Improving the fidelity of Thermus thermophilus DNA ligase
Improving the fidelity of Thermus thermophilus DNA ligase
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DOI：
10.1093/nar/24.15.3071
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发表时间：
1996-08-01
影响因子：
14.9
通讯作者：
Barany, F
中科院分区：
文献类型：
--
作者：
Luo, JY;Bergstrom, DE;Barany, F
来自嗜热栖热菌的DNA连接酶（Tth DNA连接酶）密封DNA双链体底物中的单链断裂（切口）。利用这种酶的特异性和热稳定性，在连接酶链反应（LCR）和连接酶检测反应（LDR）中区分与遗传疾病相关的单碱基突变。在此，我们描述了一种定量测定使用荧光标记的底物来研究Tth DNA连接酶的保真度。与切口的5 '侧相比，该酶对切口的3'侧上的所有单碱基错配表现出显著更大的辨别力。在切口3 '侧的所有12个可能的单碱基对错配中，仅T-G和G-T错配在23小时孵育后产生可定量水平的连接产物。Tth DNA连接酶的高保真度可以通过在区分性寡核苷酸的第三位置处引入错配碱基或通用核苷类似物来进一步改善。最后，发现两种突变Tth DNA连接酶K294 R和K294 P使用该测定具有增加的保真度。
The DNA ligase from Thermus thermophilus (Tth DNA ligase) seals single-strand breaks (nicks) in DNA duplex substrates. The specificity and thermostability of this enzyme are exploited-in the ligase chain reaction (LCR) and ligase detection reaction (LDR) to distinguish single base mutations associated with genetic diseases. Herein, we describe a quantitative assay using fluorescently labeled substrates to study the fidelity of Tth DNA ligase. The enzyme exhibits significantly greater discrimination against all single base mismatches on the 3'-side of the nick in comparison with those an the 5'-side of the nick. Among all 12 possible single base pair mismatches on the 3'-side of the nick, only T-G and G-T mismatches generated a quantifiable level of ligation products after 23 h incubation. The high fidelity of Tth DNA ligase can be improved further by introducing a mismatched base or a universal nucleoside analog at the third position of the discriminating oligonucleotide. Finally, two mutant Tth DNA ligases, K294R and K294P, were found to have increased fidelity using this assay.