Optimization and validation of a fast amplification protocol for AmpFlSTR® Profiler Plus® for rapid forensic human identification

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

“…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.…”

“…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.…”

“…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.…”

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

“…Reducing amplification processing time has been a major focus for forensic DNA testing over the past 10 years, especially through the advent of fast PCR polymerases, direct PCR, and rapid DNA testing [1][2][3][4][5][6][7][8][9][10][11][12]. But such a reduction in processing time has often been subject to an increase in cost due to reagents and/or equipment, and/or prone to a decrease in STR profile quality.…”