Wong K K, McClelland M
California Institute of Biological Research, La Jolla 92037.
Nucleic Acids Res. 1991 Mar 11;19(5):1081-5. doi: 10.1093/nar/19.5.1081.
When dCTP is replaced by methyl5-dCTP in the polymerase chain reaction some templates cannot be efficiently amplified by Taq polymerase or Vent polymerase using standard cycling parameters. However, this phenomenon can be overcome by increasing the temperature of the denaturation steps to 100 degrees C, or by adding dITP to destabilize the m5dC:dG base pairs. Once the block to amplification of m5dC-substituted DNA was overcome, methylated DNA from the 'superpolylinker' of the plasmid pSL 1180 was used as a substrate to check the methyl-sensitivity of a variety of restriction endonucleases. The m5dC-substituted DNAs should also be valuable substrates for defining the specificity of methyl-dependent endonucleases.
在聚合酶链反应中，当用甲基化5 - 脱氧胞苷三磷酸（methyl5 - dCTP）取代脱氧胞苷三磷酸（dCTP）时，一些模板无法使用标准循环参数通过Taq聚合酶或Vent聚合酶进行有效扩增。然而，这种现象可以通过将变性步骤的温度提高到100摄氏度，或者通过添加脱氧肌苷三磷酸（dITP）来破坏甲基化5 - 脱氧胞苷（m5dC）与鸟嘌呤（dG）碱基对的稳定性来克服。一旦克服了对甲基化5 - 脱氧胞苷取代DNA扩增的阻碍，就使用来自质粒pSL 1180“超级多克隆位点”的甲基化DNA作为底物，来检测各种限制性内切酶的甲基敏感性。甲基化5 - 脱氧胞苷取代的DNA对于确定甲基依赖性内切酶的特异性也应是有价值的底物。
