Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization.
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.
D001483:Base Sequence; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D013604:T-Phages / Q000201:enzymology; D013698:Templates, Genetic
D004251:DNA Transposable Elements; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes