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Summary of the October 2013 Issue of BioTechniques

The October issue of BioTechniques will feature new articles on optimizing PCR-based methylation analyses, combining RNAi and in vivo confocal microscopy to study DNA repair proteins, an improved methodology for purifying proteins at replication forks and a technique for determining the affect of reference gene choice on qPCR data analysis. In addition, the October issue will also contain our next Special Gem Series section, this one devoted to the topic of DNA sequencing.

DNA methylation is an important epigenetic modification that can influence gene expression and transcription. A number of tools have been developed to study methylation patterns, both globally and at specific genomic locations, with PCR-based approaches remaining very popular. In the October issue of BioTechniques, a group of researchers provide an in-depth review of the most commonly performed PCR-based methylation analysis techniques. Highlighting both the applications and possible pitfalls, this review should prove beneficial for all researchers interested in epigenetics and methylation analysis.

DNA repair is a crucial cellular process that occurs continuously. Studying this process requires tools that can identify sites of repair, and then specify which proteins are involved in the repair process. In a clever methodology that will be published in the October issue, a team of researchers from France detail a new approach which uses RNAi and in vivo confocal microscopy analysis of the photoconvertible Dendra2 fluorescent protein to localize and study specific DNA repair proteins. Although demonstrated with a specific repair protein, this in vivo approach will be applicable to the study of other proteins as well.

Purifying proteins can also be a challenge for biochemists. Proteins involved with DNA replication, especially those located at the replication fork, can be especially difficult to isolate and identify. Several approaches have been put forward to accomplish this and in October a team from Toronto detail their modification to an existing methodology that allows researchers to isolate these replication fork proteins under native conditions from cells.

qPCR data and interpretations are often dependent on the choice of reference genes. In some cases, a single reference gene is used to normalize a qPCR assay while in other case multiple reference genes might be used. In addition, for different assays, different reference genes might be needed. While a number of computer programs are available to help with picking good reference genes, there remain unknowns when it comes to this process. In the upcoming issue of BioTechniques, groups from Ohio State University and the University of Florida detail their results showing that at times the reference gene picked, even if it appears to be stably expressed, can influence normalization results in a negative way. The authors suggest a simple metric that can be used to quickly identify these troublesome reference genes and improve normalization of qPCR data.

DNA sequencing has come a long way since first being described in 1977. From radioactivity to automated fluorescence to next-generation nanpores, the evolution of sequencing has been nothing short of a technology revolution. In our special Gem section in October, we look at several articles from the pages of BioTechniques that influenced sequencing methodologies. This section will also have a special news feature article exploring how targeted resequencing is changing the way researchers approach genomics and studies of evolution.

Keywords: PCR, methylation, methylation-PCR, RNAi, confocal microscopy, fluorescent proteins, photoconvertible proteins, protein purification, protein tagging, replication forks, qPCR, normalization, reference genes, DNA sequencing, targeted resequencing, exome sequencing, whole genome sequencing, next-generation sequencing