Bench Marks

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 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. As one of February’s featured articles in Cold Spring Harbor Protocols, the protocol is freely available to subscribers and non-subscribers alike.

mRNA in situ hybridization is a standard laboratory technique for analyzing gene expression. In a small, transparent specimen like a zebrafish embryo, this technique is straightforward and works well. Cold Spring Harbor Protocols has a set of protocols (here, here and here) describing the method from Cecilia Moens. But what happens when you’re dealing with a larger, opaque zebrafish tissue like the adult brain? Unlike mammals, zebrafish exhibit intense ongoing neurogenesis in all areas of the central nervous system. Adult zebrafish are increasingly being used in behavioral studies as well. Because the number of antibodies useful for examining expression in zebrafish is limited, mRNA in situ hybridization is a vital tool for understanding what’s happening during these processes. In the February issue of Cold Spring Harbor Protocols, Reinhard Köster and colleagues from the Helmholtz Zentrum München provide an adaptation of the standard in situ method that deals with these larger, opaque tissues by staining them after vibratome sectioning, Analysis of Gene Expression by In Situ Hybridization on Adult Zebrafish Brain Sections. While the brain is used as the sample tissue in this protocol, it can easily be modified for analysis of other adult tissues.

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, Gregory Crawford and colleagues from Duke University 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 to subscribers and non-subscribers alike.