DNA microsatellite analysis using ion-pair reversed-phase high-performanceliquid chromatography

Genotyping based on short tandem repeat (STR) regions is used in human iden tification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab g el electrophoresis requires lengthy and labor-intensive procedures. Therefo re, alternative methods such as capillary electrophoresis or ion-pair rever sed-phase highperformance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electropho resis for STR analysis because of the reduced analysis time, and there is n o need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for t he sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC c onditions (column temperature, flow rate, percent organic modifier per minu te) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in thei r native state (double stranded) with the use of internal markers. The typi ng results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be p eak captured for further examination following such as sequencing.