自我延伸过程对木聚糖酶-CBM融合基因的影响

Influence of Self-extension Process on Fusion of Xylanase and Cellulose-binding Module

探讨了木聚糖酶-纤维素结构结合结构域(CBM)基因融合过程,在不加引物而只用两种DNA片段经PCR扩增全长融合基因,从而证明了自我延伸过程的存在,是指互补匹配的两段DNA互为引物、互为模板在聚合酶作用下合成融合基因的过程,从理论上阐明了PCR中的自我延伸过程。在传统PCR中加入3次循环的自我延伸程序(94℃×1 min,52℃×1 min,72℃×1 min),融合基因扩增量要更多。进而探讨自我延伸程序中延伸时间的影响,发现延伸时间2、3、5 min扩增的融合基因量显著增强;而延伸时间为1 min时,融合基因条带的亮度增加不明显,说明延伸时间是增加扩增量的关键因素。

This article explored the fusion process of xylanase and Cellulose - binding Module （ CBM）, and amplified the com- plete fusion gene by PCR under the conditions of no using primers and only using the gene fragments of xylanase and CBM, which proved the existence of self - extension process. Self - extension process was the synthetic process of the fusion gene under the action of polymerase and two complementary DNA fragments （mutual primers and mutual templates）, and the self- extension PCR process was clarified theoretically. Thus, adding 3 cycles of self- extension program （94 ℃ for 1 min, 52℃ for 1 min, 72 ℃ for 1 min） in the traditional PCR could obtain more fusion gene amplification amount. The influences of extended time on the fusion gene am- plification amount were studied, and it was found that the fusion gene amplification amount was significantly increased when the ex- tended time was 2, 3, 5 min ; the brightness increment of fusion gene band was not obvious as the extended time was 1 min, which indicated that extension time was a key factor increasing the amount of amplification. Key words： Fusion PCR; Self-extension process; Fusion gene