Simple and highly efficient site-specific mutagenesis, by ligation of an oligodeoxyribonucleotide into gapped heteroduplex DNA in which the template strand contains deoxyuridine.
A simple and highly efficient procedure for oligodeoxynucleotide (oligo)-directed mutagenesis has been developed. In this procedure, a gapped heteroduplex DNA is first constructed and purified. The gapped heteroduplex consists of a circular 'template' strand of DNA, which contains some misincorporated deoxyuridine nucleotides, and a complementary strand which does not contain deoxyuridine, and which lacks a defined segment. Making a specific change in the sequence of the DNA within the gapped region then only requires ligation and transformation. An oligo, exactly the same length as the gap, and with the desired sequence, is synthesized, purified, and ligated directly into the gap in the heteroduplex. When this DNA is used to transform wt (ung+) Escherichia coli, about 80% of the resulting plasmids contain the sequence determined by the synthetic oligo. One gapped heteroduplex preparation can be used for many mutagenesis experiments, so that this procedure is well-suited for producing a series of defined mutations within a defined target region flanked by sites for restriction enzyme cleavage. As the method does not require a polymerase, the effects of primer displacement and polymerase infidelity are avoided.
Terwilliger
T C
TC
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005821:Genetic Techniques; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D013698:Templates, Genetic
D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides