M Eskandarion1, M Najafi2, M Akbari Eidgahi3, A Alipour Tabrizi4, T Golmohamadi1. 1. Biochemistry Department, Tehran University of Medical Sciences, Tehran, Iran. 2. Cellular and Molecular Research Center, Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran. 3. Semnan Biotechnology Research Center, Semnan University of Medical Sciences, Semnan, Iran. 4. Iranin Legal Medicine Research Center, Legal Medicine Organization, Tehran, Iran.
Abstract
Background and Objective: Short Tandem Repeats (STR) show considerable differences among individuals in the population from which they used for identification. There are various methods for analysis of these STR loci, and capillary electrophoresis method already used as an international standard. Due to the high costs of this process, this study aimed to set up a Multiplex PCR method in some standard STR loci so that we can use its PCR product in STR analysis with different methods of HPLC, GC-Mass, and Capillary Electrophoresis. Materials and Methods: 8 typical STR loci in the identification selected according to their size in the two groups of four (CSF1PO, VWA, D18S51, PentaD and TPOX, Amelogenin, FGA, SE33) from NIST (National Institute of Standards and Technology). The above SSR primers prepared from Genbank and Monoplex PCR was designed based on their size. Then, with the changes in temperature conditions, magnesium ion, primers concentration, and setting-up, Hot Start Multiplex PCR of four markers was carried out. PCR product investigated on the agarose gel electrophoresis (3%) and the results of genotyping analyzed by Genetic Analyzer. Results: The Results showed that all STR loci under study are detectable as Monoplex PCR at a temperature of 62°-66° and 1.5 mM magnesium ion. Moreover, Multiplex PCR results showed that when the concentration of primer and temperature measured by the fixed concentration of magnesium, CSF1PO, and D18S51 loci bands are weaker than desired. Using a standard buffer and set Magnesium conditions against changes in the primer concentration and temperature, when Taq polymerase enzyme is added to test tubes at a temperature of 94°, Multiplex PCR bands are visible desirably. Capillary electrophoresis genotyping results obtained in all eight loci and the Locus FGA had the most allelic diversity and the loci TPOX and CSF1PO had the lowest allelic diversity. TPOX and CSF1PO loci had the lowest allelic frequencies, and FGA locus had the most allelic frequency. Moreover, about the determination of statistical indicators of identification using PowerStats V12 software, CSF1PO locus allocated the most RMP (0.219) and FGA locus the highest heterozygosity (100%) and the highest polymorphic rate (PIC) (0.82). Conclusion: The setup performed in this study showed that with two-step multiplex PCR procedure of four markers, PCR can be carried out for eight loci without additional real-time products that this shows proper conditions that we can use their PCR product in analyzing SRTs with different methods of HPLC, GC-Mass, and Capillary Electrophoresis. Besides, the FGA locus was raised as the best loci for identification in the study population concerning the high PD index and high polymorphism.
Background and Objective: Short Tandem Repeats (STR) show considerable differences among individuals in the population from which they used for identification. There are various methods for analysis of these STR loci, and capillary electrophoresis method already used as an international standard. Due to the high costs of this process, this study aimed to set up a Multiplex PCR method in some standard STR loci so that we can use its PCR product in STR analysis with different methods of HPLC, GC-Mass, and Capillary Electrophoresis. Materials and Methods: 8 typical STR loci in the identification selected according to their size in the two groups of four (CSF1PO, VWA, D18S51, PentaD and TPOX, Amelogenin, FGA, SE33) from NIST (National Institute of Standards and Technology). The above SSR primers prepared from Genbank and Monoplex PCR was designed based on their size. Then, with the changes in temperature conditions, magnesium ion, primers concentration, and setting-up, Hot Start Multiplex PCR of four markers was carried out. PCR product investigated on the agarose gel electrophoresis (3%) and the results of genotyping analyzed by Genetic Analyzer. Results: The Results showed that all STR loci under study are detectable as Monoplex PCR at a temperature of 62°-66° and 1.5 mM magnesium ion. Moreover, Multiplex PCR results showed that when the concentration of primer and temperature measured by the fixed concentration of magnesium, CSF1PO, and D18S51 loci bands are weaker than desired. Using a standard buffer and set Magnesium conditions against changes in the primer concentration and temperature, when Taq polymerase enzyme is added to test tubes at a temperature of 94°, Multiplex PCR bands are visible desirably. Capillary electrophoresis genotyping results obtained in all eight loci and the Locus FGA had the most allelic diversity and the loci TPOX and CSF1PO had the lowest allelic diversity. TPOX and CSF1PO loci had the lowest allelic frequencies, and FGA locus had the most allelic frequency. Moreover, about the determination of statistical indicators of identification using PowerStats V12 software, CSF1PO locus allocated the most RMP (0.219) and FGA locus the highest heterozygosity (100%) and the highest polymorphic rate (PIC) (0.82). Conclusion: The setup performed in this study showed that with two-step multiplex PCR procedure of four markers, PCR can be carried out for eight loci without additional real-time products that this shows proper conditions that we can use their PCR product in analyzing SRTs with different methods of HPLC, GC-Mass, and Capillary Electrophoresis. Besides, the FGA locus was raised as the best loci for identification in the study population concerning the high PD index and high polymorphism.