桑天牛成虫肠道细菌基因组DNA的提取及PCR反应体系优化

Genomic DNA Extraction and Optimization of PCR Reaction System of Apriona germari Intestinal Bacteria

为了建立一套适用于桑天牛肠道细菌多样性研究的PCR反应体系和程序,采用改良的十六烷基三乙基溴化铵(CTAB)法提取桑天牛成虫肠道细菌基因组DNA,通过L16(45)正交组合试验和单因素梯度试验对MgC l2、dNTP、随机引物、TaqDNA聚合酶、模板DNA的浓度和退火温度、循环次数等影响PCR扩增的重要因素进行优化。试验结果表明,采用改良的CTAB方法提取的桑天牛成虫肠道细菌DNA质量较高,适宜于PCR扩增分析。25μLPCR反应体系及反应程序中各因素优化组合为:10×Buffer 2.5μL,MgC l22.0 mmol/L,dNTP 0.2 mmol/L,随机引物0.48μmol/L,TaqDNA聚合酶0.75 U,模板DNA 75 ng;退火温度60℃,循环次数30次。

In order to establish optimal PCR （polymerase chain reaction） system and procedure for studying the diversity of Apriona germari intestinal bacteria, a modified CTAB （hexadecyl trimethyl ammonium bromide） method was employed to extract genomic DNA of the Apriona germari intestinal bacteria. Several important factors including MgCl2, dNTP, random primer, Taq polymerase and template DNA were optimized through L16（4^5） orthogonally designed experiment. Annealing temperature and number of amplification cycles were optimized through single factor experiment. The results showed that high quality genomic DNA could be obtained for PCR analyses by the modified CTAB method. The optimal reaction system for PCR analysis was as follows： a total volume of 25 μL PCR reaction mixture containing 2.5 μL of 10 × buffer, 2. 0 mmol/L of MgCl2, 0.2 mmol/L of dNTP, 0.48 μmol/L of random primer, 0.75 U of Taq polymerase, 75 ng of template DNA, and annealing temperature as 60 ℃ for 30 cycles of amplification.