Application Of Domestic Droplet Digital PCR In Forensic DNA Analysis

Objective:1.The SNP genotyping system of high-altitude adaptability 5-SNP(Single Nucleotide Polymorphism)haplotype was constructed on the domestic droplet digital PCR(dd PCR)platform,and a novel SNP genotyping detection method which is easilyused,rapid and low susceptibility to inhibitors was constructed,then its prospects in forensic application were evaluated.2.The DNA methylation level of saliva samples from Han Chinese individuals were detected using domestic dd PCR technology,then we screened out the age-related Cp G sites,and constructed an age-predictive model which was suitable for forensic applications.Methods:1.Genotyping system of the high altitude adaptive EPAS1 haplotype(rs115321619,rs73926263,rs73926264,rs73926265 and rs55981512)was established by dd PCR.Then we tested the specificity of primers and probes,evaluated its accuracy,stability,sensitivity and adaptability separately.Meanwhile,dd PCR and SNa Pshot minisequencing technology were compared in terms of inhibitor resistance ability.Finally,preliminary application of 70 samples were conducted detection.2.Firstly,we selected 11 previously reported age-related Cp G sites in saliva sample of Asian populations through literatures consulting and investigating.The sequence information of each Cp G site was obtained through UCSC genome browser,and then we designed the dd PCR primers and probes for each Cp G site using Meth Primer software.Secondly,saliva DNA from 147 Han Chinese individuals were converted by bisulfite and detected by dd PCR system.Then Pearson correlation analysis was performed to evaluate the correlation between age and methylation pattern.Thirdly,the optimal age-related Cp G sites subset was screened by stepwise regression and full subset regression,respectively.Fourthly,we applied linear regression equation to construct an age-predictive model,and validated the model’s predictive performance with MAD value.Finally,the sensitivity of the dd PCR system was evaluated.In addition,we further tested the predictive performance of the agepredictive model on 20 buccal swab samples.Results:1.The dd PCR assay only required a total run time within 2.5 h,a total of 27 samples were genotyped using dd PCR,and the consistency of all the genotyping and Sanger sequencing results was 100%.The dd PCR had exhibited a robust and reproducible option in SNP genotyping.The detection sensitivity was 0.3125 ng.The dd PCR assays also exhibited better tolerance to inhibitors than SNa Pshot.The 70 tested individuals showed good consistence with their background information.2.After correlation analysis,7 age-related Cp G sites were selected as the optimal subset: Cp G2,Cp G3,Cp G4,Cp G5,Cp G7,Cp G9 and Cp G11,respectively located on the gene of SST,CNGA3,KLF14,TSSK6,SLC12A5,FHL2 and TRIM59.Saliva samples from 147 Han Chinese individuals were randomly divided according to a ratio of 7:3,with 70% of the samples(N=102)as the training dataset and 30% of the samples(N=45)as the testing dataset.Using multiple linear regression(MLR),we achieved a mean absolute deviation(MAD)between predicted and chronological age of 3.43 years in the training dataset and 3.98 years in the testing dataset.Subsequently,detailed analysis of MAD from chronological age was carried out in 6 age groups,divided by intervals of 10 years.The results showed the prediction accuracy declined with advanced age but not affected by gender differences.Finally,using the 10-fold crossvalidation method and leave-one-out method(LOO)to evaluate the age-predictive model,the MAD were 3.76 years and 3.83 years,respectively.20 buccal swab samples resulted in highly accurate age predictions,the MAD was 3.33 years.Conclusion:1.The SNP genotyping assay based on dd PCR is accurate,rapid,easily-used and shows great resistance to PCR inhibitors,which has strong application potential in the field of rapid forensic detection,and is suitable for forensic analysis.2.Based on domestic droplet digital PCR,an age-predictive model containing 7age-related Cp G sites was constructed,exhibited high accuracy in the Han Chinese population,which laid the certain practical application value in forensic medicine.