Development of a fast PCR protocol enabling rapid generation of AmpFlSTR(R) Identifiler(R)profiles for genotyping of human DNA

Abstract

BackgroundTraditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times.MethodsFast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach.ResultsThe total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA.ConclusionsThe fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.

Assessment of Four Polymerases for Low Volume, Fast PCR Amplification of STR Loci for DNA Reference Samples

“…This range is wider than that osberved by Vallone et al (0.4-1.0ng DNA) using 10μl fast PCR reactions consisting of a combination of PyroStart (Fermentas, Glen Burnie, MD) and SpeedSTAR polymerases [8], but lower than that observed by Foster and Laurin (0.125-2.0ng DNA) using 15μl fast PCR reactions with the SpeedSTAR polymerase [1].…”

“…As has been demonstrated previously [1,5], percent stutter increases using each of the fast PCR protocols, as compared to that of standard PCR. However, use of a 20% global stutter filter or modification of the locus specific stutter thresholds in GeneMapper® ID should prevent excessive stutter peaks from being called by the software, which has already been demon Average percent stutter (n-4), pull-up and -A are displayed, as well as average number of detected stutter (n-4), pull-up, -A, low-level NSA and elevated baseline per profile.…”

“…Using fast thermal cyclers, fast PCR has been achieved in as little as 19-26 min [1,2,5]. In general, fast PCR profiles have often suffered from low level artifacts, increased n-4 stutter percentage and incomplete adenylation (-A) [1,2,5,[8][9][10].…”

“…Four commercially available products were assessed for fast PCR suitability with the Identifiler primer set using a 3μl reaction carried out on a 384-well Veriti® (Veriti; Applied Biosystems) thermal cycler: AmpliTaq Gold® Fast PCR Master Mix (AmpliTaq Gold Fast; Applied Biosystems), KAPA2G™ Fast Multiplex PCR Kit (KAPA2G; Kapa Biosystems Inc., Woburn, MA), SpeedSTAR™ HS DNA Polymerase (SpeedSTAR; Takara Bio Inc., Shiga, Japan) and Type-it Microsatellite PCR Kit (Type-it; QIAGEN, Valencia, CA). Selection of these products was based on a variety of criteria, including low cost, being a non-template adenylator, and discussions with manufacturers to identify which of their products had a high potential to perform fast PCR multiplexing, as well as some previous studies on fast PCR [1,2,5,8]. All of these products are (or contain) fast polymerases, but Type-it was not specifically designed for fast PCR.…”

“…Fast PCR and/or direct PCR are fairly recent improvements from the late 2000s/early 2010s that can be utilized by the forensic DNA community to reduce processing time and/or costs for forensic-type and reference samples [1][2][3][4][5][6][7][8][9][10]. Reduced volume PCR amplification has been shown to improve sensitivity and efficiency [11], but the more reduced the total reaction volume becomes, the more intra-locus peak balance decreases, making extremely low volume reactions likely unsuitable for anything other than single-source reference samples.…”