Zakour R A, James E A, Loeb L A
Nucleic Acids Res. 1984 Aug 24;12(16):6615-28. doi: 10.1093/nar/12.16.6615.
We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.
我们利用DNA合成的不忠实性作为定点诱变的基础。一种核酸内切酶限制片段和一种合成寡核苷酸都被用作引物。来自噬菌体T4的DNA聚合酶用于将引物末端延伸至紧邻phiX174 DNA模板上两个不同预选位置的位置。然后，来自禽成髓细胞瘤病毒的易错DNA聚合酶用于在指定位置高效插入单个非互补核苷酸。DNA序列分析证实，每个位点特异性诱变反应产生的突变噬菌体都含有与DNA复制反应期间提供的核苷酸互补的核苷酸。将讨论这种方法对克隆DNA的普遍适用性。
