Liu J S, Roesler W J, Hanson R W
Case Western Reserve University, School of Medicine.
Biotechniques. 1990 Dec;9(6):738-42.
The introduction by Kunkel of a method which uses uracil-containing single-stranded DNA as a template for site-directed mutagenesis has greatly simplified the procedure for introducing point mutations into DNA in a phagemid vector. We have extended the use of this method to introduce block mutations into specific regions of DNA, using oligonucleotide primers containing multiple nucleotide mismatches which contain new restriction sites. The oligonucleotides, with up to 15 mismatches, can specifically and stably bind to the predicted target sequence. Because of the high mutation rate and convenient selection by restriction enzyme digestion, this method of introducing site-specific block mutations into DNA is very easy and efficient. We have constructed a series of block mutations in the promoter of the gene for the cytosolic form of P-enolpyruvate carboxykinase (GTP) (EC4.1.1.32) (PEPCK) by this method showing that a block mutation in the cAMP responsive element (CRE-1) completely disrupts protein binding to CRE-1 in vitro and causes a dramatic decrease in the basal transcription from the PEPCK promoter in vivo.
昆克尔提出了一种方法，该方法使用含尿嘧啶的单链DNA作为定点诱变的模板，极大地简化了将点突变引入噬菌粒载体中DNA的程序。我们扩展了这种方法的应用，使用含有多个核苷酸错配且包含新限制酶切位点的寡核苷酸引物，将大片段突变引入DNA的特定区域。这些寡核苷酸最多有15个错配，可以特异性且稳定地与预测的靶序列结合。由于高突变率以及通过限制酶消化进行方便的筛选，这种将位点特异性大片段突变引入DNA的方法非常简便且高效。我们通过这种方法在胞质型磷酸烯醇式丙酮酸羧激酶（GTP）（EC4.1.1.32）（PEPCK）基因的启动子中构建了一系列大片段突变，结果表明，cAMP反应元件（CRE-1）中的大片段突变在体外完全破坏了蛋白质与CRE-1的结合，并在体内导致PEPCK启动子的基础转录显著下降。
