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Real-Time cdPCR Opens a Window Into Events Occurring in the First Few PCR Amplification Cycles

NCJ Number
Analytical and Bioanalytical Chemistry Volume: 407 Issue: 30 Dated: December 2015 Pages: 9061-9069
Date Published
December 2015
9 pages
The project described in this article is evaluating several commercially available dPCR (digital PCR) systems for use in certifying mass concentration in human genomic DNA reference materials.

Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as providing a potentially primary method for DNA quantification. In order to better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, the current project developed a graphical tool for evaluating and documenting the performance of PCR assays in real-time cdPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about early amplification events. The project successfully simulated ogives observed with different assays and reaction conditions using a four-stage amplification model parameterized by the probability of creating an intact 1) first generation "long" amplicon of indeterminate length from an original DNA target, 2) second generation defined-length amplicon from a long amplicon, and 3) defined-length amplicon from another defined-length amplicon. The authors are using insights from this model to optimize dPCR assay design and reaction conditions and to help validate assays proposed for use in value-assigning DNA reference materials. (Publisher abstract modified)

Date Published: December 1, 2015