Screening And Development Of An Indel Multiplex PCR System In Forensic Science

Objective: In order to provide an effective new tool for forensic DNA detection and analysis,a set of multiplex fluorescence of PCR amplification detection systems,which is suitable for the main population of China,has high individual identification ability and can be used in the DNA laboratory of forensic science.Methods: 1.We screened In Del markers for 22 main pairs of Chinese autosomal and sex chromosomes by using the human genome browser of the University of California Santa Cruz and the single nucleotide polymorphism database(NCBI db SNP)of the National Biotechnology Information center of the United States.2.A multiplex fluorescence of PCR amplification detection system consisting of four color fluorescent markers(FAM,HEX,TAMRA and ROX)was established.LIZ500 use the fifth orange fluorescent,the complex amplification system including 45 In Del loci were composed.The PCR reaction conditions,such as primer ratio concentration,the amount of DNA template and annealing temperature,were adjusted to optimize the PCR reaction conditions of the constructed In Del complex amplification system.The balanced and stable PCR product typing results were obtained,and the single tube reaction system of 45 In Del loci was realized.3.According to the electrophoretic mobility file of 45 In Dels and the requirement of Gene Mapper ID v3.2.1 software of 3130 XL genetic analyzer,panel and bin analysis documents are established.The automatic capillary electrophoresis test method based on 3130 XL genetic analyzer is established by using five color Matrix and the LIZ500 molecular internal standard.PCR amplification products were detected by 3130 XL genetic analyzer,and In Del ID loci were typed by Gene Mapper ID v3.2.1.4.The specificity,sensitivity,stability and applicability of the compound amplification system were verified by forensic science,and the forensic parameters of genetic markers were investigated.Results: 1.This subject has developed A multiplex fluorescence of PCR amplification detection system covering 45-In Dels loci of the entire genome of the human genome and sex chromosomes,and established an automatic typing method suitable for the 3130,3100 and 3500 series of genetic analyzer.2.The 45-In Dels composite amplification system was able to get complete typing when the DNA template volume was as low as 500 pg.It had specificity,good reproducibility and applicability.3.We screened In Del markers for 22 main pairs of Chinese autosomal and sex chromosomes.The allele frequency and basic forensic parameters of the 45-In Dels loci were obtained from the unrelated individuals of 200 people.The average heterozygosity of In Del loci on autosomes reached 0.56,the average personal recognition probability reached 0.60,the polymorphism information capacity(except rs2307783 loci)was more than 0.3,CPD and CPE reached 0.9999 and 0.9859,respectively.For the X chromosome In Del loci,the cumulative individual recognition probability in the female group(CPDfemale)For 0.999997,the cumulative individual recognition probability(CDPmale)in the male population was 0.999787,and the CMECTrio and CMECDuo reached 0.9978 and 0.9732,respectively.The two loci on the Y chromosome were able to reach the standard of sex determination in the samples.The single peak could be detected in the male samples,and the type peaks were not detected in the female samples,and the GD values of the two loci were 0.47 and 0.27,respectively.Conclusion: The 45-In Dels composite amplification system can provide 27 autosomes,16-X chromosomes and 2-Y chromosome polymorphisms,and the detection fragment is short.It is suitable for forensic forensic test and degradation test.It provides a new method for forensic research and practical work.