Hot papers in the field of digital PCR

Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and RT-qPCR), which are compiled and summarised on the Gene Quantification domain. The focus of this newsletter issue is:

• Hot papers in the field of digital PCR - http://digital-PCR.gene-quantification.info
• First event announcement -- qPCR & NGS 2015 Event -- "Advanced Molecular Diagnostics for Biomarker Discovery" - www.qPCR-NGS-2015.net
• "Biomolecular Detection and Quantification" -- Call for submissions - BDQ.Gene-Quantification.info
• GenEx 6 available - The most powerful tool for complex qPCR data analysis - download a free trial version - GenEx.gene-quantification.info

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Digital PCR (dPCR)
digital-PCR.gene-quantification.info

dPCR is a refinement of conventional PCR methods that can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA, or RNA). The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants, point mutations, and it is routinely used for clonal amplification of samples for "next-generation sequencing."

The first digital-PCR paper:
Quantitation of targets for PCR by use of limiting dilution
Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, Morley AA.
Biotechniques. 1992 13(3): 444-449

We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.

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