The Construction And Genetic Application Of A Multiplex Typing System With 52 SNPs In Fujian Han Population

Objective To explore the construction of SNap Shot complex typing system for 52SNPs loci and its genetic polymorphism in Fujian Han population.Methods (1)Log into SNP database(http://alfred.med.yale.edu/alfred/index.asp),52pairs of PCR amplification primers and 52 single base extension primers were designed by primer design software according to the sequence of loci.(2)Unit-point SNP detection method was constructed by unit-point SNP PCR reaction,detection of PCR products,purification of PCR products,unit-point SNP extension reaction,purification of extension products and detection of extension products.After that,with a large number of experiments,adjusting the concentration of Mg²⁺and d NTP,the ratio of primer concentration at each point,annealing temperature and cycle times,all SNP loci can be amplified well,and the SNPs complex typing system can be constructed.(3)The 52 SNPs complex typing system was used to classify 50 unrelated individuals of Fujian Han population.The allele frequencies,discrimination power(DP),polymorphism information content(PIC),observed heterozygosity(Ho),Power of exclusion of trios(PE_trio),Power of exclusion of duos(PE_duo),Total discrimination power(TDP),Cumulative probability of exclusion of trios(CPE_trio)and Cumulative probability of exclusion of duos(CPE_duo)of 52 SNPs loci were calculated by Power Stats v12.xls software.The Hardy-Weinberg equilibrium test was also carried out for 52 SNPs loci.(4)The allele frequencies of 52 SNPs in Fujian Han population and other populations were compared by SPSS 19.0 software.Results (1)52 pairs of PCR amplification primers and 52 single base extension primers were designed for SNPfor ID 52-plex.The theoretical annealing temperature of all multiplex PCR primers was 60±2?and the length of amplicon was 59-115 bp.The length of all single base extension primers ranged from 16 to92 nucleotides.(2)52 SNPs complex typing systems were successfully constructed.52 SNPs were divided into four PANELs,named PANEL1,PANEL2,PANEL3 and PANEL4,which contained 15,14,14 and 9 SNPs loci respectively.(3)The DP of52 SNPs loci in Fujian Han population was 0.1800-0.6592,the average DP was0.5265,and the TDP was 0.999999999;the PIC was 0.0905-0.3750,and the average PIC was 0.3104;the Ho was 0.10-0.60,the average Ho was 0.41;the PE_duowas 0.0045-0.1250,the average PE_duowas 0.0852;the CPE_duo was 0.990723512213;the PE_trio was 0.0464-0.2188;the average PE_trio was 0.1765;and the CPE_trio was0.999962354325.The allele frequencies of 52 SNPs loci showed no significant departure from Hardy-Weinberg equilibrium after modification by Bonferroni method.(4)Compared with other populations,there were significant differences in allele frequencies of 6,15,10,28 and 27 SNPs between Fujian Han population and Beijing Han,Uygur,Tibetan,Danish and Somali populations respectively.Six SNPs,rs1357617,rs2046361,rs1031825,rs727811,rs2076848 and rs1335873,had significant differences among the five populations and could be used in population genetics research.Conclusion In this study,we successfully constructed a 52 SNPs SNap Shot complex typing system by designing PCR and single base extension reaction primers for SNPfor ID 52-plex loci.The 52 SNPs were polymorphic in Fujian Han population and showed no significant departure from Hardy-Weinberg equilibrium.The complex typing system can be applied independently to individual identification and parentage testing of trios,and it also can be a powerful complement to parentage testing of duos.