Open AccessEvaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness
Author(s) -
Margaret C. Kline,
David L. Duewer
Publication year - 2020
Publication title -
analytical and bioanalytical chemistry/analytical and bioanalytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.86
H-Index - 166
eISSN - 1618-2650
pISSN - 1618-2642
DOI - 10.1007/s00216-020-02733-2
Subject(s) - digital polymerase chain reaction , dna , chemistry , cytosine , polymerase chain reaction , computational biology , microbiology and biotechnology , analytical chemistry (journal) , biology , chromatography , biochemistry , gene
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: A 260 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. Graphical abstract.