限制性酶切片段的TA法直接克隆与测序

报道了一种粘性末端的限制性酶切片段的直接克隆和测序的方法。对限制性酶切片段的粘性末端先用T4洲A聚合酶处理，变为平末端，然后用Taq^TM DNA聚合酶在其3′末端加上A腺苷，即可利用T／A克隆载体进行直接克隆测序。利用这种简单而快速的方法，对2个RFLP探针Psr680的限制性酶切片段(1．65kb和0．65kb)进行了测序，表明这种方法可以替代利用相应载体进行相应酶切等处理的粘性末端连接克隆测序的方法。

Direct cloning and sequencing of restricted fragments with cohesive-ends by means of TA cloning

A method is presented by which DNA restriction fragments with cohesive-ends are enzymatically blunt-ended with T4 DNA polymerase and then a single 3′ A-adenosine is added by TaqTM DNA polymerase before ligating directly into a T/A cloning vector.Using this rapid and simple method,two restriction fragments (1.65 kb and 650 bp) from a RFLP probe Psr680 were directly cloned into a T/A cloning vector,and then were sequenced.This method is an alternative method to cohesive-end ligation of restricted fragments,which uses a prepared vector containing the corresponding restrictive enzyme site.