High-quality data from a forensically relevant single-cell pipeline enabled by low PBS and proteinase K concentrations

J Forensic Sci. 2022 Mar;67(2):697-706. doi: 10.1111/1556-4029.14956. Epub 2021 Dec 22.


Interpreting forensic DNA signal is arduous since the total intensity is a cacophony of signal from noise, artifact, and allele from an unknown number of contributors (NOC). An alternate to traditional bulk-processing pipelines is a single-cell one, where the sample is collected, and each cell is sequestered resulting in n single-source, single-cell EPGs (scEPG) that must be interpreted using applicable strategies. As with all forensic DNA interpretation strategies, high quality electropherograms are required; thus, to enhance the credibility of single-cell forensics, it is necessary to produce an efficient direct-to-PCR treatment that is compatible with prevailing downstream laboratory processes. We incorporated the semi-automated micro-fluidic DEPArray™ technology into the single-cell laboratory and optimized its implementation by testing the effects of four laboratory treatments on single-cell profiles. We focused on testing effects of phosphate buffer saline (PBS) since it is an important reagent that mitigates cell rupture but is also a PCR inhibitor. Specifically, we explored the effect of decreasing PBS concentrations on five electropherogram-quality metrics from 241 leukocytes: profile drop-out, allele drop-out, allele peak heights, peak height ratios, and scEPG sloping. In an effort to improve reagent use, we also assessed two concentrations of proteinase K. The results indicate that decreasing PBS concentrations to 0.5X or 0.25X improves scEPG quality, while modest modifications to proteinase K concentrations did not significantly impact it. We, therefore, conclude that a lower than recommended proteinase K concentration coupled with a lower than recommended PBS concentration results in enhanced scEPGs within the semi-automated single-cell pipeline.

Keywords: PBS; PCR efficiency; direct-to-PCR extraction; forensic DNA; phosphate buffer saline; scEPG; single-cell electropherogram; single-cell forensic analysis.

MeSH terms

  • Alleles
  • DNA Fingerprinting* / methods
  • DNA* / analysis
  • Endopeptidase K* / genetics
  • Forensic Genetics
  • Microsatellite Repeats
  • Polymerase Chain Reaction / methods


  • DNA
  • Endopeptidase K