FIDELITY OF DNA-SYNTHESIS BY THE THERMOCOCCUS-LITORALIS DNA-POLYMERASE - AN EXTREMELY HEAT-STABLE ENZYME WITH PROOFREADING ACTIVITY
FIDELITY OF DNA-SYNTHESIS BY THE THERMOCOCCUS-LITORALIS DNA-POLYMERASE - AN EXTREMELY HEAT-STABLE ENZYME WITH PROOFREADING ACTIVITY
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DOI：
10.1093/nar/19.18.4967
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发表时间：
1991-09-25
影响因子：
14.9
通讯作者：
PITKANEN, K
中科院分区：
文献类型：
--
作者：
MATTILA, P;KORPELA, J;PITKANEN, K
我们证明了从Thermococcus litoralis分离的DNA聚合酶（Vent（TM）DNA聚合酶）是第一个报道的具有3' -> 5'校正外切核酸酶活性的热稳定DNA聚合酶。 这有助于通过聚合酶进行高度精确的体外DNA合成。 在碱基取代保真度试验中观察到的突变频率在30 x 10（-6）范围内。 这些值比其他缺乏校正活性的热稳定DNA聚合酶低5 - 10倍。 使用正向突变试验（1）分析所有DNA聚合酶错误（转换、颠换、移码突变）。 海滨热球菌DNA聚合酶的突变频率在15 - 35 x 10（-4）之间变化，比使用没有校对活性的酶获得的相应值低2 - 4倍。 我们还注意到，来自Thermococcus litoralis的DNA聚合酶的保真度响应于dNTP浓度、每个反应所用的酶单位和MgSO 4浓度相对于反应中存在的dNTP总浓度的变化。 海滨热球菌DNA聚合酶的高保真DNA体外合成为在DNA扩增应用中保持原始靶DNA序列的遗传信息完整提供了良好的可能性。
We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (Vent(TM) DNA polymerase) is the first thermostable DNA polymerase reported having a 3' --> 5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5 - 10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All clases of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15 - 35 x 10(-4) being 2 - 4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.