GENOTYPING OF A SHORT TANDEM REPEAT SYSTEM USING ION-PAIR REVERSE-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

Ion pair-reverse phase high performance liquid chromatography (IPRP HPLC) is a method that can be used for the genotyping of short tandem repeats (STRs) in human nuclear DNA. The genotyping of STRs is useful in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. IPRP HPLC offers an attractive method for STR analysis because of the short analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHOl locus on chromosome 11p15.5. The IPRP HPLC conditions (column temperature, flow rate, and percent organic modifier per minute) were optimized for the separation of PCR products in the size range of 50–434 base pairs. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double stranded) with the use of internal markers. The analysis time for the HUMTHOl locus was less than 14 minutes, and the alleles could be peak captured for further examination such as sequencing.