色谱

色谱

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毛细管电泳-激光诱导荧光检测分枝杆菌脱氧核糖核酸限制性内切酶谱

黎源倩,王国庆,米建萍,周颖,曾红燕,张朝武   

  1. West China School of Public Health, Sichuan University, Chengdu 610041, China
  • 收稿日期:2004-01-05 修回日期:1900-01-01 出版日期:2005-01-30 发布日期:1989-03-25
  • 通讯作者: 黎源倩
  • 基金资助:

Restriction Enzyme Pattern Analysis of Mycobacteria DNA by Capillary Electrophoresis with Laser

LI Yuanqian, WANG Guoqing, MI Jianping, ZHOU Ying, ZENG Hongyan, ZHANG Chaowu   

  1. West China School of Public Health, Sichuan University, Chengdu 610041, China
  • Received:2004-01-05 Revised:1900-01-01 Online:2005-01-30 Published:1989-03-25

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摘要： 建立了毛细管电泳分离-激光诱导荧光检测（CE-LIFD）分析分枝杆菌脱氧核糖核酸（DNA）限制性内切酶谱的新方法。用聚合酶链反应(PCR)扩增分枝杆菌hsp65基因的长度为439 bp的片段,该扩增片段经限制性内切酶BstEⅡ和 HaeⅢ酶切后,分别用CE-LIFD装置和常规琼脂糖电泳（AGE）对比检测酶切片段。对PCR扩增片段的酶切样品的预处理和CE条件进行了优化,获得了8种分枝杆菌DNA的限制性内切酶谱图。 DNA片段相对迁移时间的相对标准偏差（RSD）≤3.6%。结果表明,CE的分离效能明显高于AGE,是研究DNA限制性内切酶谱的更有效的检测手段。

关键词: 分枝杆菌 , 激光诱导荧光检测, 毛细管电泳, 限制性内切酶谱

Abstract: A new method for rapidly detecting restriction enzyme pattern of mycobacterium deoxyribonucleic acid (DNA) by capillary electrophoresis with laser induced fluorescence detection （CE-LIFD） was developed. Polymerase chain reaction was used to amplify a 439 bp fragment of 65000 (Mr) heat shock protein gene (hsp65) of mycobacterium. After digesting the amplification products by BstEⅡ and HaeⅢ respectively, the patterns of enzyme cleavaged products were detected by both CE-LIFD and agarose gel electrophoresis (AGE). The experimental parameters of CE were optimized. The restriction enzyme patterns of mycobacterium DNA can be detected under the optimum electrophoresis conditions: a coated capillary column with the length of 50 cm and 100 μm i.d., electrophoresis buffer of 45 mmol/L TBE (trihydroxymethyl aminomethane (Tris)-boric acid-ethylenediaminetetraacetic acid (EDTA)) and 11 kV running voltage. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of the DNA segments were less than 3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means for DNA restriction enzyme pattern analysis.

Key words: restriction enzyme pattern, laser induced fluorescence detection, mycobacterium , capillary electrophoresis