【摘 要】
：
与酶切-连接构建重组质粒方法不同,本文建立了不通过限制性内切酶酶切的重组质粒构建方法——二步PCR.第一步常规PCR扩增插入基因片断,用正向引物(IF)和反向杂合引物(IR)扩增
【机 构】
：
河南农业大学生命科学学院,郑州450002河南农业大学生命科学学院,郑州450002;农业部农业酶工程重点实验室,郑州450002
论文部分内容阅读
与酶切-连接构建重组质粒方法不同,本文建立了不通过限制性内切酶酶切的重组质粒构建方法——二步PCR.第一步常规PCR扩增插入基因片断,用正向引物(IF)和反向杂合引物(IR)扩增黑曲霉木聚糖酶Xyn基因[1],IF与基因特异性匹配,IR引物3端与基因序列匹配、5端含有19bp pET20b—端正链序列;第二步反向PCR扩增重组质粒,以扩增的Xyn基因作为正向引物,其3端含有19bp与环状pET20b模板退火、并在高保真Q5 DNA聚合酶作用下延伸,与模板另一端互补的反向引物VR协同作用,重组质粒通过PCR扩增出来.反向PCR产物经T4 DNA连接酶环化,并经DpnI限制性内切酶消解处理,而后直接转化BL21(DE3)感受态细胞.用这种方法将622bp、639bp[2]、1125bp(19)、1209bp[3]大小的基因重组pET20b中,显示出有较广泛的普适性.
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