ty1-copia, the Ty3-gypsy and the LINE (non-LTR) groups. Mixed PCR products representing the full heterogeneous pool of retrotransposo...

Resurrection of DNA function in vivo from an extinct genome.

Directory of Open Access Journals (Sweden)

Andrew J Pask

2008-05-01

Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

Genomic DNA extraction method from Annona senegalensis Pers ...

African Journals Online (AJOL)

Extraction of DNA in many plants is difficult because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, replications, amplification, as well as cloning. Modified procedure based on the hexadecyltrimethyl ammoniumbromide (CTAB) method is ...

Human Genome Research: Decoding DNA

Science.gov (United States)

dropdown arrow Site Map A-Z Index Menu Synopsis Human Genome Research: Decoding DNA Resources with of the DNA double helix during April 2003. James D. Watson, Francis Crick, and Maurice Wilkins were company Celera announced the completion of a "working draft" reference DNA sequence of the human

[Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

Science.gov (United States)

Cao, Y; Zou, K N; Huang, J P; Ma, K; Ping, Y

2017-08-01

To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine

Genomic DNA extraction method from pearl millet ( Pennisetum ...

African Journals Online (AJOL)

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to ...

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.

Science.gov (United States)

Guillaud-Bataille, Marine; Valent, Alexander; Soularue, Pascal; Perot, Christine; Inda, Maria Mar; Receveur, Aline; Smaïli, Sadek; Roest Crollius, Hugues; Bénard, Jean; Bernheim, Alain; Gidrol, Xavier; Danglot, Gisèle

2004-07-29

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

Science.gov (United States)

Lehle, Simon; Hildebrand, Dominic G.; Merz, Britta; Malak, Peter N.; Becker, Michael S.; Schmezer, Peter; Essmann, Frank; Schulze-Osthoff, Klaus; Rothfuss, Oliver

2014-01-01

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications. PMID:24371283

Omni-PolyA: a method and tool for accurate recognition of Poly(A) signals in human genomic DNA

KAUST Repository

Magana-Mora, Arturo

2017-08-15

BackgroundPolyadenylation is a critical stage of RNA processing during the formation of mature mRNA, and is present in most of the known eukaryote protein-coding transcripts and many long non-coding RNAs. The correct identification of poly(A) signals (PAS) not only helps to elucidate the 3′-end genomic boundaries of a transcribed DNA region and gene regulatory mechanisms but also gives insight into the multiple transcript isoforms resulting from alternative PAS. Although progress has been made in the in-silico prediction of genomic signals, the recognition of PAS in DNA genomic sequences remains a challenge.ResultsIn this study, we analyzed human genomic DNA sequences for the 12 most common PAS variants. Our analysis has identified a set of features that helps in the recognition of true PAS, which may be involved in the regulation of the polyadenylation process. The proposed features, in combination with a recognition model, resulted in a novel method and tool, Omni-PolyA. Omni-PolyA combines several machine learning techniques such as different classifiers in a tree-like decision structure and genetic algorithms for deriving a robust classification model. We performed a comparison between results obtained by state-of-the-art methods, deep neural networks, and Omni-PolyA. Results show that Omni-PolyA significantly reduced the average classification error rate by 35.37% in the prediction of the 12 considered PAS variants relative to the state-of-the-art results.ConclusionsThe results of our study demonstrate that Omni-PolyA is currently the most accurate model for the prediction of PAS in human and can serve as a useful complement to other PAS recognition methods. Omni-PolyA is publicly available as an online tool accessible at www.cbrc.kaust.edu.sa/omnipolya/.

Whole genome amplification of Chelex-extracted DNA from a single mite: a method for studying genetics of the predatory mite Phytoseiulus persimilis.

Science.gov (United States)

Konakandla, Bhanu; Park, Yoonseong; Margolies, David

2006-01-01

We developed and optimized a method using Chelex DNA extraction followed by whole genome amplification (WGA) to overcome problems conducting molecular genetic studies due to the limited amount of DNA obtainable from individual small organisms such as predatory mites. The DNA from a single mite, Phytoseiulus persimilis Athias-Henrot (Acari: Phytoseiidae), isolated in Chelex suspension was subjected to WGA. More than 1000-fold amplification of the DNA was achieved using as little as 0.03 ng genomic DNA template. The DNA obtained by the WGA was used for polymerase chain reaction followed by direct sequencing. From WGA DNA, nuclear DNA intergenic spacers ITS1 and ITS2 and a mitochondrial DNA 12S marker were tested in three different geographical populations of the predatory mite: California, the Netherlands, and Sicily. We found a total of four different alleles of the 12S in the Sicilian population, but no polymorphism was identified in the ITS marker. The combination of Chelex DNA extraction and WGA is thus shown to be a simple and robust technique for examining molecular markers for multiple loci by using individual mites. We conclude that the methods, Chelex extraction of DNA followed by WGA, provide a large quantity of DNA template that can be used for multiple PCR reactions useful for genetic studies requiring the genotypes of individual mites.

DNA Breaks and End Resection Measured Genome-wide by End Sequencing.

Science.gov (United States)

Canela, Andres; Sridharan, Sriram; Sciascia, Nicholas; Tubbs, Anthony; Meltzer, Paul; Sleckman, Barry P; Nussenzweig, André

2016-09-01

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice. Published by Elsevier Inc.

Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

Science.gov (United States)

Wang, Chao; Lv, Yangyong; Wang, Bin; Yin, Chao; Lin, Ying; Pan, Li

2015-01-01

The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences. PMID:25883143

A simple and efficient total genomic DNA extraction method for individual zooplankton.

Science.gov (United States)

Fazhan, Hanafiah; Waiho, Khor; Shahreza, Md Sheriff

2016-01-01

Molecular approaches are widely applied in species identification and taxonomic studies of minute zooplankton. One of the most focused zooplankton nowadays is from Subclass Copepoda. Accurate species identification of all life stages of the generally small sized copepods through molecular analysis is important, especially in taxonomic and systematic assessment of harpacticoid copepod populations and to understand their dynamics within the marine community. However, total genomic DNA (TGDNA) extraction from individual harpacticoid copepods can be problematic due to their small size and epibenthic behavior. In this research, six TGDNA extraction methods done on individual harpacticoid copepods were compared. The first new simple, feasible, efficient and consistent TGDNA extraction method was designed and compared with the commercial kit and modified available TGDNA extraction methods. The newly described TGDNA extraction method, "Incubation in PCR buffer" method, yielded good and consistent results based on the high success rate of PCR amplification (82%) compared to other methods. Coupled with its relatively consistent and economical method the "Incubation in PCR buffer" method is highly recommended in the TGDNA extraction of other minute zooplankton species.

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

OpenAIRE

Aljanabi, S M; Martinez, I

1997-01-01

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even i...

Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

Science.gov (United States)

Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

2011-06-01

This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

Science.gov (United States)

Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

2016-05-01

Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

Comparison of five DNA quantification methods

DEFF Research Database (Denmark)

Nielsen, Karsten; Mogensen, Helle Smidt; Hedman, Johannes

2008-01-01

Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than...... Quantification kit in two experiments. The measured DNA concentrations with Quantifiler were 125 and 160% higher than expected based on the manufacturers' information. When the Quantifiler human DNA standard (Raji cell line) was replaced by the commercial human DNA preparation G147A (Promega) to generate the DNA...... standard curve in the Quantifiler Human DNA Quantification kit, the DNA quantification results of the human DNA preparations were 31% higher than expected based on the manufacturers' information. The results indicate a calibration problem with the Quantifiler human DNA standard for its use...

Genome analysis methods - PGDBj Registered plant list, Marker list, QTL list, Plant DB link & Genome analysis methods | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available List Contact us PGDBj Registered plant list, Marker list, QTL list, Plant DB link & Genome analysis methods Genome analysis... methods Data detail Data name Genome analysis methods DOI 10.18908/lsdba.nbdc01194-01-005 De...scription of data contents The current status and related information of the genomic analysis about each org...anism (March, 2014). In the case of organisms carried out genomic analysis, the d...e File name: pgdbj_dna_marker_linkage_map_genome_analysis_methods_en.zip File URL: ftp://ftp.biosciencedbc.j

Measuring the Levels of Ribonucleotides Embedded in Genomic DNA.

Science.gov (United States)

Meroni, Alice; Nava, Giulia M; Sertic, Sarah; Plevani, Paolo; Muzi-Falconi, Marco; Lazzaro, Federico

2018-01-01

Ribonucleotides (rNTPs) are incorporated into genomic DNA at a relatively high frequency during replication. They have beneficial effects but, if not removed from the chromosomes, increase genomic instability. Here, we describe a fast method to easily estimate the amounts of embedded ribonucleotides into the genome. The protocol described is performed in Saccharomyces cerevisiae and allows us to quantify altered levels of rNMPs due to different mutations in the replicative polymerase ε. However, this protocol can be easily applied to cells derived from any organism.

Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici.

Science.gov (United States)

Zhang, R; Ma, Z H; Wu, B M

2015-05-01

Biotrophic fungi, such as Puccinia striiformis f. sp. tritici, because they cannot be cultured on nutrient media, to obtain adequate quantity of DNA for molecular genetic analysis, are usually propagated on living hosts, wheat plants in case of P. striiformis f. sp. tritici. The propagation process is time-, space- and labor-consuming and has been a bottleneck to molecular genetic analysis of this pathogen. In this study we evaluated multiple displacement amplification (MDA) of pathogen genomic DNA from urediospores as an alternative approach to traditional propagation of urediospores followed by DNA extraction. The quantities of pathogen genomic DNA in the products were further determined via real-time PCR with a pair of primers specific for the β-tubulin gene of P. striiformis f. sp. tritici. The amplified fragment length polymorphism (AFLP) fingerprints were also compared between the DNA products. The results demonstrated that adequate genomic DNA at fragment size larger than 23 Kb could be amplified from 20 to 30 urediospores via MDA method. The real-time PCR results suggested that although fresh urediospores collected from diseased leaves were the best, spores picked from diseased leaves stored for a prolonged period could also be used for amplification. AFLP fingerprints exhibited no significant differences between amplified DNA and DNA extracted with CTAB method, suggesting amplified DNA can represent the pathogen's genomic DNA very well. Therefore, MDA could be used to obtain genomic DNA from small precious samples (dozens of spores) for molecular genetic analysis of wheat stripe rust pathogen, and other fungi that are difficult to propagate.

Quantification and genome-wide mapping of DNA double-strand breaks.

Science.gov (United States)

Grégoire, Marie-Chantal; Massonneau, Julien; Leduc, Frédéric; Arguin, Mélina; Brazeau, Marc-André; Boissonneault, Guylain

2016-12-01

DNA double-strand breaks (DSBs) represent a major threat to the genetic integrity of the cell. Knowing both their genome-wide distribution and number is important for a better assessment of genotoxicity at a molecular level. Available methods may have underestimated the extent of DSBs as they are based on markers specific to those undergoing active repair or may not be adapted for the large diversity of naturally occurring DNA ends. We have established conditions for an efficient first step of DNA nick and gap repair (NGR) allowing specific determination of DSBs by end labeling with terminal transferase. We used DNA extracted from HeLa cells harboring an I-SceI cassette to induce a targeted nick or DSB and demonstrated by immunocapture of 3'-OH that a prior step of NGR allows specific determination of loci-specific or genome wide DSBs. This method can be applied to the global determination of DSBs using radioactive end labeling and can find several applications aimed at understanding the distribution and kinetics of DSBs formation and repair. Copyright © 2016 Elsevier B.V. All rights reserved.

Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA.

Science.gov (United States)

Skvortsova, Ksenia; Zotenko, Elena; Luu, Phuc-Loi; Gould, Cathryn M; Nair, Shalima S; Clark, Susan J; Stirzaker, Clare

2017-01-01

The discovery that 5-methylcytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) proteins has prompted wide interest in the potential role of 5hmC in reshaping the mammalian DNA methylation landscape. The gold-standard bisulphite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genome-wide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC. In this study, we provide a comparative analysis on brain DNA using three 5hmC genome-wide approaches, namely whole-genome bisulphite/oxidative bisulphite sequencing (WG Bis/OxBis-seq), Infinium HumanMethylation450 BeadChip arrays coupled with oxidative bisulphite (HM450K Bis/OxBis) and antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq). We also perform loci-specific TET-assisted bisulphite sequencing (TAB-seq) for validation of candidate regions. We show that whole-genome single-base resolution approaches are advantaged in providing precise 5hmC values but require high sequencing depth to accurately measure 5hmC, as this modification is commonly in low abundance in mammalian cells. HM450K arrays coupled with oxidative bisulphite provide a cost-effective representation of 5hmC distribution, at CpG sites with 5hmC levels >~10%. However, 5hmC analysis is restricted to the genomic location of the probes, which is an important consideration as 5hmC modification is commonly enriched at enhancer elements. Finally, we show that the widely used hMeDIP-seq method provides an efficient genome-wide profile of 5hmC and shows high correlation with WG Bis/OxBis-seq 5hmC distribution in brain DNA. However, in cell line DNA with low levels of 5hmC, hMeDIP-seq-enriched regions are not detected by WG Bis/OxBis or HM450K, either suggesting misinterpretation of 5hmC calls by hMeDIP or lack of sensitivity of the latter methods. We

Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue.

Science.gov (United States)

Liu, L; Wang, C L; Peng, W Y; Yang, J; Lan, M Q; Zhang, B; Li, J B; Zhu, Y Y; Li, C Y

2015-12-28

Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population.

Solution-based targeted genomic enrichment for precious DNA samples

Directory of Open Access Journals (Sweden)

Shearer Aiden

2012-05-01

Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

Science.gov (United States)

Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

2011-01-01

The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

Reliability and applications of statistical methods based on oligonucleotide frequencies in bacterial and archaeal genomes

DEFF Research Database (Denmark)

Bohlin, J; Skjerve, E; Ussery, David

2008-01-01

with here are mainly used to examine similarities between archaeal and bacterial DNA from different genomes. These methods compare observed genomic frequencies of fixed-sized oligonucleotides with expected values, which can be determined by genomic nucleotide content, smaller oligonucleotide frequencies......, or be based on specific statistical distributions. Advantages with these statistical methods include measurements of phylogenetic relationship with relatively small pieces of DNA sampled from almost anywhere within genomes, detection of foreign/conserved DNA, and homology searches. Our aim was to explore...... the reliability and best suited applications for some popular methods, which include relative oligonucleotide frequencies (ROF), di- to hexanucleotide zero'th order Markov methods (ZOM) and 2.order Markov chain Method (MCM). Tests were performed on distant homology searches with large DNA sequences, detection...

A CTAB Procedure Of Total Genomic DNA Extraction For Medicinal Mushrooms

International Nuclear Information System (INIS)

Azhar Mohamad; Muhammad Hussaini Mohd Mustafa; Muhammad Hanif Azhari Noor; Rosnani Abdul Rashid; Hasan Hamdani Hasan Mutaat; Meswan Meskom; Mat Rasol Awang

2014-01-01

Medicinal mushroom is defined as mushrooms used in medicine or medical research. Isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including Polymerase Chain Reaction (PCR), endonuclease restriction digestion, Southern blot analysis, and genomic library construction. The most important and prerequisite towards reliable molecular biology work is the total genomic DNA of a sample must be in good quality. Five freshly samples of medicinal mushroom were used in this work known as Auriculariapolytricha, Lentinus edode, Pleurotus sayorcaju, Sczhizopyllum commune and Ganodermalucidum. 5 mg of each sample were used to extraction the DNA, prepared in 3 replications and repeated twice. PCR based technique by using ISSR markers were used in checking the amplification ability of the total genomic extraction. A standard Doyle and Doyle protocol for genomic DNA extraction was modified in optimizing the total genomic DNA from the medicinal mushroom.The modification parameters were percentage of CTAB, incubation period and temperature. The results reveal that each sample required a certain combinations of time and period of incubation. Besides, percentage of CTAB in the buffer was found significant in giving a high yielding of extracted total genomic DNA. The extracted total genomic DNA from the medicinal mushroom yielded from 39.7 ng/ μl to 919.1 ng/ μl. The different yield among the samples found to be corresponded to polysaccharide content in the medicinal mushrooms. The objective of this works is to optimize total genomic DNA extraction of medicinal mushrooms towards a high quality intact genomic DNA for molecular activities. (author)

Facilitating the indirect detection of genomic DNA in an electrochemical DNA biosensor using magnetic nanoparticles and DNA ligase

Directory of Open Access Journals (Sweden)

Roozbeh Hushiarian

2015-12-01

This technique was found to be reliably repeatable. The indirect detection of genomic DNA using this method is significantly improved and showed high efficiency in small amounts of samples with the detection limit of 5.37 × 10−14 M.

High-throughput sequencing of three Lemnoideae (duckweeds chloroplast genomes from total DNA.

Directory of Open Access Journals (Sweden)

Wenqin Wang

Full Text Available BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

International Nuclear Information System (INIS)

Maeda, Toyoki; Chijiiwa, Yoshiharu; Tsuji, Hideo; Sakoda, Saburo; Tani, Kenzaburo; Suzuki, Tomokazu

2004-01-01

In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis

Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

Directory of Open Access Journals (Sweden)

Ito Takashi

2011-06-01

Full Text Available Abstract Background We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity. Findings We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods. Conclusions HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.

Defining functional DNA elements in the human genome

Science.gov (United States)

Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

2014-01-01

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

DNA Repair and Genome Maintenance in Bacillus subtilis

Science.gov (United States)

Lenhart, Justin S.; Schroeder, Jeremy W.; Walsh, Brian W.

2012-01-01

Summary: From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis. PMID:22933559

Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

Science.gov (United States)

Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

2012-07-01

The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

Evaluating Digital PCR for the Quantification of Human Genomic DNA: Accessible Amplifiable Targets.

Science.gov (United States)

Kline, Margaret C; Romsos, Erica L; Duewer, David L

2016-02-16

Polymerase chain reaction (PCR) multiplexed assays perform best when the input quantity of template DNA is controlled to within about a factor of √2. To help ensure that PCR assays yield consistent results over time and place, results from methods used to determine DNA quantity need to be metrologically traceable to a common reference. Many DNA quantitation systems can be accurately calibrated with solutions of DNA in aqueous buffer. Since they do not require external calibration, end-point limiting dilution technologies, collectively termed "digital PCR (dPCR)", have been proposed as suitable for value assigning such DNA calibrants. The performance characteristics of several commercially available dPCR systems have recently been documented using plasmid, viral, or fragmented genomic DNA; dPCR performance with more complex materials, such as human genomic DNA, has been less studied. With the goal of providing a human genomic reference material traceably certified for mass concentration, we are investigating the measurement characteristics of several dPCR systems. We here report results of measurements from multiple PCR assays, on four human genomic DNAs treated with four endonuclease restriction enzymes using both chamber and droplet dPCR platforms. We conclude that dPCR does not estimate the absolute number of PCR targets in a given volume but rather the number of accessible and amplifiable targets. While enzymatic restriction of human genomic DNA increases accessibility for some assays, in well-optimized PCR assays it can reduce the number of amplifiable targets and increase assay variability relative to uncut sample.

Characterization of noncoding regulatory DNA in the human genome.

Science.gov (United States)

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Studies on the Interaction between Zinc-Hydroxybenzoite Complex and Genomic DNA

Directory of Open Access Journals (Sweden)

Hacali Necefoglu

2006-04-01

Full Text Available Zinc-Hydroxybenzoite ([Zn (H206] (p-HO-C6H4COO22H20 complex which wassynthesized and characterized by instrumental methods and the DNA samples which hadbeen isolated from cattle were allowed to interact at 37 oC for different time periods. Theinteraction of genomic DNA with this complex has been followed by agarose gelelectrophoresis at 50 V for 2 h. When DNA samples were allowed to interact with this metalcomplex, it was found that band intensities changed with the concentrations of the complex.In the result of interaction between this complex and genomic DNA samples, it wasdetermined that the intensities of bands were changed at the different concentrations of thecomplex. The brightness of the bands was increased and mobility of the bands wasdecreased, indicating the occurrence of increased covalent binding of the metal complexwith DNA. In this study it was concluded that the damage effect of ascorbate was reducedby Zinc-Hydroxybenzoite.

A direct detection of Escherichia coli genomic DNA using gold nanoprobes

Directory of Open Access Journals (Sweden)

Padmavathy

2012-02-01

Full Text Available Abstract Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to

DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction.

Science.gov (United States)

Focke, Felix; Haase, Ilka; Fischer, Markus

2011-01-26

Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.

Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

Directory of Open Access Journals (Sweden)

Mohammed Bakkali

Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

DNA Extraction Protocols for Whole-Genome Sequencing in Marine Organisms.

Science.gov (United States)

Panova, Marina; Aronsson, Henrik; Cameron, R Andrew; Dahl, Peter; Godhe, Anna; Lind, Ulrika; Ortega-Martinez, Olga; Pereyra, Ricardo; Tesson, Sylvie V M; Wrange, Anna-Lisa; Blomberg, Anders; Johannesson, Kerstin

2016-01-01

The marine environment harbors a large proportion of the total biodiversity on this planet, including the majority of the earths' different phyla and classes. Studying the genomes of marine organisms can bring interesting insights into genome evolution. Today, almost all marine organismal groups are understudied with respect to their genomes. One potential reason is that extraction of high-quality DNA in sufficient amounts is challenging for many marine species. This is due to high polysaccharide content, polyphenols and other secondary metabolites that will inhibit downstream DNA library preparations. Consequently, protocols developed for vertebrates and plants do not always perform well for invertebrates and algae. In addition, many marine species have large population sizes and, as a consequence, highly variable genomes. Thus, to facilitate the sequence read assembly process during genome sequencing, it is desirable to obtain enough DNA from a single individual, which is a challenge in many species of invertebrates and algae. Here, we present DNA extraction protocols for seven marine species (four invertebrates, two algae, and a marine yeast), optimized to provide sufficient DNA quality and yield for de novo genome sequencing projects.

Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

Directory of Open Access Journals (Sweden)

Maruoka Shuichiro

2008-12-01

Full Text Available Abstract Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK, a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05 after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models

Genome-wide DNA methylation patterns and transcription analysis in sheep muscle.

Directory of Open Access Journals (Sweden)

Christine Couldrey

Full Text Available DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS. While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

Directory of Open Access Journals (Sweden)

Sarah M Hykin

Full Text Available 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

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

Science.gov (United States)

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

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Science.gov (United States)

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human.

Science.gov (United States)

Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

2017-02-16

DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation.

A simple and inexpensive method for genomic restriction mapping analysis

International Nuclear Information System (INIS)

Huang, C.H.; Lam, V.M.S.; Tam, J.W.O.

1988-01-01

The Southern blotting procedure for the transfer of DNA fragments from agarose gels to nitrocellulose membranes has revolutionized nucleic acid detection methods, and it forms the cornerstone of research in molecular biology. Basically, the method involves the denaturation of DNA fragments that have been separated on an agarose gel, the immobilization of the fragments by transfer to a nitrocellulose membrane, and the identification of the fragments of interest through hybridization to /sup 32/P-labeled probes and autoradiography. While the method is sensitive and applicable to both genomic and cloned DNA, it suffers from the disadvantages of being time consuming and expensive, and fragments of greater than 15 kb are difficult to transfer. Moreover, although theoretically the nitrocellulose membrane can be washed and hybridized repeatedly using different probes, in practice, the membrane becomes brittle and difficult to handle after a few cycles. A direct hybridization method for pure DNA clones was developed in 1975 but has not been widely exploited. The authors report here a modification of their procedure as applied to genomic DNA. The method is simple, rapid, and inexpensive, and it does not involve transfer to nitrocellulose membranes

Effects of storage temperature on the quantity and integrity of genomic DNA extracted from mice tissues: A comparison of recovery methods

Directory of Open Access Journals (Sweden)

Huda H. Al-Griw

2017-08-01

Full Text Available Efficient extraction of genomic DNA (gDNA from biological materials found in harsh environments is the first step for successful forensic DNA profiling. This study aimed to evaluate two methods for DNA recovery from animal tissues (livers, muscles, focusing on the best storage temperature for DNA yield in term of quality, quantity, and integrity for use in several downstream molecular techniques. Six male Swiss albino mice were sacrificed, liver and muscle tissues (n=32 were then harvested and stored for one week in different temperatures, -20C, 4C, 25C and 40C. The conditioned animal tissues were used for DNA extraction by Chelex-100 method or NucleoSpin Blood and Tissue kit. The extracted gDNA was visualized on 1.5% agarose gel electrophoresis to determine the quality of gDNA and analysed spectrophotometrically to determine the DNA concentration and the purity. Both methods, Chelex-100 and NucleoSpin Blood and Tissue kit found to be appropriate for yielding high quantity of gDNA, with the Chelex100 method yielding a greater quantity (P < 0.045 than the kit. At -20C, 4C, and 25C temperatures, the concentration of DNA yield was numerically lower than at 40C. The NucleoSpin Blood and Tissue kit produced a higher (P=0.031 purity product than the Chelex-100 method, particularly for muscle tissues. The Chelex-100 method is cheap, fast, effective, and is a crucial tool for yielding DNA from animal tissues (livers, muscles exposed to harsh environment with little limitations.

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA

DEFF Research Database (Denmark)

Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte

2014-01-01

Chip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better......Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies......, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap...

Whole genome DNA methylation: beyond genes silencing

OpenAIRE

Tirado-Magallanes, Roberto; Rebbani, Khadija; Lim, Ricky; Pradhan, Sriharsa; Benoukraf, Touati

2016-01-01

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation at near base pair level resolution, far beyond that of the kilobase-long canonical CpG islands that initially revealed the biological relevance of this covalent DNA modification. The latest high-resolution studies have revealed a role for very punctual DNA methylation in chromatin plasticity, gene regulation and splicing. Here, we aim to outline the ...

Non liquid nitrogen-based-method for isolation of DNA from ...

African Journals Online (AJOL)

A simple, efficient, reliable and cost-effective method for isolation of total genomic DNA from fungi, suitable for polymerase chain reaction (PCR) amplification and other molecular applications was described. The main advantages of the method are: (1) does not require the use of liquid nitrogen for preparation of fungi DNA; ...

Reciprocal Regulation between DNA-PKcs and Snail1 Conferring Genomic Instability

International Nuclear Information System (INIS)

Seo, Haeng Ran; Lee, Hae June; Jin, Yeung Bae; Bae, Sang Woo; Lee, Yun Sil; Kim, Nam Hee; Kim, Hyun Sil; Nam, Hyung Wook; Yook, Jong In

2010-01-01

Although the roles of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) involving non-homologous end joining (NHEJ) of DNA repair are well recognized, the biological mechanisms and regulators by which DNA-PKcs regulate genomic instability are not clearly defined. We show herein that DNA-PKcs activity resulting from DNA damage caused by ionizing radiation (IR) phosphorylates Snail1 at serine 100, which results in increased Snail1 expression and its function by inhibition of GSK-3-mediated phosphorylation. Furthermore, Snail1 phosphorylated at serine 100 can reciprocally inhibit kinase activity of DNA-PKcs, resulting in an inhibition to recruit DNA-PKcs or Ku70/80 to a DNA double-strand break site, and ultimately inhibition of DNA repair activity. The impairment of repair activity by a direct interaction between Snail1 and DNA-PKcs increases the resistance to DNA damaging agents, such as IR, and genomic instability. Our findings provide a novel cellular mechanism for induction of genomic instability by reciprocal regulation of DNA-PKcs and Snail1

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA.

Science.gov (United States)

Mattagajasingh, Ilwola; Mukherjee, Arup Kumar; Das, Premananda

2006-01-01

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

Science.gov (United States)

Doudican, Nicole A; Song, Binwei; Shadel, Gerald S; Doetsch, Paul W

2005-06-01

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

Genome-Wide DNA Methylation Profiles of Phlegm-Dampness Constitution

Directory of Open Access Journals (Sweden)

Haiqiang Yao

2018-03-01

Full Text Available Background/Aims: Metabolic diseases are leading health concerns in today’s global society. In traditional Chinese medicine (TCM, one body type studied is the phlegm-dampness constitution (PC, which predisposes individuals to complex metabolic disorders. Genomic studies have revealed the potential metabolic disorders and the molecular features of PC. The role of epigenetics in the regulation of PC, however, is unknown. Methods: We analyzed a genome-wide DNA methylation in 12 volunteers using Illumina Infinium Human Methylation450 BeadChip on peripheral blood mononuclear cells (PBMCs. Eight volunteers had PC and 4 had balanced constitutions. Results: Methylation data indicated a genome-scale hyper-methylation pattern in PC. We located 288 differentially methylated probes (DMPs. A total of 256 genes were mapped, and some of these were metabolic-related. SQSTM1, DLGAP2 and DAB1 indicated diabetes mellitus; HOXC4 and SMPD3, obesity; and GRWD1 and ATP10A, insulin resistance. According to Ingenuity Pathway Analysis (IPA, differentially methylated genes were abundant in multiple metabolic pathways. Conclusion: Our results suggest the potential risk for metabolic disorders in individuals with PC. We also explain the clinical characteristics of PC with DNA methylation features.

Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data.

Directory of Open Access Journals (Sweden)

Can Alkan

2007-09-01

Full Text Available The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%-5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data.

Science.gov (United States)

Alkan, Can; Ventura, Mario; Archidiacono, Nicoletta; Rocchi, Mariano; Sahinalp, S Cenk; Eichler, Evan E

2007-09-01

The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%-5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

Large-scale chromosome folding versus genomic DNA sequences: A discrete double Fourier transform technique.

Science.gov (United States)

Chechetkin, V R; Lobzin, V V

2017-08-07

Using state-of-the-art techniques combining imaging methods and high-throughput genomic mapping tools leaded to the significant progress in detailing chromosome architecture of various organisms. However, a gap still remains between the rapidly growing structural data on the chromosome folding and the large-scale genome organization. Could a part of information on the chromosome folding be obtained directly from underlying genomic DNA sequences abundantly stored in the databanks? To answer this question, we developed an original discrete double Fourier transform (DDFT). DDFT serves for the detection of large-scale genome regularities associated with domains/units at the different levels of hierarchical chromosome folding. The method is versatile and can be applied to both genomic DNA sequences and corresponding physico-chemical parameters such as base-pairing free energy. The latter characteristic is closely related to the replication and transcription and can also be used for the assessment of temperature or supercoiling effects on the chromosome folding. We tested the method on the genome of E. coli K-12 and found good correspondence with the annotated domains/units established experimentally. As a brief illustration of further abilities of DDFT, the study of large-scale genome organization for bacteriophage PHIX174 and bacterium Caulobacter crescentus was also added. The combined experimental, modeling, and bioinformatic DDFT analysis should yield more complete knowledge on the chromosome architecture and genome organization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of strategies for the isolation of PCR-compatible, genomic DNA from a municipal biogas plants.

Science.gov (United States)

Weiss, Agnes; Jérôme, Valérie; Freitag, Ruth

2007-06-15

The goal of the project was the extraction of PCR-compatible genomic DNA representative of the entire microbial community from municipal biogas plant samples (mash, bioreactor content, process water, liquid fertilizer). For the initial isolation of representative DNA from the respective lysates, methods were used that employed adsorption, extraction, or precipitation to specifically enrich the DNA. Since no dedicated method for biogas plant samples was available, preference was given to kits/methods suited to samples that resembled either the bioreactor feed, e.g. foodstuffs, or those intended for environmental samples including wastewater. None of the methods succeeded in preparing DNA that was directly PCR-compatible. Instead the DNA was found to still contain considerable amounts of difficult-to-remove enzyme inhibitors (presumably humic acids) that hindered the PCR reaction. Based on the isolation method that gave the highest yield/purity for all sample types, subsequent purification was attempted by agarose gel electrophoresis followed by electroelution, spermine precipitation, or dialysis through nitrocellulose membrane. A combination of phenol/chloroform extraction followed by purification via dialysis constituted the most efficient sample treatment. When such DNA preparations were diluted 1:100 they did no longer inhibit PCR reactions, while they still contained sufficient genomic DNA to allow specific amplification of specific target sequences.

Next Generation DNA Sequencing and the Future of Genomic Medicine

OpenAIRE

Anderson, Matthew W.; Schrijver, Iris

2010-01-01

In the years since the first complete human genome sequence was reported, there has been a rapid development of technologies to facilitate high-throughput sequence analysis of DNA (termed “next-generation” sequencing). These novel approaches to DNA sequencing offer the promise of complete genomic analysis at a cost feasible for routine clinical diagnostics. However, the ability to more thoroughly interrogate genomic sequence raises a number of important issues with regard to result interpreta...

An efficient method for DNA extraction from Cladosporioid fungi.

Science.gov (United States)

Moslem, M A; Bahkali, A H; Abd-Elsalam, K A; Wit, P J G M

2010-11-23

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

Science.gov (United States)

Ahmed, Wareed; Sala, Claudia; Hegde, Shubhada R; Jha, Rajiv Kumar; Cole, Stewart T; Nagaraja, Valakunja

2017-05-01

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

ATM signaling and genomic stability in response to DNA damage

International Nuclear Information System (INIS)

Lavin, Martin F.; Birrell, Geoff; Chen, Philip; Kozlov, Sergei; Scott, Shaun; Gueven, Nuri

2005-01-01

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed

Genomic gigantism: DNA loss is slow in mountain grasshoppers.

Science.gov (United States)

Bensasson, D; Petrov, D A; Zhang, D X; Hartl, D L; Hewitt, G M

2001-02-01

Several studies have shown DNA loss to be inversely correlated with genome size in animals. These studies include a comparison between Drosophila and the cricket, Laupala, but there has been no assessment of DNA loss in insects with very large genomes. Podisma pedestris, the brown mountain grasshopper, has a genome over 100 times as large as that of Drosophila and 10 times as large as that of Laupala. We used 58 paralogous nuclear pseudogenes of mitochondrial origin to study the characteristics of insertion, deletion, and point substitution in P. pedestris and Italopodisma. In animals, these pseudogenes are "dead on arrival"; they are abundant in many different eukaryotes, and their mitochondrial origin simplifies the identification of point substitutions accumulated in nuclear pseudogene lineages. There appears to be a mononucleotide repeat within the 643-bp pseudogene sequence studied that acts as a strong hot spot for insertions or deletions (indels). Because the data for other insect species did not contain such an unusual region, hot spots were excluded from species comparisons. The rate of DNA loss relative to point substitution appears to be considerably and significantly lower in the grasshoppers studied than in Drosophila or Laupala. This suggests that the inverse correlation between genome size and the rate of DNA loss can be extended to comparisons between insects with large or gigantic genomes (i.e., Laupala and Podisma). The low rate of DNA loss implies that in grasshoppers, the accumulation of point mutations is a more potent force for obscuring ancient pseudogenes than their loss through indel accumulation, whereas the reverse is true for Drosophila. The main factor contributing to the difference in the rates of DNA loss estimated for grasshoppers, crickets, and Drosophila appears to be deletion size. Large deletions are relatively rare in Podisma and Italopodisma.

An efficient method for DNA extraction from Cladosporioid fungi

OpenAIRE

Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de, P.J.G.M.

2010-01-01

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A260/A280 ratio ranging between 1.6 and 2.0. Isolated genomic DNA w...

Single-tube linear DNA amplification for genome-wide studies using a few thousand cells

NARCIS (Netherlands)

Shankaranarayanan, P.; Mendoza-Parra, M.A.; Gool, van W.; Trindade, L.M.; Gronemeyer, H.

2012-01-01

Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled

Genomic analysis of murine DNA-dependent protein kinase

International Nuclear Information System (INIS)

Fujimori, A.; Abe, M.

2003-01-01

Full text: The gene of catalytic subunit of DNA dependent protein kinase is responsible gene for SCID mice. The molecules play a critical role in non-homologous end joining including the V(D)J recombination. Contribution of the molecules to the difference of radiosensitivity and the susceptibility to cancer has been suggested. Here we show the entire nucleotide sequence of approximately 193 kbp and 84 kbp genomic regions encoding the entire DNA-PKcs gene in the mouse and chicken respectively. Retroposon was found in the intron 51 of mouse genomic DNA-PKcs gene but in human and chicken. Comparative analysis of these two species strongly suggested that only two genes, DNA-PKcs and MCM4, exist in the region of both species. Several conserved sequences and cis elements, however, were predicted. Recently, the orthologous region for the human DNA-PKcs locus was completed. The results of further comparative study will be discussed

The mitochondrial and plastid genomes of Volvox carteri: bloated molecules rich in repetitive DNA

Directory of Open Access Journals (Sweden)

Lee Robert W

2009-03-01

Full Text Available Abstract Background The magnitude of noncoding DNA in organelle genomes can vary significantly; it is argued that much of this variation is attributable to the dissemination of selfish DNA. The results of a previous study indicate that the mitochondrial DNA (mtDNA of the green alga Volvox carteri abounds with palindromic repeats, which appear to be selfish elements. We became interested in the evolution and distribution of these repeats when, during a cursory exploration of the V. carteri nuclear DNA (nucDNA and plastid DNA (ptDNA sequences, we found palindromic repeats with similar structural features to those of the mtDNA. Upon this discovery, we decided to investigate the diversity and evolutionary implications of these palindromic elements by sequencing and characterizing large portions of mtDNA and ptDNA and then comparing these data to the V. carteri draft nuclear genome sequence. Results We sequenced 30 and 420 kilobases (kb of the mitochondrial and plastid genomes of V. carteri, respectively – resulting in partial assemblies of these genomes. The mitochondrial genome is the most bloated green-algal mtDNA observed to date: ~61% of the sequence is noncoding, most of which is comprised of short palindromic repeats spread throughout the intergenic and intronic regions. The plastid genome is the largest (>420 kb and most expanded (>80% noncoding ptDNA sequence yet discovered, with a myriad of palindromic repeats in the noncoding regions, which have a similar size and secondary structure to those of the mtDNA. We found that 15 kb (~0.01% of the nuclear genome are homologous to the palindromic elements of the mtDNA, and 50 kb (~0.05% are homologous to those of the ptDNA. Conclusion Selfish elements in the form of short palindromic repeats have propagated in the V. carteri mtDNA and ptDNA, resulting in the distension of these genomes. Copies of these same repeats are also found in a small fraction of the nucDNA, but appear to be inert in this

TALENs: customizable molecular DNA scissors for genome engineering of plants.

Science.gov (United States)

Chen, Kunling; Gao, Caixia

2013-06-20

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases (TALENs), consisting of an engineered specific (TALE) DNA binding domain and a Fok I cleavage domain, are newly developed versatile reagents for genome engineering in different organisms. Because of the simplicity of the DNA recognition code and their modular assembly, TALENs can act as customizable molecular DNA scissors inducing double-strand breaks (DSBs) at given genomic location. Thus, they provide a valuable approach to targeted genome modifications such as mutations, insertions, replacements or chromosome rearrangements. In this article, we review the development of TALENs, and summarize the principles and tools for TALEN-mediated gene targeting in plant cells, as well as current and potential strategies for use in plant research and crop improvement. Copyright © 2013. Published by Elsevier Ltd.

Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

Directory of Open Access Journals (Sweden)

Rihong Zhai

2012-01-01

Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

Science.gov (United States)

Bitner, Rex M.; Koller, Susan C.

2002-06-01

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

International Nuclear Information System (INIS)

Tak, Yu Kyung; Kim, Won Young; Kim, Min Jung; Han, Eunyoung; Han, Myun Soo; Kim, Jong Jin; Kim, Wook; Lee, Jong Eun; Song, Joon Myong

2012-01-01

Highlights: ► Genomic DNA quantification were performed using a quantum dot-labeled Alu sequence. ► This probe provided PCR-free determination of human genomic DNA. ► Qdot-labeled Alu probe-hybridized genomic DNAs had a 2.5-femtogram detection limit. ► Qdot-labeled Alu sequence was used to assess DNA samples for human identification. - Abstract: Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

Science.gov (United States)

Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

2018-04-20

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp.).

Science.gov (United States)

Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

2013-01-01

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

Genomic and Molecular Landscape of DNA Damage Repair Deficiency across The Cancer Genome Atlas

Directory of Open Access Journals (Sweden)

Theo A. Knijnenburg

2018-04-01

Full Text Available Summary: DNA damage repair (DDR pathways modulate cancer risk, progression, and therapeutic response. We systematically analyzed somatic alterations to provide a comprehensive view of DDR deficiency across 33 cancer types. Mutations with accompanying loss of heterozygosity were observed in over 1/3 of DDR genes, including TP53 and BRCA1/2. Other prevalent alterations included epigenetic silencing of the direct repair genes EXO5, MGMT, and ALKBH3 in ∼20% of samples. Homologous recombination deficiency (HRD was present at varying frequency in many cancer types, most notably ovarian cancer. However, in contrast to ovarian cancer, HRD was associated with worse outcomes in several other cancers. Protein structure-based analyses allowed us to predict functional consequences of rare, recurrent DDR mutations. A new machine-learning-based classifier developed from gene expression data allowed us to identify alterations that phenocopy deleterious TP53 mutations. These frequent DDR gene alterations in many human cancers have functional consequences that may determine cancer progression and guide therapy. : Knijnenburg et al. present The Cancer Genome Atlas (TCGA Pan-Cancer analysis of DNA damage repair (DDR deficiency in cancer. They use integrative genomic and molecular analyses to identify frequent DDR alterations across 33 cancer types, correlate gene- and pathway-level alterations with genome-wide measures of genome instability and impaired function, and demonstrate the prognostic utility of DDR deficiency scores. Keywords: The Cancer Genome Atlas PanCanAtlas project, DNA damage repair, somatic mutations, somatic copy-number alterations, epigenetic silencing, DNA damage footprints, mutational signatures, integrative statistical analysis, protein structure analysis

Rapid and efficient extraction of genomic DNA from different phytopathogenic fungi using DNAzol reagent.

Science.gov (United States)

Guo, Jian-Rong; Schnieder, F; Abd-Elsalam, K A; Verreet, J A

2005-01-01

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 microg g(-1) dry mycelia and the A(260)/A(280) ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A(260)/A(280) ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.

Automated Processing of 2-D Gel Electrophoretograms of Genomic DNA for Hunting Pathogenic DNA Molecular Changes.

Science.gov (United States)

Takahashi; Nakazawa; Watanabe; Konagaya

1999-01-01

We have developed the automated processing algorithms for 2-dimensional (2-D) electrophoretograms of genomic DNA based on RLGS (Restriction Landmark Genomic Scanning) method, which scans the restriction enzyme recognition sites as the landmark and maps them onto a 2-D electrophoresis gel. Our powerful processing algorithms realize the automated spot recognition from RLGS electrophoretograms and the automated comparison of a huge number of such images. In the final stage of the automated processing, a master spot pattern, on which all the spots in the RLGS images are mapped at once, can be obtained. The spot pattern variations which seemed to be specific to the pathogenic DNA molecular changes can be easily detected by simply looking over the master spot pattern. When we applied our algorithms to the analysis of 33 RLGS images derived from human colon tissues, we successfully detected several colon tumor specific spot pattern changes.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

Science.gov (United States)

Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

Science.gov (United States)

Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

2016-08-01

Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

Undermethylated DNA as a source of microsatellites from a conifer genome.

Science.gov (United States)

Zhou, Y; Bui, T; Auckland, L D; Williams, C G

2002-02-01

Developing microsatellites from the large, highly duplicated conifer genome requires special tools. To improve the efficiency of developing Pinus taeda L. microsatellites, undermethylated (UM) DNA fragments were used to construct a microsatellite-enriched copy library. A methylation-sensitive restriction enzyme, McrBC, was used to enrich for UM DNA before library construction. Digested DNA fragments larger than 9 kb were then excised and digested with RsaI and used to construct nine dinucleotide and trinucleotide libraries. A total of 1016 microsatellite-positive clones were detected among 11 904 clones and 620 of these were unique. Of 245 primer sets that produced a PCR product, 113 could be developed as UM microsatellite markers and 70 were polymorphic. Inheritance and marker informativeness were tested for a random sample of 36 polymorphic markers using a three-generation outbred pedigree. Thirty-one microsatellites (86%) had single-locus inheritance despite the highly duplicated nature of the P. taeda genome. Nineteen UM microsatellites had highly informative intercross mating type configurations. Allele number and frequency were estimated for eleven UM microsatellites using a population survey. Allele numbers for these UM microsatellites ranged from 3 to 12 with an average of 5.7 alleles/locus. Frequencies for the 63 alleles were mostly in the low-common range; only 14 of the 63 were in the rare allele (q < 0.05) class. Enriching for UM DNA was an efficient method for developing polymorphic microsatellites from a large plant genome.

Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

Directory of Open Access Journals (Sweden)

Minsoung Rhee

Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

Whole-genome methylation caller designed for methyl- DNA ...

African Journals Online (AJOL)

etchie

2013-02-20

Feb 20, 2013 ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, Hidden ... its response to environmental cues. .... have a great potential to become the most cost-effective ... hg18 reference genome (set to 0 if not present in retrieved reads). ..... DNA methylation patterns and epigenetic memory.

DNA extraction method for PCR in mycorrhizal fungi.

Science.gov (United States)

Manian, S; Sreenivasaprasad, S; Mills, P R

2001-10-01

To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.

Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

Science.gov (United States)

Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

1998-08-28

The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

Evaluation of different sources of DNA for use in genome wide studies and forensic application.

Science.gov (United States)

Al Safar, Habiba S; Abidi, Fatima H; Khazanehdari, Kamal A; Dadour, Ian R; Tay, Guan K

2011-02-01

In the field of epidemiology, Genome-Wide Association Studies (GWAS) are commonly used to identify genetic predispositions of many human diseases. Large repositories housing biological specimens for clinical and genetic investigations have been established to store material and data for these studies. The logistics of specimen collection and sample storage can be onerous, and new strategies have to be explored. This study examines three different DNA sources (namely, degraded genomic DNA, amplified degraded genomic DNA and amplified extracted DNA from FTA card) for GWAS using the Illumina platform. No significant difference in call rate was detected between amplified degraded genomic DNA extracted from whole blood and amplified DNA retrieved from FTA™ cards. However, using unamplified-degraded genomic DNA reduced the call rate to a mean of 42.6% compared to amplified DNA extracted from FTA card (mean of 96.6%). This study establishes the utility of FTA™ cards as a viable storage matrix for cells from which DNA can be extracted to perform GWAS analysis.

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

Directory of Open Access Journals (Sweden)

Haque Kashif A

2005-09-01

Full Text Available Abstract Background Whole genome amplification (WGA promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA quantity. We evaluated the performance of multiple displacement amplification (MDA WGA using gDNA extracted from lymphoblastoid cell lines (N = 27 with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR. Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel and N = 49 SNP (TaqMan® genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. Results The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. Conclusion The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of

Isolating silkworm genomic DNA without liquid nitrogen suitable for ...

African Journals Online (AJOL)

Genomic DNA was isolated from posterior silk gland of silkworms, Antheraea assama. Absolute alcohol was used as tissue fixing solution instead of grinding in liquid nitrogen, which yielded high molecular weight DNA (>40 kb). Samples yielded similar amount of DNA when fixed in absolute alcohol (400 μmg/g of silk gland ...

Analysis of the giant genomes of Fritillaria (Liliaceae) indicates that a lack of DNA removal characterizes extreme expansions in genome size.

Science.gov (United States)

Kelly, Laura J; Renny-Byfield, Simon; Pellicer, Jaume; Macas, Jiří; Novák, Petr; Neumann, Pavel; Lysak, Martin A; Day, Peter D; Berger, Madeleine; Fay, Michael F; Nichols, Richard A; Leitch, Andrew R; Leitch, Ilia J

2015-10-01

Plants exhibit an extraordinary range of genome sizes, varying by > 2000-fold between the smallest and largest recorded values. In the absence of polyploidy, changes in the amount of repetitive DNA (transposable elements and tandem repeats) are primarily responsible for genome size differences between species. However, there is ongoing debate regarding the relative importance of amplification of repetitive DNA versus its deletion in governing genome size. Using data from 454 sequencing, we analysed the most repetitive fraction of some of the largest known genomes for diploid plant species, from members of Fritillaria. We revealed that genomic expansion has not resulted from the recent massive amplification of just a handful of repeat families, as shown in species with smaller genomes. Instead, the bulk of these immense genomes is composed of highly heterogeneous, relatively low-abundance repeat-derived DNA, supporting a scenario where amplified repeats continually accumulate due to infrequent DNA removal. Our results indicate that a lack of deletion and low turnover of repetitive DNA are major contributors to the evolution of extremely large genomes and show that their size cannot simply be accounted for by the activity of a small number of high-abundance repeat families. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Modified protocol for genomic DNA extraction from newly plucked feathers of lophura leucomelana hamiltoni (Galliformes) for genetic studies and its endo-restriction analysis

International Nuclear Information System (INIS)

Andleeb, S.; Shamim, S.; Minhas, R.A.

2012-01-01

A rapid and accurate protocol was used first time to isolate the high-quality genomic DNA from newly plucked feathers of Lophura leucomelana. Two different lysis protocols were used depending on the feather size and it was observed that 55 deg. C for 3 to 4 days showed better results of feathers lysis as compared with the 37 deg. C for overnight with gentle shaking. Purification of genomic DNA was also performed with phenol: chloroform: isoamyl alcohol and 100% absolute ethanol precipitation methods. By using this protocol, a significant amount of high-quality genomic DNA was obtained and the purity of DNA was analyzed through endo-restriction analysis. Genomic DNA isolated with this modified method will be used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing and the evolutionary relationships among the other pheasants. (author)

A novel rat genomic simple repeat DNA with RNA-homology shows triplex (H-DNA)-like structure and tissue-specific RNA expression

International Nuclear Information System (INIS)

Dey, Indranil; Rath, Pramod C.

2005-01-01

Mammalian genome contains a wide variety of repetitive DNA sequences of relatively unknown function. We report a novel 227 bp simple repeat DNA (3.3 DNA) with a d {(GA) 7 A (AG) 7 } dinucleotide mirror repeat from the rat (Rattus norvegicus) genome. 3.3 DNA showed 75-85% homology with several eukaryotic mRNAs due to (GA/CU) n dinucleotide repeats by nBlast search and a dispersed distribution in the rat genome by Southern blot hybridization with [ 32 P]3.3 DNA. The d {(GA) 7 A (AG) 7 } mirror repeat formed a triplex (H-DNA)-like structure in vitro. Two large RNAs of 9.1 and 7.5 kb were detected by [ 32 P]3.3 DNA in rat brain by Northern blot hybridization indicating expression of such simple sequence repeats at RNA level in vivo. Further, several cDNAs were isolated from a rat cDNA library by [ 32 P]3.3 DNA probe. Three such cDNAs showed tissue-specific RNA expression in rat. pRT 4.1 cDNA showed strong expression of a 2.39 kb RNA in brain and spleen, pRT 5.5 cDNA showed strong expression of a 2.8 kb RNA in brain and a 3.9 kb RNA in lungs, and pRT 11.4 cDNA showed weak expression of a 2.4 kb RNA in lungs. Thus, genomic simple sequence repeats containing d (GA/CT) n dinucleotides are transcriptionally expressed and regulated in rat tissues. Such d (GA/CT) n dinucleotide repeats may form structural elements (e.g., triplex) which may be sites for functional regulation of genomic coding sequences as well as RNAs. This may be a general function of such transcriptionally active simple sequence repeats widely dispersed in mammalian genome

Comparison of Six DNA Extraction Procedures and the Application of Plastid DNA Enrichment Methods in Selected Non-photosynthetic Plants

Directory of Open Access Journals (Sweden)

Shin-Yi Shyu

2013-12-01

Full Text Available Genomic DNA was isolated using three DNA extraction commercial kits and three CTAB-based methods for two non-photosynthetic plants, Balanophora japonica and Mitrastemon kanehirai. The quality of the isolated DNA was evaluated and subjected to following restriction enzyme digestions. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998 performed well in isolating DNA from both species for restriction enzyme digestion. In addition, we succeeded to enrich plastid DNA content by using the methods depending on a high salt buffer to deplete nuclear material. The ‘high salt’ methods based on protocol presented by Milligan (1989 were able to increase plastid DNA effectively and significantly reduce nuclear DNA from M. kanehirai. The plastid DNA enrichment protocols are inexpensive and not time-consuming, and may be applicable to other non-photosynthetic plants.

DMS-Seq for In Vivo Genome-wide Mapping of Protein-DNA Interactions and Nucleosome Centers.

Science.gov (United States)

Umeyama, Taichi; Ito, Takashi

2017-10-03

Protein-DNA interactions provide the basis for chromatin structure and gene regulation. Comprehensive identification of protein-occupied sites is thus vital to an in-depth understanding of genome function. Dimethyl sulfate (DMS) is a chemical probe that has long been used to detect footprints of DNA-bound proteins in vitro and in vivo. Here, we describe a genomic footprinting method, dimethyl sulfate sequencing (DMS-seq), which exploits the cell-permeable nature of DMS to obviate the need for nuclear isolation. This feature makes DMS-seq simple in practice and removes the potential risk of protein re-localization during nuclear isolation. DMS-seq successfully detects transcription factors bound to cis-regulatory elements and non-canonical chromatin particles in nucleosome-free regions. Furthermore, an unexpected preference of DMS confers on DMS-seq a unique potential to directly detect nucleosome centers without using genetic manipulation. We expect that DMS-seq will serve as a characteristic method for genome-wide interrogation of in vivo protein-DNA interactions. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Energy Technology Data Exchange (ETDEWEB)

Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

2010-05-07

Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

Directory of Open Access Journals (Sweden)

Tran Duc

2010-05-01

Full Text Available Abstract Background Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

Science.gov (United States)

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

A MITE-based genotyping method to reveal hundreds of DNA polymorphisms in an animal genome after a few generations of artificial selection

Directory of Open Access Journals (Sweden)

Tetreau Guillaume

2008-10-01

Full Text Available Abstract Background For most organisms, developing hundreds of genetic markers spanning the whole genome still requires excessive if not unrealistic efforts. In this context, there is an obvious need for methodologies allowing the low-cost, fast and high-throughput genotyping of virtually any species, such as the Diversity Arrays Technology (DArT. One of the crucial steps of the DArT technique is the genome complexity reduction, which allows obtaining a genomic representation characteristic of the studied DNA sample and necessary for subsequent genotyping. In this article, using the mosquito Aedes aegypti as a study model, we describe a new genome complexity reduction method taking advantage of the abundance of miniature inverted repeat transposable elements (MITEs in the genome of this species. Results Ae. aegypti genomic representations were produced following a two-step procedure: (1 restriction digestion of the genomic DNA and simultaneous ligation of a specific adaptor to compatible ends, and (2 amplification of restriction fragments containing a particular MITE element called Pony using two primers, one annealing to the adaptor sequence and one annealing to a conserved sequence motif of the Pony element. Using this protocol, we constructed a library comprising more than 6,000 DArT clones, of which at least 5.70% were highly reliable polymorphic markers for two closely related mosquito strains separated by only a few generations of artificial selection. Within this dataset, linkage disequilibrium was low, and marker redundancy was evaluated at 2.86% only. Most of the detected genetic variability was observed between the two studied mosquito strains, but individuals of the same strain could still be clearly distinguished. Conclusion The new complexity reduction method was particularly efficient to reveal genetic polymorphisms in Ae. egypti. Overall, our results testify of the flexibility of the DArT genotyping technique and open new

Order and correlations in genomic DNA sequences. The spectral approach

International Nuclear Information System (INIS)

Lobzin, Vasilii V; Chechetkin, Vladimir R

2000-01-01

The structural analysis of genomic DNA sequences is discussed in the framework of the spectral approach, which is sufficiently universal due to the reciprocal correspondence and mutual complementarity of Fourier transform length scales. The spectral characteristics of random sequences of the same nucleotide composition possess the property of self-averaging for relatively short sequences of length M≥100-300. Comparison with the characteristics of random sequences determines the statistical significance of the structural features observed. Apart from traditional applications to the search for hidden periodicities, spectral methods are also efficient in studying mutual correlations in DNA sequences. By combining spectra for structure factors and correlation functions, not only integral correlations can be estimated but also their origin identified. Using the structural spectral entropy approach, the regularity of a sequence can be quantitatively assessed. A brief introduction to the problem is also presented and other major methods of DNA sequence analysis described. (reviews of topical problems)

Functional role of a highly repetitive DNA sequence in anchorage of the mouse genome.

Science.gov (United States)

Neuer-Nitsche, B; Lu, X N; Werner, D

1988-09-12

The major portion of the eukaryotic genome consists of various categories of repetitive DNA sequences which have been studied with respect to their base compositions, organizations, copy numbers, transcription and species specificities; their biological roles, however, are still unclear. A novel quality of a highly repetitive mouse DNA sequence is described which points to a functional role: All copies (approximately 50,000 per haploid genome) of this DNA sequence reside on genomic Alu I DNA fragments each associated with nuclear polypeptides that are not released from DNA by proteinase K, SDS and phenol extraction. By this quality the repetitive DNA sequence is classified as a member of the sub-set of DNA sequences involved in tight DNA-polypeptide complexes which have been previously shown to be components of the subnuclear structure termed 'nuclear matrix'. From these results it has to be concluded that the repetitive DNA sequence characterized in this report represents or comprises a signal for a large number of site specific attachment points of the mouse genome in the nuclear matrix.

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

Energy Technology Data Exchange (ETDEWEB)

McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L.; dePamphilis, Claude W.

2005-08-26

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses.

Science.gov (United States)

Yamamoto, F; Yamamoto, M

2004-07-01

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme NotI in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15 x 2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.

DNA fingerprinting of Mycobacterium tuberculosis: from phage typing to whole-genome sequencing.

Science.gov (United States)

Schürch, Anita C; van Soolingen, Dick

2012-06-01

Current typing methods for Mycobacterium tuberculosis complex evolved from simple phenotypic approaches like phage typing and drug susceptibility profiling to DNA-based strain typing methods, such as IS6110-restriction fragment length polymorphisms (RFLP) and variable number of tandem repeats (VNTR) typing. Examples of the usefulness of molecular typing are source case finding and epidemiological linkage of tuberculosis (TB) cases, international transmission of MDR/XDR-TB, the discrimination between endogenous reactivation and exogenous re-infection as a cause of relapses after curative treatment of tuberculosis, the evidence of multiple M. tuberculosis infections, and the disclosure of laboratory cross-contaminations. Simultaneously, phylogenetic analyses were developed based on single nucleotide polymorphisms (SNPs), genomic deletions usually referred to as regions of difference (RDs) and spoligotyping which served both strain typing and phylogenetic analysis. National and international initiatives that rely on the application of these typing methods have brought significant insight into the molecular epidemiology of tuberculosis. However, current DNA fingerprinting methods have important limitations. They can often not distinguish between genetically closely related strains and the turn-over of these markers is variable. Moreover, the suitability of most DNA typing methods for phylogenetic reconstruction is limited as they show a high propensity of convergent evolution or misinfer genetic distances. In order to fully explore the possibilities of genotyping in the molecular epidemiology of tuberculosis and to study the phylogeny of the causative bacteria reliably, the application of whole-genome sequencing (WGS) analysis for all M. tuberculosis isolates is the optimal, although currently still a costly solution. In the last years WGS for typing of pathogens has been explored and yielded important additional information on strain diversity in comparison to the

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

Directory of Open Access Journals (Sweden)

Flores Kevin

2012-09-01

Full Text Available Abstract Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee and Nasonia vitripennis (jewel wasp analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice

Study on the relationship between DNA-PKcs and genomic instability and hyper-radiosensitivity

International Nuclear Information System (INIS)

Yang Kang; Zhu Jiayun; Ding Nan; Li Junhong; Hu Wentao; Su Fengtao; He Jinpeng; Li Sha

2010-01-01

To investigate the relationship between DNA-PKcs and genome instability and hyper-radiosensitivity, human glioma cell lines M059K and M059J, as a model expressing wild-type DNA-PKcs and a model defective in DNA-PKcs activity, were exposed to low doses of X-rays. Cells survival fractions were assessed by colony-forming assay and Cytochalasin-B micronucleus assay was employed to detect the genomic instability happening in each single irradiated colony. It has been found that as the post-incubation time increased, M059K cells expressing wild-type DNA-PKcs exhibited low-dose hyper-radiosensitivity and showed a similar genomic instability after 0.2 Gy and 0.6 Gy irradiations, but the M059J cells lacking in DNA-PKcs didn't present low-dose hyper-radiosensitivity and showed a higher genomic instability of 0.6 Gy than that of 0.2 Gy. The results indicate that DNA-PKcs may act as one of the key factors that lead to low-dose hyper-radiosensitivity. (authors)

Comparison of protocols for genomic DNA extraction from 'velame ...

African Journals Online (AJOL)

usuario

2013-07-24

Jul 24, 2013 ... involving C. linearifolius, we compared the efficiency of six protocols for genomic DNA extraction previously ... phytic, with diverse aspect and floristics, average rainfall between ..... The variation observed for DNA concentrations estimated with .... performed with protocol 1 (data not shown), or still, bands.

DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

Science.gov (United States)

Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

2017-01-25

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G 2 /M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in

Studies on the effects of persistent RNA priming on DNA replication and genomic stability

OpenAIRE

Stuckey, Ruth

2014-01-01

[EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

DEFF Research Database (Denmark)

Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan

2009-01-01

MOTIVATION: The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary...... determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than...

DNA rearrangements from γ-irradiated normal human fibroblasts preferentially occur in transcribed regions of the genome

International Nuclear Information System (INIS)

Forrester, H.B.; Radford, I.R.

2003-01-01

Full text: DNA rearrangement events leading to chromosomal aberrations are central to ionizing radiation-induced cell death. Although DNA double-strand breaks are probably the lesion that initiates formation of chromosomal aberrations, little is understood about the molecular mechanisms that generate and modulate DNA rearrangement. Examination of the sequences that flank sites of DNA rearrangement may provide information regarding the processes and enzymes involved in rearrangement events. Accordingly, we developed a method using inverse PCR that allows the detection and sequencing of putative radiation-induced DNA rearrangements in defined regions of the human genome. The method can detect single copies of a rearrangement event that has occurred in a particular region of the genome and, therefore, DNA rearrangement detection does not require survival and continued multiplication of the affected cell. Ionizing radiation-induced DNA rearrangements were detected in several different regions of the genome of human fibroblast cells that were exposed to 30 Gy of γ-irradiation and then incubated for 24 hours at 37 deg C. There was a 3- to 5-fold increase in the number of products amplified from irradiated as compared with control cells in the target regions 5' to the C-MYC, CDKN1A, RB1, and FGFR2 genes. Sequences were examined from 121 DNA rearrangements. Approximately half of the PCR products were derived from possible inter-chromosomal rearrangements and the remainder were from intra-chromosomal events. A high proportion of the sequences that rearranged with target regions were located in genes, suggesting that rearrangements may occur preferentially in transcribed regions. Eighty-four percent of the sequences examined by reverse transcriptase PCR were from transcribed sequences in IMR-90 cells. The distribution of DNA rearrangements within the target regions is non-random and homology occurs between the sequences involved in rearrangements in some cases but is not

DNA Oncogenic Virus-Induced Oxidative Stress, Genomic Damage, and Aberrant Epigenetic Alterations

Directory of Open Access Journals (Sweden)

Mankgopo Magdeline Kgatle

2017-01-01

Full Text Available Approximately 20% of human cancers is attributable to DNA oncogenic viruses such as human papillomavirus (HPV, hepatitis B virus (HBV, and Epstein-Barr virus (EBV. Unrepaired DNA damage is the most common and overlapping feature of these DNA oncogenic viruses and a source of genomic instability and tumour development. Sustained DNA damage results from unceasing production of reactive oxygen species and activation of inflammasome cascades that trigger genomic changes and increased propensity of epigenetic alterations. Accumulation of epigenetic alterations may interfere with genome-wide cellular signalling machineries and promote malignant transformation leading to cancer development. Untangling and understanding the underlying mechanisms that promote these detrimental effects remain the major objectives for ongoing research and hope for effective virus-induced cancer therapy. Here, we review current literature with an emphasis on how DNA damage influences HPV, HVB, and EBV replication and epigenetic alterations that are associated with carcinogenesis.

The DNA-encoded nucleosome organization of a eukaryotic genome.

Science.gov (United States)

Kaplan, Noam; Moore, Irene K; Fondufe-Mittendorf, Yvonne; Gossett, Andrea J; Tillo, Desiree; Field, Yair; LeProust, Emily M; Hughes, Timothy R; Lieb, Jason D; Widom, Jonathan; Segal, Eran

2009-03-19

Nucleosome organization is critical for gene regulation. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for approximately 40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes have a central role in determining the organization of nucleosomes in vivo.

Criminal Genomic Pragmatism: Prisoners' Representations of DNA Technology and Biosecurity

Science.gov (United States)

Machado, Helena; Silva, Susana

2012-01-01

Background. Within the context of the use of DNA technology in crime investigation, biosecurity is perceived by different stakeholders according to their particular rationalities and interests. Very little is known about prisoners' perceptions and assessments of the uses of DNA technology in solving crime. Aim. To propose a conceptual model that serves to analyse and interpret prisoners' representations of DNA technology and biosecurity. Methods. A qualitative study using an interpretative approach based on 31 semi-structured tape-recorded interviews was carried out between May and September 2009, involving male inmates in three prisons located in the north of Portugal. The content analysis focused on the following topics: the meanings attributed to DNA and assessments of the risks and benefits of the uses of DNA technology and databasing in forensic applications. Results. DNA was described as a record of identity, an exceptional material, and a powerful biometric identifier. The interviewees believed that DNA can be planted to incriminate suspects. Convicted offenders argued for the need to extend the criteria for the inclusion of DNA profiles in forensic databases and to restrict the removal of profiles. Conclusions. The conceptual model entitled criminal genomic pragmatism allows for an understanding of the views of prison inmates regarding DNA technology and biosecurity. PMID:22791960

Criminal Genomic Pragmatism: Prisoners' Representations of DNA Technology and Biosecurity

Directory of Open Access Journals (Sweden)

Helena Machado

2012-01-01

Full Text Available Background. Within the context of the use of DNA technology in crime investigation, biosecurity is perceived by different stakeholders according to their particular rationalities and interests. Very little is known about prisoners’ perceptions and assessments of the uses of DNA technology in solving crime. Aim. To propose a conceptual model that serves to analyse and interpret prisoners’ representations of DNA technology and biosecurity. Methods. A qualitative study using an interpretative approach based on 31 semi-structured tape-recorded interviews was carried out between May and September 2009, involving male inmates in three prisons located in the north of Portugal. The content analysis focused on the following topics: the meanings attributed to DNA and assessments of the risks and benefits of the uses of DNA technology and databasing in forensic applications. Results. DNA was described as a record of identity, an exceptional material, and a powerful biometric identifier. The interviewees believed that DNA can be planted to incriminate suspects. Convicted offenders argued for the need to extend the criteria for the inclusion of DNA profiles in forensic databases and to restrict the removal of profiles. Conclusions. The conceptual model entitled criminal genomic pragmatism allows for an understanding of the views of prison inmates regarding DNA technology and biosecurity.

Relationships between 16S-23S rRNA gene internal transcribed spacer DNA and genomic DNA similarities in the taxonomy of phototrophic bacteria

International Nuclear Information System (INIS)

Okamura, K; Hisada, T; Takata, K; Hiraishi, A

2013-01-01

Rapid and accurate identification of microbial species is essential task in microbiology and biotechnology. In prokaryotic systematics, genomic DNA-DNA hybridization is the ultimate tool to determine genetic relationships among bacterial strains at the species level. However, a practical problem in this assay is that the experimental procedure is laborious and time-consuming. In recent years, information on the 16S-23S rRNA gene internal transcribed spacer (ITS) region has been used to classify bacterial strains at the species and intraspecies levels. It is unclear how much information on the ITS region can reflect the genome that contain it. In this study, therefore, we evaluate the quantitative relationship between ITS DNA and entire genomic DNA similarities. For this, we determined ITS sequences of several species of anoxygenic phototrophic bacteria belonging to the order Rhizobiales, and compared with DNA-DNA relatedness among these species. There was a high correlation between the two genetic markers. Based on the regression analysis of this relationship, 70% DNA-DNA relatedness corresponded to 92% ITS sequence similarity. This suggests the usefulness of the ITS sequence similarity as a criterion for determining the genospecies of the phototrophic bacteria. To avoid the effects of polymorphism bias of ITS on similarities, PCR products from all loci of ITS were used directly as genetic probes for comparison. The results of ITS DNA-DNA hybridization coincided well with those of genomic DNA-DNA relatedness. These collective data indicate that the whole ITS DNA-DNA similarity can be used as an alternative to genomic DNA-DNA similarity.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

Science.gov (United States)

Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

2011-08-01

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA.

Science.gov (United States)

Shahal, Tamar; Gilat, Noa; Michaeli, Yael; Redy-Keisar, Orit; Shabat, Doron; Ebenstein, Yuval

2014-08-19

5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

Suicidal function of DNA methylation in age-related genome disintegration.

Science.gov (United States)

Mazin, Alexander L

2009-10-01

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

Science.gov (United States)

Cai, Yi; Tsai, Hsing-Chen; Yen, Ray-Whay Chiu; Zhang, Yang W; Kong, Xiangqian; Wang, Wei; Xia, Limin; Baylin, Stephen B

2017-04-01

Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential. © 2017 Cai et al.; Published by Cold Spring Harbor Laboratory Press.

In situ genomic DNA extraction for PCR analysis of regions of interest in four plant species and one filamentous fungi

Directory of Open Access Journals (Sweden)

Luis E. Rojas

2014-07-01

Full Text Available The extraction methods of genomic DNA are usually laborious and hazardous to human health and the environment by the use of organic solvents (chloroform and phenol. In this work a protocol for in situ extraction of genomic DNA by alkaline lysis is validated. It was used in order to amplify regions of DNA in four species of plants and fungi by polymerase chain reaction (PCR. From plant material of Saccharum officinarum L., Carica papaya L. and Digitalis purpurea L. it was possible to extend different regions of the genome through PCR. Furthermore, it was possible to amplify a fragment of avr-4 gene DNA purified from lyophilized mycelium of Mycosphaerella fijiensis. Additionally, it was possible to amplify the region ap24 transgene inserted into the genome of banana cv. `Grande naine' (Musa AAA. Key words: alkaline lysis, Carica papaya L., Digitalis purpurea L., Musa, Saccharum officinarum L.

Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

Directory of Open Access Journals (Sweden)

Zhongai Li

2015-01-01

Conclusions: NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

cDNA structure, genomic organization and expression patterns of ...

African Journals Online (AJOL)

Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations.

Science.gov (United States)

Bonaïti, Catherine; Parayre, Sandrine; Irlinger, Françoise

2006-03-15

Cheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol-chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription-PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices.

Whole genome DNA copy number changes identified by high density oligonucleotide arrays

Directory of Open Access Journals (Sweden)

Huang Jing

2004-05-01

Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

Potassium hydroxide-ethylene diamine tetraacetic acid method for the rapid preparation of small-scale PCR template DNA from actinobacteria.

Science.gov (United States)

Sun, Zhibin; Huang, Yan; Wang, Yanzhuo; Zhao, Yuguo; Cui, Zhongli

2014-01-01

Genomic DNA extraction from Gram-positive bacteria is a laborious and time-consuming process. A rapid and convenient method was established to extract genomic DNA from a single colony as a PCR template. KOH-EDTA is used as a lysis buffer to disrupt the cell envelope, releasing genomic DNA, and Tris-HCl (pH = 4) is then added to neutralize the lysate. The lysate can be used directly as a template for PCR amplification. 16S rDNA was successfully amplified from Gram-positive bacteria from the genera of Bacillus, Streptomyces, Micromonospora, Nonomuraea, Microbispora, and Staphylococcus. Amplification of the trpB gene indicated that this method could also be applied to the amplification of functional genes. Compared to colony PCR methods without KOH-EDTA, this method is extremely fast and efficient, and it is applicable to high-throughput PCR amplifications.

Genome-wide prediction of cis-regulatory regions using supervised deep learning methods.

Science.gov (United States)

Li, Yifeng; Shi, Wenqiang; Wasserman, Wyeth W

2018-05-31

In the human genome, 98% of DNA sequences are non-protein-coding regions that were previously disregarded as junk DNA. In fact, non-coding regions host a variety of cis-regulatory regions which precisely control the expression of genes. Thus, Identifying active cis-regulatory regions in the human genome is critical for understanding gene regulation and assessing the impact of genetic variation on phenotype. The developments of high-throughput sequencing and machine learning technologies make it possible to predict cis-regulatory regions genome wide. Based on rich data resources such as the Encyclopedia of DNA Elements (ENCODE) and the Functional Annotation of the Mammalian Genome (FANTOM) projects, we introduce DECRES based on supervised deep learning approaches for the identification of enhancer and promoter regions in the human genome. Due to their ability to discover patterns in large and complex data, the introduction of deep learning methods enables a significant advance in our knowledge of the genomic locations of cis-regulatory regions. Using models for well-characterized cell lines, we identify key experimental features that contribute to the predictive performance. Applying DECRES, we delineate locations of 300,000 candidate enhancers genome wide (6.8% of the genome, of which 40,000 are supported by bidirectional transcription data), and 26,000 candidate promoters (0.6% of the genome). The predicted annotations of cis-regulatory regions will provide broad utility for genome interpretation from functional genomics to clinical applications. The DECRES model demonstrates potentials of deep learning technologies when combined with high-throughput sequencing data, and inspires the development of other advanced neural network models for further improvement of genome annotations.

Continued colonization of the human genome by mitochondrial DNA.

Directory of Open Access Journals (Sweden)

Miria Ricchetti

2004-09-01

Full Text Available Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4-6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

HPCE quantification of 5-methyl-2'-deoxycytidine in genomic DNA: methodological optimization for chestnut and other woody species.

Science.gov (United States)

Hasbún, Rodrigo; Valledor, Luís; Rodríguez, José L; Santamaria, Estrella; Ríos, Darcy; Sanchez, Manuel; Cañal, María J; Rodríguez, Roberto

2008-01-01

Quantification of deoxynucleosides using micellar high-performance capillary electrophoresis (HPCE) is an efficient, fast and inexpensive evaluation method of genomic DNA methylation. This approach has been demonstrated to be more sensitive and specific than other methods for the quantification of DNA methylation content. However, effective detection and quantification of 5-methyl-2'-deoxycytidine depend of the sample characteristics. Previous works have revealed that in most woody species, the quality and quantity of RNA-free DNA extracted that is suitable for analysis by means of HPCE varies among species of the same gender, among tissues taken from the same tree, and vary in the same tissue depending on the different seasons of the year. The aim of this work is to establish a quantification method of genomic DNA methylation that lends itself to use in different Castanea sativa Mill. materials, and in other angiosperm and gymnosperm woody species. Using a DNA extraction kit based in silica membrane has increased the resolutive capacity of the method. Under these conditions, it can be analyzed different organs or tissues of angiosperms and gymnosperms, regardless of their state of development. We emphasized the importance of samples free of nucleosides, although, in the contrary case, the method ensures the effective separation of deoxynucleosides and identification of 5-methyl-2'-deoxycytidine.

Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genome sequencing method

Directory of Open Access Journals (Sweden)

Bingfu Guo

2016-07-01

Full Text Available Molecular characterization of sequences flanking exogenous fragment insertions is essential for safety assessment and labeling of genetically modified organisms (GMO. In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS method. About 21 Gb sequence data (~21× coverage for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundary of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767-50543792 and Chr17: 7980527-7980541 in these two transgenic lines. Identification of the genomic insertion site of the G2-EPSPS and GAT transgenes will facilitate the use of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS is a cost-effective and rapid method of identifying sites of T-DNA insertions and flanking sequences in soybean.

Norgal: extraction and de novo assembly of mitochondrial DNA from whole-genome sequencing data.

Science.gov (United States)

Al-Nakeeb, Kosai; Petersen, Thomas Nordahl; Sicheritz-Pontén, Thomas

2017-11-21

Whole-genome sequencing (WGS) projects provide short read nucleotide sequences from nuclear and possibly organelle DNA depending on the source of origin. Mitochondrial DNA is present in animals and fungi, while plants contain DNA from both mitochondria and chloroplasts. Current techniques for separating organelle reads from nuclear reads in WGS data require full reference or partial seed sequences for assembling. Norgal (de Novo ORGAneLle extractor) avoids this requirement by identifying a high frequency subset of k-mers that are predominantly of mitochondrial origin and performing a de novo assembly on a subset of reads that contains these k-mers. The method was applied to WGS data from a panda, brown algae seaweed, butterfly and filamentous fungus. We were able to extract full circular mitochondrial genomes and obtained sequence identities to the reference sequences in the range from 98.5 to 99.5%. We also assembled the chloroplasts of grape vines and cucumbers using Norgal together with seed-based de novo assemblers. Norgal is a pipeline that can extract and assemble full or partial mitochondrial and chloroplast genomes from WGS short reads without prior knowledge. The program is available at: https://bitbucket.org/kosaidtu/norgal .

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

Science.gov (United States)

Christen, Matthias; Del Medico, Luca; Christen, Heinz; Christen, Beat

2017-01-01

Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Genome Partitioner: A web tool for multi-level partitioning of large-scale DNA constructs for synthetic biology applications.

Directory of Open Access Journals (Sweden)

Matthias Christen

Full Text Available Recent advances in lower-cost DNA synthesis techniques have enabled new innovations in the field of synthetic biology. Still, efficient design and higher-order assembly of genome-scale DNA constructs remains a labor-intensive process. Given the complexity, computer assisted design tools that fragment large DNA sequences into fabricable DNA blocks are needed to pave the way towards streamlined assembly of biological systems. Here, we present the Genome Partitioner software implemented as a web-based interface that permits multi-level partitioning of genome-scale DNA designs. Without the need for specialized computing skills, biologists can submit their DNA designs to a fully automated pipeline that generates the optimal retrosynthetic route for higher-order DNA assembly. To test the algorithm, we partitioned a 783 kb Caulobacter crescentus genome design. We validated the partitioning strategy by assembling a 20 kb test segment encompassing a difficult to synthesize DNA sequence. Successful assembly from 1 kb subblocks into the 20 kb segment highlights the effectiveness of the Genome Partitioner for reducing synthesis costs and timelines for higher-order DNA assembly. The Genome Partitioner is broadly applicable to translate DNA designs into ready to order sequences that can be assembled with standardized protocols, thus offering new opportunities to harness the diversity of microbial genomes for synthetic biology applications. The Genome Partitioner web tool can be accessed at https://christenlab.ethz.ch/GenomePartitioner.

Genome-wide alterations of the DNA replication program during tumor progression

Science.gov (United States)

Arneodo, A.; Goldar, A.; Argoul, F.; Hyrien, O.; Audit, B.

2016-08-01

Oncogenic stress is a major driving force in the early stages of cancer development. Recent experimental findings reveal that, in precancerous lesions and cancers, activated oncogenes may induce stalling and dissociation of DNA replication forks resulting in DNA damage. Replication timing is emerging as an important epigenetic feature that recapitulates several genomic, epigenetic and functional specificities of even closely related cell types. There is increasing evidence that chromosome rearrangements, the hallmark of many cancer genomes, are intimately associated with the DNA replication program and that epigenetic replication timing changes often precede chromosomic rearrangements. The recent development of a novel methodology to map replication fork polarity using deep sequencing of Okazaki fragments has provided new and complementary genome-wide replication profiling data. We review the results of a wavelet-based multi-scale analysis of genomic and epigenetic data including replication profiles along human chromosomes. These results provide new insight into the spatio-temporal replication program and its dynamics during differentiation. Here our goal is to bring to cancer research, the experimental protocols and computational methodologies for replication program profiling, and also the modeling of the spatio-temporal replication program. To illustrate our purpose, we report very preliminary results obtained for the chronic myelogeneous leukemia, the archetype model of cancer. Finally, we discuss promising perspectives on using genome-wide DNA replication profiling as a novel efficient tool for cancer diagnosis, prognosis and personalized treatment.

Global DNA Methylation in the Chestnut Blight Fungus Cryphonectria parasitica and Genome-Wide Changes in DNA Methylation Accompanied with Sectorization

Directory of Open Access Journals (Sweden)

Kum-Kang So

2018-02-01

Full Text Available Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase, demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

Directory of Open Access Journals (Sweden)

Ben Beheshti

2003-01-01

Full Text Available Conventional comparative genomic hybridization (CGH profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1 no amplifications evident, 2 a small MYCN amplicon as the only detectable imbalance, and 3 a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma.

Optimized pH method for DNA elution from buccal cells collected in Whatman FTA cards.

Science.gov (United States)

Lema, Carolina; Kohl-White, Kendra; Lewis, Laurie R; Dao, Dat D

2006-01-01

DNA is the most accessible biologic material for obtaining information from the human genome because of its molecular stability and its presence in every nucleated cell. Currently, single nucleotide polymorphism genotyping and DNA methylation are the main DNA-based approaches to deriving genomic and epigenomic disease biomarkers. Upon the discontinuation of the Schleicher & Schuell IsoCode product (Dassel, Germany), which was a treated paper system to elute DNA from several biologic sources for polymerase chain reaction (PCR) analysis, a high-yielding DNA elution method was imperative. We describe here an improved procedure of the not fully validated Whatman pH-based elution protocol. Our DNA elution procedure from buccal cells collected in Whatman FTA cards (Whatman Inc., Florham Park, NJ) yielded approximately 4 microg of DNA from a 6-mm FTA card punch and was successfully applied for HLA-DQB1 genotyping. The genotypes showed complete concordance with data obtained from blood of the same subjects. The achieved high DNA yield from buccal cells suggests a potential cost-effective tool for genomic and epigenomic disease biomarkers development.

The Genomic Pattern of tDNA Operon Expression in E. coli.

Directory of Open Access Journals (Sweden)

2005-06-01

Full Text Available In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates.

Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner.

Science.gov (United States)

Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan; Brent, Michael R

2009-07-01

The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than heuristics. We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners. To validate these results with natural sequences, we performed cross-species alignment using orthologous transcripts from human, mouse and rat. We found that aligner accuracy is heavily dependent on sequence identity. For sequences with 100% identity, Pairagon achieved accuracy levels of >99.6%, with one quarter of the errors of any other aligner. Furthermore, for human/mouse alignments, which are only 85% identical, Pairagon achieved 87% accuracy, higher than any other aligner. Pairagon source and executables are freely available at http://mblab.wustl.edu/software/pairagon/

An improved protocol and a new grinding device for extraction of genomic DNA from microorganisms by a two-step extraction procedure.

Science.gov (United States)

Zhang, S S; Chen, D; Lu, Q

2012-05-21

Current protocols to extract genomic DNA from microorganisms are still laborious, tedious and costly, especially for the species with thick cell walls. In order to improve the effectiveness of extracting DNA from microbial samples, a novel protocol, defined as two-step extraction method, along with an improved tissue-grinding device, was developed. The protocol included two steps, disruption of microbial cells or spores by grinding the sample together with silica sand in a new device and extraction of DNA with an effective buffer containing cell lysis chemicals. The device was prepared by using a commercial electric mini-grinder, adapted with a grinding stone, and a sample cup processed by lathing from a polytetrafluoroethylene rod. We tested the method with vegetative cells of four microbial species and two microbial spores that have thick cell walls and are therefore hard to process; these included Escherichia coli JM109, Bacillus subtilis WB600, Sacchromyces cerevisiae INVSc1, Trichoderma viride AS3.3711, and the spores of S. cerevisiae and T. viride, respectively, representing Gram-positive bacteria, Gram-negative bacteria, yeast, filamentous fungi. We found that this new method and device extracted usable quantities of genomic DNA from the samples. The DNA fragments that were extracted exceeded 23 kb. The target sequences up to about 5 kb were successfully and exclusively amplified by PCR using extracted DNA as the template. In addition, the DNA extraction was finalized within 1.5 h. Thus, we conclude that this two-step extraction method is an effective and improved protocol for extraction of genomic DNA from microbial samples.

The detection of large deletions or duplications in genomic DNA.

Science.gov (United States)

Armour, J A L; Barton, D E; Cockburn, D J; Taylor, G R

2002-11-01

While methods for the detection of point mutations and small insertions or deletions in genomic DNA are well established, the detection of larger (>100 bp) genomic duplications or deletions can be more difficult. Most mutation scanning methods use PCR as a first step, but the subsequent analyses are usually qualitative rather than quantitative. Gene dosage methods based on PCR need to be quantitative (i.e., they should report molar quantities of starting material) or semi-quantitative (i.e., they should report gene dosage relative to an internal standard). Without some sort of quantitation, heterozygous deletions and duplications may be overlooked and therefore be under-ascertained. Gene dosage methods provide the additional benefit of reporting allele drop-out in the PCR. This could impact on SNP surveys, where large-scale genotyping may miss null alleles. Here we review recent developments in techniques for the detection of this type of mutation and compare their relative strengths and weaknesses. We emphasize that comprehensive mutation analysis should include scanning for large insertions and deletions and duplications. Copyright 2002 Wiley-Liss, Inc.

Epigenetic control of mobile DNA as an interface between experience and genome change

Directory of Open Access Journals (Sweden)

James A. Shapiro

2014-04-01

Full Text Available Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration.

BuD, a helix–loop–helix DNA-binding domain for genome modification

Energy Technology Data Exchange (ETDEWEB)

Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

2014-07-01

Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

A method to evaluate genome-wide methylation in archival formalin-fixed, paraffin-embedded ovarian epithelial cells.

Directory of Open Access Journals (Sweden)

Qiling Li

Full Text Available The use of DNA from archival formalin and paraffin embedded (FFPE tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue.Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E.Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample.We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

Science.gov (United States)

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Concentrating and labeling genomic DNA in a nanofluidic array

DEFF Research Database (Denmark)

Marie, Rodolphe; Pedersen, Jonas Nyvold; Mir, Kalim U.

2018-01-01

, however, hinder the polymerase activity. We demonstrate a device and a protocol for the enzymatic labeling of genomic DNA arranged in a dense array of single molecules without attaching the enzyme or the DNA to a surface. DNA molecules accumulate in a dense array of pits embedded within a nanoslit due...... to entropic trapping. We then perform ϕ29 polymerase extension from single-strand nicks created on the trapped molecules to incorporate fluorescent nucleotides into the DNA. The array of entropic traps can be loaded with λ-DNA molecules to more than 90% of capacity at a flow rate of 10 pL min-1. The final...

Comparison of Different Drying Methods for Recovery of Mushroom DNA.

Science.gov (United States)

Wang, Shouxian; Liu, Yu; Xu, Jianping

2017-06-07

Several methods have been reported for drying mushroom specimens for population genetic, taxonomic, and phylogenetic studies. However, most methods have not been directly compared for their effectiveness in preserving mushroom DNA. In this study, we compared silica gel drying at ambient temperature and oven drying at seven different temperatures. Two mushroom species representing two types of fruiting bodies were examined: the fleshy button mushroom Agaricus bisporus and the leathery shelf fungus Trametes versicolor. For each species dried with the eight methods, we assessed the mushroom water loss rate, the quality and quantity of extracted DNA, and the effectiveness of using the extracted DNA as a template for PCR amplification of two DNA fragments (ITS and a single copy gene). Dried specimens from all tested methods yielded sufficient DNA for PCR amplification of the two genes in both species. However, differences among the methods for the two species were found in: (i) the time required by different drying methods for the fresh mushroom tissue to reach a stable weight; and (ii) the relative quality and quantity of the extracted genomic DNA. Among these methods, oven drying at 70 °C for 3-4 h seemed the most efficient for preserving field mushroom samples for subsequent molecular work.

Instability of plastid DNA in the nuclear genome.

Directory of Open Access Journals (Sweden)

Anna E Sheppard

2009-01-01

Full Text Available Functional gene transfer from the plastid (chloroplast and mitochondrial genomes to the nucleus has been an important driving force in eukaryotic evolution. Non-functional DNA transfer is far more frequent, and the frequency of such transfers from the plastid to the nucleus has been determined experimentally in tobacco using transplastomic lines containing, in their plastid genome, a kanamycin resistance gene (neo readymade for nuclear expression. Contrary to expectations, non-Mendelian segregation of the kanamycin resistance phenotype is seen in progeny of some lines in which neo has been transferred to the nuclear genome. Here, we provide a detailed analysis of the instability of kanamycin resistance in nine of these lines, and we show that it is due to deletion of neo. Four lines showed instability with variation between progeny derived from different areas of the same plant, suggesting a loss of neo during somatic cell division. One line showed a consistent reduction in the proportion of kanamycin-resistant progeny, suggesting a loss of neo during meiosis, and the remaining four lines were relatively stable. To avoid genomic enlargement, the high frequency of plastid DNA integration into the nuclear genome necessitates a counterbalancing removal process. This is the first demonstration of such loss involving a high proportion of recent nuclear integrants. We propose that insertion, deletion, and rearrangement of plastid sequences in the nuclear genome are important evolutionary processes in the generation of novel nuclear genes. This work is also relevant in the context of transgenic plant research and crop production, because similar processes to those described here may be involved in the loss of plant transgenes.

Improvement in the amine glass platform by bubbling method for a DNA microarray

Directory of Open Access Journals (Sweden)

Jee SH

2015-10-01

Full Text Available Seung Hyun Jee,1 Jong Won Kim,2 Ji Hyeong Lee,2 Young Soo Yoon11Department of Chemical and Biological Engineering, Gachon University, Seongnam, Gyeonggi, Republic of Korea; 2Genomics Clinical Research Institute, LabGenomics Co., Ltd., Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of KoreaAbstract: A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool. Keywords: DNA microarray, glass platform, bubbling method, self-assambled monolayer

Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

Science.gov (United States)

Rudd, K E; Miller, W; Ostell, J; Benson, D A

1990-01-25

We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects.

In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

Directory of Open Access Journals (Sweden)

Janet L Smith

2015-05-01

Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

DEFF Research Database (Denmark)

Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi

2016-01-01

and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing...... methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved...

Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

DEFF Research Database (Denmark)

Winkel, Bo G; Hollegaard, Mads V; Olesen, Morten S

2011-01-01

BACKGROUND: The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted...

RICD: A rice indica cDNA database resource for rice functional genomics

Directory of Open Access Journals (Sweden)

Zhang Qifa

2008-11-01

Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

DEFF Research Database (Denmark)

Knudsen, Berith Elkær; Bergmark, Lasse; Munk, Patrick

2016-01-01

that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10......Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required...... for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial...

G-Quadruplexes Involving Both Strands of Genomic DNA Are Highly Abundant and Colocalize with Functional Sites in the Human Genome.

Directory of Open Access Journals (Sweden)

Andrzej S Kudlicki

Full Text Available The G-quadruplex is a non-canonical DNA structure biologically significant in DNA replication, transcription and telomere stability. To date, only G4s with all guanines originating from the same strand of DNA have been considered in the context of the human nuclear genome. Here, I discuss interstrand topological configurations of G-quadruplex DNA, consisting of guanines from both strands of genomic DNA; an algorithm is presented for predicting such structures. I have identified over 550,000 non-overlapping interstrand G-quadruplex forming sequences in the human genome--significantly more than intrastrand configurations. Functional analysis of interstrand G-quadruplex sites shows strong association with transcription initiation, the results are consistent with the XPB and XPD transcriptional helicases binding only to G-quadruplex DNA with interstrand topology. Interstrand quadruplexes are also enriched in origin of replication sites. Several topology classes of interstrand quadruplex-forming sequences are possible, and different topologies are enriched in different types of structural elements. The list of interstrand quadruplex forming sequences, and the computer program used for their prediction are available at the web address http://moment.utmb.edu/allquads.

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

DEFF Research Database (Denmark)

Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

2015-01-01

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro......Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway...... that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...... facilitating the advanced engineering of robust cell factories....

Comparison of manual methods of extracting genomic DNA from dried blood spots collected on different cards: implications for clinical practice.

Science.gov (United States)

Molteni, C G; Terranova, L; Zampiero, A; Galeone, C; Principi, N; Esposito, S

2013-01-01

Isolating genomic DNA from blood samples is essential when studying the associations between genetic variants and susceptibility to a given clinical condition, or its severity. This study of three extraction techniques and two types of commercially available cards involved 219 children attending our outpatient pediatric clinic for follow-up laboratory tests after they had been hospitalised. An aliquot of venous blood was drawn into plastic tubes without additives and, after several inversions, 80 microL were put on circles of common paper cards and Whatman FTA-treated cards. Three extraction methods were compared: the Qiagen Investigator, Gensolve, and Masterpure. The best method in terms of final DNA yield was Masterpure, which led to a significantly higher yield regardless of the type of card (p less than 0.001), followed by Qiagen Investigator and Gensolve. Masterpure was also the best in terms of price, seemed to be simple and reliable, and required less hands-on time than other techniques. These conclusions support the use of Masterpure in studies that evaluate the associations between genetic variants and the severity or prevalence of infectious diseases.

The study of genomic DNA adsorption and subsequent interactions using total internal reflection ellipsometry.

Science.gov (United States)

Nabok, Alexei; Tsargorodskaya, Anna; Davis, Frank; Higson, Séamus P J

2007-10-31

The adsorption of genomic DNA and subsequent interactions between adsorbed and solvated DNA was studied using a novel sensitive optical method of total internal reflection ellipsometry (TIRE), which combines spectroscopic ellipsometry with surface plasmon resonance (SPR). Single strands of DNA of two species of fish (herring and salmon) were electrostatically adsorbed on top of polyethylenimine films deposited upon gold coated glass slides. The ellipsometric spectra were recorded and data fitting utilized to extract optical parameters (thickness and refractive index) of adsorbed DNA layers. The further adsorption of single stranded DNA from an identical source, i.e. herring ss-DNA on herring ss-DNA or salmon ss-DNA on salmon ss-DNA, on the surface was observed to give rise to substantial film thickness increases at the surface of about 20-21 nm. Conversely adsorption of DNA from alternate species, i.e. salmon ss-DNA on herring ss-DNA or herring ss-DNA on salmon ss-DNA, yielded much smaller changes in thickness of 3-5 nm. AFM studies of the surface roughness of adsorbed layers were in line with the TIRE data.

Links between DNA methylation and nucleosome occupancy in the human genome.

Science.gov (United States)

Collings, Clayton K; Anderson, John N

2017-01-01

DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

Direct extraction of genomic DNA from maize with aqueous ionic liquid buffer systems for applications in genetically modified organisms analysis.

Science.gov (United States)

Gonzalez García, Eric; Ressmann, Anna K; Gaertner, Peter; Zirbs, Ronald; Mach, Robert L; Krska, Rudolf; Bica, Katharina; Brunner, Kurt

2014-12-01

To date, the extraction of genomic DNA is considered a bottleneck in the process of genetically modified organisms (GMOs) detection. Conventional DNA isolation methods are associated with long extraction times and multiple pipetting and centrifugation steps, which makes the entire procedure not only tedious and complicated but also prone to sample cross-contamination. In recent times, ionic liquids have emerged as innovative solvents for biomass processing, due to their outstanding properties for dissolution of biomass and biopolymers. In this study, a novel, easily applicable, and time-efficient method for the direct extraction of genomic DNA from biomass based on aqueous-ionic liquid solutions was developed. The straightforward protocol relies on extraction of maize in a 10 % solution of ionic liquids in aqueous phosphate buffer for 5 min at room temperature, followed by a denaturation step at 95 °C for 10 min and a simple filtration to remove residual biopolymers. A set of 22 ionic liquids was tested in a buffer system and 1-ethyl-3-methylimidazolium dimethylphosphate, as well as the environmentally benign choline formate, were identified as ideal candidates. With this strategy, the quality of the genomic DNA extracted was significantly improved and the extraction protocol was notably simplified compared with a well-established method.

A Portrait of Ribosomal DNA Contacts with Hi-C Reveals 5S and 45S rDNA Anchoring Points in the Folded Human Genome.

Science.gov (United States)

Yu, Shoukai; Lemos, Bernardo

2016-12-31

Ribosomal RNAs (rRNAs) account for >60% of all RNAs in eukaryotic cells and are encoded in the ribosomal DNA (rDNA) arrays. The rRNAs are produced from two sets of loci: the 5S rDNA array resides exclusively on human chromosome 1, whereas the 45S rDNA array resides on the short arm of five human acrocentric chromosomes. The 45S rDNA gives origin to the nucleolus, the nuclear organelle that is the site of ribosome biogenesis. Intriguingly, 5S and 45S rDNA arrays exhibit correlated copy number variation in lymphoblastoid cells (LCLs). Here we examined the genomic architecture and repeat content of the 5S and 45S rDNA arrays in multiple human genome assemblies (including PacBio MHAP assembly) and ascertained contacts between the rDNA arrays and the rest of the genome using Hi-C datasets from two human cell lines (erythroleukemia K562 and lymphoblastoid cells). Our analyses revealed that 5S and 45S arrays each have thousands of contacts in the folded genome, with rDNA-associated regions and genes dispersed across all chromosomes. The rDNA contact map displayed conserved and disparate features between two cell lines, and pointed to specific chromosomes, genomic regions, and genes with evidence of spatial proximity to the rDNA arrays; the data also showed a lack of direct physical interaction between the 5S and 45S rDNA arrays. Finally, the analysis identified an intriguing organization in the 5S array with Alu and 5S elements adjacent to one another and organized in opposite orientation along the array. Portraits of genome folding centered on the ribosomal DNA array could help understand the emergence of concerted variation, the control of 5S and 45S expression, as well as provide insights into an organelle that contributes to the spatial localization of human chromosomes during interphase. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

Science.gov (United States)

Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

2017-05-15

5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

Directory of Open Access Journals (Sweden)

Farkhondeh Saba

2017-01-01

Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

Directory of Open Access Journals (Sweden)

Farkhondeh Saba

2016-09-01

Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

Genomic footprinting in mammalian cells with ultraviolet light

International Nuclear Information System (INIS)

Becker, M.M.; Wang, Z.; Grossmann, G.; Becherer, K.A.

1989-01-01

A simple and accurate genomic primer extension method has been developed to detect ultraviolet footprinting patterns of regulatory protein-DNA interactions in mammalian genomic DNA. The technique can also detect footprinting or sequencing patterns introduced into genomic DNA by other methods. Purified genomic DNA, containing either damaged bases or strand breaks introduced by footprinting or sequencing reactions, is first cut with a convenient restriction enzyme to reduce its molecular weight. A highly radioactive single-stranded DNA primer that is complementary to a region of genomic DNA whose sequence or footprint one wishes to examine is then mixed with 50 micrograms of restriction enzyme-cut genomic DNA. The primer is approximately 100 bases long and contains 85 radioactive phosphates, each of specific activity 3000 Ci/mmol (1 Ci = 37 GBq). A simple and fast method for preparing such primers is described. Following brief heat denaturation at 100 degrees C, the solution of genomic DNA and primer is cooled to 74 degrees C and a second solution containing Taq polymerase (Thermus aquaticus DNA polymerase) and the four deoxynucleotide triphosphates is added to initiate primer extension of genomic DNA. Taq polymerase extends genomic hybridized primer until its polymerization reaction is terminated either by a damaged base or strand break in genomic DNA or by the addition of dideoxynucleotide triphosphates in the polymerization reaction. The concurrent primer hybridization-extension reaction is terminated after 5 hr and unhybridized primer is digested away by mung bean nuclease. Primer-extended genomic DNA is then denatured and electrophoresed on a polyacrylamide sequencing gel, and radioactive primer extension products are revealed by autoradiography

A quantitative comparison of single-cell whole genome amplification methods.

Directory of Open Access Journals (Sweden)

Charles F A de Bourcy

Full Text Available Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA, Multiple Annealing and Looping Based Amplification Cycles (MALBAC, and the PicoPLEX single-cell WGA kit (NEB-WGA. We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

A DNA-based pattern classifier with in vitro learning and associative recall for genomic characterization and biosensing without explicit sequence knowledge.

Science.gov (United States)

Lee, Ju Seok; Chen, Junghuei; Deaton, Russell; Kim, Jin-Woo

2014-01-01

Genetic material extracted from in situ microbial communities has high promise as an indicator of biological system status. However, the challenge is to access genomic information from all organisms at the population or community scale to monitor the biosystem's state. Hence, there is a need for a better diagnostic tool that provides a holistic view of a biosystem's genomic status. Here, we introduce an in vitro methodology for genomic pattern classification of biological samples that taps large amounts of genetic information from all genes present and uses that information to detect changes in genomic patterns and classify them. We developed a biosensing protocol, termed Biological Memory, that has in vitro computational capabilities to "learn" and "store" genomic sequence information directly from genomic samples without knowledge of their explicit sequences, and that discovers differences in vitro between previously unknown inputs and learned memory molecules. The Memory protocol was designed and optimized based upon (1) common in vitro recombinant DNA operations using 20-base random probes, including polymerization, nuclease digestion, and magnetic bead separation, to capture a snapshot of the genomic state of a biological sample as a DNA memory and (2) the thermal stability of DNA duplexes between new input and the memory to detect similarities and differences. For efficient read out, a microarray was used as an output method. When the microarray-based Memory protocol was implemented to test its capability and sensitivity using genomic DNA from two model bacterial strains, i.e., Escherichia coli K12 and Bacillus subtilis, results indicate that the Memory protocol can "learn" input DNA, "recall" similar DNA, differentiate between dissimilar DNA, and detect relatively small concentration differences in samples. This study demonstrated not only the in vitro information processing capabilities of DNA, but also its promise as a genomic pattern classifier that could

Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

International Nuclear Information System (INIS)

Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

2005-01-01

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [ 14 C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [ 14 C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 (micro)g of DNA. This sensitivity may allow the method to be used for clinical applications

Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

Energy Technology Data Exchange (ETDEWEB)

Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

2005-12-29

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

Science.gov (United States)

Duewer, David L; Kline, Margaret C; Romsos, Erica L; Toman, Blaza

2018-05-01

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Inverse PCR-based method for isolating novel SINEs from genome.

Science.gov (United States)

Han, Yawei; Chen, Liping; Guan, Lihong; He, Shunping

2014-04-01

Short interspersed elements (SINEs) are moderately repetitive DNA sequences in eukaryotic genomes. Although eukaryotic genomes contain numerous SINEs copy, it is very difficult and laborious to isolate and identify them by the reported methods. In this study, the inverse PCR was successfully applied to isolate SINEs from Opsariichthys bidens genome in Eastern Asian Cyprinid. A group of SINEs derived from tRNA(Ala) molecular had been identified, which were named Opsar according to Opsariichthys. SINEs characteristics were exhibited in Opsar, which contained a tRNA(Ala)-derived region at the 5' end, a tRNA-unrelated region, and AT-rich region at the 3' end. The tRNA-derived region of Opsar shared 76 % sequence similarity with tRNA(Ala) gene. This result indicated that Opsar could derive from the inactive or pseudogene of tRNA(Ala). The reliability of method was tested by obtaining C-SINE, Ct-SINE, and M-SINEs from Ctenopharyngodon idellus, Megalobrama amblycephala, and Cyprinus carpio genomes. This method is simpler than the previously reported, which successfully omitted many steps, such as preparation of probes, construction of genomic libraries, and hybridization.

Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

Science.gov (United States)

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2016-01-01

Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen

Directory of Open Access Journals (Sweden)

Gilda ESLAMI

2017-06-01

Full Text Available Background: One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata.Methods: The larvae were collected from the sheep’s visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI, and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively.Results: The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same.Conclusion: Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common.

Evolutionary analyses of entire genomes do not support the association of mtDNA mutations with Ras/MAPK pathway syndromes.

Directory of Open Access Journals (Sweden)

Alberto Gómez-Carballa

Full Text Available BACKGROUND: There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS and related disorders (such as LEOPARD, neurofibromatosis type 1, although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM, which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45, most of them classified as NS patients (n = 42. METHODS/PRINCIPAL FINDINGS: The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg patterns of a typical Iberian dataset (including hgs H, T, J, and U. Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5 are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. CONCLUSIONS/SIGNIFICANCE: As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS.

Evolutionary Analyses of Entire Genomes Do Not Support the Association of mtDNA Mutations with Ras/MAPK Pathway Syndromes

Science.gov (United States)

Cerezo, María; Balboa, Emilia; Heredia, Claudia; Castro-Feijóo, Lidia; Rica, Itxaso; Barreiro, Jesús; Eirís, Jesús; Cabanas, Paloma; Martínez-Soto, Isabel; Fernández-Toral, Joaquín; Castro-Gago, Manuel; Pombo, Manuel; Carracedo, Ángel; Barros, Francisco

2011-01-01

Background There are several known autosomal genes responsible for Ras/MAPK pathway syndromes, including Noonan syndrome (NS) and related disorders (such as LEOPARD, neurofibromatosis type 1), although mutations of these genes do not explain all cases. Due to the important role played by the mitochondrion in the energetic metabolism of cardiac muscle, it was recently proposed that variation in the mitochondrial DNA (mtDNA) genome could be a risk factor in the Noonan phenotype and in hypertrophic cardiomyopathy (HCM), which is a common clinical feature in Ras/MAPK pathway syndromes. In order to test these hypotheses, we sequenced entire mtDNA genomes in the largest series of patients suffering from Ras/MAPK pathway syndromes analyzed to date (n = 45), most of them classified as NS patients (n = 42). Methods/Principal Findings The results indicate that the observed mtDNA lineages were mostly of European ancestry, reproducing in a nutshell the expected haplogroup (hg) patterns of a typical Iberian dataset (including hgs H, T, J, and U). Three new branches of the mtDNA phylogeny (H1j1, U5b1e, and L2a5) are described for the first time, but none of these are likely to be related to NS or Ras/MAPK pathway syndromes when observed under an evolutionary perspective. Patterns of variation in tRNA and protein genes, as well as redundant, private and heteroplasmic variants, in the mtDNA genomes of patients were as expected when compared with the patterns inferred from a worldwide mtDNA phylogeny based on more than 8700 entire genomes. Moreover, most of the mtDNA variants found in patients had already been reported in healthy individuals and constitute common polymorphisms in human population groups. Conclusions/Significance As a whole, the observed mtDNA genome variation in the NS patients was difficult to reconcile with previous findings that indicated a pathogenic role of mtDNA variants in NS. PMID:21526175

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

Science.gov (United States)

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

Role of oxidative DNA damage in genome instability and cancer

International Nuclear Information System (INIS)

Bignami, M.; Kunkel, T.

2009-01-01

Inactivation of mismatch repair (MMR) is associated with a dramatic genomic instability that is observed experimentally as a mutator phenotype and micro satellite instability (MSI). It has been implicit that the massive genetic instability in MMR defective cells simply reflects the accumulation of spontaneous DNA polymerase errors during DNA replication. We recently identified oxidation damage, a common threat to DNA integrity to which purines are very susceptible, as an important cofactor in this genetic instability

To beat or not to beat a tick: comparison of DNA extraction methods for ticks (Ixodes scapularis

Directory of Open Access Journals (Sweden)

Alyssa D. Ammazzalorso

2015-08-01

Full Text Available Background. Blacklegged ticks (Ixodes scapularis are important disease vectors in the United States, known to transmit a variety of pathogens to humans, including bacteria, protozoa, and viruses. Their importance as a disease vector necessitates reliable and comparable methods for extracting microbial DNA from ticks. Furthermore, to explore the population genetics or genomics of this tick, appropriate DNA extraction techniques are needed for both the vector and its microbes. Although a few studies have investigated different methods of DNA isolation from ticks, they are limited in the number and types of DNA extraction and lack species-specific quantification of DNA yield.Methods. Here we determined the most efficient and consistent method of DNA extraction from two different developmental stages of I. scapularis—nymph and adult—that are the most important for disease transmission. We used various methods of physical disruption of the hard, chitinous exoskeleton, as well as commercial and non-commercial DNA isolation kits. To gauge the effectiveness of these methods, we quantified the DNA yield and confirmed the DNA quality via PCR of both tick and microbial genetic material.Results. DNA extraction using the Thermo GeneJET Genomic DNA Purification Kit resulted in the highest DNA yields and the most consistent PCR amplification when combined with either cutting or bead beating with select matrices across life stages. DNA isolation methods using ammonium hydroxide as well as the MoBio PowerSoil kit also produced strong and successful PCR amplification, but only for females.Discussion. We contrasted a variety of readily available methods of DNA extraction from single individual blacklegged ticks and presented the results through a quantitative and qualitative assessment.

Evaluation and comparison of FTA card and CTAB DNA extraction methods for non-agricultural taxa 1

OpenAIRE

Siegel, Chloe S.; Stevenson, Florence O.; Zimmer, Elizabeth A.

2017-01-01

Premise of the study: An efficient, effective DNA extraction method is necessary for comprehensive analysis of plant genomes. This study analyzed the quality of DNA obtained using paper FTA cards prepared directly in the field when compared to the more traditional cetyltrimethylammonium bromide (CTAB)?based extraction methods from silica-dried samples. Methods: DNA was extracted using FTA cards according to the manufacturer?s protocol. In parallel, CTAB-based extractions were done using the a...

Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

Directory of Open Access Journals (Sweden)

Carr Steven M

2007-09-01

Full Text Available Abstract Background Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by

Dragon polya spotter: Predictor of poly(A) motifs within human genomic DNA sequences

KAUST Repository

Kalkatawi, Manal M.

2011-11-15

Motivation: Recognition of poly(A) signals in mRNA is relatively straightforward due to the presence of easily recognizable polyadenylic acid tail. However, the task of identifying poly(A) motifs in the primary genomic DNA sequence that correspond to poly(A) signals in mRNA is a far more challenging problem. Recognition of poly(A) signals is important for better gene annotation and understanding of the gene regulation mechanisms. In this work, we present one such poly(A) motif prediction method based on properties of human genomic DNA sequence surrounding a poly(A) motif. These properties include thermodynamic, physico-chemical and statistical characteristics. For predictions, we developed Artificial Neural Network and Random Forest models. These models are trained to recognize 12 most common poly(A) motifs in human DNA. Our predictors are available as a free web-based tool accessible at http://cbrc.kaust.edu.sa/dps. Compared with other reported predictors, our models achieve higher sensitivity and specificity and furthermore provide a consistent level of accuracy for 12 poly(A) motif variants. The Author(s) 2011. Published by Oxford University Press. All rights reserved.

Genome-wide survey of repetitive DNA elements in the button mushroom Agaricus bisporus

NARCIS (Netherlands)

Foulongne-Oriol, M.; Murat, C.; Castanera, R.; Ramírez, L.; Sonnenberg, A.S.M.

2013-01-01

Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Science.gov (United States)

Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

2013-01-01

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Wyrobek, Andrew J.

2009-01-18

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization. During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.

Crucial optimization steps in getting premier quality of Aquilaria malaccensis genomic DNA for molecular activities

International Nuclear Information System (INIS)

Muhammad Hanif Azhari; Azhar Mohamad

2013-01-01

Gaharu resin is derived from Aquilaria sp. or Agar wood tree in a tropical ecosystem. In Malaysia the Aquilaria species especially A. malaccensis in danger of extinction in the wild due to illegal logging as its resin is highly used for the production of greatly valued incense throughout Asia. A significant tool in fingerprinting the species is through molecular activities of Polymerase Chain Reaction (PCR) application on which requires total genomic DNA as a starting material. This paper, described optimizations of both fresh and dried samples derived from A. malaccensis for genomic DNA. Three main parameters for the optimization were temperature (60, 65, and 70 degree Celsius), incubation period (30, 60, 90 minutes) and concentration of CTAB (1 %, 3 %, 5 %). The experimental design in these work resulted a total of 46 combinations of the parameters in which 0.5 g samples was used in each combination. Nano-drop spectrometer was used in detecting the quantitative genomic DNA at ng/ μl. In fresh samples, incubation temperatures at 65 degree Celsius for 60 minutes in 3 % were yielded 723.2 ng/ μl genomic DNA. Whereas, for dried samples, incubation temperature at 70 degree Celsius for 90 minutes in 5 % CTAB were yielded 70.2 ng/ μl of genomic DNA. Spectrometer reading at OD280/ 260 was 1.9 for both type of samples. The isolated genomic DNA is useful for the molecular activities to identify specific plants between the same species or among the Aquilaria species. (author)

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

Science.gov (United States)

Li, Chunmei; Yu, Zhilong; Fu, Yusi; Pang, Yuhong; Huang, Yanyi

2017-04-26

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

Science.gov (United States)

Tycko, Josh; Myer, Vic E.; Hsu, Patrick D.

2016-01-01

Summary Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

Science.gov (United States)

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

Directory of Open Access Journals (Sweden)

Karolina Chwialkowska

2017-11-01

Full Text Available Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq. We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation

G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

John A Capra

2010-07-01

Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

Biased distribution of DNA uptake sequences towards genome maintenance genes

DEFF Research Database (Denmark)

Davidsen, T.; Rodland, E.A.; Lagesen, K.

2004-01-01

Repeated sequence signatures are characteristic features of all genomic DNA. We have made a rigorous search for repeat genomic sequences in the human pathogens Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae and found that by far the most frequent 9-10mers residing within...... in these organisms. Pasteurella multocida also displayed high frequencies of a putative DUS identical to that previously identified in H. influenzae and with a skewed distribution towards genome maintenance genes, indicating that this bacterium might be transformation competent under certain conditions....

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

Science.gov (United States)

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

Ribosomal DNA sequence heterogeneity reflects intraspecies phylogenies and predicts genome structure in two contrasting yeast species.

Science.gov (United States)

West, Claire; James, Stephen A; Davey, Robert P; Dicks, Jo; Roberts, Ian N

2014-07-01

The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

Science.gov (United States)

Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

2002-01-01

DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

Optimization of conditions to extract high quality DNA for PCR analysis from whole blood using SDS-proteinase K method

Directory of Open Access Journals (Sweden)

Wajhul Qamar

2017-11-01

Full Text Available In case of studies associated with human genetics, genomics, and pharmacogenetics the genomic DNA is extracted from the buccal cells, whole blood etc. Several methods are exploited by the researchers to extract DNA from the whole blood. One of these methods, which utilizes cell lysis and proteolytic properties of sodium dodecyl sulfate (SDS and proteinase K respectively, might also be called SDS-PK method. It does not include any hazardous chemicals such as phenol or chloroform and is inexpensive. However, several researchers report the same method with different formulas and conditions. During our experiments with whole blood DNA extraction we experienced problems such as protein contamination, DNA purity and yield when followed some SDS-PK protocols reported elsewhere. A260/A280 and A260/A230 ratios along with PCR amplification give a clear idea about the procedure that was followed to extract the DNA. In an effort to increase the DNA purity from human whole blood, we pointed out some steps of the protocol that play a crucial role in determining the extraction of high quality DNA.

An improved method for genomic DNA extraction from strawberry leaves Otimização de um método para extração de DNA genômico a partir de folhas de morangueiro

Directory of Open Access Journals (Sweden)

Claudinéia Ferreira Nunes

2011-08-01

Full Text Available Several extraction methods of genomic DNA for identification and characterization of genetic diversity in different plant species are routinely applied during molecular analysis. However, the presence of undesirable compounds such as polyphenols and polysaccharides is one of the biggest problems faced during the isolation and purification of high quality DNA in plants. Therefore, achievement of fast and accurate methods for DNA extraction is crucial in order to produce pure samples. Leaves of strawberry genotypes (Fragaria ananassa have high contents of polysaccharides and polyphenols which increase the sample viscosity and decrease the DNA quality, interfering with the PCR performance. Thereby, in this study we evaluated the quality and amount of genomic DNA extracted from young leaves of strawberry after tissue lyophilization and maceration in presence of polivinilpirrolidone (PVP. The CTAB method was used as reference procedure and it was modified to improve the DNA extraction. The modifications consisted of tissue lyophilization overnight until it was completely freeze-dried and addition of PVP during the tissue maceration in liquid nitrogen. The results showed the efficiency and reliability of the modified method compared to the unmodified method, indicating that combination of lyophilization and PVP improve the quality and amount of the DNA extracted from strawberry leaves.Vários métodos de extração de DNA genômico para a identificação e caracterização da diversidade genética em diferentes espécies de plantas são rotineiramente aplicados durante a análise molecular. Entretanto, a presença de compostos indesejáveis, tais como polifenóis e polissacarídeos, é um dos maiores problemas que ocorrem durante o isolamento e purificação de DNA de alta qualidade em plantas. Dessa forma, o sucesso no desenvolvimento de métodos de extração de DNA rápidos e acurados é crucial para produzir amostras puras. Folhas de genótipos de

Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

Science.gov (United States)

Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

2017-01-01

The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae.

Science.gov (United States)

Macas, Jiří; Novák, Petr; Pellicer, Jaume; Čížková, Jana; Koblížková, Andrea; Neumann, Pavel; Fuková, Iva; Doležel, Jaroslav; Kelly, Laura J; Leitch, Ilia J

2015-01-01

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae.

Directory of Open Access Journals (Sweden)

Jiří Macas

Full Text Available The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55-83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57% of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%. Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.

Novel DNA sequence detection method based on fluorescence energy transfer

International Nuclear Information System (INIS)

Kobayashi, S.; Tamiya, E.; Karube, I.

1987-01-01

Recently the detection of specific DNA sequence, DNA analysis, has been becoming more important for diagnosis of viral genomes causing infections disease and human sequences related to inherited disorders. These methods typically involve electrophoresis, the immobilization of DNA on a solid support, hybridization to a complementary probe, the detection using labeled with /sup 32/P or nonisotopically with a biotin-avidin-enzyme system, and so on. These techniques are highly effective, but they are very time-consuming and expensive. A principle of fluorescene energy transfer is that the light energy from an excited donor (fluorophore) is transferred to an acceptor (fluorophore), if the acceptor exists in the vicinity of the donor and the excitation spectrum of donor overlaps the emission spectrum of acceptor. In this study, the fluorescence energy transfer was applied to the detection of specific DNA sequence using the hybridization method. The analyte, single-stranded DNA labeled with the donor fluorophore is hybridized to a probe DNA labeled with the acceptor. Because of the complementary DNA duplex formation, two fluorophores became to be closed to each other, and the fluorescence energy transfer was occurred

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

Energy Technology Data Exchange (ETDEWEB)

Peng, Jamy C. [Univ. of California, Berkeley, CA (United States)

2007-01-01

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) that binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in

Selection of the most suitable method for the extraction of DNA from foods and feeds for species identification

Directory of Open Access Journals (Sweden)

Michaela Nesvadbová

2010-01-01

Full Text Available High quality and purity of DNA isolated from food and feed is essential for species identification and has unpredictable influences an effect of analysis. In this study, the efficiency of eight different methods for DNA isolation was investigated. For DNA extraction, the raw chicken meet, ham, sausages, tinned lunch meat, pate, tinned feeds for dogs, complete granulated feeds for dogs and chicken flour were used. Kits of several different producers, i.e.: NucleoSpin Food (Marchery-Nagel, Wizard Genomic DNA Purification Kit (Promega, Invisorb Spin Food Kit I (Invitek, Wizard SV Genomic DNA Purification System (Promega, JetQuick Tissue DNA Spin Kit (Genomed, RNA Blue (Top-Bio, JetQuick Blood & Cell Culture Kit (Genomed, QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit (Qiagen were employed in the study. Gel agarose electrophoresis for primary verification of DNA quality was performed. The isolates were subsequently assessed for quantity and quality using by spectrophotometer Nanodrop 2000 (Thermo Scientific. To verify of template usability and quality of isolated DNA, the polymerase chain reaction (PCR was used.Differences between isolated DNA from tinned products and meat, ham, sausage, granulated dog feed and chicken flour were found. In tinned food and feed, the DNA was more degraded, DNA content and DNA purity was lower and also PCR amplification was the most difficult. Overall DNA yield and quality have important influence on PCR products amplification. The best results were obtained with NucleoSpin Food and JetQuick Tissue DNA Spin Kit. DNA extracted by these methods proved highest yields, purity and template quality in all foods and feeds and the results of PCR analysis are excellent reproducible. Analyses showed that results depended on different food or feed using and dif­fe­rent isolation system.The results of this work will be utilized to choose the suitable isolating kit for educational course, which is designed for students and

Genomic comparisons of Brucella spp. and closely related bacteria using base compositional and proteome based methods

DEFF Research Database (Denmark)

Bohlin, Jon; Snipen, Lars; Cloeckaert, Axel

2010-01-01

BACKGROUND: Classification of bacteria within the genus Brucella has been difficult due in part to considerable genomic homogeneity between the different species and biovars, in spite of clear differences in phenotypes. Therefore, many different methods have been used to assess Brucella taxonomy....... In the current work, we examine 32 sequenced genomes from genus Brucella representing the six classical species, as well as more recently described species, using bioinformatical methods. Comparisons were made at the level of genomic DNA using oligonucleotide based methods (Markov chain based genomic signatures...... between the oligonucleotide based methods used. Whilst the Markov chain based genomic signatures grouped the different species in genus Brucella according to host preference, the codon and amino acid frequencies based methods reflected small differences between the Brucella species. Only minor differences...

Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes

Directory of Open Access Journals (Sweden)

Christina L. Zheng

2014-11-01

Full Text Available Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs arising in an XPC−/− background. XPC−/− cells lack global genome nucleotide excision repair (GG-NER, thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

Detection of genomic variation by selection of a 9 mb DNA region and high throughput sequencing.

Directory of Open Access Journals (Sweden)

Sergey I Nikolaev

Full Text Available Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb and 7 (1.1 Mb from an individual from the International HapMap Project (NA12872. We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage > or = 4-fold, and 97.9% concordant in regions with coverage > or = 15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

Genomic DNA-based absolute quantification of gene expression in Vitis.

Science.gov (United States)

Gambetta, Gregory A; McElrone, Andrew J; Matthews, Mark A

2013-07-01

Many studies in which gene expression is quantified by polymerase chain reaction represent the expression of a gene of interest (GOI) relative to that of a reference gene (RG). Relative expression is founded on the assumptions that RG expression is stable across samples, treatments, organs, etc., and that reaction efficiencies of the GOI and RG are equal; assumptions which are often faulty. The true variability in RG expression and actual reaction efficiencies are seldom determined experimentally. Here we present a rapid and robust method for absolute quantification of expression in Vitis where varying concentrations of genomic DNA were used to construct GOI standard curves. This methodology was utilized to absolutely quantify and determine the variability of the previously validated RG ubiquitin (VvUbi) across three test studies in three different tissues (roots, leaves and berries). In addition, in each study a GOI was absolutely quantified. Data sets resulting from relative and absolute methods of quantification were compared and the differences were striking. VvUbi expression was significantly different in magnitude between test studies and variable among individual samples. Absolute quantification consistently reduced the coefficients of variation of the GOIs by more than half, often resulting in differences in statistical significance and in some cases even changing the fundamental nature of the result. Utilizing genomic DNA-based absolute quantification is fast and efficient. Through eliminating error introduced by assuming RG stability and equal reaction efficiencies between the RG and GOI this methodology produces less variation, increased accuracy and greater statistical power. © 2012 Scandinavian Plant Physiology Society.

Download - PGDBj Registered plant list, Marker list, QTL list, Plant DB link & Genome analysis methods | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available List Contact us PGDBj Registered plant list, Marker list, QTL list, Plant DB link & Genome analysis methods ...t_db_link_en.zip (36.3 KB) - 6 Genome analysis methods pgdbj_dna_marker_linkage_map_genome_analysis_methods_... of This Database Site Policy | Contact Us Download - PGDBj Registered plant list, Marker list, QTL list, Plant DB link & Genome analysis methods | LSDB Archive ...

AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

Science.gov (United States)

Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

2006-05-01

The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability.

The Statistical Segment Length of DNA: Opportunities for Biomechanical Modeling in Polymer Physics and Next-Generation Genomics.

Science.gov (United States)

Dorfman, Kevin D

2018-02-01

The development of bright bisintercalating dyes for deoxyribonucleic acid (DNA) in the 1990s, most notably YOYO-1, revolutionized the field of polymer physics in the ensuing years. These dyes, in conjunction with modern molecular biology techniques, permit the facile observation of polymer dynamics via fluorescence microscopy and thus direct tests of different theories of polymer dynamics. At the same time, they have played a key role in advancing an emerging next-generation method known as genome mapping in nanochannels. The effect of intercalation on the bending energy of DNA as embodied by a change in its statistical segment length (or, alternatively, its persistence length) has been the subject of significant controversy. The precise value of the statistical segment length is critical for the proper interpretation of polymer physics experiments and controls the phenomena underlying the aforementioned genomics technology. In this perspective, we briefly review the model of DNA as a wormlike chain and a trio of methods (light scattering, optical or magnetic tweezers, and atomic force microscopy (AFM)) that have been used to determine the statistical segment length of DNA. We then outline the disagreement in the literature over the role of bisintercalation on the bending energy of DNA, and how a multiscale biomechanical approach could provide an important model for this scientifically and technologically relevant problem.

Genome-wide DNA methylation profiling in cultured eutopic and ectopic endometrial stromal cells.

Directory of Open Access Journals (Sweden)

Yoshiaki Yamagata

Full Text Available The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa and without endometriosis (euESCb and ovarian endometrial cysts (choESC. Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2, which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.

The structures of bovine herpesvirus 1 virion and concatemeric DNA: implications for cleavage and packaging of herpesvirus genomes

International Nuclear Information System (INIS)

Schynts, Frederic; McVoy, Michael A.; Meurens, Francois; Detry, Bruno; Epstein, Alberto L.; Thiry, Etienne

2003-01-01

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I L ) segments have been reported for some class D herpesviruses. Inefficient formation of class D I L genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I L genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I L orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I L genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur

Gross genomic damage measured by DNA image cytometry independently predicts gastric cancer patient survival

NARCIS (Netherlands)

Belien, J.A.M.; Buffart, T.E.; Gill, A.; Broeckaert, M.A.M.; Quirke, P.; Meijer, G.A.; Grabsch, H.

2009-01-01

BACKGROUND: DNA aneuploidy reflects gross genomic changes. It can be measured by flow cytometry (FCM-DNA) or image cytometry (ICM-DNA). In gastric cancer, the prevalence of DNA aneuploidy has been reported to range from 27 to 100%, with conflicting associations with clinicopathological variables.

Human β satellite DNA: Genomic organization and sequence definition of a class of highly repetitive tandem DNA

International Nuclear Information System (INIS)

Waye, J.S.; Willard, H.F.

1989-01-01

The authors describe a class of human repetitive DNA, called β satellite, that, at a most fundamental level, exists as tandem arrays of diverged ∼68-base-pair monomer repeat units. The monomer units are organized as distinct subsets, each characterized by a multimeric higher-order repeat unit that is tandemly reiterated and represents a recent unit of amplification. They have cloned, characterized, and determined the sequence of two β satellite higher-order repeat units: one located on chromosome 9, the other on the acrocentric chromosomes (13, 14, 15, 21, and 22) and perhaps other sites in the genome. Analysis by pulsed-field gel electrophoresis reveals that these tandem arrays are localized in large domains that are marked by restriction fragment length polymorphisms. In total, β-satellite sequences comprise several million base pairs of DNA in the human genome. Analysis of this DNA family should permit insights into the nature of chromosome-specific and nonspecific modes of satellite DNA evolution and provide useful tools for probing the molecular organization and concerted evolution of the acrocentric chromosomes

A simple, rapid and efficient method for the extraction of genomic ...

African Journals Online (AJOL)

The isolation of intact, high-molecular-mass genomic DNA is essential for many molecular biology applications including long range PCR, endonuclease restriction digestion, southern blot analysis, and genomic library construction. Many protocols are available for the extraction of DNA from plant material, but obtain it is ...

A Network of Multi-Tasking Proteins at the DNA Replication Fork Preserves Genome Stability.

Directory of Open Access Journals (Sweden)

2005-12-01

Full Text Available To elucidate the network that maintains high fidelity genome replication, we have introduced two conditional mutant alleles of DNA2, an essential DNA replication gene, into each of the approximately 4,700 viable yeast deletion mutants and determined the fitness of the double mutants. Fifty-six DNA2-interacting genes were identified. Clustering analysis of genomic synthetic lethality profiles of each of 43 of the DNA2-interacting genes defines a network (consisting of 322 genes and 876 interactions whose topology provides clues as to how replication proteins coordinate regulation and repair to protect genome integrity. The results also shed new light on the functions of the query gene DNA2, which, despite many years of study, remain controversial, especially its proposed role in Okazaki fragment processing and the nature of its in vivo substrates. Because of the multifunctional nature of virtually all proteins at the replication fork, the meaning of any single genetic interaction is inherently ambiguous. The multiplexing nature of the current studies, however, combined with follow-up supporting experiments, reveals most if not all of the unique pathways requiring Dna2p. These include not only Okazaki fragment processing and DNA repair but also chromatin dynamics.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

Science.gov (United States)

Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.

Science.gov (United States)

Shin, Jongoh; Lee, Namil; Cho, Suhyung; Cho, Byung-Kwan

2018-01-01

Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.

Evaluation of five methods for total DNA extraction from western corn rootworm beetles.

Directory of Open Access Journals (Sweden)

Hong Chen

Full Text Available BACKGROUND: DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction. METHODOLOGY/PRINCIPAL FINDINGS: From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol reagent, Puregene solutions and DNeasy column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue at much lower cost and less degradation as revealed on agarose gels. The DNeasy kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle's genome, and all samples showed successful amplifications. CONCLUSION/SIGNIFICANCE: These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.

Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, J.C.; Stevsner, Tinna; Bohr, Vilhelm A. (National Cancer Institute, NIH, Bethesda, MD (USA). Division of Cancer Treatment, Laboratory of Molecular Pharmacology); Mattern, M.R. (Smith Kline Beecham Pharmaceuticals, King of Prussia, PA (USA). Department of Biomolecular Discovery)

1991-09-01

The effects were studied of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. The inhibitors were tested of DNA poly-merase {alpha} and {delta} (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topo-isomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, the effects were tested of the potential topoisomerase I activator, {beta}-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; {beta}-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair. (author). 36 refs.; 3 figs.; 2 tabs.

Extensive variation in the density and distribution of DNA polymorphism in sorghum genomes.

Directory of Open Access Journals (Sweden)

Joseph Evans

Full Text Available Sorghum genotypes currently used for grain production in the United States were developed from African landraces that were imported starting in the mid-to-late 19(th century. Farmers and plant breeders selected genotypes for grain production with reduced plant height, early flowering, increased grain yield, adaptation to drought, and improved resistance to lodging, diseases and pests. DNA polymorphisms that distinguish three historically important grain sorghum genotypes, BTx623, BTx642 and Tx7000, were characterized by genome sequencing, genotyping by sequencing, genetic mapping, and pedigree-based haplotype analysis. The distribution and density of DNA polymorphisms in the sequenced genomes varied widely, in part because the lines were derived through breeding and selection from diverse Kafir, Durra, and Caudatum race accessions. Genomic DNA spanning dw1 (SBI-09 and dw3 (SBI-07 had identical haplotypes due to selection for reduced height. Lower SNP density in genes located in pericentromeric regions compared with genes located in euchromatic regions is consistent with background selection in these regions of low recombination. SNP density was higher in euchromatic DNA and varied >100-fold in contiguous intervals that spanned up to 300 Kbp. The localized variation in DNA polymorphism density occurred throughout euchromatic regions where recombination is elevated, however, polymorphism density was not correlated with gene density or DNA methylation. Overall, sorghum chromosomes contain distal euchromatic regions characterized by extensive, localized variation in DNA polymorphism density, and large pericentromeric regions of low gene density, diversity, and recombination.

Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

Science.gov (United States)

Lee, Patrick K H; Men, Yujie; Wang, Shanquan; He, Jianzhong; Alvarez-Cohen, Lisa

2015-02-03

Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

Genome-Wide Expression of MicroRNAs Is Regulated by DNA Methylation in Hepatocarcinogenesis

Directory of Open Access Journals (Sweden)

Jing Shen

2015-01-01

Full Text Available Background. Previous studies, including ours, have examined the regulation of microRNAs (miRNAs by DNA methylation, but whether this regulation occurs at a genome-wide level in hepatocellular carcinoma (HCC is unclear. Subjects/Methods. Using a two-phase study design, we conducted genome-wide screening for DNA methylation and miRNA expression to explore the potential role of methylation alterations in miRNAs regulation. Results. We found that expressions of 25 miRNAs were statistically significantly different between tumor and nontumor tissues and perfectly differentiated HCC tumor from nontumor. Six miRNAs were overexpressed, and 19 were repressed in tumors. Among 133 miRNAs with inverse correlations between methylation and expression, 8 miRNAs (6% showed statistically significant differences in expression between tumor and nontumor tissues. Six miRNAs were validated in 56 additional paired HCC tissues, and significant inverse correlations were observed for miR-125b and miR-199a, which is consistent with the inactive chromatin pattern found in HepG2 cells. Conclusion. These data suggest that the expressions of miR-125b and miR-199a are dramatically regulated by DNA hypermethylation that plays a key role in hepatocarcinogenesis.

Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

Science.gov (United States)

Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

2016-04-07

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. Copyright © 2016 Vrljicak et al.

Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health.

Science.gov (United States)

Hazkani-Covo, Einat; Martin, William F

2017-05-01

Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

"Isogaba Maware": quality control of genome DNA by checkpoints.

Science.gov (United States)

Kitazono, A; Matsumoto, T

1998-05-01

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.

Science.gov (United States)

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.

Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome.

Science.gov (United States)

Robicheau, Brent M; Susko, Edward; Harrigan, Amye M; Snyder, Marlene

2017-02-01

Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

Directory of Open Access Journals (Sweden)

Bekim Sadikovic

Full Text Available Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.

DNA dynamics is likely to be a factor in the genomic nucleotide repeats expansions related to diseases.

Directory of Open Access Journals (Sweden)

Boian S Alexandrov

Full Text Available Trinucleotide repeats sequences (TRS represent a common type of genomic DNA motif whose expansion is associated with a large number of human diseases. The driving molecular mechanisms of the TRS ongoing dynamic expansion across generations and within tissues and its influence on genomic DNA functions are not well understood. Here we report results for a novel and notable collective breathing behavior of genomic DNA of tandem TRS, leading to propensity for large local DNA transient openings at physiological temperature. Our Langevin molecular dynamics (LMD and Markov Chain Monte Carlo (MCMC simulations demonstrate that the patterns of openings of various TRSs depend specifically on their length. The collective propensity for DNA strand separation of repeated sequences serves as a precursor for outsized intermediate bubble states independently of the G/C-content. We report that repeats have the potential to interfere with the binding of transcription factors to their consensus sequence by altered DNA breathing dynamics in proximity of the binding sites. These observations might influence ongoing attempts to use LMD and MCMC simulations for TRS-related modeling of genomic DNA functionality in elucidating the common denominators of the dynamic TRS expansion mutation with potential therapeutic applications.

Study in mutation of alfalfa genome DNA due to low energy N+ implantation using RAPD

International Nuclear Information System (INIS)

Chen Roulei; Song Daojun; Yu Zengliang; Li Yufeng; Liang Yunzhang

2001-01-01

After implanted by various dosage N + beams, germination rate of alfalfa seeds appears to be saddle line with dosage increasing. The authors have studied in mutation of genome DNA due to low energy N + implantation, and concluded that 30 differential DNA fragments have been amplified by 8 primers (S 41 , S 42 , S 45 , S 46 , S 50 , S 52 , S 56 , S 58 ) in 100 primers, moreover, number of differential DNA fragments between CK and treatments increases with dosage. Consequently, low energy ion implantation can cause mutation of alfalfa genome DNA. The more dosage it is, the more mutation alfalfa will be

Ancient genomes

OpenAIRE

Hoelzel, A Rus

2005-01-01

Ever since its invention, the polymerase chain reaction has been the method of choice for work with ancient DNA. In an application of modern genomic methods to material from the Pleistocene, a recent study has instead undertaken to clone and sequence a portion of the ancient genome of the cave bear.

Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

Directory of Open Access Journals (Sweden)

David S. Shin

2014-01-01

Full Text Available As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine.

Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

International Nuclear Information System (INIS)

Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong

2012-01-01

DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

Epigenetic changes of Arabidopsis genome associated with altered DNA methyltransferase and demethylase expressions after gamma irradiation

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji Eun; Cho, Eun Ju; Kim, Ji Hong; Chung, Byung Yeoup; Kim, Jin Hong [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2012-05-15

DNA methylation at carbon 5 of cytosines is a hall mark of epigenetic inactivation and heterochromatin in both plants and mammals. In Arabidopsis, DNA methylation has two roles that protect the genome from selfish DNA elements and regulate gene expression. Plant genome has three types of DNA methyltransferase, METHYLTRANSFERASE 1 (MET1), DOMAINREARRANGED METHYLASE (DRM) and CHROMOMETHYLASE 3 (CMT3) that are capable of methylating CG, CHG (where H is A, T, or C) and CHH sites, respectively. MET1 is a maintenance DNA methyltransferase that controls CG methylation. Two members of the DRM family, DRM1 and DRM2, are responsible for de novo methylation of CG, CHG, and CHH sites but show a preference for CHH sites. Finally, CMT3 principally carries out CHG methylation and is involved in both de novo methylation and maintenance. Alternatively, active DNA demethylation may occur through the glycosylase activity by removing the methylcytosines from DNA. It may have essential roles in preventing transcriptional silencing of transgenes and endogenous genes and in activating the expression of imprinted genes. DNA demetylation in Arabidopsis is mediated by the DEMETER (DME) family of bifunctional DNA glycosylase. Three targets of DME are MEA (MEDEA), FWA (FLOWERING WAGENINGEN), and FIS2 (FERTILIZATION INDEPENDENT SEED 2). The DME family contains DEMETER-LIKE 2 (DML2), DML3, and REPRESSOR OF SILENING 1 (ROS1). DNA demetylation by ROS1, DML2, and DML3 protect the hypermethylation of specific genome loci. ROS1 is necessary to suppress the promoter methylation and the silencing of endogenous genes. In contrast, the function of DML2 and DML3 has not been reported. Several recent studies have suggested that epigenetic alterations such as change in DNA methylation and histone modification should be caused in plant genomes upon exposure to ionizing radiation. However, there is a lack of data exploring the underlying mechanisms. Therefore, the present study aims to characterize and

Comparison of DNA Quantification Methods for Next Generation Sequencing.

Science.gov (United States)

Robin, Jérôme D; Ludlow, Andrew T; LaRanger, Ryan; Wright, Woodring E; Shay, Jerry W

2016-04-06

Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library's heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.

Chemical biology on the genome.

Science.gov (United States)

Balasubramanian, Shankar

2014-08-15

In this article I discuss studies towards understanding the structure and function of DNA in the context of genomes from the perspective of a chemist. The first area I describe concerns the studies that led to the invention and subsequent development of a method for sequencing DNA on a genome scale at high speed and low cost, now known as Solexa/Illumina sequencing. The second theme will feature the four-stranded DNA structure known as a G-quadruplex with a focus on its fundamental properties, its presence in cellular genomic DNA and the prospects for targeting such a structure in cels with small molecules. The final topic for discussion is naturally occurring chemically modified DNA bases with an emphasis on chemistry for decoding (or sequencing) such modifications in genomic DNA. The genome is a fruitful topic to be further elucidated by the creation and application of chemical approaches. Copyright © 2014 Elsevier Ltd. All rights reserved.

Genome-wide nucleosome occupancy and DNA methylation profiling of four human cell lines

Directory of Open Access Journals (Sweden)

Aaron L. Statham

2015-03-01

Full Text Available DNA methylation and nucleosome positioning are two key mechanisms that contribute to the epigenetic control of gene expression. During carcinogenesis, the expression of many genes is altered alongside extensive changes in the epigenome, with repressed genes often being associated with local DNA hypermethylation and gain of nucleosomes at their promoters. However the spectrum of alterations that occur at distal regulatory regions has not been extensively studied. To address this we used Nucleosome Occupancy and Methylation sequencing (NOMe-seq to compare the genome-wide DNA methylation and nucleosome occupancy profiles between normal and cancer cell line models of the breast and prostate. Here we describe the bioinformatic pipeline and methods that we developed for the processing and analysis of the NOMe-seq data published by (Taberlay et al., 2014 [1] and deposited in the Gene Expression Omnibus with accession GSE57498.

In situ genomic DNA extraction for PCR analysis of regions of interest in four plant species and one filamentous fungi