Nucleic Acids Research, 1989, Vol. 17, No. 2 723-733
© 1989
MOLECULAR BIOLOGY |
Construction of mutant and chimeric genes using the polymerase chain reaction
Department of Cell Biology and The Kaplan Cancer Center, New York University School of Medicine New York, NY 10016, USA
In the polymerase chain reaction (PCR) the specific amplification of a small segment of DNA within a complex DNA sample is effected by repeated cycles of DNA denaturation and enzymatic synthesis primed by two oligonucleotides complementary to regions within opposite strands of the DNA. In this report a simple and efficient method is described in which PCR methodology is used to introduce specific mutations into a double stranded DNA molecule. In this procedure a supercoiled plasmid DNA serves as template for a PCR in which a primer bearing the mutated sequence is incorporated into the amplified product. The presence of convenient restriction sites in the mutagenic primer and in the amplified DNA permit direct replacement of a wild type DNA segment with the mutated segment by treating the PCR mixture with the appropriate restriction endonucleases followed by DNA ligase. Using this procedure, a single amino acid replacement, a 16 amino acid deletion and a replacement of four amino acids with a twelve amino acid segment from another membrane protein were introduced into the amino terminal signal segment of rat hepatic cytochrome P450b (P450IIB1).
+Present address: Laboratoire de Neurobiologie, Ecole Normale Superieure, 46 rue d'UIm 75005, Paris, FRANCE
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