The Establishment And Application Of Fluorescent Multiplex Set For Five Ministr Loci

Objective：Short tandem repeat sequences present in gene and genesoutside the region of the human genome. Contains a large number of geneticmarkers.Currently in the Construction of a genetic linkage map, thediagnosis of genetic disease has a very important role. To provide strongtechnical support for the solution to the problem of personal identificationand paternity testing in forensic applications.However, in the actualprosecution case, often a DNA sample degradation due to various reasons,broken, lost fragments and the sample size is too small, etc. At this point, theapplication of STR technology often can not successfully typing, severelyrestricted the accuracy, timeliness, authority of the forensic cases detectionThe United States re-designed primers to make it closer to the core repeatsequence flanking region in the events of September11, thus making theproduct fragment was amplified by PCR product than conventional STRgreatly reduced, to solve the many inadequacies of the STR technology, anduse to individual identification of the victims, called miniSTR technology.STR loci in the CODIS system is not applicable to miniSTR technology.Therefore, looking for more miniSTR loci outside of the CODIS system can greatly enhance the detection performance, and statistical parameters of itsgene frequency in the population survey to provide data support for theestablishment of the Chinese population miniSTR kit. This article develop afluorescent labeled multiplex amplification system which is consisted of5miniSTR loci-D1S1676，D2S441，D3S4529，D22S1045，amelogenin，and then analyze the genetic polymorphism from a sample of200unrelatedHan Chinese in Chongqing.Methods：Literature review, D1S1676, D2S441, D3S4529, D22S1045,amelogenin of five miniSTR loci, primer sequences, Once again, we use theorthogonal experimental method to optimize the template concentration,Mg2+concentration, primer concentration and the amount of the enzyme, theoptimal annealing temperature conditions, set28,29,30,31,32times thenumber of PCR cycles tested. We apply the NCBI database to proofreading,the primer premier5.0, autodimerv1software redesign the primer. Primersof D1S1676，D2S441，D3S4529，D22S1045，amelogenin were labeled with5’TAMRA，5’6-FAM，5’HEX，5’TET， respectively. By optimal zing，the multiplex amplification system was established. Data were collected bythe3130gene analyzer， and then genotyped by calculating the length of theamplification fragments using Gene Mapper v3.2.1software. The5fluorescent labeled miniSTR multiplex amplification system weredeveloped. Genotyped of200unrelated Han Chinese in Chongqing wereobtained. Results：Every loci of the5fluorescent labeled miniSTR multiplexamplification system can be genotyped clearly.9，9，8，9，2alleles and103genotypes were obtained from the genotyped data from blood sample of200unrelated Han Chinese in Chongqing， and the distribution of the genotypeswas according with the Hardy-Weinberg equilibrium. The heterozygosis andthe polymorphism information content(PIC) of the5loci in Chongqing Hanpopulation were0.71，0.765，0.73，0.71and0.660041，0.798329，0.753177，0.797888，respectively. The combined PE is0.9248004and the combinedDP is0.9999551.Conclusions：Each loci(D1S1676，D2S441，D3S4529，D22S1045，amelogenin of the established5fluorescent labeled miniSTR multiplexamplification system can be genotyped accurately and clearly。ChongqingHan population of the five loci genetic data of unrelated individuals to showtheir higher individual recognition rate and the probability of paternityexclusion in Chongqing Han,it can be used as basis materials for study ofpopulation genetics and forensic appliance。...