Su T Z, el-Gewely M R
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Gene. 1988 Sep 15;69(1):81-9. doi: 10.1016/0378-1119(88)90380-0.
A method to introduce multiple mutations and to reconstruct genes, using a single oligodeoxyribonucleotide and DNA polymerase with high processivity, such as modified T7 DNA polymerase [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 84 (1987a) 4767-4771], is described. A eukaryotic cDNA, coding for porcine growth hormone (pGH), was reconstructed in this study to delete 75 bp and to introduce a G----A transition. The deletion removes 75 bp and brings an ATG just upstream from the codon for the first amino acid in the mature protein. Moreover, the G----A substitution creates a new PvuII restriction site to facilitate further manipulation of the gene. Maximum mutation frequency with this multisite-directed mutagenesis is reached within 15 min with an efficiency approaching 50%, when using the modified T7 DNA polymerase. No multisite-directed mutants were obtained when T4 DNA polymerase or Klenow (large) fragment of DNA polymerase I were used. The described method is also applicable to simple single site-directed mutations as well as to more complex gene reconstruction strategies.
本文描述了一种利用单链寡脱氧核糖核苷酸和具有高持续合成能力的DNA聚合酶（如修饰的T7 DNA聚合酶 [塔博尔和理查森，《美国国家科学院院刊》84 (1987a) 4767 - 4771]）引入多个突变并重建基因的方法。在本研究中，对编码猪生长激素（pGH）的真核cDNA进行了重建，以删除75个碱基对并引入一个G→A的转换。该缺失去除了75个碱基对，并在成熟蛋白第一个氨基酸的密码子上游引入了一个ATG。此外，G→A替换产生了一个新的PvuII限制性酶切位点，便于对该基因进行进一步操作。使用修饰的T7 DNA聚合酶时，在15分钟内即可达到这种多位点定向诱变的最大突变频率，效率接近50%。当使用T4 DNA聚合酶或DNA聚合酶I的Klenow（大片段）时，未获得多位点定向突变体。所描述的方法也适用于简单的单位点定向突变以及更复杂的基因重建策略。
