Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms

Abstract

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.

Multiplex STR amplification sensitivity in a silicon microchip

“…Not surprisingly, in one of the 125 pg containing triplicate samples several dropouts were observed, all of which were longer than 250 bp. The results presented here are in accordance with the comparative study presented by Butts and Vallone 41 in which six different benchtop thermal cycling platforms were tested for their potential to perform rapid PCR amplification in a forensic setting. Using an identical primer set, the sensitivity presented here, using the on-chip amplification, is slightly better then what was reported for these six thermal cyclers.…”

Results of the 2018 Rapid DNA Maturity Assessment

“…Over the last decade, there have been many advances to reduce time within the DNA typing process and the laboratory‐sized footprint . These advances include rapid PCR protocols for short tandem repeat (STR) typing , advancements in microfluidic efforts for PCR , and separation and detection . All these advancements aided in the several parallel efforts made to integrate the forensic STR analysis workflow and utilize a simple “swab in, answer out” process within a single instrument .…”

The future of forensic DNA analysis

“…There are technological limits with any technique, which in the case of PCR amplification is a combination of the polymerase biochemistry, primer binding kinetics and the thermal cycling device. The fastest full 15-locus STR profiles generated to-date require 14 min of multiplex PCR amplification with 28 cycles of heating and cooling, which can lead to DNA profiles being produced in a laboratory setting in less than an hour [32]. While instruments may exist to heat and cool small volumes of liquid more quickly, sufficient time for polymerase extension of multiple DNA templates is required to obtain useful DNA profiles.…”