线粒体DNA的PCR-RFLP分型法医学应用研究

A study on genetic polymorphism of mtDNA by PCR-RFLP and applications in forensic practice

目的建立PCR RFLP技术检测mtDNA序列多态性的方法,调查武汉汉族人群mtDNAHVⅠ区段限制性片段长度多态性,并对PCR RFLP技术在毛干、指甲等生物检材的个体识别案件中的应用进行评估。方法应用nest PCR技术和RFLP技术建立检测mtDNAHVⅠ区段限制性片段长度多态性的方法,调查150例武汉汉族人群无关个体,同时对实际案件中的毛干、陈旧骨骼、水浸血痕等不同种类、不同保存时间和条件的生物检材进行检测。结果RsaⅠ酶切检出4种表型,频率分别为0.760、0.167、0.066和0.007,遗传差异度(GD值)为0.393,随机匹配概率(P值)为0.607。应用PCR RFLP对毛干、20年陈旧骨骼、水浸血痕进行了检测以及在个体识别及母子关系亲子鉴定案例中应用。结论用PCR RFLP法检测mtDNA序列多态性在法医物证检验中具有应用价值。

Objective To develop a method of sequencing mitochondrial DNA(mtDNA)using PCR-RFLP by which the genetic polymorphism of RFLPs in HV-Ⅰregion of the mtDNA was studied in population of Han nationality and evaluate applicability of the method by investigating the RFLP of mtDNA HV-Ⅰwith samples such as hair shafts and fingernails.Methods 15997-16401nt region of the mtDNA in 150 unrelated individuals of Han population in Wu Han area was amplified by PCR.The PCR products were digested by restriction endonuclease RsaⅠand electrophorezied by PAGE,followed by silver staining.Using PCR-RFLP method,we have genotyped material such as hair shafts,old bone fragments,water-immersed blood stains and samples preserved under various conditions.Results Among the 150 unrelated individuals,4 genotypes were detected using restriction endonuclease RsaⅠ.The genotype frequencies were 0.760,0.167,0.066 and 0.007,respectively.Genetic diversity(GD)was 0.393 and random match probability(P)was 0.607.The method was also successfully applied to the genotyping of mtDNA in hair shafts,bone fragments preserved for 20 years and water-immersed bloodstains and in cases of paternity testing.Conclusion The gene haplotyping of mtDNA by PCR-RFLP are applicable to cases in forensic science.