| Objective: Due to the unique mode of inheritance, X chromosome bearsthe potential to efficiently complement the analysis of autosomal in forensicscience. Single nucleotide polymorphisms (SNPs) are a type of polymorphisminvolving variation of one single base pair that occurs at appreciable geneticsequence in human genome. SNPs have large amount, low mutation rates andcan be genotyped with high-throughput technologies. Especially, they can beanalyzed after PCR amplification of very short DNA regions, which makesthem ideal markers for analyzing degraded DNA. Among large number ofdifferent SNP typing technologies, single base extention technic which baseson the commercial SNaPshot reagent kit can detect a dozen or more SNPssimultaneously. Meanwhile, it has a high sensitivity and easy to bepopularized. While, there is not enough research and application on X-SNPs inthe forensic field now. The objective of this study is to select a sufficientnumber of X chromosome SNP loci that of high polymorphism, and thendevelop a highly efficient DNA typing strategy for analyzing all locisimultaneously by using multiplex PCR in association with fluorescentlabelled single base extension (SBE) technique, and perform a seriesexperiments to assess the forensic application of the methods.Methods:1Loci selection:20X-SNPs loci that met the criteria of highpolymorphism and uniformly distribution were selected using the populationdata of Hapmap.2Primer design: Primers for PCR amplifying and single base extentionwere designed using primer designing software Primer Premier5.0and Oligo6.0according to the reference sequence from dbSNP (http://www.ncbi.nlm.nih.gov/snp/). 3Construction of multiplex typing system: The multiplex PCR systemwith all SNPs was developed, then purified by Exonuclease (Exo I) andshrimp alkaline phosphatase (SAP), in order to remove the primers andunincorporated dNTP. The SBE reaction was performed by SNaPshot kit andthe excess ddNTPs were removed by addition of SAP. The SBE product wasdetected by ABI3130Genetic analyzer, and the data was collected by DataCollection V3.0software, fragment size determination and genotyping of allthe SNPs loci were done with Genemapper V3.2simultaneously. Themultiplex amplification conditions such as the concentration of primer, Mg2+,dNTP, anneal temperature, cycle times were optimized, in order to balance thePCR product of each locus.4Population genetic investigation and forensic application: Populationgenetic investigation was performed on217healthy unrelated individuals ofHan population in Hebei (112females and105males) using this SNPsmultiplex typing system. Forensic genetic parameters and linkagedisequilibrium were calculated. A series of experiments were performed tovalidate the sensitivity,species specificity and concordance of different tissuefrom a same body. Application value in paternity testing and highly degradedsamples were evaluated.Results:1By using the designed primers, the lengths of the PCR products rangedfrom61to99bp and the lengths of the SBE primers were between20and68nucleotides.2A SNPs multiplex typing system including20X-SNPs with highheterozygosity was developed. A balanced amplification was performedbetween each of the loci.3The population genetics data of217unrelated HeBei Han individualsfor the20X-SNPs loci was collected. No deviation from Hardy-Weinbergequilibrium was observed in the female samples. All the105male have uniquehaplotypes. No population differentiation was found between female and malefrequencies, and allele frequencies from female and male sample were pooled in20loci. The minor allele frequencies of all the loci were larger than0.3and75%of them were more than0.4, which indicates a high polymorphism. Themean heterozygosity (HET) was0.486. The mean polymorphism informationcomponent (PIC) was0.367. The mean power of discrimination for females(PDfemale) was0.617and the power of discrimination for males (PDmale) was0.486. The mean exclusion chance (MEC) for ChrX markers in trios involvingdaughters and MEC for ChrX markers in father/daugther dous wererespectively0.367and0.243. Linkage disequilibrium was tested. Aftermultiple comparison, all the value of︱D′︳were less than0.5. The pairwiseLD analyses revealed an association between rs4826645and rs1988916(p=0︱D′︳=0.3642r2=0.0943), rs5951622and rs5963947(p=0.00001︱D′︳=0.3848r2=0.0848), rs757018and rs2522169(p=0.00023︱D′︳=0.2741r2=0.0606), even after Bonferroni correction (p≤0.05/190). The distancebetween them are respectively35.1Mb,19.4Mb and105.0Mb。4A complete SNP profile was obtained for9948standard DNA from1ng.Species specific study results showed that no signal was detected in otherspecies. No mutation was found in kinship test and the results verified theunique genetic mode of ChrX. The genotypes of the different tissues in thesame cadaver were identical. Compared with nvestigator Argus-X12PCRAssay kit, the system produced a higher success rate for genetyping withdegraded DNA samples.Conclusions: We successfully developed a SNPs multiplex typing systemincluding20X-SNPs loci. The population genetics data indicated that with ahigh heterozygosity, the20X-SNPs loci were suitable for paternityidentification and personal identification in forensic practice. |
PDF Full Text Request