VALIDATION OF MULTIPLEX POLYMORPHIC STR AMPLIFICATION SETS DEVELOPED FOR PERSONAL IDENTIFICATION APPLICATIONS

Polymorphic short tandem repeat (STR) loci, which typically consist of variations in the number of 3-7 base pair repeats present at a site, provide an effective means of personal identification. Typing can be a ccomplished by amplification of genomic DNA using the polymerase chain reaction (PCR) and locus-specific primers, separation of amplified al leles using gel electrophoresis and their display using silver stainin g or fluorescent detection. Primers for several STR loci can be combin ed in a single multiplex reaction so typing of multiple loci can be ac complished rapidly and with less DNA than required if each locus were analyzed separately. Before such multiplex systems are used in forensi c or paternity applications, it is desirable that they undergo testing for their reliability. This study evaluates the performance of two ST R tripler systems, one containing the loci HUMCSF1PO, HUMTPOX, and HLT MTHO1, and the other containing HUMHPRTB, HUMFESFPS, and HUMVWFA31. Pr otocols for amplification of these two triplexes, and their correspond ing monoplexes, were evaluated for sensitivity of detection, resistanc e to changes in the annealing temperature of the amplification protoco l, and the ability to identify the minority contributor in amplificati on of mixed samples. In addition, five laboratories determined the all eles of twenty DNA samples, each extracted by one of four different ex traction methods. The results illustrate that the two STR tripler syst ems and the monoplex systems contained within them can be used with as little as 0.25 ng of DNA template. Both triplexes amplified with 100% success using the Perkin Elmer Model 480 thermal cycler. With the Gen eAmp 9600 System, the CTT tripler amplified with 100% success and the HFv tripler in 95.6% of attempts. These experiments meet many requirem ents for use in validation of DNA typing systems for forensic cases an d paternity identification.