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 30^th 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

Solution nuclear magnetic resonance (NMR) spectroscopy is widely applied to the analysis of complex mixtures of organic compounds such as biological fluids and tissue extracts. However, targeted profiling approaches with reliable compound quantification are still hard to accomplish due to signal overlap and other interferences. In the May 2013 issue of BioTechniques, a team of researchers present a tool for automated compound quantification from pre-processed 1D and 2D heteronuclear single quantum coherence (HSQC) NMR spectral data and concomitant validation of results. The performance of this new tool is tested on a urinary spike-in dataset and compared with other quantification strategies.

In a Benchmark article slated for the May issue, a team from Seoul National University describe a facile, efficient, cost-effective and rapid purification method for chemically labeled oligonucleotide. This method eliminates the time-consuming and inefficient conventional procedures, such as ethanol precipitation and size-exclusion chromatography, of un-reacted fluorophore removal that is required to retrieve the labeled DNA once DNA-fluorophore conjugation reaction is performed. Using n-butanol saturated with water, un-reacted free fluorophores in the reaction mixture are partitioned to organic phase while the labeled DNA remains in aqueous phase. This phase extraction method is very simple and fast, and multiple samples can be handled at the same time, thereby raising the prospect of being used as a high-throughput labeling strategy.

In an effort to address the issue of quantification for antibody assays with protein microarrays, a team of researchers from Arizona State University describes the development of a Microarray Nonlinear Calibration (MiNC) method that quantifies antibody binding to the surface of microarray spots.  MiNC significantly increased the linear dynamic range and reduced assay variations. In a proof of concept experiment, serological analysis of guinea pig Mycobacterium tuberculosis models showed that a larger number of candidates were identified with MiNC, which is consistent with the improved assay performance of protein microarrays.

DNA hybridization capture combined with next generation sequencing can be used to determine the DNA sequences of hundreds of target genes across hundreds of individuals in a single experiment. However, the approach has thus far only been successfully applied to sequences from closely related individuals. In the May issue, researchers from the College of Charleston and University of York detail a series of modifications that extend the reach of the method to comparisons among evolutionarily highly divergent organisms which has important implications for comparative biology.

Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. In a Report slated for May, researchers at the University of Pennsylvania describe a multiplexed Pairwise Agonist Scanning (PAS) method of measuring platelet inside-out responses to all pairs of 6 platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE2) used at their EC50 concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y1, P2Y12, TP, and IP receptors. The 3-color flow cytometry method simultaneously measured integrin αIIbβ3 activation with PAC-1 antibody, P-Selectin exposure (via alpha granule release) with anti-P-selectin, and phosphatidylserine exposure with annexinV. This method has the potential for efficiently scanning for patient-specific responses across a broad agonist-receptor space.

Keywords: next-generation sequencing, flow cytometry, metabolite analysis, metabolomics, NMR, mass spectrometry, target capture, sequence capture, antibodies, protein microarrays, linear calibration, nucleotide labeling, nucleotide purification, phase extraction

Isolating endogenous protein complexes is important for understanding how proteins interact and function within cells. When isolating such complexes, it is often critical to use gentle conditions and perform experiments as rapidly as possible. Two articles in this issue of BioTechniques report on new tools to assist in the isolation of tagged proteins. First, a team of researchers detail their modification to an existing peptide that enables the rapid elution of protein-A tagged molecules in less than 15 minutes. In comparison to the previous protein-A peptide, the author’s newly modified reagent is significantly faster (15 minutes compared to several hours for elution), works under milder conditions, and can be removed following elution using a simple spin column procedure. This new peptide should enhance workflows for researchers purifying proteins, and proteins complexes, with protein-A tags.

The second April article focused on protein tagging and isolation describes a new calcium responsive tag.  Here, the authors have developed a calcium-stimulus responsive synthetic peptide tag based on the naturally occurring repeat-in-toxin (RTX) domain, which can be used for protein precipitation and purification under gentle, calcium-induced conditions. This novel tag provides researchers a new, milder alternative when looking for a stimulus responsive tag for their protein isolations.

Protein interaction methods such as BRET and FRET rely on transient transfection of potential protein interaction partners. Transient transfections can result in over-expression of interacting partners, as well as non-homogeneous expression, complicating the interpretation of protein interaction assays. But in April, a team of researchers present a new method for the rapid generation of stable cell lines that provide single copy, isogenic expression of proteins for BRET interaction analyses.

Determining the stoichiometry of membrane proteins has traditionally been done through stochastic GFP photobleaching using total internal reflection (TIRF) microscopy. However, TIRF microscopy requires specialized equipment and knowledge, limiting usage. In the April 2013 issue of BioTechniques, a group of researchers describe the application of laser scanning confocal microscopy with a membrane sheet preparation to assess membrane protein oligomerization and distribution through GFP photobleaching. The simplicity of the author’s technique should enable GFP photobleaching experiments to be performed on membrane proteins using many different epifluorescence microscopy systems, thus significantly extending the potential use of this technique.

Generating single stranded DNA for molecular biology applications can be a challenge for scientists, but in the April 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.

Finally, the April issue will also feature a special Tech News examining the growing toolkit for engineering the genome. Here, the latest techniques (including ZFNs, TALENs, CRISPRs, MAGE, and homologous recombination) will be highlighted and compared with the intent of providing readers with a clearer understanding of the possibilities and potential of each techniques through specific examples. In addition to the Tech News, our other regular features such as the Scientist Profile, BioSpotlights, Troubleshooting Forum and Citations will also be included in the April issue.

Keywords: genome engineering, protein isolation and purification, chromatography, calcium-responsive tags, protein tags, ZFN, TALEN, MAGE, TIRF, confocal microscopy, rolling circle amplification (RCA), ribozymes, single-stranded DNA markers, GFP photobleaching, single molecule analysis, BRET, FRET, protein-A, generation of stable cell lines, transfection

Analysis of membrane proteins is critical since these biomolecules enable a cell to communicate and interpret their external environment. In this issue of BioTechniques, a group of researchers describe a new technique aimed at understanding the distribution, localization and trafficking of membrane proteins. The researchers from Helsinki detail a biochemical technique that takes advantage of a cold-adapted trypsin enzyme and a fluorescent dye to label intracellular pools of proteins prior to incorporation in the membrane. In this way, researchers can study trafficking of proteins to the cell membrane in response to chemical signals or other extracellular cues.

Chromatography can be an important step in the purification of proteins, including those found on the membrane. One approach advanced to improve protein purification is the creation of stimulus-responsive protein tags. These tags become active under specific conditions and then can then be used to precipitate the target protein. In a Report slated for the March issue, a team of researchers describes a new calcium-responsive precipitation tag that can be easily cleaved following protein isolation. The value of this new tag is that by being calcium-responsive it will enable bioseparations under more gentle conditions than previously possible using other stimulus-responsive tag approaches.

With the recent publications from the ENCODE consortium describing various regulatory elements within the genome, interest in understanding gene regulation and transcription factor binding is very high at the moment. One issue for researchers curious to predict where within a particular sequence a transcription factor might bind prior to an experiment is the lack of integrated computational tools to make such binding site predictions. In the March issue of BioTechniques, a team of researchers describes an integrated computer platform for the analysis of transcription factor binding sites and tools to visualize those sites within sequences of interest. This new suite of applications provides a “one-stop” location for computational analysis of transcription factor binding.

‘Omics datasets (think transcriptomics, proteomics, metabolomics, ect), especially those generated by approach such as mass spectrometry and NMR, tend to be both large and complex, but also often missing values or datapoints. This large number of non-random missing values can often make data analysis methods, such as the commonly used principal component analysis (PCA) technique, less effective when it comes to pattern visualization. To overcome this limitation and extend analysis/interpretation of these large-scale datasets, researchers at the Pacific Northwest National Laboratory describe the use of sequential projection pursuit PCA as an alternative to PCA that works even in the presence of missing data without the need for imputation.

Something special is starting in the March issue of BioTechniques – we will be celebrating our 30^th anniversary as a publication with a unique series of four special sections containing articles published in the pages of this journal that have truly shaped the way research is done in the lab. We are calling these articles “BioTechniques Gems”, and eight will be highlighted in 2013 in celebration of 30 years of BioTechniques. In March, our first two Gem articles in the category of biochemical methods will be revealed. Both articles will be reprinted in full, as they appeared originally, along with a special commentary on each article, its history and significance within the field of biochemical methods. Look for future Gem sections on PCR, imaging and DNA sequencing in the months to come.

Keywords: chromatography, sequencing, mass spectrometry, network analysis, , diagnostics, GFP, protein purification and separation, fluorescence microscopy, membrane proteins, membrane trafficking, data analysis, ‘omics approaches and technologies, protein tag, in vivo imaging, DNA/RNA/protein isolation, fluorescent probes

When it comes to exploring genomes, copy number variations, SNPs and insertions/deletions are critical landmarks. While sequencing today is faster and deeper than ever before, defining a large-scale genomic deletion, along with exact breakpoints, is not an easy task for users of NGS systems. The reason for this is that NGS systems, while producing millions of reads, generally produce short read lengths. However, as a team from Switzerland report in the February issue of BioTechniques, both the single molecule system of Pacific Biosciences as well as the Illumina NGS platform can be used for deletion mapping. The authors demonstrate the ability to map deletions from several kilobases up to several megabases, and define a set of parameters that can be used to align and determine break points for these deletions. While care must still be taken when dealing with cases where identical sequences flank the breakpoint, the approach presented here should enable more efficient and faster deletion analysis in the future.

Cell migration is crucial for many biological and developmental processes. Analyzing patterns of cell migration, especially collective migration patterns, can be useful in determining migration cues as well as the molecular mechanisms behind cell migration. To this end, a team of researchers report a new software platform for the analysis of cell migration patterns. This tool is capable of analyzing the width of streams of cells rapidly, providing researchers the chance to study correlated cell movements and assess the molecular signals involved in such migrations.

Quantitative PCR (qPCR) is a sensitive approach for the determination of sample copy number. Amplification relies on 2 primers surrounding a sequence of interest. But what if instead of two primers, four primers were used? And what if the polymerase had stand displacement activity? This is the idea behind the polymerase chain displacement reaction (PCDR) developed by a team of researchers and described in the February issue of BioTechniques.    In PCDR, four primers are employed in the reaction – amplification is initiated from both the outer primers and the inner primers and by using a polymerase with strand displacement activity, PCDR enables increased template amplification per cycle compared to the standard two primer reaction and thus enhanced sensitivity in qPCR applications.

Identifying microRNAs that play a role in human disease can be a challenge. Although transcriptome analysis can identify differentially expressed microRNAs, their specific roles in disease progression and biology are unclear. In an attempt to design an assay that can identify differentially expressed microRNAs and their effects, a team of researchers from the Johns Hopkins University created a methodology incorporating the lentiviral expression of different microRNAs with qPCR to assess functionally related increases in specific microRNA abundance. The approach, detailed in an article slated for the February issue of BioTechniques, provides researchers with a new high-throughput methodology for assessing microRNAs and their cellular roles.

The $1000 genome continues to be a goal for many in the sequencing community. But how realistic is this? What is the cost associate with data storage and analysis? How much is the researcher’s time worth? In a special Tech News in February, contributing writer Jeffrey Perkel explores the landscape of genome sequencing, and poses the question: we will ever hit the $1000 genome, and what could hold us back.

Keywords:  genomics, qPCR, miRNA, lentiviral vectors, next-generation sequencing, bioinformatics, genome analysis, CNV, SNP analysis, polymerase, cell migration/invasion, peptides, bioseperations, protein analysis

Analysis of membrane proteins is critical as these biomolecules enable a cell to communicate with the surrounding environment. In this issue of BioTechniques, two groups of researchers describe new techniques aimed at understanding the distribution and localization of membrane proteins. In the first article, researchers from Helsinki detail a biochemical technique that takes advantage of a cold-adapted trypsin enzyme and a fluorescent dye to label pools of proteins prior to incorporation in the membrane. In this way, researchers can study trafficking of these proteins to the membrane in response to signals or other cellular cues. The second article is from a group of scientists from Chile describing their development of a cell-free assay capable of determining the stoichiometry of plasma membrane proteins. Here, a methodological modification is introduced allowing stochastic GFP photobleaching to be used with any epifluorescence microscope rather than exclusively needing a total internal fluorescence microscopy, making the technique more accessible and affordable.

Chromatography can be an important step in the purification of proteins, including those found at the membrane. One approach being advanced to improve protein purification is the creation of stimulus-responsive protein tags that activate under specific conditions to enable precipitation of a target protein. In a Report in the January issue, a group of researchers describe a new calcium-responsive precipitation tag that can be easily cleaved following protein isolation. This new tag enables bioseparations under more gentle conditions than previously possible using other approaches.

‘Omics datasets, such as those generated by mass spectrometry, lead to large complex datasets. The problem is that these datasets also include a large number of non-random missing values, making data analysis tools such as principal component analysis (PCA) less effective in pattern visualization. To overcome this limitation and extend analysis of new large-scale datasets, researchers at the Pacific Northwest National Laboratory describe the use of sequential projection pursuit PCA as an alternative to PCA that works even in the presence of missing data without imputation.

Viral diagnostics often requires the quantitative amplification of viral particles from very low starting concentrations, a challenge for traditional PCR techniques. In a Report slated for the January issue, researchers from Oxfordshire, UK describe the development of a PCR technique called Polymerase Chain Displacement Reaction (PCDR) which takes advantage of multiple nested primers to first extend a new strand initiating from an outer primer which in turn then displaces the inner primer. The result of using the multiple combinations of primers suggested in PCDR is the 10-fold increase in sensitivity, enhancing the potential of PCR in viral diagnostics, as well as other applications focused on low copy number materials, in the future.

Keywords: chromatography, sequencing, mass spectrometry, network analysis, PCR, diagnostics, GFP, protein purification and separation, fluorescence microscopy, membrane proteins, membrane trafficking, data analysis, ‘omics approaches and technologies, protein tag

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

Stable recombinant protein production often requires the use of an antibiotic to select protein producing cell lines. In an October Report, authors from the Karolinska Institute in Stockholm, Sweden detail a new selection scheme for protein producing cells, one that relies on a cell’s adhesive properties rather than antibiotic resistance. The authors demonstrate that by fusing a protein of interest to SCARA5, a scavenger receptor of the SR family, the resulting cells, which produce the protein of interest, show improved adhesion to plastic surfaces. Using this knowledge, the authors then demonstrate that such cells can be selected for by simple washing steps as opposed to more expensive antibiotics and media. This approach should enhance the speed and lower the costs of generating protein producing cell lines.

Understanding the dynamics of protein folding and unfolding is critical to a deeper knowledge of protein structure and function relationships. In another Report slated for the October 2012 issue of BioTechniques, a team of researchers detail a novel approach to examining protein unfolding using a quantitative real-time thermocycler. By using fluorophores and modifying standard real-time PCR protocols, the authors were able to assess protein unfolding in presence of a wide range of denaturants in real time. This new approach should provide enhance the collection of protein denaturation kinetic data for biochemists.

Profiling gene expression patterns (transcriptome analysis) has become increasingly important in understanding genome regulation. Recent years have seen a number of new and improved tools and methods capable of evaluating gene expression on a global scale. Enriching for coding transcripts and non-coding regulatory RNAs is an important first step in any transcriptome analysis as the majority of the RNA in a sample is often ribosomal. In a new Report, authors from Sandia Laboratories describe the use of hydroxyapatite chromatography for the enrichment of mRNA transcripts and regulatory RNAs. The approach joins other methods, including duplex specific nuclease normalization, as tools to enhance transcriptome analysis studies.

Array-CGH enables researchers to search for, and study, genetic variation as well as profile genomic alterations and integrity. Now, a team of researchers from Germany report that arresting cells prior to array-CGH analysis leads to better data. The authors used drugs to arrest cell populations in G1 phase prior to array-CGH analysis, finding that this led in turn led to consistent and smoother genomic profiles enabling more reliable detection of copy number variations. This simple modification should have a large impact on analysis of copy number variations in a variety of species.

In another Report slated for October, researchers from the University of Southern California describe a new approach for the separation of DNA fragments. Unlike traditional approaches where DNA is immobilized on a surface, the authors immobilized an intercalator capable of binding DNA onto microspheres. As the intercalator will only interact with double stranded DNA (dsDNA), this new approach can be used to isolate dsDNA from single stranded DNA in a mixture.

In addition to these research articles, October will also feature a Tech News article devoted to the growing field of genome engineering. Using a variety of new methods and tools, researchers are modifying large stretches of DNA in an effort to better understand genome structure and function. Our feature article in October looks at many of these approaches, and what may come in the future in this rapidly expanding field.

Keywords: DNA isolation, microarrays, array-CGH, sequencing, genome engineering, cloning, mutagenesis, copy number variation, transcription analysis, transcriptomics, chromatography, protein analysis, protein kinetics, protein production, cell analysis, stable cell line production

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