Research On Rapid Typing Of Forensic DNA

DNA genetic marker is of great value in personal identification,paternity test,chimerism analysis and other related forensic tests.The traditional multiplex detection methods of DNA genetic marker rely on special instruments and the open-tube method requires post-PCR sample handling,which is easily causing assay contamination.The workflow of closed-tube uniplex detection methods increase with the detection sites increasing.Therefore,it is necessary to estabilish a homogeneous,multiplex,rapid,accurate and convenient method for DNA genetic marker analysis.In chapter one,we briefly reviewed DNA forensic typing,then described the development of DNA genetic marker.Finally,we summarized the content and innovation of this study.In chapter two,we established 14 multicolor melting curve analysis(MMCA)assays to detect 10 short tandem repeats(STR)loci(from combined DNA index system)and one sex determination loci,the cumulative power of discrimination(CDP)and the cumulative probability of exclusion(CDE)of this assay are 0.999999999920 and 0.99945,which has high resolution rate.We evaluated this assay by genotyping 170 unrelated individuals,each loci’s allele frequencies are in agreement with the literature.The entire assay could be finished within 2.5 h,which achieves one-step and rapid detection of STR.In chapter three,we established a multiplex single nucleotide polymorphism(SNP)detection assay using MMCA and the tailed primers.We increased the number of targets that can be detected in each MMCA detection.This closed-tube assay can genotype 9 SNPs in one reaction,the entire assay could be finished within 2.5 h.With the use of tailed primers,our multiplex asymmetric detection assay has high-test sensitivity,which can detect as low as 0.05 ng/μL gDNA(amount 15 copies/μL).This indicates MMCA has huge potential in multi-target detection.We evaluated this assay by genotyping 238 unrelated individuals,each loci’s allele frequencies are in agreement with the literature,the CDP of this assay is 0.9997,which has high resolution rate.As a homogeneous,convenient,and multiplex detection assay,this assay can promote the new generation DNA genetic marker’s database establishment and has good applicating prospect in forensic DN A typing.In chapter four,we established a 10-plex deletion and insertion polymorphism(DIP)detection assay using MeltArray.This multiplex DIP detection assay can genotype 10 DIPs in one reaction.The closed-tube assay has high-test sensitivity,which can detect as low as 0.05 ng/μL gDNA.The entire assay could be finished within 3.5 h.We evaluated this assay by genotyping 238 unrelated individuals,each loci’s allele frequencies are in agreement with the literature,the CDP of this assay is 0.99994,which has high resolution rate.In chapter five,we gave the summary and prospect of this dissertation.