HIGH-FIDELITY AMPLIFICATION USING A THERMOSTABLE DNA-POLYMERASE ISOLATED FROM PYROCOCCUS-FURIOSUS
HIGH-FIDELITY AMPLIFICATION USING A THERMOSTABLE DNA-POLYMERASE ISOLATED FROM PYROCOCCUS-FURIOSUS
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DOI：
10.1016/0378-1119(91)90480-y
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发表时间：
1991-12-01
期刊：
影响因子：
3.5
通讯作者：
MATHUR, EJ
中科院分区：
文献类型：
--
作者：
LUNDBERG, KS;SHOEMAKER, DD;MATHUR, EJ
从嗜热古细菌中分离到一个具有3‘-5’-核酸外切酶(校对)活性的耐热DNA聚合酶。为了测试它的保真度，我们利用了一种在聚合酶链式反应(PCR)过程中直接在体外测量DNA聚合酶保真度的遗传测试方法。我们的结果表明，用从P.Furiosus中纯化的DNA聚合酶进行的聚合酶扩增得到的扩增产物的突变数量不到Taq DNA聚合酶的10%。PCR保真度分析是基于使用PFU或Taq DNA聚合酶扩增和克隆LacI、LACO和LacZ-α基因序列(lacIOZ-α)。LacI基因中的某些突变使Lac抑制蛋白失活，并允许表达β-Gal。当电镀在显色底物上时，这些LacI突变体表现出蓝色斑块的表型。这些研究表明，在LacI基因的182个已知可检测位点上，PFU DNA聚合酶的每个核苷酸的错误率为1.6×10(-6)，比Taq DNA聚合酶的2.0×10(-5)的错误率提高了10倍以上。10(5)-倍增。
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.