tailieunhanh - Efficient site directed in vitro mutagenesis using ampicillin selection

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60- 90%) mutagenesis was obtained | 1990 Oxford University Press Nucleic Acids Research Vol. 18 No. 12 3439 Efficient site directed in vitro mutagenesis using ampicillin selection Martin and David Promega Corporation Madison Wl 53711 USA Received April 11 1990 Accepted May 25 1990 ABSTRACT A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector highly efficient 60 - 90 mutagenesis was obtained. INTRODUCTION Site-directed in vitro mutagenesis is a valuable technique for among other things the study of critical amino acid residues involved in enzymatic activity the study of DNA promoter and enhancer function and structure the study of residues important in protein folding the study of the structure of DNA binding sites for proteins the study of functions of particular residues or domains in protein stability the creation of mutant proteins with increased stability or resistance to environmental agents the study of effects of removing sites for protein modification such as phosphorylation or glycosylation and for engineering of expression clones. Hutchison et al. 1 introduced a general method to obtain sitespecific changes in DNA sequences using single-stranded DNA ssDNA and a synthetic oligonucleotide. The oligonucleotide is complementary to the single-stranded template DNA except for a region of mismatch in the center. Following hybridization the oligonucleotide is extended with DNA polymerase to create a double-strand structure. The nick is sealed and the .