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. 1984 Aug 24;12(16):6615–6628. doi: 10.1093/nar/12.16.6615

Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization.

R A Zakour, E A James, L A Loeb
PMCID: PMC320100  PMID: 6089123

Abstract

We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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