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Exploring Digital PCR as a primary method for the quantification of human genomic DNA: Ogive plots and models

Published

Author(s)

David L. Duewer, Margaret C. Kline, Erica L. Romsos

Abstract

Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as a potentially primary method for DNA quantification. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in high molecular mass human genomic DNA reference materials. We previously demonstrated that dPCR assays do not estimate the absolute number of PCR targets originally present in these ma-terials but only the number of targets that are accessible and amplifiable. Incomplete target accessibility may cause differ-ent dPCR assays to underestimate DNA quantity to a greater or lesser extent. To more fully characterize among-assay bias-es, we are developing a suite of PCR assays that appear to have fully accessible nucleotide target sequences. This report describes a graphical tool for evaluating and documenting the performance of these assays in real-time dPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about events during the initial few amplification cycles. We have successfully simulated ogives observed with different as-says and reaction conditions using a four-stage amplification model.
Citation
Analytical Chemistry

Keywords

Human genomic DNA quantification, digital PCR, amplification model

Citation

Duewer, D. , Kline, M. and Romsos, E. (2015), Exploring Digital PCR as a primary method for the quantification of human genomic DNA: Ogive plots and models, Analytical Chemistry, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=918233 (Accessed August 12, 2022)
Created October 4, 2015, Updated September 25, 2020