【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439bp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。更多还原

【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 65 000 (M_r) 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.更多还原