Pat Wojtkiewicz
North Louisiana Crimalistics Laboratory, 1115 Brooks Street, Shreveport, LA 71101

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Multiplex PCR has made DNA analysis more efficient by reducing the number of amplifications and polyacrylamide gels necessary to analyze cases. Crime laboratories usually analyze one or two evidence samples and two references in typical cases. Although not a serious inconvenience, this results in having two or more cases on a single gel. However, if several multiplexes could be run concurrently (side-by-side) on a single gel, then results from each case could be conveniently stored in one location. This process could be further improved if the PCR setup and amplification could be done for several multiplexes simultaneously. The objective of this investigation was to develop a protocol by which samples could be concurrently amplified and then run on one polyacrylamide gel. The triplexes, CTT and FFV, were amplified using conditions recommended by Promega. A third triplex (amelogenin, F13B, and LPL) was developed by empirically determining an optimum ratio of the three primer sets. All amplifications were carried out using a hot-start procedure. After amplification, the PCR products were run on a 3.0% Metaphor XR agarose gel to quantify the amplification yield. The yield values were used to determine the amount to load on the analytical polyacrylamide gel. Sample loading was staggered to allow all three triplexes to arrive at the bottom of the gel at the same time. The electrophoresis was carried out by limiting Watts and temperature. The gels were silver stained at the conclusion of the run, then scanned to evaluate the results by computer imaging. The results were clear for all phenotypes tested. Alleles that exhibit microheterogeneity, such as the TH01 9.3 and the F13A01 3.2 were examined closely to evaluate the resolution of the electrophoresis system. The TH01 9.3 allele is easily resolved from the TH01 9 allele, as is the F13A01 3.2 from the F13A01 4. Potentially, a more difficult problem is resolving the TH01 9.3 and TH01 10 alleles, where the size difference is a single base. These two alleles migrate very closely, but can be resolved into four bands with this system. An important point is regulating the amount of DNA loaded on the gel so that all bands stain with equal intensity. Mixtures of DNA were evaluated to identify potential problems that could be encountered by using a minimum amount of DNA on the polyacrylamide gel.