George R. Riley, PhD, Teresa Johnston, BS, Tia Aulinska, PhD and Howard Coleman, BS, BA
GeneLex Corporation, 2203 Airport Way South, Suite 350, Seattle, WA 98134
We have evaluated the species specificity of the CSF1PO/TPOX/TH01 (CTT) multiplex kit (Promega) and the D1S80 kit (Perkin Elmer) as part of validation for both forensic and paternity casework. In addition, the prototype F13A01/FESFPS/vWF (Ffv) STR multiplex kit (Promega) was evaluated.
The species specificity of the CTT kit (Promega) and the D1S80 kit (Perkin Elmer) were tested using DNAs extracted from 27 different species, including: 14 non-human primates composed of higher order primates and both Old and New World lower order primates, 8 non-primate species, and 5 microbial species. Standard validation specimens included genomic DNAs from various tissues and stains from a number of individuals, specimens obtained externally for interlab comparison, DNA from mixed specimens, degraded DNA, specimens from non-probative evidence and from previously adjudicated cases. Consistency was tested both by repetitive amplification of individual samples, and testing different tissue types. The ability to discern mixed samples was tested both using actual mixed specimens and series of mixed neat DNAs. Assay sensitivity was tested both by serial DNA dilution and testing the ability to amplify degraded DNA. Many of these DNA specimens were typed with the Polymarker kit for comparison and to confirm D1S80 and STR results.
PCR amplifications with the D1S80 kit (Perkin Elmer) and the CTT and Ffv kits (Promega) were as described by the manufacturer. D1S80 amplicon was separated on 0.4mm nondenaturing GeneAmp Detection Gel (Perkin Elmer) proprietary polyacrylamide in TBE and STR amplicons were separated on 0.4mm PAGE-urea gels. Amplified alleles were detected by silver staining (Promega).
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