The Genetic Polymorphisms Of The X-STR And The SNPs In The Flanking Sequence And Their Significance In The Identification Of The Particular Genetic Relationships And The Mixed Stains

Objective:In the forensic identifications,the identification of the mixed stain from two persons and the particular genetic relationships(such as the mother/son or daughter,father/daughter,grandmother/granddaughter,half sisters with the same father etc.)were the pressing problems.Currently the statistical analysis of the mixed stain mainly adopts the likelihood ratio of interpretation which could be divided into the likelihood ratio method using the unrestricted combinatorial approach and the likelihood ratio method using the restricted combinatorial approach to the basis of whether the peak height or area of alleles were taken into account or not.This method was affected greatly by sample concentration and proportion of the mixed sample,and also need to consider the influence on the stutter band,the allelic drop-out and the low copy number to the results.It could not achieve the purpose of identifying the different individuals in the mixed sample.It was the key to establish the method of DNA typing for different individuals to solve the problem of the individual identification of the mixed sample.In the above identification of complex genetic relationships,when there weremore than 50% same alleles in the X-STR genotyping results from two samples,it can not be excluded or not affirm the relationship.If the judgement whether the same alleles in the two samples weree present by random chance or genetic could be made,the probability of exclusion or certainty of genetic relationships will be greatly improved.X chromosome was diploid in female and haploid in male.X-STR was great significance of the identification of complex genetic relationships of X chromosome,especially in the identification of individuals.In this study,the fragment containing the DXS7423 and about 2000 bp in its upper and down region respectively will be sequenced to detect the distribution of SNPs in the population.Based on differences in individual SNPs,the DNA typing method aimed at different individuals was established by PCR-SSP,in order to solve the problem of identification in different individuals from the mixed sample.In parentage testing,according to the differences and similarities in the SNPs in the upper and down of the XSTR,it was the basis that whether the same X-STR alleles in two samples were present because of random chance or genetic reason which will be a new indicator in some identifications of complex genetic relationships.The genetic polymorphism of twelve STR loci on X chromosome in Manchu population of Liaoning Province was investigated,which result could contribute to individual identification and paternity testing in forensic applications and also provide a basic data for population genetics.Methods:Manchu 772(male 514,female 258)unrelated healthy blood samples in Liaoning Province,three generations of four families(grandmother,father,mother and granddaughter)and three generations of five families(grandmother,father,mother and two fraternal granddaughters)were detected.DNA standards(9947A)and sterile deionized water were used as positive and negative controls respectively.Genomic DNA was extracted by phenol chloroform method.DNA fragments containing the DXS7423 and the upper and down about 2000 bp region were amplified,and the typing results of the SNPs were obtained by sequencing.The comparison between the male and female groups was performed.The allele frequencies,haplotype frequencies,and forensic parameters of SNPs were calculated.The differences of SNPs were compared and the probability of the coincident genotype of DXS7423 caused by random chance or genetic reason.According to different results of SNPs typing,DXS7423 of different individuals in mixed samples were amplified by PCR-SSP.Investigator Argus X-12 kit was used to investigate the genetic polymorphism of twelve STR loci of X chromosome in Manchu population of Liaoning area.The allele frequencies,haplotype frequencies,and forensic parameters of twelve STR loci were calculated.Results:There were four SNPs in the upper and down about 2000 bp region of DXS7423.The allele frequency distributions of the four SNPs loci were in the Hardy-Weinberg equilibrium.And there was no significant difference in the distribution of alleles between males and females.The allele frequencies of the four SNPs loci were rs762822(G0.3067/A0.6933),rs546652(T0.2333/G0.7667),rs17279108(A0.7867/T0.2133)and rs529211(C0.7400/G0.2600).The polymorphisms of four SNP loci were good,which will be used in the identification and paternity cases.The PCR-SSP method could be used to distinguish the different components of the two males in mixed sample according to the SNPs genotypes.The sequence results of separated components were in accord with the STR genotypes tested by the commercial kit.It indicates that this method may be used to distinguish the individual characters of the mixed sample.According to the individual differences of SNPs genotypes,there were 53.52% possibilities to distinguish different individuals after comparison.According to this principle to speculate,if enough X-STR can be screened,then the identification of different individuals in the mixed sample can be achieved by the multiplex amplification.The comparison of the pedigree test results showed that if mother/son or daughter,father/daughter,grandmother/granddaughter,half sisters with the same father share the same alleles,it was supported that these alleles were obtained by genetic.Taking the unrelated females and unrelated males as the alleged mother(or grandmother)or alleged father,there was 53.52% possibilities to exclude the genetic relationship after comparing the SNPs in the STR flanking sequence.The polymorphism distribution of twelve X-STR loci in 772 individuals were in Hardy-Weinberg equilibrium which were powerful in personal identification and paternity of exclusion.Conclusion:According to the differences between the SNPs in the X-STR flanking sequence,the PCR-SSP method could be used to distinguish the mixed samples which the suspects were known and gain the individual X-STR genotypes respectively.In some complex relationship identification,the SNPs which were in the STR flanking sequence whit the same alleles could provide a new basis of excluding or identifying the genetic relationship.The polymorphism of twelve X chromosome STR loci in Manchu population was high,which was valuable in forensic and could be used in identification and paternity testing.