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,再克隆于其他表达载体中.
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.
刘定燮,骆抗先.应用T/A策略克隆乙肝病毒pC/C及C基因[J].生物化学与生物物理进展,2000,27(6):662-663
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