TWGDAM Validation of a Nine-Locus and a Four-Locus Fluorescent STR Multiplex System

Abstract

The Gene Print® PowerPlex™ 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex™ 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO® Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from bloodstrains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 × 108 and 1 in 2658 for the PowerPlex™ 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 × 1011. The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex™ 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.