Cariello N F, Swenberg J A, Skopek T R
University of North Carolina, Pathology Department, Chapel Hill 27599.
Nucleic Acids Res. 1991 Aug 11;19(15):4193-8. doi: 10.1093/nar/19.15.4193.
DNA synthesis fidelities of two thermostable DNA polymerases, Thermus aquaticus (Taq) and Thermococcus litoralis (Tli, also known as Vent), and a non-thermostable enzyme, a modified T7 DNA polymerase (Sequenase), were determined by analyzing polymerase chain reaction (PCR) products using denaturing gradient gel electrophoresis (DGGE). The error rates were 4.4, 8.9, and 2.4 x 10(-5) errors/bp for modified T7, Taq, and Tli polymerase, respectively. Reducing the nucleotide triphosphate concentration for Tli polymerase during PCR did not alter the fidelity. The ability of DGGE to detect a mutant present at several percent in a wild type population is related to the polymerase fidelity. To examine the sensitivity of mutant detection, human genomic DNA containing a 1% fraction of a known base pair substitution mutant was PCR-amplified with the three enzymes using primers that flank the mutant sequence. The PCR products were analyzed by DGGE. The signal from the mutant present at 1% was visible in the samples amplified with modified T7 and Tli polymerase, but the higher error rate of Taq polymerase did not permit visualization of the signal in DNA amplified with Taq polymerase.
通过使用变性梯度凝胶电泳（DGGE）分析聚合酶链反应（PCR）产物，测定了两种耐热DNA聚合酶——嗜热水生栖热菌（Taq）和嗜热栖热放线菌（Tli，也称为Vent）以及一种非耐热酶——修饰的T7 DNA聚合酶（测序酶）的DNA合成保真度。修饰的T7、Taq和Tli聚合酶的错误率分别为4.4、8.9和2.4×10⁻⁵个错误/碱基对。在PCR过程中降低Tli聚合酶的三磷酸核苷酸浓度不会改变保真度。DGGE检测野生型群体中百分之几的突变体的能力与聚合酶保真度有关。为了检测突变体检测的灵敏度，使用位于突变序列两侧的引物，用这三种酶对含有1%已知碱基对替代突变体的人类基因组DNA进行PCR扩增。通过DGGE分析PCR产物。在用修饰的T7和Tli聚合酶扩增的样品中，1%的突变体信号可见，但Taq聚合酶较高的错误率使得在用Taq聚合酶扩增的DNA中无法看到该信号。
