Summary of the December 2012 issue of BioTechniques
The December 2012 issue of BioTechniques will feature research articles highlighting new methods for sequencing small genomes using third-generation sequencing technologies, normalizing cDNA to enhance transcriptome analysis, the description of a novel peptide for efficient bioseparations, and a new biochemical approach for analysis of membrane protein insertion. The December issue will also contain a special focus section devoted to new approaches and tools for genetic engineering, as well as a Tech News feature article exploring the latest techniques and instrumentation developments in the field of mass spectrometry.
Next-generation sequencing platforms are opening new avenues for researchers and inspiring methods developers to find new applications for this technology. This issue of BioTechniques features two articles describing enhancements in next-generation sequencing (NGS) methodologies that should further expand the sequencing application base. In most instances, prior to sequencing, researchers are required to carefully construct sequencing libraries, which are then used as starting material. To enable very rapid sequencing of DNA it would be beneficial if samples could be directly sequenced without the requirement of library creation. In a Report in the December issue, a group of researchers from the Hinxton, UK describe the direct sequencing of small bacterial and viral genomes using the Pacific Biosciences RS sequencer. Requiring very little input DNA (another benefit compared with library construction) and only a couple hours to complete, this new approach is rapid and sample efficient. Although the sequencing metrics will likely be enhanced in the future with further refinement, the method has great potential in situations where rapid identification of an unknown virus is necessary.
The second article in December focused on enhancing NGS applications describes a procedure for enriching transcriptome diversity during RNA-seq. RNA-seq has advanced tremendously in recent years, allowing for the interrogation of RNAs and their regulatory roles in the cell. Here, a group from Livermore CA report on the use of hydroxyapatite column chromatography prior to RNA-seq for the normalization of cDNA. Their results demonstrate that this normalization leads to more even representation of RNAs present in samples (from high to low abundance) when compared to standard approaches.
Chromatography is also an important step in the purification of proteins. One approach that has been advanced to improve protein purification is the creation of stimulus-responsive protein tags that are activated under specific conditions to enable precipitation of proteins. In a Report in the December issue, a group of researchers describe a new stimulus-responsive precipitation tag that is calcium responsive and can be easily cleaved following protein isolation. This new tag enables bioseparations under more gentle conditions than previously possible using other approaches.
Determining the rate and number of surface expressed membrane proteins can be challenging. While direct visualization is possible through microscopy, this is often not quantitative in nature. In a Benchmark article in December, a team from Helsinki, Finland presents a biochemical method for quantitative assessment of de novo insertion of membrane proteins using a cold-active trypsin and biotin. The method can be used to monitor protein trafficking and is amenable to both cells in culture and tissue slices.
The December issue will also contain a special focus section called “Tools to Engineer the Genome”. In this section, a feature article will examine the latest in nuclease-based genome modification approaches as well as approaches for genome alteration involving homologous recombination, while a research Report will detail a new technique for the generation of nucleotide libraries using lambda exonuclease.
Keywords: Next-generation sequencing (NGS), cloning, chromatography, mass spectrometry, proteomics, protein tags, biochemistry, calcium responsive elements, membrane trafficking, biotin, trypsin cleavage reactions, membrane proteins, RNA-seq, whole genome sequencing, genetic engineering, ZFN, TALEN, homologous recombination, directed evolution