Comparison of polymerases used for amplification of mitochondrial DNA from challenging hairs and hairs of various treatments. (May 2021) - Damaso, Natalie
Abstract: Forensic DNA analysis of hair evidence typically involves the amplification and sequencing of the control region (CR) of the mitochondrial genome (mtgenome). In compromised hair samples, such as shed hairs, the number of mtgenome copies could be low; thus, it is imperative that the polymerase used in PCR is efficient to ensure maximum amplification. Considering this, the first phase of this study compared the yields obtained from 12 polymerases (sourced from a range of commercial companies) when amplifying the CR, hypervariable (HV) region II (HV2), and hypervariable subregion II-B (HV2B). This initial assessment was performed using mitochondrial DNA (mtDNA) extracted from 2 cm of hair adjacent to the root from three donors of different self-reported ancestries and hair color/texture. PrimeSTAR HS and KAPA HiFi HotStart consistently generated significantly higher amplicon yields ( p < 0.05, ~5-fold increase) for most regions than AmpliTaq Gold DNA polymerase (the polymerase validated for use in most forensic laboratories). The second phase of this project was focused on assessing the broad utility of these top two performing polymerases for amplifying two regions of the mtgenome (CR and HV2B) from hair samples representing diverse self-reported ancestral origins (European, Latin American, African American, Asian, and Native American), characteristics/treatments (bleached, dyed, and chemically straightened), and anatomical origins (e.g., head and pubic region) (n = 41). These regions were chosen as they are the most challenging to amplify and sequence in compromised hair samples due to length (i.e., the CR is ~1.2 kb) and repeat structure (i.e., the polycytosine stretch within HV2B). The results indicated that regardless of sample type, PrimeSTAR HS and KAPA HiFi HotStart polymerases outperformed ( p < 0.05) AmpliTaq Gold DNA polymerase (averaging 11- and 8-fold increased yields, respectively). The results from this study highlight that enhanced commercially available polymerases appear to significantly improve the amplification of mtDNA from challenging hair samples. Highlights: mtDNA amplification yields were compared for 12 polymerases when analyzing human hair shafts. Best performing polymerases were further assessed with challenging hair samples. KAPA HiFi HotStart and PrimeSTAR® HS consistently outperformed the validated polymerase.