改进的PCR法克隆带小棒链霉菌cefD基因

Cloning of S.clavuligerus cefD Gene by Improved PCR

以 GC含量高达 74%的带小棒链霉菌 ( S.clavuligerus)染色体 DNA为模板 ,从引物设计、退火温度、有机辅剂及 DNA聚合酶等方面改善 PCR反应体系和扩增条件 ,成功地克隆得到了异青霉素异构酶的 cef D基因。改进后的 PCR体系中 φDMSO=0 .0 5并加入适量的 Taq DNA聚合酶 ,退火温度比常规选择高一些 ,从而克服了常规 PCR法扩增链霉菌基因遇到的二级结构复杂、引物与模板难以配对及引物间和引物内部易形成碱基配对等问题。改进后的 PCR法提高了链霉菌基因克隆的效率 ,也可以广泛应用于高

Although the template was the chromosomal DNA of S.clavuligerus with a GC content greater than 74 percent, through optimizing the PCR amplification by improving the reaction system and condition, such as the design of the primers, annealing temperature, organic solvent and the composition of DNA polymerase, the target gene of IPN isomerase ( cefD ) was specifically amplified. Successful modifications are the addition of φ DMSO =0.05 to the standard reaction, the usage of 2μL Taq DNA polymerase in 50μL reaction system, the preference of higher annealing temperature. Such improvements can deal with many problems in standard PCR amplification, such as some secondary structures in the template, the base mispair within the primer or between two primers and the difficulty of the base pair between the primer and the template. Improved PCR reaction enhances the efficiency of the gene clone in Streptomyces and it can be widely available for amplification of a GC rich template.