High-Throughput Analysis of Gene Regulation, DNA Synthesis in CSH Protocols|
COLD SPRING HARBOR, N.Y. (Mon., Feb. 1, 2010) – Mapping DNase I hypersensitive sites has long been the standard method for identifying genetic regulatory elements such as promoters, enhancers, silencers, insulators, and locus control regions. Sequences that are nucleosome-depleted, presumably to provide access for transcription factors, are selectively digested by DNase I. Traditional low-throughput methods use Southern blots to then identify these hypersensitive sites. In the February issue of Cold Spring Harbor Protocols (www.cshprotocols.org/TOCs/toc2_10.dtl), Gregory Crawford and colleagues from Duke University (www.genome.duke.edu/people/faculty/crawford/) present DNase-seq: A High-Resolution Technique for Mapping Active Gene Regulatory Elements Across the Genome from Mammalian Cells. DNase-seq is a high-throughput method that identifies DNase I hypersensitive sites across the whole genome by capturing DNase-digested fragments and applying next-generation sequencing techniques. In a single experiment, DNase-seq can identify most active regulatory regions from potentially any cell type, from any species with a sequenced genome. As one of February’s featured articles, it is freely available on the journal’s website (cshprotocols.cshlp.org/cgi/content/full/2010/2/pdb.prot5384).
The incorporation of thymidine analogues, such as 5-bromo-2?-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair and other aspects of DNA metabolism. In Genome-Wide Analysis of DNA Synthesis by BrdU Immunoprecipitation on Tiling Microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae, Oscar Aparicio and colleagues from the University of Southern California (www.usc.edu/programs/pibbs/site/faculty/aparicio_o.htm) couple BrdU immunoprecipitation with DNA microarrays to enable genome-wide identification of BrdU-labeled chromosomal DNA. BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression. The article is featured in the February issue of Cold Spring Harbor Protocols (www.cshprotocols.org/TOCs/toc2_10.dtl) and is freely available on the journal’s website (cshprotocols.cshlp.org/cgi/content/full/2010/2/pdb.prot53685).
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Cold Spring Harbor Protocols (www.cshprotocols.org) is a monthly peer-reviewed journal of methods used in a wide range of biology laboratories. It is structured to be highly interactive, with each protocol cross-linked to related methods, descriptive information panels, and illustrative material to maximize the total information available to investigators. Each protocol is clearly presented and designed for easy use at the bench—complete with reagents, equipment, and recipe lists. Life science researchers can access the entire collection via institutional site licenses, and can add their suggestions and comments to further refine the techniques.
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