基于正交试验设计优化真菌DNA提取的CTAB法

摘要

[目的] 从统计学的角度明确CTAB法提取真菌DNA过程中各因素的影响,确定CTAB法提取真菌DNA的最优方案.[方法] 本试验以菌体量、破壁、抽提、沉淀为考察因素,以核糖体RNA(rRNA)内转录间隔区(ITS)和28S rRNA基因序列的PCR反应扩增率为指标,用SPSS软件设计混合水平正交试验L25(5×34)并进行方差分析.[结果] 采用CTAB法提取真菌基因组DNA时,菌体细胞破壁和沉淀DNA这两个步骤对真菌DNA提取效果的影响最大,其次是抽提步骤,在100 mg范围内,菌体量对DNA的提取效果影响不大.[结论] 采用CTAB法提取DNA时,挑取40~80 mg菌体,用带有无菌塑料钻头的手电钻研磨进行菌体细胞的破壁,研磨时可先加入液氮冷冻后再进行研磨,或直接在冰上研磨,酚∶氯仿∶异戊醇(25∶24∶1)抽提1次,等体积异丙醇沉淀DNA,可以得到较好的DNA提取效果.%[Objective] This study aimed to statistically clarify the influence of different factors during extracting fungal genomic DNA using cetyltrimethyl ammonium Bromide (CTAB) method to determined the optimal scheme.[Method] The orthogonal experiment L25(5×34)was designed and the variance analysis was performed using the software SPSS,with mycelial amount,wall breaking,phenol-chloroform extraction and precipitation of DNA as factors,and the amplification percentage of PCR reactions of the internal transcribed spacer (ITS) and 28S rRNA gene sequences of ribosomal RNA (rRNA) being used to indicate the DNA quality.[Result] The results showed that wall breaking and precipitation of DNA had the most significant effect on fungal DNA extraction,followed by phenol-chloroform extraction,while the mycelial amount at the range of 100 mg had little effect.[Conclusion] A better way to extract fungal DNA using CTAB was determined as follows: picking up 40~80 mg mycelia,grinding the mycelia using an electric hand drill equipped with plastic grinding aiguilles (grinding after freezing by liquid nitrogen or on ice directly),extract DNA once using phenol∶chloroform∶isoamylol (25∶24∶1),and precipitate DNA with equivalent isopropanol.