Investigation Of Genetic Polymorphism Of The Four MiniSTR Loci In Chongqing

Objective: DNA Short tandem repeat(STR),due to its high degree ofgenetic polymorphisms an simplicity of methods, becomes the mainresearch methods of forensic science, especially in paternity testing andpersonal identification. Probably because of the extensive application ofSTR, expose some of the shortcomings of its, which is the limitations of thesamples(must be a certain number quantity of DNA), and in micro-materialevidence. Usually the CODIS system STR fragments is large than200bp,soit is difficult to amplification in some samples, and so we reduce the size ofthe STR, without affecting the STR repeat region, be able to increase theexamine of the STR, so that is why we use MiniSTR.. MiniSTRamplification primers were designed closer to the core repeat sequencesflanking sequences, so that the entire size of the amplification productsonly150kb or so, which would reduce the difficulty of DNA extraction toimprove the DNA detection rate. Because the core sequence did not change,so MiniSTR locus still maintain the genetic polymorphism of the STRfragments, with all the advantages of STR. Also, MiniSTR kit used in somemajor disaster, like911. highlighting the advantages of small fragments of personal identification. And MiniSTR loci have been DNA typingEuropean Working Group (EDNAP) is defined as a new generation ofgenetic markers. In2006, AB company introduced a commercial of theMiniSTR kit minifiler, including the sex genes, including nine CODISsystem, but because the CODIS system is limited, and most of theinappropriate redesign the primers, Therefore, the development of sitesoutside of the CODIS system to become the focus of the study. China hasalso been the sites in the CODIS system, indicates that the regions aresignificantly different in Central China, Hunan and other places, so there isthe need for systematic investigation of different parts of the non-CODIS,the lack of Chongqing area. In order to provide a research for domestic kit,the multiplex including D1S1627, D2S441, D10S1248, D22S1045，the fourMiniSTR locus for four-color fluorescent labeled, and for the production ofthe allelic ladder, and investigate the genetic polymorphism of fourMiniSTR in Chongqing city.Methods: Using chelex100to the extract170Chongqingregion-independent healthy individuals the whole gene group of DNA.willbe of PCR amplification system for10μL, will be the reaction system,primer concentrations, Mg2+concentration, cycle number, annealingtemperature, of dNTP, of DNA Taq enzyme are carried out optimized toidentify the most suitable reaction conditions, and then polyacrylamide gelelectrophoresis (PAGE) separation of amplification products by silver staining, and select the appropriate allele after gel DNA recovery, recycledgoods, again amplified purification, re-polyacrylamide gel electrophoresisand sequencing, will be named in accordance with the InternationalTribunal for blood genetics (ISFH) recommended naming will be adjustedby adjusting the concentration of the primers electropherogram peak, toreach consistent. The Allele ladder of the laboratory-made are used toanalyse the samples of genetic research. Mark the primers D10S1248,D2S441and D1S1627, D22S1045upstream primer5’end andTAMRA,6-FAM, HEX, respectively. Homemade allele of standard materialand primers with the above electrophoresis in the ABI3130sequencer,genotyping analysisResults: fluorescence labeled multiplex typing system to build asuccessful. The10μL the multiplex PCR system:10×PCR buffer:1μl,1mmol/L; of MgCl2:0.6μl,25mmol/L; of dNTP:1μl,2.5mmol/L;D1S1627andD2S441, upstream and downstream the primers0.20μl,10μmol/L; D10S1248, D22S1045, downstream primer each0.3μl,10μmol/L of Taq DNA polymerase0.15μl L (5U/μl) template DNA1μl(1ng/μL), sterilized double distilled water2.55μl. PCR amplificationparameters: thermal cycling parameters for95°C: predenaturation3min,denaturation94℃30s, annealing59℃30s, extension at72℃for60s,32cycles of72℃15min. Every loci of the3fluorescent labeled MiniSTRmultiplex amplification system can be genotyped clearly.6,9,9,9alleles and 107genotypes were obtained from the genotyped data from blood sampleof170unrelated Han Chinese in Chongqing, and the distribution of thegenotypes was according with the Hardy-Weinberg equilibrium. Theheterozygosity and the polymorphism information content(PIC) of the5loci in Chongqing Han population were0.816，0.764，0.741，0.70and0.816，0.764，0.741，0.70,respectively. The combined PE is0.9999802, andthe combined DP is0.950167.Conclusions: Allele ladder of the laboratory-mad can be used asMiniSTR loci typing, forensic personal identification and paternity testapplication can be used to improve the survey of non-CODIS the systemMiniSTR loci gene frequencies. And the fluorescence labeled multiplextyping system could be the foundation of the commercial MiniSTR kit.