Misincorporation during DNA synthesis, analyzed by gel electrophoresis.
Misincorporation during DNA synthesis, analyzed by gel electrophoresis.
复制标题
通过凝胶电泳分析 DNA 合成过程中的错误掺入。
DOI：
10.1093/nar/12.7.3155
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发表时间：
1984
影响因子：
14.9
通讯作者：
Beattie,KL
中科院分区：
文献类型：
--
作者：
Hillebrand,GG;McCluskey,AH;Abbott,KA;Revich,GG;Beattie,KL
A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template–primer. In this method elongation of a [5′–12P]primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these corditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by antoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to ‘minus’ reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity ofE.coliDNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.
