Optimizing denaturing HPLC as a robust technique for identification of Short Tandem Repeats (STR) in forensic medicine.

Short Tandem Repeats (STRs) are defined as short lengths of 2-7 base pairs spreading through human genome which due to their highly diverse individually distribution are widely applied for identity detection and other forensic medicine purposes. Burdening considerable costs by the conventional methods such as capillary electrophoresis, we aimed to compare concomitant usage of multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) as cheap, fast, highly accurate, and more accessible methods, with capillary electrophoresis (CE) to evaluate their potential for early screening of STRs. The present study randomly included 20 blood samples from the subjects referred to forensic medicine of Semnan, Iran. According to the size and allele frequency, we selected 8 major STR loci including CSF1PO, VWA, D18S51, TPOX, Amelogenin, FGA, SE33, and Penta D. A quad-STR multiplex PCR was performed for each locus and the PCR products were then analyzed using DHPLC machine and compared with the basic genetic properties obtained by capillary electrophoresis. By optimizing the PCR and DHPLC conditions, our findings suggest this strategy as an effective method for STR detection. The genotypes were determined using size of loci which led to comparable results with capillary electrophoresis confirming an insignificant variation in the detection of TOPX, Amelogenin, CSF1PO, and D18S5 (p = 0.331), but discrepant results for FGA and VWA loci (p = 0.002). Our study proposed DHPLC method as an effective screening method to characterize TOPX, Amelogenin, CSF1PO, and D18S51 as frequently used STR loci during identity detection in forensic medicine.

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