Abstract:
:The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.
journal_name
Genome Resjournal_title
Genome researchauthors
Heyman JA,Cornthwaite J,Foncerrada L,Gilmore JR,Gontang E,Hartman KJ,Hernandez CL,Hood R,Hull HM,Lee WY,Marcil R,Marsh EJ,Mudd KM,Patino MJ,Purcell TJ,Rowland JJ,Sindici ML,Hoeffler JPsubject
Has Abstractpub_date
1999-04-01 00:00:00pages
383-92issue
4eissn
1088-9051issn
1549-5469journal_volume
9pub_type
杂志文章相关文献
GENOME RESEARCH文献大全abstract::Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unma...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.156034.113
更新日期:2013-11-01 00:00:00
abstract::Next-generation sequencing technologies have made it possible to sequence targeted regions of the human genome in hundreds of individuals. Deep sequencing represents a powerful approach for the discovery of the complete spectrum of DNA sequence variants in functionally important genomic intervals. Current methods for ...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.100040.109
更新日期:2010-04-01 00:00:00
abstract::Population genetics has evolved from a theory-driven field with little empirical data into a data-driven discipline in which genome-scale data sets test the limits of available models and computational analysis methods. In humans and a few model organisms, analyses of whole-genome sequence polymorphism data are curren...
journal_title:Genome research
pub_type: 杂志文章,评审
doi:10.1101/gr.079509.108
更新日期:2010-03-01 00:00:00
abstract::Lemurs and the other strepsirrhine primates are of great interest to the primate genomics community due to their phylogenetic placement as the sister lineage to all other primates. Previous attempts to resolve the phylogeny of lemurs employed limited mitochondrial or small nuclear data sets, with many relationships po...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.7265208
更新日期:2008-03-01 00:00:00
abstract::Large copy number variants (CNVs) have been recently found as structural polymorphisms of the human genome of still unknown biological significance. CNVs are significantly enriched in regions with segmental duplications or low-copy repeats (LCRs). Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused ...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.073197.107
更新日期:2008-05-01 00:00:00
abstract::It appears that, for many genes, the two alleles possessed by an individual may produce different amounts of transcript. When such allelic differences in transcription are observed for some individuals but not others, a plausible explanation is genetic variation in the cis-acting elements that regulate the gene in que...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.5663007
更新日期:2007-01-01 00:00:00
abstract::Translocations are known to affect the expression of genes at the breakpoints and, in the case of unbalanced translocations, alter the gene copy number. However, a comprehensive understanding of the functional impact of this class of variation is lacking. Here, we have studied the effect of balanced chromosomal rearra...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.103622.109
更新日期:2010-05-01 00:00:00
abstract::Short tandem repeats (STRs) have a wide range of applications, including medical genetics, forensics, and genetic genealogy. High-throughput sequencing (HTS) has the potential to profile hundreds of thousands of STR loci. However, mainstream bioinformatics pipelines are inadequate for the task. These pipelines treat S...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.135780.111
更新日期:2012-06-01 00:00:00
abstract::Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions. We decided to measure mRNA decay rates in two human ce...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.1272403
更新日期:2003-08-01 00:00:00
abstract::Dystroglycan is a laminin binding protein, which provides a structural link between the subsarcolemmal cytoskeleton and the extracellular matrix. It is also involved in the organization of basement membranes. So far the genomic organization of the dystroglycan gene DAG1 has not been completely investigated. Here we re...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.10.3.295
更新日期:2000-03-01 00:00:00
abstract::In positional cloning the initial assignment of a gene to a specific chromosomal locus is followed by physical mapping of the critical region. The construction of a high-resolution physical map still involves considerable effort. However, new high-resolution fluorescence in situ hybridization (FISH) techniques have fa...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.6.10.1002
更新日期:1996-10-01 00:00:00
abstract::Retroposed copies (RPCs) of genes are functional (intronless paralogs) or nonfunctional (processed pseudogenes) copies derived from mRNA through a process of retrotransposition. Previous studies found that gene families involved in mRNA translation or nuclear function were more likely to have large numbers of RPCs. He...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.893803
更新日期:2003-05-01 00:00:00
abstract::DNA methylation at the promoter of a gene is presumed to render it silent, yet a sizable fraction of genes with methylated proximal promoters exhibit elevated expression. Here, we show, through extensive analysis of the methylome and transcriptome in 34 tissues, that in many such cases, transcription is initiated by a...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.212050.116
更新日期:2017-04-01 00:00:00
abstract::In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato frui...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.080127.108
更新日期:2008-10-01 00:00:00
abstract::Clusters of functionally related genes can be disrupted by a single copy number variant (CNV). We demonstrate that the simultaneous disruption of multiple functionally related genes is a frequent and significant characteristic of de novo CNVs in patients with developmental disorders (P = 1 × 10(-3)). Using three diffe...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.184325.114
更新日期:2015-06-01 00:00:00
abstract::Repetitive DNA is a significant component of eukaryotic genomes. We have developed a strategy to efficiently and accurately sequence repetitive DNA in the nematode Caenorhabditis elegans using integrated artificial transposons and automated fluorescent sequencing. Mapping and assembly tools represent important compone...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.7.5.551
更新日期:1997-05-01 00:00:00
abstract::Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs)...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.107995.110
更新日期:2010-11-01 00:00:00
abstract::The nuclear space is not a homogeneous biochemical environment. Many studies have demonstrated that the transcriptional activity of a gene is linked to its positioning within the nuclear space. Following the discovery of lamin-associated domains (LADs), which are transcriptionally repressed chromatin regions, the nonr...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.215186.116
更新日期:2017-07-01 00:00:00
abstract::We have developed a novel quantitative method for rapidly assessing the CpG methylation density of a DNA region in mammalian cells. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with primers containing two dam sites (GATC). The purified PCR products are then incubated with 14C-la...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.202501
更新日期:2002-01-01 00:00:00
abstract::The mammalian cell nucleus contains numerous discrete suborganelles named nuclear bodies. While recruitment of specific genomic regions into these large ribonucleoprotein (RNP) complexes critically contributes to higher-order functional chromatin organization, such regions remain ill-defined. We have developed the hig...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.237073.118
更新日期:2018-11-01 00:00:00
abstract::Whole-genome sequencing using massively parallel sequencing technologies enables accurate detection of somatic rearrangements in cancer. Pinpointing large numbers of rearrangement breakpoints to base-pair resolution allows analysis of rearrangement microhomology and genomic location for every sample. Here we analyze 9...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.141382.112
更新日期:2013-02-01 00:00:00
abstract::The gastrointestinal microbiome undergoes shifts in species and strain abundances, yet dynamics involving closely related microorganisms remain largely unknown because most methods cannot resolve them. We developed new metagenomic methods and utilized them to track species and strain level variations in microbial comm...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.142315.112
更新日期:2013-01-01 00:00:00
abstract::The densities of transposable elements (TEs) in the human genome display substantial variation both within individual chromosomes and among chromosome types (autosomes and the two sex chromosomes). Finding an explanation for this variability has been challenging, especially in light of genome landscapes unique to the ...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.099044.109
更新日期:2010-05-01 00:00:00
abstract::Chromosomal aberrations have been thought to be random events. However, recent findings introduce a new paradigm in which certain DNA segments have the potential to adopt unusual conformations that lead to genomic instability and nonrandom chromosomal rearrangement. One of the best-studied examples is the palindromic ...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.079244.108
更新日期:2009-02-01 00:00:00
abstract::High-throughput sequencing of cDNA (RNA-seq) is a widely deployed transcriptome profiling and annotation technique, but questions about the performance of different protocols and platforms remain. We used a newly developed pool of 96 synthetic RNAs with various lengths, and GC content covering a 2(20) concentration ra...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.121095.111
更新日期:2011-09-01 00:00:00
abstract::We have constructed a detailed map of the genomic region containing the ETS-variant gene 6 (ETV6), involved in translocations and deletions associated with hematologic malignancies. Thirty-eight cosmids were characterized belonging to two contigs spanning 340 kb, and an EcoRl restriction map was developed. The gap bet...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.6.5.404
更新日期:1996-05-01 00:00:00
abstract::Insertion of the human non-LTR retrotransposon LINE-1 (L1) into chromosomal DNA is thought to be initiated by a mechanism called target-primed reverse transcription (TPRT). This mechanism readily accounts for the attachment of the 3'-end of an L1 copy to the genomic target, but the subsequent integration steps leading...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.3421505
更新日期:2005-06-01 00:00:00
abstract::In this study we quantify the features of meiotic recombination on the long arm of human chromosome 21. We constructed a 67. 3-centimorgan (cM) high-resolution, comprehensive, and accurate genetic linkage map of chromosome 21q using 187 highly polymorphic markers covering almost the entire long arm; 46 loci, consistin...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.138100
更新日期:2000-09-01 00:00:00
abstract::Current methods struggle to reconstruct and visualize the genomic relationships of large numbers of bacterial genomes. GrapeTree facilitates the analyses of large numbers of allelic profiles by a static "GrapeTree Layout" algorithm that supports interactive visualizations of large trees within a web browser window. Gr...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.232397.117
更新日期:2018-09-01 00:00:00
abstract::RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accuratel...
journal_title:Genome research
pub_type: 杂志文章
doi:10.1101/gr.234807.118
更新日期:2019-11-01 00:00:00