Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary...

Capillary electrophoresis (CE) is an analytical technique which provides rapid, high resolution analysis of amplified DNA fragments produced by the polymerase chain reaction (PCR). In this study, two internal standards are used as size markers to bracket und precisely size PCR products. The technique is applied to typing PCR products from the short tandem repeat locus HUMTH01. HUMTH01 consists of five to seven major alleles in the size range of 179-203 bp, with each allele four bp apart. Using this genetic marker, a population containing 97 individuals was examined with both polyacrylamide gel electrophoresis and CE. Identical genotypes were obtained with both techniques demonstrating the reliability of CE in DNA typing applications. The DNA analysis took place in sets of 10 with a calibration of the CE being performed between each set of samples. For the 97 samples examined, the pooled standard deviation was 0.3 bp. The observed genotype frequencies determined from the sample set did not deviate significantly from Hardy-Weinberg expectations. From these CE results, we conclude that HUMTH01 PCR products can be accurately and precisely sized by capillary electrophoresis using the method described.