Slupphaug G, Alseth I, Eftedal I, Volden G, Krokan H E
UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
Anal Biochem. 1993 May 15;211(1):164-9. doi: 10.1006/abio.1993.1248.
Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.
在PCR扩增过程中，常用掺入dUMP而非dTMP的方法来控制残留污染。我们测试了四种热稳定DNA聚合酶在PCR中利用dUTP作为底物的能力。在存在dUTP而非dTTP的情况下，嗜热水生栖热菌DNA聚合酶对产物的扩增效果良好，但其他三种热稳定DNA聚合酶的扩增效率极低。后者的原因如下：(a) 相对于dTMP，dUMP的掺入率较低；(b) 对DNA中dUMP的校对增加；(c) 通过在存在dUTP的情况下进行测序反应证实，在dUMP残基处相对终止；(d) 商业酶制剂中存在热稳定dUTPase活性。最后一点仅适用于激烈热球菌DNA聚合酶。本研究表明，各种热稳定DNA聚合酶利用dTTP和dUTP的效率差异很大，因此DNA聚合酶的选择对于DNA扩增可能至关重要。
