Towards developing forensically relevant single-cell pipelines by incorporating direct-to-PCR extraction: compatibility, signal quality, and allele detection

Int J Legal Med. 2021 May;135(3):727-738. doi: 10.1007/s00414-021-02503-4. Epub 2021 Jan 23.


Current analysis of forensic DNA stains relies on the probabilistic interpretation of bulk-processed samples that represent mixed profiles consisting of an unknown number of potentially partial representations of each contributor. Single-cell methods, in contrast, offer a solution to the forensic DNA mixture problem by incorporating a step that separates cells before extraction. A forensically relevant single-cell pipeline relies on efficient direct-to-PCR extractions that are compatible with standard downstream forensic reagents. Here we demonstrate the feasibility of implementing single-cell pipelines into the forensic process by exploring four metrics of electropherogram (EPG) signal quality-i.e., allele detection rates, peak heights, peak height ratios, and peak height balance across low- to high-molecular-weight short tandem repeat (STR) markers-obtained with four direct-to-PCR extraction treatments and a common post-PCR laboratory procedure. Each treatment was used to extract DNA from 102 single buccal cells, whereupon the amplification reagents were immediately added to the tube and the DNA was amplified/injected using post-PCR conditions known to elicit a limit of detection (LoD) of one DNA molecule. The results show that most cells, regardless of extraction treatment, rendered EPGs with at least a 50% true positive allele detection rate and that allele drop-out was not cell independent. Statistical tests demonstrated that extraction treatments significantly impacted all metrics of EPG quality, where the Arcturus® PicoPure™ extraction method resulted in the lowest median allele drop-out rate, highest median average peak height, highest median average peak height ratio, and least negative median values of EPG sloping for GlobalFiler™ STR loci amplified at half volume. We, therefore, conclude the feasibility of implementing single-cell pipelines for casework purposes and demonstrate that inferential systems assuming cell independence will not be appropriate in the probabilistic interpretation of a collection of single-cell EPGs.

Keywords: Direct-to-PCR; Forensic DNA; Forensic DNA Mixtures; Human identification; Single-cell forensic DNA.

MeSH terms

  • Alleles*
  • DNA / analysis*
  • DNA / isolation & purification*
  • DNA Fingerprinting / methods*
  • Electrophoresis, Capillary
  • Humans
  • Limit of Detection
  • Microsatellite Repeats
  • Mouth Mucosa
  • Polymerase Chain Reaction / methods*
  • Single-Cell Analysis*


  • DNA