Validation of Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

Abstract

This project was a continuation of a previous project that examined various polymerases to assess their suitability for low volume, fast PCR amplification of forensic STR loci. The primary focus of this project is to develop multiple protocols using the product selected from the previous study (KAPA2G™ Fast Multiplex PCR Kit) along with primer sets and non-fast thermal cyclers commonly used in the forensic community in order to substantially reduce PCR time without added costs or equipment. Ultimately, 3µl fast PCR protocols were validated for the PowerPlex® 16 HS System, AmpFℓSTR® Identifiler® and Identifiler® Plus PCR Amplification Kit primer sets on a 384-well Veriti® thermal cycler (43-51min), as well as 5μl and 6μl Identifiler® fast reactions on a GeneAmp® PCR System 9700 thermal cycler (51min). Two-step PCR cycling utilizing a combined annealing/extension step was successfully incorporated into the fast PCR protocols for all primer sets, except PowerPlex® 16 HS. These protocols were validated for use with forensic reference-type samples, including buccal swabs or Buccal DNA Collectors™, based on optimal DNA input range (sensitivity, reproducibility, inter-and intra-locus peak balance, stutter, pull-up, incomplete adenylation (-A), and baseline assessment), stochastic, precision, stutter, automation, contamination, lot-to-lot variation and storage condition studies. Overall, a 56-73% reduction in amplification time was achieved compared to low volume, standard PCR amplification, along with STR profile pass rates of 95-98% (n=86-89) using these low volume, fast PCR protocols, all of which had an optimal DNA input range of 0.375-1.5ng. Higher percent stutter was observed compared to that of standard PCR, but non-specific amplification, incomplete non-template adenylation, and poor intra-locus peak balance were not problematic compared to other previous studies involving fast PCR.

Alternative direct‐to‐amplification cell lysis techniques for forensically relevant non‐sperm cells

“…Finally, the interlocus balance was examined by calculating the CV of LPH:TPH for each sample and then averaging for each experimental group. Based on the available literature for this method, a well‐balanced profile would exhibit a CV of 0.35 or less [42]. Peak heights across loci within a given profile should be similar in order to assure that all allele peaks are sufficiently above the analytical threshold so that true homozygosity can be confidently determined, as well as to enable more accurate differentiation of peaks from different individuals in a DNA mixture [46, 47].…”

“…By measuring interlocus balance, peak height variation for each locus was compared to the average peak height across the entire profile. The desired CV should be ≤0.35, indicating that the peak heights at a specific locus do not differ more than 35% from those at other loci in the DNA profile [42].…”

Alternative direct‐to‐amplification sperm cell lysis techniques for sexual assault sample processing

“…The standard deviation of all LPH:TPH ratios was then calculated and divided by the mean of those ratios to calculate the CV. Based on the available literature, ideal CV values when using this method are ≤0.35, which signifies the peak heights at any given locus vary no more than 35% from peak heights at other loci within the same DNA profile [55]. For the mean peak heights, the mean percentage of STR alleles detected, and the mean CV of LPH:TPH, an ANOVA was performed to compare the results of the control method to the six additional lysis methods evaluated (= 0.05).…”

“…The standard deviation of all LPH:TPH ratios was then calculated and divided by the mean of those ratios to calculate the CV. Based on the available literature, ideal CV values when using this method are ≤0.35, which signifies the peak heights at any given locus vary no more than 35% from peak heights at other loci within the same DNA profile [55].…”

“…This technique could also be used for direct amplification of semen, which may be needed for microdevice-based, rapid F I G U R E 4 Mean interlocus balance (CV of LPH:TPH) for samples processed with the seven alternative lysis methods (n = 10). The optimal CV (≤0.35) is represented by the red line [55]. STR profiles of samples lysed with the HTX method exhibited significantly higher CV (i.e., worse interlocus balance) than all other lysis methods tested (p < 0.00005).…”