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Rapid PCR Protocols for Forensic DNA Typing on Six Thermal Cycling Platforms

Published

Author(s)

Peter Vallone, Erica Romsos

Abstract

Rapid PCR protocols for the amplification of typing short tandem repeat multiplexes were evaluated on 6 different thermal cyclers. PCR primers from the commercially available multiplex short tandem repeat typing kit Identifiler were used to target 15 STR loci plus the sex-typing maker Amelogenin. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to a little as 14 minutes. Previously described 2- step and 3-step thermal cycling protocols were evaluated for the 6 thermal cyclers on 95 unique single-source DNA extracts. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus 4 stutter ratios on averages were 30 % to 40 % higher than for standard Identifiler PCR conditions. Non-specific 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 pg 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 databasing laboratory.
Citation
Electrophoresis

Keywords

forensic DNA, multiplex PCR , rapid PCR, STR, thermal cycler

Citation

Vallone, P. and Romsos, E. (2014), Rapid PCR Protocols for Forensic DNA Typing on Six Thermal Cycling Platforms, Electrophoresis, [online], https://doi.org/10.1002/elps.201400179, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=916080 (Accessed August 12, 2022)
Created August 21, 2014, Updated October 15, 2021