ACCURATE SIZE COMPARISON OF SHORT TANDEM REPEAT ALLELES AMPLIFIED BY PCR

A strategy is presented for classifying complex short tandem repeat (S TR) alleles by size. Such alleles can differ in length by only 1 bp. T he HUMACTBP2 locus was used as a model. Dye-labeled, PCR-amplified all eles were analyzed on an automated DNA sequencer with laser-induced fl uorescence detection and fragment-sizing software. Between-gel allele sizes calculated against an in-lane allelic ladder or viral DNA size s tandard were too imprecise to distinguish a 1-bp difference. However, the size difference between a sample allele and its matching ladder al lele provided a reliable criterion for size classification. The mean d ifference +/-3 SDs was 0.5 bp, and so an individual result within this interval signified a match. Statistically, 99.7% of the results shoul d lie within +/-3 SDs with virtually no chance of encountering the 9-S D difference from the mean necessary to misclassify an allele by 1 bp. The method was valid for sample alleles sized against the allelic lad der and for both sample and ladder alleles sized against the viral DNA standard. A correction for the effect of different dye labels on mobi lity rt as included in the calculations.