A quantitative method to assess DNA extraction efficiency

Abstract

Obtaining an accepted minimum amount of extracted and purified DNA is critical for generating complete forensic short tandem repeat (STR) profiles. Certain sample types encountered in forensic laboratories may pose challenges in obtaining the desired target amount of extracted DNA, which can be compounded when an extraction yields low recovery, resulting in a loss of the genetic material necessary for downstream forensic DNA typing. While multiple studies have evaluated the impact of extraction chemistries on yield, they often focus on “end-to-end” profiling success rather than quantifying the discrete loss of DNA during the extraction step. This study presents a quantitative framework using digital PCR (dPCR) to benchmark extraction efficiency by comparing known amounts of DNA pre- and post-extraction. Extraction efficiency was evaluated using silica spin column and magnetic resin-based protocols across five input amounts, with three sample types: whole blood, human cells, and pre-extracted DNA. Results demonstrate significant differences in efficiency between protocols for cellular samples, with both methods exhibiting increased variability at the 1 ng threshold. This data also revealed that previously extracted DNA (SRM 2372a) fails to accurately represent cellular extraction dynamics, serving instead as a control for purification-related loss (e.g., column retention) rather than lysis efficiency.