The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein/bait interactions that drive biological processes. Analysis via mass spectrometry rather than western blotting extends the identification of interactors, often allowing detection of thousands of proteins from complex mixtures. But this increased sensitivity can lead to problems distinguishing specific interactions from background noise. In the March issue of Cold Spring Harbor Protocols, Shao-En Ong from the Broad Institute of MIT and Harvard presents Unbiased Identification of Protein/Bait Interactions Using Biochemical Enrichment and Quantitative Proteomics. This method uses quantitative proteomics approaches to compare enrichment with the bait of interest against samples using control baits to allow sensitive detection and discrimination of specific protein/bait interactions. As one of March’s featured articles, it is freely available to subscribers and non-subscribers alike.

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.

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.

The introduction of high-throughput laboratory methods has greatly increased the pace of research into the genetics of complex diseases. Instead of focusing only on one or a few coding variants in a small sample of individuals, the ability to accurately and efficiently genotype many individuals and to cover more of the variation within individual genes has resulted in genetic studies with greater statistical power. Laboratory Methods for High-Throughput Genotyping, from Howard Edenberg and Yunlong Liu at the University of Indiana, presents an overview of the commonly used methods for high-throughput single-nucleotide polymorphism (SNP) genotyping for different stages of genetic studies and briefly reviews some of the high-throughput sequencing methods just coming into use. The authors also discuss recent developments in “next-generation” sequencing that will enable other kinds of studies. The article is excerpted from the recently published Genetics of Complex Human Diseases laboratory manual. It is featured in the November issue of Cold Spring Harbor Protocols, and like all our featured articles, is freely available to subscribers and non-subscribers alike.

High-throughput whole-genome analysis is becoming a standard laboratory approach for investigating cellular processes. Next-generation sequencing is replacing microarrays as the technique of choice for genome-scale analysis, because it offers advantages in both sensitivity and scale. The June issue of Cold Spring Harbor Protocols features Native Chromatin Preparation and Illumina/Solexa Library Construction from Keji Zhao and colleagues at the National Heart, Lung and Blood Institute. The article describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Solexa/Illumina Genome Analyzer. Step-by-step instructions are given for purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.

One of the great advantages of CSH Protocols over other online methods sources is that we have access to material from Cold Spring Harbor Laboratory’s cutting edge laboratory courses. November’s issue of CSH Protocols features material from the Molecular Embryology of the Mouse Course (as noted earlier), and the first set of many upcoming protocols from the Proteomics Course.

Course directors Andrew Link and Josh LaBaer have done a stellar job putting together a new laboratory manual based on the course. Proteomics will be out in December, but in the meantime, we’re publishing methods from the manual in advance in CSH Protocols. November’s issue brings a set of seven protocols covering Construction of Nucleic Acid Programmable Protein Arrays (NAPPA).

NAPPA differs from other protein array approaches in that proteins are translated in situ on the array surface, removing the need for individual protein purification. From the introduction:

This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains.

Protocols are available for preparing slides and cultures, isolating DNA, labeling and arraying DNA, expressing proteins, detecting proteins and detecting DNA.

With the sequencing of the human genome came the startling revelation that the number of copies of a given gene can vary widely between individuals. This Copy Number Variation (or CNV), contributes to our species’ genetic diversity but it has also been linked to genetic diseases. This month’s issue of Cold Spring Harbor Protocols features a new method for detecting copy number variation. Like all of our monthly featured protocols, it’s freely accessible for subscribers and non-subscribers alike.

Copy Number Variation Detection Via High-Density SNP Genotyping describes the use of PennCNV, a new computational tool for CNV detection in data from genomic arrays. Developed in the laboratory of Maja Bucan at the University of Pennsylvania, the software is freely available for download. Analysis with PennCNV will provide a more comprehensive understanding of genome variation and will aid in studies seeking the causes of genetic diseases. More information on PennCNV can be found in this Genome Research article, PennCNV: An integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data.

This month’s issue of CSH Protocols features an article by Andrew Salinger and Monica Justice, detailing a technique for Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU) (article is freely available as one of our featured protocols). Back in the ancient days of my graduate school work, the idea of doing large scale forward genetics in mouse was unthinkable. Who had the space, let alone the funding and personnel to keep and track all of those cages? It was always one of those reasons we grumbled about the Drosophila labs, and the incredibly cool tools they had at their disposal. Over the years, the techniques were refined, and now, according to Justice, screens like this are an “established as part of a mouse geneticist’s toolkit,” and can be effective even in labs with very limited amounts of mouse space. So it’s nice to see this incredibly productive method readily available for use in mouse. Now if we can just do something about that pesky internal development that’s so limiting to imaging experiments…..

The March issue of CSH Protocols has two featured (freely available) protocols on high-throughput methods for studying gene regulation.

The first method approaches regulatory analysis through epigenetic mechanisms. Methylated CpG Island Amplification and Microarray (MCAM) for High-Throughput Analysis of DNA Methylation, developed by Marcos Estecio and Jean-Pierre Issa of the MD Anderson Cancer Center, and Pearlly Yan and Tim Huang of the Ohio State University Comprehensive Cancer Center is a rapid, genome-wide method for identifying regions where methylation is occurring. This protocol has proven successful for proven successful for use in comparing normal tissues and tumors, helping researchers better understand the factors responsible for cancer.

The second protocol looks at the binding of regulatory proteins to DNA and their role in transcriptional regulation. The method, DNA Immunoprecipitation (DIP) for the Determination of DNA-binding Specificity, allows researchers to determine the specific DNA sequence that a regulatory protein binds. The technique allows for rapid screening of the entire genome for these binding sites, which gives insight into which genes these protein factors control.