P. Sean Walsh, Katherine Lazaruk, Jeanette Wallin and James Robertson
Perkin-Elmer Applied Biosystems, Foster City, CA

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The development of a complete system for typing multiplex STR loci involves several important considerations to assure robust, specific amplification and simple genotyping and interpretation across multicolor detection platforms. Our laboratory is currently developing fluorescent triplex STR systems for using in forensic casework. The loci are combined into triplex groups that are defined by the dye color used, as follows: FGA, vWA and D3S1358 (Blue); CSF1PO, TH01, TPOX and Amelogenin (Green); D7S820, D13S317 and D5S818 (Yellow); D18S51, D21S11 and D8S1179 (Green). For each of these triplex systems, primer modification is often necessary to achieve maximum balance between the loci, specific amplification, and maximum A addition; examples of each will be discussed. The role of AmpliTaq Gold™ DNA polymerase in providing high specificity and the optimization of the PCR reaction to accommodate this enzyme will also be presented. The goal in optimizing each triplex is to integrate the primer design, PCR Mix and thermal cycling conditions so that the conditions for each triplex are the same, and so that the results give the desired sensitivity (at least 1 ng), specificity, and locus balance. The progress to date for each triplex system will be presented.

Another challenge in developing a complete system is to ensure compatibility of results between the different multicolor detection platforms (ABI PRISM™ 373, 377, 310 instruments) so that genotyping is accurate and simple. Because alleles can size differently on different detection platforms, allelic ladders are used to standardize the results such that genotypes can be compared between the instruments. Using size bins based on the allelic ladder sizes (for each gel or group of capillary electrophoresis runs) allows for completely discrete allele designations for all of the STR loci, even when the alleles differ in size by only one bp (as for the TH01 9.3/10 alleles). Results will be presented indicating that alleles of the same length that differ in sequence do not show size mobility shifts relative to the allelic ladder when run under denaturing conditions. Sequence analysis of vWA alleles having an unusual repeat structure and the relationship to the proportion of observed stutter band will also be presented. Sequence results from alleles contained in the allelic ladder will also be summarized.