This NIJ-funded research examined the effects of PCR (Polymerase Chain Reaction) inhibitors on the DNA polymerase enzyme from alternative Thermus acuaticus (Taq) mutants, and it explored an approach to eliminate the need to extract and purify DNA prior to PCR, which decreases the time required, lowers the cost, and increases the efficiency of forensic DNA typing.
The project, which was conducted by DNA Polymerase Technologies, developed novel, genetically engineered mutants of Taq DNA polymerase that are highly resistant to PCR inhibitors. In addition, buffers and PCR enhancer cocktails (PECs) were optimized to be compatible with the novel Taq polymerases. Protocols for STR genotyping of crude samples were designed to be compatible with the primers and cycling conditions of both PowerPlex 16 HS (Promega) and AmpFLSTR Identifiler Plus, the two most commonly used kits in forensic DNA labs. The master mix provided with each kit was replaced with the project's master mix containing the alternative Taq mutants, along with an optimized buffer and PEC additions. In comparative tests of direct STR typing with challenging crude samples, the master mix outperformed comparable mixes from name-brand suppliers.