应用T/A策略克隆乙肝病毒pC/C及C基因  

Cloning of pC/C and C Gene of Hepatitis B Virus by T/A Strategy.

作　　者：刘定燮[1] 骆抗先[1] 

机构地区：[1]广州第一军医大学南方医院感染内科实验室,广州510515

出　　处：《生物化学与生物物理进展》2000年第6期662-663,共2页Progress In Biochemistry and Biophysics

摘　　要：以克隆乙型肝炎病毒 pC/C及C基因为例 ,报道了在DNA重组中 ,当目的基因与载体末端不匹配时可采取的一新方法 .用内切酶切取的基因片段为平端时 ,可在含dATP的反应体系中 ,用Taq酶的末端转移酶活性在其 3′末端加上单个碱基 (dA)的突出尾 ;基因片段为 3′凹端时 ,可在含 4种dNTP的体系中 ,利用Taq酶的聚合酶活性先将其末端补平 ,再经末端转移酶活性在其 3′末端加dA尾 ;末端经此修饰的基因片段可亚克隆至T载体中 。A new strategy of cloning uncompatible DNA ends was reported here with the example of cloning of pC/C and C gene of hepatitis B virus. When the terminals of DNA fragments digested by restriction enzymes are blunt ends, they can be formed into tails with a single 3′ adenosine overhanged by modified with template independent terminal transferase activity of Taq polymerase in reaction buffer containing dATP. Furthermore, if the DNA fragments have 5′ protruding sticky ends, these terminus can be filled into blunt ends with 5′ to 3′ Taq polymerase activity in the existence of dNTP, and then again added a single adenosine at their 3′ ends by the transferase activity of Taq polymerase. The fragments modified as such above mentioned can be easily subcloned into T vector.

关 键 词：DNA重组 T载体 TAQ DNA聚合 乙型肝炎病毒 

分 类 号：R373.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]