Exploring Digital PCR as a primary method for the quantification of human genomic DNA: Ogive plots and models
David L. Duewer, Margaret C. Kline, Erica Romsos
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. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in human genomic DNA reference materials. To better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, we have 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. We have 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. We 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.
, Kline, M.
and Romsos, E.
Exploring Digital PCR as a primary method for the quantification of human genomic DNA: Ogive plots and models, Analytical and Bioanalytical Chemistry, [online], https://doi.org/10.1007/s00216-015-9073-8, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=918233
(Accessed September 23, 2023)