Showing papers on "genomic DNA published in 2015"
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TL;DR: It is found that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells.
Abstract: Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to sequence both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification. We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells. Applications of our integrated sequencing approach could range from gaining insights into cancer evolution and heterogeneity to understanding the transcriptional consequences of copy number variations in healthy and diseased tissues.
447 citations
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TL;DR: The M methylC-sequencing (MethylC-seq) library preparation method is described, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution.
Abstract: Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein-DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.
260 citations
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TL;DR: To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, a single-guide RNA (sgRNA) was designed that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus.
Abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA.
183 citations
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TL;DR: It is shown that simulations of the binding kinetics are both necessary and sufficient to design nucleic acid probe systems with consistently high specificity as they enable the discovery of an optimal combination of thermodynamic parameters.
Abstract: The use of kinetic simulations to guide the design of competitive hybridization probe systems is shown to enable high selectivity for single-nucleotide variants. Using this approach across 44 cancer mutation/wild-type sequence pairs showed between a 200- and 3,000-fold higher binding affinity than the corresponding wild-type sequence. In combination with PCR amplification this method enabled the detection of a 1% concentration of variant alleles in human genomic DNA.
128 citations
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TL;DR: The chemistry of DPC formation in cells is outlined, recent efforts to identify the cross-linked proteins by mass spectrometry are described, and various methodologies for preparing DNA strands containing structurally defined, site specific DPC lesions are discussed.
Abstract: ConspectusNoncovalent DNA–protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in th...
127 citations
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TL;DR: Four methods, comprising three modified silica based commercial kits and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.
Abstract: Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.
121 citations
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TL;DR: This work analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias to conventional sequencing methods.
Abstract: Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed “reads” are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.
115 citations
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TL;DR: It is concluded that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.
Abstract: Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.
110 citations
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TL;DR: This method provides generally well-supported relationships at interspecific and intergeneric levels that agree with results from more standard phylogenetic analyses of commonly used markers, and it is proposed that this methodology may prove especially useful in groups where there is little genetic differentiation in standard phylogenetics markers.
Abstract: A large proportion of genomic information, particularly repetitive elements, is usually ignored when researchers are using next-generation sequencing. Here we demonstrate the usefulness of this repetitive fraction in phylogenetic analyses, utilizing comparative graph-based clustering of next-generation sequence reads, which results in abundance estimates of different classes of genomic repeats. Phylogenetic trees are then inferred based on the genome-wide abundance of different repeat types treated as continuously varying characters; such repeats are scattered across chromosomes and in angiosperms can constitute a majority of nuclear genomic DNA. In six diverse examples, five angiosperms and one insect, this method provides generally well-supported relationships at interspecific and intergeneric levels that agree with results from more standard phylogenetic analyses of commonly used markers. We propose that this methodology may prove especially useful in groups where there is little genetic differentiation in standard phylogenetic markers. At the same time as providing data for phylogenetic inference, this method additionally yields a wealth of data for comparative studies of genome evolution.(Repetitive DNA; continuous characters; genomics; next-generation sequencing; phylogenetics; molecular systematics.)
110 citations
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TL;DR: It is demonstrated that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies.
Abstract: Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
104 citations
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TL;DR: High-throughput sequencing of fragments in DNA from the yeast Saccharomyces cerevisiae revealed widespread ribonucleotide distribution, with a strong preference for cytidine and guanosine, and identified hotspots of ribon nucleotide incorporation in nuclear and mitochondrial DNA.
Abstract: Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed ribose-seq, a method for capturing unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments in DNA from the yeast Saccharomyces cerevisiae revealed widespread ribonucleotide distribution, with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly synthesized leading strand of nuclear DNA and were present upstream of (G+C)-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA.
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01 Jan 2015
TL;DR: The method involves disruption of fungal cell by bead beating in a homogenizer, followed by RNase treatment, phenol: chloroform: Isoamyl alcohol extraction and precipitation with isopropanol, which yielded good quality and quantity of DNA.
Abstract: We report a rapid and efficient method of genomic DNA extraction from filamentous fungi with high-throughput potential. The method involves disruption of fungal cell by bead beating in a homogenizer, followed by RNase treatment, phenol: chloroform: Isoamyl alcohol extraction and precipitation with isopropanol. The method does not involve any enzymatic digestion and it can be completed within 2.5 hours. The method yielded good quality and quantity (60 μg – 230 μg/200 mg of wet fungal mass) of the DNA. Being a closed system of gDNA extraction, our method has been found to be useful in avoiding the laboratory borne contamination during DNA extraction. The extracted DNA was found to be suitable for PCR based molecular methods like single and multicopy gene amplification and RAPD analysis.
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TL;DR: Saazer et al. as discussed by the authors provided draft genomes for two hop cultivars [cv.Saazer (SZ) and cv. Shinshu Wase (SW)] and a Japanese wildhop [H. lupulus var. cordifolius; also known as Karahanasou(KR)].
Abstract: Thefemaleflowerofhop(Humuluslupulusvar.lupulus)isanessential ingredient that gives characteristic aroma, bitter-nessanddurability/stabilitytobeer.However,themoleculargeneticbasisforidentifyingDNAmarkersinhopforbreedingand to study its domestication has been poorly established.Here, we provide draft genomes for two hop cultivars [cv.Saazer (SZ) and cv. Shinshu Wase (SW)] and a Japanese wildhop [H. lupulus var. cordifolius; also known as Karahanasou(KR)]. Sequencing and de novo assembly of genomic DNAfrom heterozygous SW plants generated scaffolds with atotal size of 2.05Gb, corresponding to approximately 80%of the estimated genome size of hop (2.57Gb). The scaffoldscontained 41,228 putative protein-encoding genes. Thegenome sequences for SZ and KR were constructed by align-ing their short sequence reads to the SW reference genomeandthenreplacingthenucleotidesatsinglenucleotidepoly-morphism (SNP) sites. De novo RNA sequencing (RNA-Seq)analysis of SW revealed the developmental regulation ofgenes involved in specialized metabolic processes thatimpact taste and flavor in beer. Application of a novel bio-informatics tool, phylogenetic comparative RNA-Seq (PCP-Seq), which is based on read depth of genomic DNAs andRNAs, enabled the identification of genes related to the bio-synthesis of aromas and flavors that are enriched in SWcomparedtoKR.Ourresultsnotonlysuggestthesignificanceof historical human selection process for enhancing aromaandbitternessbiosynthesesinhopcultivars,butalsoserveascrucial information for breeding varieties with high qualityand yield.Keywords: Genomics Hop Humulus Specialized metab-olism Transcriptome.Abbreviations: BCCA, branched-chain amino acid; BWA,Burrows–Wheeler aligner; CCL, cytosolic CoA ligase; CNV,copy number variation; FPKM, fragments per kilobase ofexon model per million mapped reads; FPP, farnesyldiphosphate; gDNA, genomic DNA; GO, gene ontology;GPP, geranyl diphosphate; GPPS, GPP, geranyl diphosphatesynthase; ispF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphatesynthase; LSU, large subunit; LTR, long terminal repeat; MP,mate-pair; MTS, monoterpene synthase; NGS, next gener-ation sequencing; OMT, O-methyltransferase; PA, proantho-cyanidin; PCP-Seq, phylogenetic comparative RNAsequencing; PE, paired-end; qRT-PCR, quantitative reversetranscription–PCR; rbcL, ribulose-1,5-bisphosphate carboxyl-ase large subunit; RNA-Seq, RNA sequencing; SNP, single nu-cleotide polymorphism; VPS, valerophenone synthase; WGS,whole-genome sequencing.The nucleotide sequences described in this paper have beensubmitted to the DNA Databank of Japan (DDBJ) as aBioProject (hop: Humulus lupulus) with the accession number(PRJDB3233) (Supplementary information).
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TL;DR: A nanobiosensor based on gold nanoparticles (AuNPs) and oligonucleotide probe was designed for the visual detection of Brucella spp.
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TL;DR: Through the injection of Cas9 protein instead of mRNA into embryos, the CRISPR-Cas RNA-guided system is used for novel genetically modified mouse and rat models and fewer off-target effects of Cas 9 are observed and point mutation knock-in efficiency is increased.
Abstract: The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis.
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TL;DR: It is demonstrated that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system, providing insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISpr- Cas9 in genome engineering applications.
Abstract: The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. IMPORTANCE Bacteria were recently found to have a form of adaptive immunity, the CRISPR-Cas systems, which use nucleic acid pairing to recognize and cleave genomic DNA of invaders such as bacteriophage. Historic work with tailed phages has shown that phage DNA is often modified by covalent attachment of large chemical groups. Here we demonstrate that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system. This finding provides insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISPR-Cas9 in genome engineering applications.
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TL;DR: It is found that the TaKaRa PrimeSTAR GXL DNA polymerase can amplify almost all amplicons with different sizes and Tm values under identical PCR conditions, which provided useful results for sequencing research focused on large genomic regions.
Abstract: Long-range PCR remains a flexible, fast, efficient and cost-effective choice for sequencing candidate genomic regions in a small number of samples, especially when combined with next-generation sequencing (NGS) platforms. Several long-range DNA polymerases are advertised as being able to amplify up to 15 kb or longer genomic DNA. However, their real-world performance characteristics and their suitability for NGS remain unclear. We evaluated six long-range DNA polymerases (Invitrogen SequalPrep, Invitrogen AccuPrime, TaKaRa PrimeSTAR GXL, TaKaRa LA Taq Hot Start, KAPA Long Range HotStart and QIAGEN LongRange PCR Polymerase) to amplify three amplicons, with sizes of 12.9 kb, 9.7 kb, and 5.8 kb, respectively. Subsequently, we used the PrimeSTAR enzyme to amplify entire BRCA1 (83.2 kb) and BRCA2 (84.2 kb) genes from nine subjects and sequenced them on an Illumina MiSeq sequencer. We found that the TaKaRa PrimeSTAR GXL DNA polymerase can amplify almost all amplicons with different sizes and Tm values under identical PCR conditions. Other enzymes require alteration of PCR conditions to obtain optimal performance. From the MiSeq run, we identified multiple intronic and exonic single-nucleotide variations (SNVs), including one mutation (c.5946delT in BRCA2) in a positive control. Our study provided useful results for sequencing research focused on large genomic regions.
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TL;DR: The demonstration of the universal presence of DNA adenine methylation constitutes the first and essential step toward understanding of m6dA functions in higher eukaryotes.
Abstract: DNA adenine methylation (N6-methyl-2′-deoxyadenosine, m6dA) plays important functional roles in prokaryotes and protists, including regulation of gene transcription, DNA replication and repair, and the restriction-modification system. However, there is no definitive evidence supporting the presence of DNA adenine methylation in genomic DNA of higher eukaryotes, such as mammals and plants, where DNA cytosine methylation (5-methylcytosine) instead is well recognized as an important epigenetic mark that has regulatory roles in various biological processes. In the current study, we developed a Dpn I cleavage coupled with size-exclusion ultrafiltration method, with which we discovered the wide-spread existence of m6dA in genomic DNA of higher eukaryotes, including human cells, rat tissues, and plants besides bacteria and protists by employing high-resolution mass spectrometry analysis. And the contents of m6dA vary in different cell types with the range of 0.00006–0.00077% (m6dA dA−1). Moreover, similar to N6-methyladenosine (m6A) in RNA, m6dA contents significantly decreased in type 2 diabetes mellitus (T2DM) patients compared to control subjects, indicating m6dA plays important roles in the pathogenesis of T2DM as m6A. In addition, knockdown of cellular fat mass and obesity-associated (FTO) protein increased the m6dA content, while overexpression of cellular FTO decreased m6dA content in DNA, suggesting m6dA and m6A may share the same demethylase of FTO. The demonstration of the universal presence of DNA adenine methylation constitutes the first and essential step toward understanding of m6dA functions in higher eukaryotes.
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TL;DR: Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio‐decaffeination, remediation of caffeine‐contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.
Abstract: The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.
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TL;DR: It is shown that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment.
Abstract: : The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user-specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment. Predetermined genomic DNA segments ranging from several-hundred base pairs to 1 Mbp can be precisely deleted at frequencies of 1% to 10%, with no apparent correlation between the size of the deleted fragment and the deletion frequency. The high efficiency of this technique holds promise for large genomic deletions that could be useful in generation of cell and animal models with engineered chromosomes. Keywords : CRISPR/Cas9; large genomic deletion; HPRT
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TL;DR: This platform is potentially an enabling tool to permit multiple genomic measurements performed on the same single cells and opens new opportunities to tackle a range of fundamental biology questions including non-genetic cell-to-cell variability, epigenetic regulation, and stem cell fate control.
Abstract: Despite the recent advance of single-cell gene expression analyses, co-measurement of both genomic and transcriptional signatures at the single-cell level has not been realized. However such analysis is necessary in order to accurately delineate how genetic information is transcribed, expressed, and regulated to give rise to an enormously diverse range of cell phenotypes. Here we report on a microfluidics-facilitated approach that allows for controlled separation of cytoplasmic and nuclear contents of a single cell followed by on-chip amplification of genomic DNA and cytoplasmic mRNA. When coupled with off-chip polymerase chain reaction, gel electrophoresis and Sanger sequencing, a panel of genes and transcripts from the same single cell can be co-detected and sequenced. This platform is potentially an enabling tool to permit multiple genomic measurements performed on the same single cells and opens new opportunities to tackle a range of fundamental biology questions including non-genetic cell-to-cell variability, epigenetic regulation, and stem cell fate control. It also helps address clinical challenges such as diagnosing intra-tumor heterogeneity and dissecting complex cellular immune responses.
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TL;DR: A next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor.
Abstract: Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC) and N(4)-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.
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TL;DR: Drop-Phase is a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution, and effective at long genomic distances (200 kb).
Abstract: Determining the chromosomal phase of pairs of sequence variants – the arrangement of specific alleles as haplotypes – is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11–116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).
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TL;DR: Methylome maps are generated for four sample types at single-base resolution, a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.
Abstract: Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors. We perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures. We generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.
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13 Apr 2015
TL;DR: In this paper, the authors proposed a method for site-specific sequence modification of a target genomic DNA region in cells comprising: transfecting the cells by electroporation with a composition comprising a DNA oligo and a DNA digesting agent wherein the donor DNA comprises: (i) a homologous region comprising nucleic acid sequence homologously to the target DNA region and (ii) a sequence modification region.
Abstract: Compositions and methods concern the sequence modification of an endogenous genomic DNA region. Certain aspects relate to a method for site-specific sequence modification of a target genomic DNA region in cells comprising: transfecting the cells by electroporation with a composition comprising (a) a DNA oligo and (b) a DNA digesting agent wherein the donor DNA comprises: (i) a homologous region comprising nucleic acid sequence homologous to the target genomic DNA region and (ii) a sequence modification region; and wherein the genomic DNA sequence is modified specifically at the target genomic DNA region.
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TL;DR: It is reported here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells, which likely facilitates nucleocapsid disassembly (uncoating) to release the genomic DNA for conversion to CCCDNA.
Abstract: Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. For reasons yet to be understood, HBV is apparently unable to make CCC DNA in normal mouse hepatocytes in the liver. We report here that HBV CCC DNA was formed efficiently in an immortalized mouse hepatocyte cell line, AML12HBV10, and this is associated with destabilization of mature NCs in these cells. These results suggest that destabilization of mature HBV NCs in AML12HBV10 cells facilitates efficient NC uncoating and subsequent CCC DNA formation. They further implicate NC uncoating as an important step in CCC DNA formation that is subject to host regulation and potentially a critical determinant of host range and/or cell tropism of HBV.
IMPORTANCE Persistent infection by hepatitis B virus (HBV), afflicting hundreds of millions worldwide, is sustained by the episomal viral covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes. CCC DNA is converted from the viral genomic (precursor) DNA contained in cytoplasmic viral nucleocapsids. The conversion process remains ill defined, but host cell factors are thought to play an essential role. In particular, HBV fails to make CCC DNA in normal mouse hepatocytes despite the presence of large amounts of nucleocapsids containing the precursor viral DNA. We have found that in an immortalized mouse hepatocyte cell line, HBV is able to make abundant amounts of CCC DNA. This ability correlates with increased instability of viral nucleocapsids in these cells, which likely facilitates nucleocapsid disassembly (uncoating) to release the genomic DNA for conversion to CCC DNA. Our studies have thus revealed a novel mechanism of controlling viral persistence via regulating nucleocapsid disassembly.
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TL;DR: Whole-genome sequencing of variants of single-copy rrn strains revealed duplications of large stretches of genomic DNA, which presumably leads to elevated genomic instability in Escherichia coli.
Abstract: Single-copy rrn strains facilitate genetic ribosomal studies in Escherichia coli. Consecutive markerless deletion of rrn operons resulted in slower growth upon inactivation of the fourth copy, which was reversed by supplying transfer RNA genes encoded in rrn operons in trans. Removal of the sixth, penultimate rrn copy led to a reduced growth rate due to limited rrn gene dosage. Whole-genome sequencing of variants of single-copy rrn strains revealed duplications of large stretches of genomic DNA. The combination of selective pressure, resulting from the decreased growth rate, and the six identical remaining scar sequences, facilitating homologous recombination events, presumably leads to elevated genomic instability.
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TL;DR: It is demonstrated that the levels of 5-hmC are dramatically reduced in human breast cancer tissue compared with those in normal tissue.
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TL;DR: Comparative genomic analysis with nine closely related fungal species in Hypocreales provided insight into the evolutionary relationship of P. lilacinum and significant up-regulation of subtilisin-like serine protease genes in presence of nematode eggs in quantitative real-time analyses suggested potential role of Serine proteases in nematodes pathogenesis.
Abstract: The fungus Purpureocillium lilacinum is widely known as a biological control agent against plant parasitic nematodes. This research article consists of genomic annotation of the first draft of whole genome sequence of P. lilacinum. The study aims to decipher the putative genetic components of the fungus involved in nematode pathogenesis by performing comparative genomic analysis with nine closely related fungal species in Hypocreales. de novo genomic assembly was done and a total of 301 scaffolds were constructed for P. lilacinum genomic DNA. By employing structural genome prediction models, 13, 266 genes coding for proteins were predicted in the genome. Approximately 73 % of the predicted genes were functionally annotated using Blastp, InterProScan and Gene Ontology. A 14.7 % fraction of the predicted genes shared significant homology with genes in the Pathogen Host Interactions (PHI) database. The phylogenomic analysis carried out using maximum likelihood RAxML algorithm provided insight into the evolutionary relationship of P. lilacinum. In congruence with other closely related species in the Hypocreales namely, Metarhizium spp., Pochonia chlamydosporia, Cordyceps militaris, Trichoderma reesei and Fusarium spp., P. lilacinum has large gene sets coding for G-protein coupled receptors (GPCRs), proteases, glycoside hydrolases and carbohydrate esterases that are required for degradation of nematode-egg shell components. Screening of the genome by Antibiotics & Secondary Metabolite Analysis Shell (AntiSMASH) pipeline indicated that the genome potentially codes for a variety of secondary metabolites, possibly required for adaptation to heterogeneous lifestyles reported for P. lilacinum. Significant up-regulation of subtilisin-like serine protease genes in presence of nematode eggs in quantitative real-time analyses suggested potential role of serine proteases in nematode pathogenesis. The data offer a better understanding of Purpureocillium lilacinum genome and will enhance our understanding on the molecular mechanism involved in nematophagy.
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TL;DR: It is concluded that 5-hmC is not present in biologically relevant quantities within plant genomic DNA.
Abstract: 5-Hydroxymethylcytosine (5-hmC) is an intermediate in active demethylation in metazoans, as well as a potentially stable epigenetic mark. Previous reports investigating 5-hydroxymethylcytosine in plants have reached conflicting conclusions. We systematically investigated whether 5-hmC is present in plant DNA using a range of methods. Using the model organism Arabidopsis thaliana, in addition to other plant species, we assayed the amount or distribution of 5-hydroxymethylcytosine by thin-layer chromatography, immunoprecipitation-chip, ELISA, enzymatic radiolabeling, and mass spectrometry. The failure to observe 5-hydroxymethylcytosine by thin-layer chromatography established an upper bound for the possible fraction of the nucleotide in plant DNA. Antibody-based methods suggested that there were low levels of 5-hmC in plant DNA, but these experiments were potentially confounded by cross-reactivity with the abundant base 5-methylcytosine. Enzymatic radiolabeling and mass spectrometry, the most sensitive methods for detection that we used, failed to detect 5-hydroxymethylcytosine in A. thaliana genomic DNA isolated from a number of different tissue types and genetic backgrounds. Taken together, our results led us to conclude that 5-hmC is not present in biologically relevant quantities within plant genomic DNA.