Fast Multiplexed Polymerase Chain Reaction for Conventional and Microfluidic Short Tandem Repeat Analysis

Abstract

The time required for short tandem repeat (STR) amplification is determined by the temperature ramp rates of the thermal cycler, the components of the reaction mix, and the properties of the reaction vessel. Multiplex amplifications in microfluidic biochip-based and conventional tube-based thermal cyclers have been demonstrated in 17.3 and 19 min, respectively. Optimized 28-cycle amplification protocols generated alleles with signal strengths above calling thresholds, heterozygous peak height ratios of greater than 0.65, and incomplete nontemplate nucleotide addition and stutter of less than 15%. Full CODIS-compatible profiles were generated using the Profiler Plus ID, COfiler and Identifiler primer sets. PCR performance over a wide range of DNA template levels from 0.006 to 4 ng was characterized by separation and detection on a microfluidic electrophoresis system, Genebench-FX. The fast multiplex PCR approach has the potential to reduce process time and cost for STR analysis and enables development of a fully integrated microfluidic forensic DNA analysis system.

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

“…Therefore, it was a goal of this study to demonstrate to other laboratories that thermal cyclers that are commonly used in forensic DNA testing are suitable for fast PCR. Fast thermal cyclers can achieve shorter amplification times (as low as 19 min [2]) than the non-fast ones used here (43min to >1hr on the Veriti), but the need for such a short amplification may not be justified for a particular laboratory and, therefore, may not warrant such an expense.…”

“…A longer denaturation time was attempted, as suggested by Giese et al, [2] and Walsh et al, [20], as were a 3-step PCR cycle (which helped to remedy low PHR with AmpliTaq Gold) and longer annealing/extension times (which was helpful with KAPA2G), but none of these, or other potential solutions, resolved the frequently low PHR. Similar to AmpliTaq Gold Fast as well, -A was not always prevented using a 10min final extension, so 13min was utilized.…”

“…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.…”