Evaluating Digital PCR for the Quantification of Human Nuclear DNA: Determining Target Strandedness
Margaret C. Kline, David L. Duewer
Digital polymerase chain reaction (dPCR) methodology has been asserted to be a "potentially primary" analytical approach for assigning DNA concentration. Currently available chamber dPCR (cdPCR) and droplet dPCR (ddPCR) platforms are thought capable of providing accurate, metrologically traceable results for DNA tissue extracts without having to be calibrated with a DNA standard. The essence of dPCR measurements is the independent dispersal of fragments into multiple reaction partitions, amplifying fragments containing a target nucleotide sequence until the signal from all partitions containing at least one such fragment rises above threshold, and then determining the proportion of partitions with an above-threshold signal. Should originally double-stranded DNA (dsDNA) fragments be converted into two single-strands (ssDNA) prior to dispersal, the dPCR measurements could be biased high by as much as a factor of two. Therefore, realizing dPCR's metrological potential requires analytical methods for determining the proportion of ssDNA in nominally dsDNA samples. We have investigated several plausible approaches to this determination: A260 Ratio, dPCR Ratio, cdPCR Staircase, and ddPCR Enzyme. In our hands, only ddPCR Enzyme provides adequately accurate (true and precise) estimate for relatively small ssDNA proportions. This Type II restriction endonuclease-based approach compares ddPCR assay results for native samples and aliquots of those samples that have been enzyme cut within the assay's target sequence. We here present four (enzyme, assay) pairs that provide consistent results for human nuclear DNA materials derived from 12 sources. The proportion of ssDNA consistently differs by as much as 50% between assays, apparently related to the guanine-cytosine (GC) content of the fragment near the target sequence. While materials extracted by us have no more than 6% ssDNA content even after long storage, a commercially obtained PCR assay calibrant contains more than 15% ss
and Duewer, D.
Evaluating Digital PCR for the Quantification of Human Nuclear DNA: Determining Target Strandedness, Analytical and Bioanalytical Chemistry, [online], https://doi.org/10.1007/s00216-020-02733-2, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=929734
(Accessed September 26, 2023)