CAJ | 학술논문

目的：基于等位基因特异性PCR（allele-specific PCR，AS-PCR）技术，建立一种三色荧光标记复合扩增检测线粒体DNA（mtDNA） SNP的方法。方法基于AS-PCR原理，选择20个mtDNA编码区SNP位点，分为两组，分别标记FAM和HEX荧光，每个位点设计具有长度差异的两条上游（下游）等位基因特异性引物以及一条下游（上游）通用引物。结合AS-PCR技术和毛细管电泳，检测200份无关个体血样。各位点随机选取至少3个样本进行直接测序验证，并进行单倍型频率调查。结果200份血样均得到清晰分型，各位点的检测结果与直接测序结果完全一致。10μL体系下，DNA最低检测浓度为0.2 pg，当模板量为0.5~5 pg时能得到较为理想的分型图谱。在200名无关个体中，共分出15种单倍型，单倍型多样性为0.9060。结论 AS-PCR技术是一种简单、快速且有效的mtDNA SNP分型方法，适用于法庭科学检验的需求。
목적：기우등위기인특이성PCR（allele-specific PCR，AS-PCR）기술，건립일충삼색형광표기복합확증검측선립체DNA（mtDNA） SNP적방법。방법기우AS-PCR원리，선택20개mtDNA편마구SNP위점，분위량조，분별표기FAM화HEX형광，매개위점설계구유장도차이적량조상유（하유）등위기인특이성인물이급일조하유（상유）통용인물。결합AS-PCR기술화모세관전영，검측200빈무관개체혈양。각위점수궤선취지소3개양본진행직접측서험증，병진행단배형빈솔조사。결과200빈혈양균득도청석분형，각위점적검측결과여직접측서결과완전일치。10μL체계하，DNA최저검측농도위0.2 pg，당모판량위0.5~5 pg시능득도교위이상적분형도보。재200명무관개체중，공분출15충단배형，단배형다양성위0.9060。결론 AS-PCR기술시일충간단、쾌속차유효적mtDNA SNP분형방법，괄용우법정과학검험적수구。
Objective To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluo-rescence labeling for mitochondrial DNA (mtDNA) SNP typing. Methods Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided in-to 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood sam-ples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three ran-dom samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated. Results Distinct electropherograms of 200 blood samples were obtained suc-cessfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10μL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0. Conclusion AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.