Efficacy and limits of genotyping low copy number DNA samples by multiplex PCR of STR loci

Abstract

In this validation study, we have evaluated the efficacy and the validity of the SGM Plus test using an amplification regime of 34 cycles. We obtained valid DNA typing results from pristine extracts with an extremely low DNA content. In this context, the aspects of single cell PCR typing were also evaluated. In these experiments, the allele dropout phenomenon was clearly demonstrated. From actual casework samples, we obtained conclusive DNA profiles from highly purified extracts of bone and teeth that failed to demonstrate typing results using the standard PCR protocol of 28 cycles. Moreover, low copy number (LCN) DNA typing offered us the possibility to reanalyse crime samples that failed to produce a conclusive profile after 28 cycles. Unfortunately, several complications accompany ultrasensitive PCR amplification. During our validation studies, we have observed increased risk of contamination, allelic dropout, locus dropout and heightened stutters. Analyses of heterozygote balance, between-loci balance and stutter heights, show that the 34-cycle PCR has its own characteristic features. We finally show that reamplification of SGM Plus PCR products by an extra 6 PCR cycles offers a promising new alternative if too little of the original sample extract is left for a complete reanalysis.