Construction of mutant and chimeric genes using the polymerase chain reaction.
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).
Vallette
F
F
Department of Cell Biology, New York University School of Medicine 10016.
Mege
E
E
Reiss
A
A
Adesnik
M
M
D000818:Animals; D001483:Base Sequence; D002678:Chimera; D002872:Chromosome Deletion; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D005785:Gene Conversion; D005813:Genes, Synthetic; D015345:Oligonucleotide Probes; D051381:Rats; D019914:Taq Polymerase
D015345:Oligonucleotide Probes; D003577:Cytochrome P-450 Enzyme System; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase