This report documents our evaluation of three digital PCR (dPCR) based methods for estimating the proportion, if any, of single-stranded DNA (ssDNA) in a sample that is believed to be mostly double-stranded DNA (dsDNA). These methods are: 1) a modification of a published real time chamber-digital PCR (cdPCR) method, 2) comparing droplet-digital (ddPCR) results for native samples with those of an aliquot that has been gently heat-denatured, and 3) comparing ddPCR results for native samples with those of an aliquot in which dsDNA entities have been enzymatically rendered non amplifiable. The cdPCR method requires use of exceptionally efficient PCR assays and appears to be insensitive to ssDNA proportions less than about 8%. The denaturation/native comparison does not provide a unique estimate of ssDNA proportion but rather an upper limit. The enzymatic method requires careful choice and evaluation of restriction enzyme and PCR assay but has the potential to provide metrologically traceable estimates of the proportion of ssDNA.
Special Publication (NIST SP) - 1200-27
chamber digital PCR (cdPCR), denaturation, deoxyribose nucleic acid (DNA) double stranded DNA (dsDNA), droplet digital PCR (ddPCR), human nuclear DNA (nDNA), single stranded DNA (ssDNA), Standard Reference Material (SRM), SRM 2372 Human DNA Quantitation Standard, SRM 2372a Human DNA Quantitation Standard