Nucleic Acids Research, 1989, Vol. 17, No. 16 6545-6551
© 1989
ENZYMOLOGY |
A simple method for site-directed mutagenesis using the polymerase chain reaction
Department of Molecular Biology, University of Southern California Los Angeles, CA 90089-1340, USA 1Department of Biochemistry, University of California Berkeley, CA 94720, USA
*To whom correspondence should be addressed
Received June 5, 1989. Revised July 12, 1989. Accepted July 12, 1989.
We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.
+ Present address: Department of Biological Sciences, University of California, Santa Barbara, CA 93106, USA
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K. K. H. Poon, J. C.-L. Chu, and S.-L. Wong Roles of Glucitol in the GutR-mediated Transcription Activation Process in Bacillus subtilis. GLUCITOL INDUCES GutR TO CHANGE ITS CONFORMATION AND TO BIND ATP J. Biol. Chem., August 3, 2001; 276(32): 29819 - 29825. [Abstract] [Full Text] [PDF]
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J. Carmichael, J. Chatellier, A. Woolfson, C. Milstein, A. R. Fersht, and D. C. Rubinsztein Bacterial and yeast chaperones reduce both aggregate formation and cell death in mammalian cell models of Huntington's disease PNAS, August 15, 2000; 97(17): 9701 - 9705. [Abstract] [Full Text] [PDF]
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