Chang G J, Johnson B J, Trent D W
Division of Vector-Borne Viral Diseases, Centers for Disease Control, Fort Collins, CO 80522.
DNA. 1988 Apr;7(3):211-7. doi: 10.1089/dna.1988.7.211.
A simple and efficient mutagenesis procedure is described which uses both the 3'----5' exonuclease and 5'----3' polymerase activities of T4 DNA polymerase. Different types of mutation-deletion, insertion, and substitution-can be introduced into the DNA in a single reaction. The technique uses recombinant M13 single-stranded DNA and two complementary DNA oligonucleotides to target and control the extent of deletions catalyzed by T4 DNA polymerase. The second oligonucleotide not only directs ligation, but also serves as a template for insertion or substitution of nucleotides by T4 polymerase. Mutant phages in a genetically pure form can be obtained at high efficiency, allowing their characterization directly by nucleotide sequencing without prior enrichment, plaque purification, and screening. We tested the versatility of this method by manipulating five regions of cDNA encoding the structural proteins of eastern equine encephalitis virus.
本文描述了一种简单高效的诱变程序，该程序利用了T4 DNA聚合酶的3'→5'核酸外切酶和5'→3'聚合酶活性。不同类型的突变——缺失、插入和替换——可在单一反应中引入到DNA中。该技术使用重组M13单链DNA和两个互补的DNA寡核苷酸来靶向并控制T4 DNA聚合酶催化的缺失范围。第二个寡核苷酸不仅指导连接，还作为T4聚合酶插入或替换核苷酸的模板。可以高效获得基因纯合形式的突变噬菌体，无需事先富集、噬菌斑纯化和筛选，即可直接通过核苷酸测序对其进行表征。我们通过操作编码东部马脑炎病毒结构蛋白的cDNA的五个区域，测试了该方法的通用性。
