Pritchard C E, Southern E M
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Nucleic Acids Res. 1997 Sep 1;25(17):3403-7. doi: 10.1093/nar/25.17.3403.
The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets. Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases. Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers. The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer. Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction. In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers. The results are relevant to mechanisms of ligation.
双链DNA中的切口需通过DNA连接酶进行封闭,其对沃森-克里克碱基配对的要求是区分DNA靶标中单个碱基突变的寡核苷酸连接测定法的基础。在模型系统中进行的实验表明,不同连接酶能够连接的寡核苷酸的最短长度各不相同。嗜热栖热菌(Tth)DNA连接酶无法连接任何长度为六或更短的寡核苷酸,而T4 DNA连接酶和T7 DNA连接酶都能够连接六聚体。Tth DNA连接酶进行寡核苷酸连接的速率在七聚体和九聚体之间增加。在连接末端远端导致双链因磨损而缩短的错配会减缓连接反应。就Tth DNA连接酶而言,在切口5'端的第七和第八位的错配会完全抑制八聚体的连接。这些结果与连接机制相关。