法医学杂志

法医学杂志 ›› 2005, Vol. 0 ›› Issue (4): 265-270.

• 论文 • 上一篇    下一篇

DHPLC-mtDNA控制区多态性分析系统的建立

孙宏钰,蔡贵庆,陆惠玲,刘超,陈丽娴,李向阳,伍新尧   

  1. 中山大学基础医学院法医学系,中山大学基础医学院法医学系,中山大学基础医学院法医学系,广州市刑事科学技术研究所,中山大学基础医学院法医学系,广州市公安局东山区分局,中山大学基础医学院法医学系 广东广州510089 ,广东广州510089 ,广东广州510089 ,广东广州510030 ,广东广州510089 ,广东广州510080 ,广东广州510089
  • 发布日期:2005-08-25 出版日期:2005-08-28

The Establishment of Analytic System of DHPLC-mtDNA Control Region Polymorphism

SUN HONG-YU, CAI GUI-QING, LU HUI-LIN, ET AL. (DEPARTMENT OF FORENSIC MEDICINE, SCHOOL OF PRECLINICAL MEDICINE, ZHONGSHAN UNIVERSITY, GUANGZHOU 510089, CHINA)   

  • Online:2005-08-25 Published:2005-08-28

被引次数

2

摘要： 目的基于变性高效液相色谱技术,建立一种不需测序和杂交的新的mtDNA控制区多态性分析系统。方法mtDNA控制区序列(包括HVⅠ,HVⅡ和HVⅢ)被分为4个扩增片段,采用配对分析突变检测模式进行DHPLC分析。对DHPLC检测条件(包括柱温和洗脱梯度等)进行优化。对100个不同类型差异序列的组合配对以检验该方法的检测效力。结果10组序列相同的样本配对DHPLC图谱均只显示1个样品峰。对序列相差1个碱基～7个碱基、插入(/缺失)1个碱基、插入(/缺失)2个碱基等类型的90个扩增片段组合,用DHPLC进行分析,均得到≥2个样本峰的DHPLC图谱,序列差异检出率达100%。该技术可检测的异质性DNA成分的最小百分含量为10%。结论DHPLC-mtDNA控制区多态性分析系统快速、经济和实用,在检测mtDNA异质性方面较直接测序更灵敏。

关键词: 线粒体DNA(mtDNA), 变性高效液相色谱(DHPLC), 多态性, 异质性

Abstract: Objective To establish a new method for analyzing the polymorphism of mtDNA control region based on DHPLC (denaturing high performance liquid chromatography) without sequencing or hybridizing. Methods The fragment of mtDNA control region (including HVI, HVII and HVIII) was divided into 4 amplicons. Conditions for PCR and DHPLC (including the melt temperature and gradient, etc) were optimized. 100 pairs of mixed samples were analyzed using DHPLC to evaluate the system′s ability to detect different sequences. Results Only 1 elutive peak was obtained for the 10 pairs of samples mixed with identical sequences. More than 2 peaks were captured for the 90 pairs of samples mixed with sequence differences of 1～6 bp, 1 and 2 bases del (/ins), respectively. The capability of detecting different sequence is 100%. The least distinguishable ratio of the heteroplasmic DNAs is 10%. Conclusion The analytic system presented here is rapid, efficient and economic. It is more effective to detect the heteroplasmy than direct sequencing.

Key words: mitochondrial DNA (mtDNA), denaturing high performance liquid chromatography (DHPLC), polymorphism, heteroplasmy