A Simple Method for Site-Directed Mutagenesis with Double-Stranded Plasmid DNA
Oligonucleotide-directed site-specific mutagenesis is a powerful tool to explore protein structure—function relationships. The single-stranded (M13) method (1,2), the polymerase chain reaction (PCR) (3, 4, 5, 6), and the double-stranded plasmids method (7, 8, 9) are three basic procedures for these purposes. The single-stranded method developed by Zoller and Smith (10) has been modified to achieve a higher yield of mutants. Recently, the PCR method has become an easy and popular technique for site-directed mutagenesis. Both advantages and disadvantages of these methods have been discussed thoroughly (11,12). Limitations of these methods include the availability of restriction sites for subcloning and the instability of large inserts in M13 vectors (13), the low fidelity of Taq polymerase, the cost of multiple primers in the PCR protocols, and the low mutant yields with the double-stranded plasmid method.
KeywordsPolymerase Chain Reaction Method Klenow Fragment Polymerase Chain Reaction Protocol Mutagenic Primer Mutant Plasmid
- 7.Vlasuk, G. P. and Inouye, S. (1983) Site-specific mutagenesis vising synthetic oligodeoxyribonucleotides as mutagens, in Experimental Manipulation of Gene Expression, Appendix 2 (Inouye, M., ed.), Academic, New York, pp. 291–303.Google Scholar
- 12.Inouye, S. and Inouye M. (1987) Oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA, in Synthesis and Applications of DNA and RNA (Narang, S. A., ed.), Academic, New York, pp. 181–206.Google Scholar
- 16.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar