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

AbstractCurrent 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 DN A 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, r endered 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 signific...
Source: International Journal of Legal Medicine - Category: Medical Law Source Type: research