Eskandarion M, Najafi M, Akbari Eidgahi M, Alipour Tabrizi A, Golmohamadi T
Biochemistry Department, Tehran University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Research Center, Biochemistry Department, Iran University of Medical Sciences, Tehran, Iran.
J Med Life. 2015;8(Spec Iss 4):180-185.
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. 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. 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). 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.
短串联重复序列（STR）在用于个体识别的人群中个体间存在显著差异。有多种分析这些STR位点的方法，毛细管电泳法已被用作国际标准。由于该过程成本高昂，本研究旨在建立一种针对某些标准STR位点的多重PCR方法，以便我们能够将其PCR产物用于采用高效液相色谱法、气相色谱 - 质谱法和毛细管电泳法等不同方法的STR分析。根据美国国家标准与技术研究院（NIST）的标准，从两组四个典型的用于识别的STR位点（CSF1PO、VWA、D18S51、五聚体D和TPOX、牙釉蛋白、FGA、SE33）中，依据其大小进行选择。上述SSR引物从Genbank中获取，并根据其大小设计单重PCR。然后，随着温度条件、镁离子、引物浓度的变化以及设置，对四个标记进行热启动多重PCR。PCR产物在3%的琼脂糖凝胶电泳上进行检测，并通过基因分析仪分析基因分型结果。结果表明，在所研究的所有STR位点在62° - 66°的温度和1.5 mM镁离子条件下作为单重PCR均可检测到。此外，多重PCR结果表明，当在固定镁离子浓度下测量引物浓度和温度时，CSF1PO和D18S51位点的条带比预期的弱。使用标准缓冲液并针对引物浓度和温度的变化设置镁离子条件，当在94°的温度下将Taq聚合酶加入试管时，多重PCR条带清晰可见。在所有八个位点均获得了毛细管电泳基因分型结果，其中FGA位点的等位基因多样性最高，TPOX和CSF1PO位点的等位基因多样性最低。TPOX和CSF1PO位点的等位基因频率最低，FGA位点的等位基因频率最高。此外，关于使用PowerStats V12软件确定识别的统计指标，CSF1PO位点的随机匹配概率（RMP）最高（0.219），FGA位点的杂合度最高（100%）且多态性率（PIC）最高（0.82）。本研究中进行的设置表明，通过四个标记的两步多重PCR程序，可以对八个位点进行PCR，无需额外的实时产物，这表明在适当的条件下，我们能够将其PCR产物用于采用高效液相色谱法、气相色谱 - 质谱法和毛细管电泳法等不同方法分析STR。此外，就高PD指数和高多态性而言，FGA位点在研究人群中被确定为最佳识别位点。
