Lundberg K S, Shoemaker D D, Adams M W, Short J M, Sorge J A, Mathur E J
Division of Research and Development, Stratagene, Inc., La Jolla, CA 92037.
Gene. 1991 Dec 1;108(1):1-6. doi: 10.1016/0378-1119(91)90480-y.
A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.
一种具有相关3'至5'核酸外切酶（校对）活性的耐热DNA聚合酶已从嗜热古细菌激烈火球菌（Pfu）中分离出来。为了测试其保真度，我们利用了一种遗传测定法，该方法可在聚合酶链反应（PCR）过程中直接在体外测量DNA聚合酶的保真度。我们的结果表明，用从激烈火球菌中纯化的DNA聚合酶进行PCR，产生的扩增产物所含的突变数量比用Taq DNA聚合酶进行类似扩增所获得的突变数量少不到10%。PCR保真度测定法基于使用Pfu或Taq DNA聚合酶对lacI、lacO和lacZα基因序列（lacIOZα）进行扩增和克隆。lacI基因内的某些突变会使Lac阻遏蛋白失活，并允许β半乳糖苷酶的表达。当接种在显色底物上时，这些LacI-突变体表现出蓝色菌斑表型。这些研究表明，对于Pfu DNA聚合酶，在lacI基因的182个已知可检测位点中每核苷酸诱导的错误率为1.6×10-6，在大约105倍扩增后，比Taq DNA聚合酶的2.0×10-5错误率有超过十倍的改善。
