Weiner M P
Stratagene Cloning Systems, La Jolla, CA 92037.
Biotechniques. 1993 Sep;15(3):502-5.
A method that allows the directional cloning of blunt-ended polymerase chain reaction (PCR) fragments is described. One PCR primer must be 5' phosphorylated. Extra bases are not required on either PCR primer. A linearized vector is enzymatically processed to contain a single 5'-terminal phosphate. The monophosphorylated vector is amenable to recombinant-insertion during ligation when the fragment is in the correct orientation. Increased recombinant yield results from incubating the monophosphorylated vector with a restriction enzyme (SrfI) that relinearizes nonrecombinant plasmids during the ligation reaction.
本文描述了一种用于定向克隆平端聚合酶链反应（PCR）片段的方法。其中一个PCR引物必须进行5'磷酸化修饰。两个PCR引物均无需额外碱基。将线性化载体进行酶处理，使其含有单个5'-末端磷酸基团。当片段方向正确时，单磷酸化载体在连接过程中易于进行重组插入。通过将单磷酸化载体与一种限制酶（SrfI）一起孵育，可提高重组产率，该限制酶在连接反应过程中使非重组质粒重新线性化。
