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Evaluating Digital PCR for the Quantification of Human Nuclear DNA: Factors Influencing Target Strandedness



Margaret C. Kline, David L. Duewer


The presence of single stranded DNA (ssDNA) in an extract of nominally double stranded DNA (dsDNA) can lead digital polymerase chain reaction (dPCR) measurements to overestimate the mass concentration of DNA in a sample by up to a factor of two. Motivated by measured ssDNA proportions of nearly 40 % in commercially obtained extracts, we have investigated some of the possible drivers of high ssDNA content in these materials to inform the development of future DNA reference materials. Our primary focus has been on the extraction methods: aqueous solution salt out, silica column, magnetic particles, and ion exchange resin. While many studies have compared various implementations of these methods with regard to the resources required and the quantity, purity, and amplifiability of the extracted DNA, none have compared the strandedness of extracts produced nor have they examined the impact of the methods on the ratio between the human mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) entity content, mtDNA/nDNA. In our hands, salt- out extraction of DNA from human blood-related tissue induces less dsDNA strand separation than the silica column and magnetic particle methods evaluated. Salt-out is also more efficient at extracting nDNA. It is therefore our method of choice for producing human-source nDNA reference materials for evaluation by and use with dPCR techniques. However, salt-out may be less efficient for and/or more damaging to mtDNA then are the silica column and magnetic particle methods. Further characterization is required before the accuracy of mtDNA/nDNA ratio measurements can be assured.
Special Publication (NIST SP) - 1200-28
Report Number


digital polymerase chain reaction (dPCR), double stranded DNA (dsDNA), mitochondrial DNA (mtDNA), nuclear DNA (nDNA), salt out extraction, single stranded DNA (ssDNA)


Kline, M. and Duewer, D. (2021), Evaluating Digital PCR for the Quantification of Human Nuclear DNA: Factors Influencing Target Strandedness, Special Publication (NIST SP), National Institute of Standards and Technology, Gaithersburg, MD, [online],, (Accessed July 22, 2024)


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Created February 10, 2021, Updated October 12, 2021