The Analysis Of Mitochondrial DNA Polymorphism Using PCR-SSCP And PCR-RFLP Methods And Its Application In Forensic Medicine Research

IntroductionDNA genetic markers in domestic and international forensic laboratories fall into two categories:nuclear genome DNA and mitochondrial DNA(mtDNA).Compared with the nuclear DNA,mtDNA has many advantages,such as high copy number,low demand for the quality and quatity of the samples.These features are very suitable for forensic biological samples,which has low copy number or was seriously degraded, such as bones,hair,nails,and trace bloodstains.Therefore,mtDNA is increasingly widely appilied in the forensic science.In addition,mtDNA don't only meet the two basic requirements,but also has become increasingly used in many various aspects of forensic science.In recent years,with the development of mtDNA,the detection ability of mitochondrial DNA,to a large extent,depends on the sensitivity of detection methods.Therefore,it is very important to choose a suitable analysis techonology.Sequencing is the most widely use in many detection methods.Compared with sequencing,SSCP and RFLP techonology are suitable for routine laboratory.The feature of mtDNA,the special sructure,high copy number,and stronger resistance for the degraded samples play an important role in the forensic special biological samples.In order to set up an analysis technology for grass root laboratories and improve the resolving power of mitochondrial DNA,the study will use PCR-SSCP and PCR-RFLP method to analysis mitochondrial DNA polymorphism and provide basic data for the population genetics.Methods1.200μl blood samples of 150 Chinese Han unrelated individuals and 30 cases of true triple pedigree,5 cases of the main visceral organs(heart,liver,spleen,lung,kidney, brain)0.5cm×0.5cm×0.5cm and five cases of formalin-fixed paraffin-embedded tisses(1cm×0.5cm×6μm) are provided by the Faculty of Forensic Medicine in China Medical University.2.Use primer 5.0 to synthesis gene specific primer,apply PCR-SSCP and RFLP technology to analyse 150 unrelated Chinese Han individuals and determine genotypes.Result1.In 150 unrelated Liaoning Han individuals,9 haplotypes were found by PCR-SSCP,DP=0.8809;3 genotypes were found by RsaI-RFLP techonology, DP=0.1073;8 genotypes were found by MnlI-RFLP technology,DP=0.6698.12 genotypes were found by RsaI combined with MnlI-RFLP techonology,DP=0.7078.33 genotypes were found by combining PCR-SSCP with PCR-RFLP techonology, DP=0.925.2.The various normal tissues have the same band pattern in the 5 cases.3.In no instance,mother displayed alleles different from those of child within each trio in 30 family trios.4.5 paraffin-embedded tissues had the same alleles as those of corresponding normal tissue.Conclusion1.PCR-RFLP and PCR-SSCP method,the preliminary screening method of forensic evidence,is simple,rapid,economic and suitable for the general lab.2.The mtDNA HVI16106-16297 region has a good polymorphism distribution in Liaoning Han and have a high application value in forensic medicine.3.The mtDNA,compared with STR,is more suitable for formaldehyde-fixed and paraffin-embedded tissues analysis.