Validation of AmpFISTR ProfilerÔ STR Multiplex for use in Forensic Casework
James P. Ross, Demris A. Lee, Jeanne
M. Willard, Richard E. Wilson and Mitchell M. Holland
Armed Forces DNA Identification Laboratory, Armed Forces Medical Examiner, Armed Forces
Institute of Pathology, Rockville, MD
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PCR-based STR typing is a widely used tool in forensic DNA typing. Recently, a fluorescent based "Mega" plex has become commercially available. The AmpFISTR ProfilerÔ amplification kit (PE-ABD) is a complete STR typing kit that utilizes AmpliTaq GoldÔ DNA Polymerase. Profiler consists of nine tetrameric STR loci (5-Fam –D3S1358, vWA, FGA; JOE-TH01, TPOX, CFS1PO; NED-D5S818, D13S317, D7S820) which are co-amplified with the X-Y homologous gene Amelogenin (JOE) for sex determination giving a combined frequency of 1 in 3 billion.
AFDIL has completed validation studies on the Profiler system according to TWGDAM guidelines for use in forensic DNA typing. The STR system was validated on the ABI PRISMÔ 377 DNA Sequencer using 36 cm WTR 5% Long Ranger®, 6M urea gels. A variety of studies were performed which included inter and intra gel sizing comparisons, sensitivity assays, mixture studies, reproducibility, standard specimen studies, and analysis of non-probative casework samples. To determine the sensitivity of the system, dilutions ranging from 10.0 ng to 80 pg were analyzed in duplicate. Both bloodstain mixtures and mixtures of extracted DNA were evaluated at different ratios (1:1 to 1:20) in order to determine a lower limit of mixture detection. In order to evaluate the reproducibility and consistency of different specimens from the same individual, hair, buccal scrapes, blood and semen specimens were collected from 5 individuals. These samples were either organically extracted or extracted using the Chelex® 100 method and subsequently analyzed by 3 different individuals. Specimens from 5 different non-probative cases, which were previously typed using a variety of STR loci, were used to evaluate the systems performance on typical casework specimens. These specimens included tissue, fresh bone, paraffin blocks/cytological slides and bloodstains. The results from the non-probative case samples were automatically genotyped using Genotyper® software (PE-ABD). Samples from the validation study were also run on the ABI 310 Genetic Analyzer for comparison purposes. A summary of the results for these studies will be presented.
The opinions and assertions expressed herein are solely those of the authors and are not to be construed as official or as the views of the United States Department of Defense or the United States Department of the Army.
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