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Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness

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Abstract

Digital polymerase chain reaction (dPCR) methodology has been asserted to be a “potentially primary” analytical approach for assigning DNA concentration. 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. Realizing dPCR’s metrological potential therefore requires analytical methods for determining the proportion of ssDNA in nominally dsDNA samples. To meet this need, we have investigated several approaches to this determination: A260 ratio, dPCR ratio, cdPCR staircase, and ddPCR enzyme. In our hands, only the endonuclease-based approach provides adequately accurate estimates for relatively small ssDNA proportions. We present four (enzyme, assay) pairs that provide self-consistent results for human nuclear DNA extracts. However, the proportion of ssDNA differs by as much as 50% between assays, apparently related to the guanine-cytosine (GC) content of the fragment near the assay’s 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 ≈18% ssDNA.

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Data availability

The summary ddPCR and cdPCR data used are presented as Tables S1 and S2 in the ESM. The aA, aB, and aC materials are components A, B, and C of SRM 2372a and are available for purchase from NIST through https://www.nist.gov/srm. The spreadsheet-based cdPCR Staircase analysis system is available on request from the corresponding author.

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Funding

This work was supported in part by the NIST Special Programs Office project Forensic DNA.

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Authors and Affiliations

  1. Biomolecular Measurement Division, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8314, USA

    Margaret C. Kline

  2. Chemical Sciences Division, Material Measurement Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Stop 8390, Gaithersburg, MD, 20899-8390, USA

    David L. Duewer

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  1. Margaret C. Kline
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Correspondence to David L. Duewer.

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The authors declare that they have no conflict of interest nor competing interests.

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All work presented has been reviewed and approved by the National Institute of Standards and Technology Human Subjects Protections Office. This study was determined to be “not human subjects research” (often referred to as research not involving human subjects) as defined in U. S. Department of Commerce Regulations, 15 CFR 27, also known as the Common Rule (45 CFR 46, Subpart A), for the Protection of Human Subjects.by the NIST Human Subjects Protection Office and therefore not subject to oversight by the NIST Institutional Review Board.

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Kline, M.C., Duewer, D.L. Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness. Anal Bioanal Chem 412, 4749–4760 (2020). https://doi.org/10.1007/s00216-020-02733-2

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  • DOI: https://doi.org/10.1007/s00216-020-02733-2

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