Development of a normalized extraction to further aid in fast, high-throughput processing of forensic DNA reference samples

Abstract

The goal of this project was to develop a “normalized” extraction procedure to be used in conjunction with previously validated 3μL fast PCR reactions (42–51min utilizing KAPA2G™ Fast Multiplex PCR Kit) and alternative capillary electrophoresis (24–28min injection using POP-6™ Polymer and a 22cm array). This was the final phase of a workflow overhaul for the database unit at Cellmark Forensics to achieve a substantial reduction in processing time for forensic DNA database samples without incurring significant added costs and/or the need for new instrumentation, while still generating high quality STR profiles. Extraction normalization aimed to consistently yield a small range of DNA concentrations, thereby eliminating the need for sample quantification and dilution. This was specifically achieved using the ChargeSwitch® Forensic DNA Purification Kit and a reduction in extraction bead quantity, thereby forcing an increase in bead binding efficiency. Following development of this extraction procedure, an evaluation ensued to assess the combination of normalized extraction, 3μL fast PCR (with PowerPlex 16 HS, Identifiler Plus and Identifiler primer sets), and alternative CE detection – further referred to as new “first pass” procedures. These modifications resulted in a 37% reduction in processing time and were evaluated via an in depth validation, from which nearly 2000 STR profiles were generated, of which 554 profiles from 77 swab donors and 210 profiles from 35 buccal collector donors specifically arose from the new first pass procedures. This validation demonstrates the robustness of these processes for buccal swabs and Buccal DNA Collectors™ using the three primer sets evaluated and their ability to generate high quality STR profiles with 95–99% and 88–91% pass rates, respectively.