Diaz R S, Sabino E C
Laboratório de Retrovirologia, Universidade Federal de São Paulo, Brasil.
Braz J Med Biol Res. 1998 Oct;31(10):1239-42. doi: 10.1590/s0100-879x1998001000001.
For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.
对于聚合酶链反应（PCR）的某些应用，可能有必要考虑复制的准确性。使PCR便于用户使用的突破是嗜热栖热菌（Taq）DNA聚合酶的商业化，这种酶能在DNA变性所需的高温下存活。当需要更高保真度时，开发具有固有的3'至5'核酸外切酶校对活性（Taq聚合酶缺乏这种活性）的酶将是一种改进。我们使用正向突变试验来比较Taq聚合酶和嗜热栖热放线杆菌（ULTMA）DNA聚合酶（一种具有校对活性的酶）的保真度。即使使用最佳缓冲条件、热循环参数和循环次数（分别读取约3000个碱基后，ULTMA和Taq的错误率分别为0.2%和0.13%），我们也没有发现这两种酶在保真度上有显著差异。我们得出结论，就测序目的而言，使用含有固有3'至5'核酸外切酶活性的DNA聚合酶进行DNA扩增没有差异。也许PCR的特异性和保真度是受靶序列性质以及每个PCR组分影响的复杂问题。
