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Summary of the June 2013 Issue of BioTechniques

The June 2013 issue of BioTechniques will feature articles describing: (i) an improved method for sequence capture of protein coding genes, (ii) a technique for producing single stranded DNA products using deoxyribozymes and rolling circle amplification, (iii) a novel approach to determining transcript numbers from single neurons using digital PCR, and (iv) a newly developed protein microarray to assess influenza antibody responses. The June issue will also feature the second installment of the BioTechniques 30th Anniversary Gem article collection. This second section in the Gem series will highlight two PCR articles from the pages of BioTechniques that led to new applications for PCR in the lab.

Targeted resequencing remains an important application for next-generation sequencing. Since not all researchers have the capability, or the need, to sequence whole genomes, the having the capability to rapidly isolate and sequence specific sections of a target genome remains an important. But what is missing for some researchers is the ability to also target unknown genes, especially within those organisms where reference genomes do not exist. Now, a team of researchers from the College of Charleston report a new approach to target capture in the June issue of BioTechniques that enables isolation of unknown sequences that are up to 40% divergent from the bait probes. This method will allow researchers to isolate sequences up to 300 million years divergent from a target probe, enabling the rapid study of genomes that previously would have required whole genome sequencing or low-throughput degenerate PCR approaches to interrogate.

Generating single stranded DNA for molecular biology applications can be a challenge for scientists, but in the June issue, a team from Yale University describes a new approach to obtaining ssDNA of specific lengths. By incorporating deoxyribozyme sequences into templates for rolling circle amplification, the authors clearly show that single stranded DNAs of single and multiple-unit lengths with defined sizes and precise termini can be obtained. The authors go on to demonstrate the utility of their deoxyribozyme incorporated sequences by using the technique to generate a set of ssDNA markers.

Digital PCR is a technique that enables the direct quantification of nucleic acids in a sample. The application of digital PCR to single cell samples has been at the forefront of many recent insights into the nature of gene expression in cells. In a Report slated for the June issue, a team of scientists from the University of Szeged in Hungary describes a new approach to analyze the transcript content of single neurons following patch clamping using digital PCR. This methodology not only provides a quantitative indication of gene expression within a single neuron, but also allows direct observations of neuron function via patch clamp analysis, providing a more complete picture of the relationship between gene expression and phenotype.

The ability to rapidly analyze antibody responses to influenza infection is critical to better understanding the type of infection and possible treatment. In an effort to provide the broadest understanding of influenza infection, a team of researchers from the Infectious Disease Research Institute have developed and validated a new hemagglutinin protein microarray capable of analyzing patient sera for influenza antibody responses. This high-throughput platform will provide assessments of antibody responses to influenze disease and vaccination.

In addition to the above articles, the June 2013 issue of BioTechniques will also feature our usual collection of features (Citations, BioSpotlights, Troubleshooting Forum, Scientist Profile, and From the Editor) as well as a Tech News article examining epigenetic regulation of gene expression and our growing understanding of the functional role that large non-coding RNAs play in the cell.

Special Section for June 2013 issue of BioTechniques: PCR Gems from the past 30 years. A highlight of the June 2013 issue of BioTechniques is the inclusion of our second 30th Anniversary Gem article section, this one focusing on PCR. Many modern PCR methodologies owe their widespread dissemination to BioTechniques. In the 1990s, BioTechniques was an essential resource in educating scientists about the latest applications for this critical molecular biology method. To honor of our strong PCR tradition, the editors combed through BioTechniques issues of the past looking for the most influential PCR-focused articles published in these pages during the past 30 years. In the end, two were selected. The first PCR Gem article was published in 1997 by Carl Wittwer and colleagues and range of the scope of qPCR techniques. At this time, real-time PCR was starting to take off but the methodologies were limited to Taqman probes. Wittwer and his colleagues proposed the use of a simple dye called SYBR green to monitor incorporation and production of amplified template in real-time. Today, SYBR green has become a critical component in the real-time PCR toolkit – an approach that will continue to be used by researchers from a long time to come. Our second PCR Gem article comes from 1991, and represents the first description, and the intellectual foundation, for a technique that would come to be known as digital PCR. Skyes et al. described an approach for limiting dilution of a sample prior to PCR where a single molecule could be amplified per reaction. If enough reactions are performed, a researcher could obtain a quantitative measure of the number of molecules in that sample – a approach that is growing even bigger in 2013 as more researchers dive into the world of single cell analysis. Both PCR Gem articles will be re-printed within the June issue along with special commentaries from leading researchers detailing the impact these methods had within molecular biology labs.

Keywords:  lincRNA, next-generation sequencing, targeted resequencing, digital PCR, real-time PCR, qPCR, SYBR green, patch clamping, single neuron analysis, ssDNA, rolling circle amplification (RCA), protein microarray, antibody analysis, ELISA , ribozymes