法医学杂志

法医学杂志 ›› 2014, Vol. 30 ›› Issue (2): 96-100.DOI: 10.3969/j.issn.1004-5619.2014.02.005

• 论著 • 上一篇    下一篇

AS-PCR技术检测20个mtDNA SNP位点及单倍型频率

聂燕钗1，张  晨2，刘亚楠2，黄江平2，焦海涛3，吴  丹2，周怀谷2   

  1. （1. 复旦大学上海医学院法医学系，上海 200032； 2. 上海市公安局物证鉴定中心 法医物证学现场应用技术公安部重点实验室 上海市现场物证重点实验室，上海 200083； 3. 上海锦博生物技术有限公司，上海 200433）
  • 发布日期:2014-04-25 出版日期:2014-04-28
  • 通讯作者: 周怀谷，男，博士，主任法医师，主要从事法医DNA分析技术的应用和研究；E-mail： hgzhou803@hotmail.com
  • 作者简介:聂燕钗（1988—），女，福建福州人，硕士研究生，主要从事法医DNA分析技术的应用和研究；E-mail：nieycs@gmail. com
  • 基金资助:

    公安部科技基础工作专项资助项目（2011GABJC006）

AS-PCR Assay for 20 mtDNA SNP Typing and Haplotype Frequency

NIE YAN-CHAI1, ZHANG CHEN2, LIU YA-NAN2, HUANG JIANG-PING2, JIAO HAI-TAO3, WU DAN2, ZHOU HUAI-GU2   

  1. （1. Department of Forensic Medicine, Shanghai Medical College, Fudan University, Shanghai 200032, China; 2. Shanghai Key Laboratory of Crime Scene Evidence, Key Laboratory of Forensic Evidence and Science Technology, Ministry of Public Security, Institute of Forensic Science, Shanghai Public Security Bureau, Shanghai 200083, China; 3. Shanghai Gene-Bio Technology CO. LTD., Shanghai 200433, China）
  • Online:2014-04-25 Published:2014-04-28

被引次数

1

摘要： 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。10 μL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0.906 0。 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。

关键词: 法医遗传学, 多态性, 单核苷酸, 等位基因, 聚合酶链反应, 单倍型

Abstract: Objective To develop a multiplex allele-specific PCR （AS-PCR） assay with three-color fluorescence 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 into 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 samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random 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 successfully. 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 10 μL. 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.

Key words: forensic genetics, polymorphism, single nucleotide, alleles, polymerase chain reaction, haplotype

中图分类号: