Development Of The Multiplex PCR System Of Non-CODIS STRs And Its Application In Forensic Genetics

Objective1.To Screen autosomal supplementary STR loci and establish a multiplex PCR amplification system.2.To evaluate the effectiveness of the established system of forensic medicine application system and investigate its genetic parameters,and evaluate the application value of the system in forensic research and practice of forensic expertise.Methods1.Loci screening:By searching relevant databases and literature,non-CODIS STR loci with good polymorphisms in Han Chinese populations were screened as a supplement.Then,the design of primer was implemented based on a software,Primer Premier 5.0,and a tool,Primer-BLAST,from the NCBI database.Finally,the amplification of single-point and agarose gel electrophoresis were conducted to the designed primers for the verification of amplification efficiency belonging to single STR locus primers.2.Construction of fluorescent multiplex PCR amplification system:There selected 24 loci and a sex identification one were divided into four groups according to the size of amplified fragments,and fluorescent dyes,containing FAM,HEX,TAMRA and ROX,were employed to respectively mark the 5’ends of forward primers of each locus.Moreover,reaction conditions were gradually optimized for the multiplex amplification PCR system,including the concentration of primer and Taq enzyme,the annealing temperature,times of cycle amplification and the final extension time et al.After this,a 3500XL genetic analyzer was utilized to the detection of the amplified products.Each loci was named according to the rules of the International Society of Forensic Genetics(ISFG)and the Sanger sequencing results,and the allelic ladder were prepared at the same time.The Panel and Bin files were written and imported based on the requirements of GeneMapper ID-X software.3.Validation of the multiplex PCR system for 24 autosomal STR loci:According to the requirements of the ISFG and the Scientific Working Group on DNA Analysis Methods(SWGDAM),the sensitivity,species specificity,accuracy and the balance degree were conducted.The system was utilized to analyze the genetic data from 216 healthy and unrelated individuals in the Han population of China,and the genetic parameters were calculated to measure the capability of this system in paternity testing.Results1.In this study,23 non-CODIS STR loci,1 CODIS system core locus and 1 sex identification loci were screened for forensic applications,and a fluorescent composite amplification detection system containing 25 STR loci was successfully constructed.Complete and clear genotyping results were obtained from 25 loci,and non-specific amplification products without interference typing could achieve accurate typing.2.The forensic validation results of this study on the constructed multiplex amplification fluorescent detection system showed that the complete typing of 25 loci could still be obtained at template DNA was as low as 0.125ng;the non-human-derived genome DNA samples will not interfere with the typing results in the species-specific studies;the consistent typing results were still obtained after mixing with inhibitors of different concentrations;and the main and minor components can obtain complete typing results in the mixed samples were less than 1:3;All sample alleles were within ±0.50 bp of the corresponding allele in the allelic ladder,,within the range recognizable by the 3500XL genetic analyzer.3.In this study,the genetic investigation showed that a total of 241 alleles with alleles ranging from 0.0045 to 0.4773 were observed in 216 unrelated individual samples,and the heterozygosity of the 24 autosomal STR loci ranges from 0.6455 to 0.9545;the polymorphic information content(PIC)ranges from 0.6230 to 0.9010,and the discrimination power ranges from 0.8299 to 0.9752,cumulative discrimination power is greater than 1-1.24×1-10-27,cumulative probability of exclusion is greater than 1-5.72×1-10-12.ConclusionsIn this study,a multiplex amplified fluorescence detection system including 24 autosomal STR loci and a sex identification loci Amologenin was successfully constructed,23 of them are non-CODIS STR loci and include 4 miniSTR loci.The typing results of this system are accurate and reliable,with a sensitivity of 0.125ng,has good species-specificity,and strong resistance to inhibition.It provides new genetic markers and detection methods for forensic research and forensic identification practice,which can supplement more genetic information for parental identification cases that cannot be solved by conventional STR kits,improve system efficiency,and provide new technical reserves for the field of forensic...