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Evaluation of Promega GenePrint™ STR Multiplex PCR Parameters for the GAP 9600Cecelia Crouse, PhD1, Debra Glidewell, BS1, Sue Rogers, BS2
and Stephanie Evans, BS1 One of the most challenging goals when performing the polymerase chain reaction (PCR) is the optimization of amplification parameters in order to maximize the quantity and quality of specific DNA target sequences and to minimize the generation of non-specific amplification products. In order to optimize the amplification of more than one DNA locus in a single reaction, a compromise among the select systems is often necessary. The amplification of DNA sequences using the Promega GenePrint™ STR Multiplex Systems has been designed for use on the Perkin-Elmer 9600 thermal cyclers. Recently, revised PCR programs designed for use on the Perkin-Elmer 9600 thermal cyclers (CTT) and F13A1/FESFPS/vWF (FFV) STR systems were introduced for the GAP 9600 which includes using programmed ramp times instead of default ramp times and the addition of an oil overlay for each microamp tube. Both the Palm Beach Sheriff's Office (PBSO) and the Alabama Department of Forensic Sciences (ADFS) have conducted extensive validation studies based on the original CTT PCR protocols. The results of these studies show that there is virtually no difference between the original and revised CTT PCR parameters as determined by 1) sensitivity assays with as little as 250pg of DNA; 2) analysis of mixed stain samples in which allele data is readily evident at all three CTT loci at a 90:10 ratio; and 3) non-probative sexual assault analysis in which there is female-to-male DNA carry-over. In contrast to the CTT PCR protocol comparisons, the revised FFV PCR parameters offer much greater specificity and sensitivity than the original program. Data comparing the original and new CTT PCR protocols on a variety of forensic-type specimens will be presented. Go to proceedings home page
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