STR Typing With High Performance Liquid Chromatography

Objective The purpose of our work was to study the STR typing method with HPLC(high performance liquid chromatography) and to comprehend the rules of STR typing with HPLC. Methods Firstly, all alleles of D10S2325 were found and mixed to construct an allele marker. Secondly, the HPLC conditions (column temperature, analyzing time) to separate all of the alleles were optimized. The stability of our method was studied. Thirdly, STR was typed by comparing their retention time of HPLC with the allele ladder. Our method was validated by typing same samples with PAGE(polyacrylamide gel electrophoresis). Lastly, the sensitivity to type single sample and the ability to type mixed sample of our method were tested. Results The method of STR typing with HPLC was successfully established.The optimized temperature was 50℃ and the analyzing time was about 8 minutes. It was observed that analyzing STR with HPLC was stable in the same batch of samples but variable among different batch of samples, especially after making up the buffer newly, so that the allele ladder should be compared with sample in the same batch. It was also observed that the retention time was affected regularly by the variation of the sample concentration. The affection was revised by an equation of liner regression. The result of typing 20 samples with HPLC was in accordance with that of PAGE. This method was able to type single sample contained at least 6.25ng DNA template and mixed samples with the ratio of 1 to 1. Conclusion The method of typing STR with HPLC established by us was successful. It was necessary to consider both the chromatographic conditions and the variation of retention time caused by the sample concentration when we analyzed STR with HPLC.