This report describes the procedures and outcome of efforts to increase the accuracy of multiplex STR genotyping of low copy number (LCN) DNA evidence (100 pg or less), using degenerate oligonucleotide-primed PCR (DOP-PCR), which is one form of whole genome amplification (WGA).
The method developed significantly improved STR allele success with LCN DNA samples when compared to traditional STR analysis without preamplification. It produced strong partial or full profiles in many cases in which little to no STR data was previously obtained. Data quality was generally equivalent or superior to traditional STR analysis. The proposed method will provide the forensic DNA community with a relatively easy, inexpensive alternative for analyzing compromised and/or LCN DNA evidence. Data from preliminary studies that used the DOP-PCR preamplification technique showed small increases in STR allele amplification; however, stochastic issues that affected data interpretation were prevalent. This led to the development of a modified DOP-PCR technique (dcDOP-PCR). Experiments included varying the number of nonspecific cycles in the initial phase of the DOP-PCR reaction, as well as altering the degeneracy of the DOP-PCR primer and adding proofreading polymerases to the reaction mixture. Data generated under these experimental conditions were compared to data collected by using the standard DOP-PCR approach, which includes a 6N degenerate primer, five nonspecific cycles and Taq polymerase only. The 10N degenerate primer, 12 nonspecific cycles, and the addition of DeepVent proofreading enzyme in the DOP-PCR reaction all significantly increased the number of alleles successfully amplified and detected. Further, these modifications, when combined, lowered the rate of sporadic additional allele occurrence (drop-in) when compared to the previous DOP-PCR results. Additionally, intra-locus heterozygote peak ratios were consistently 0.6 or greater for most LCN DNA samples examined. 11 tables, 21 figures, 55 references, and a listing of publications that disseminate the research findings