直接用于PCR反应的芽胞杆菌基因组DNA抽提改良方法的建立

Development of an improved method for extraction of genomic DNA from bacillus,used for PCR directly

目的建立一种直接用于PCR反应的芽胞杆菌基因组DNA提取的改良方法。方法用十六烷基三甲基溴化铵(Cetyltrimethyl ammonium bromide,CTAB)-溶菌酶-冻融裂解法提取蜡样芽胞杆菌、苏云金芽胞杆菌、纳豆芽胞杆菌基因组DNA,用紫外分光光度计测定提取的基因组DNA在230、260、280 nm波长下的A值,计算DNA浓度,并以提取的基因组DNA为模板进行PCR扩增。结果采用改良CTAB法提取的基因组DNA A260/A280值均在1.8～2.0之间,A260/A230值均大于2.0,DNA浓度均大于110μg/ml;PCR扩增产物均可见1 500 bp的清晰条带,浓度较高,未见其他特异条带。结论 CTAB-溶菌酶-冻融裂解法提取芽胞杆菌基因组DNA简单、高效,并可用于PCR反应,适用于临床分子生物学检验。

Objective To develop an improved method for extraction of genomic DNA from bacillus, which can be used directly for PCR. Methods Genomic DNAs were extracted from Bacillus cereus, Bacillus thuringiensis and Bacillus natto by cetyl trimethyl ammonium bromide （CTAB）-lysozyme-frozen-thaw lysis method and determined for Avalues at wavelengths of 230, 260 and 280 nm by utralviolet spectrophotometer, based on which the DNA concentrations of extracted samples were calculated. The extracted DNAs were used as templates for PCR. Results The A260/A280 values of DNAs extracted by the improved CTAB method were 1.8 2. 0, while the A260/A230 values were more than 2. 0, and the DNA concentrations were more than 110 μg/ml. PCR product showed clear bands each at length of 1 500 bp and a high concentration, while no other specific bands were observed, and all the bands were clear. Conclusion CTAB-lysozyme-frozen-thaw lysis method was simple and effective for extraction of genomic DNA from bacillus and might be used for PCR, which was suitable for molecular biological test in clinic.