一种简单高效的5’-突出末端平端化方法

A Simple,Efficient Method for 5’-Overhang Blunting with Pfu DNA Polymerase

目的:报道一种利用Pfu DNA聚合酶延伸法补平限制片段5’-突出末端的平端化方法。方法:Pfu DNA聚合酶是用于PCR扩增的常规高保真DNA聚合酶,可在DNA模板和dNTPs存在条件下,沿5’→3’方向催化寡聚核苷酸聚合。由于终产物为平末端,可利用这一特点进行限制片段5’-突出末端补平。本文采用这一方法消除pAN7-1潮霉素抗性标记末端的XbaⅠ位点,以便载体构建中能够利用这一常见位点。pAN7-1先用XbaⅠ酶切成线性化载体,产生的5’-突出末端再用Pfu DNA聚合酶延伸法补平,通过平端化载体自连后XbaⅠ位点的消除来评价Pfu DNA聚合酶的平端化效果。结果:随机挑取3个重组子提取质粒均不能再用XbaⅠ切开,表明XbaⅠ位点成功消除。结论:Pfu DNA聚合酶具有高效率及高保真特性,因此本法简单高效而且经济适用。

Objective： A novel method for 5＇ - overhang blunting with Pfu DNA polymerase was re.ported in this paper. Methods： Pfu DNA polymerase is conventional high- fidelity DNA pelymerase used for PCR, catalyzing the polymerization of nucleotides into duplex DNA in 5＇ to 3＇ direction in the presence of DNA template and dNTPs. Its final products are blunt - ended, the feature of which can be utilized for 5＇ - overhang filling- in of restriction fragments. In this paper,we used this method to eliminate the restriction site Xba Ⅰ from filamentous fungal marker vector pAN7 - 1 so as to utilize this common restriction site in vector construction, pAN7- 1 was linearized through Xba Ⅰ digestion and its 5＇ - over- hang was filled - in through Pfu DNA polymerase elongation. Xba I elimination of pAN7 - 1 was obtained by serf - ligation of the blunted linearized vector, by which the efficiency of 5＇ - overhang filling - in with Pfu DNA polymerase was evaluated. Results： Three recombinmants randomly picked were not able to be digested by Xba Ⅰ , indicating the restriction site Xba Ⅰ was successfully eliminated. Conclusion： Pfu DNA polymerase is of high efficiency and high fidelity, so this method is efficient and accurate, simple and economical.