| Abstract: | A model system is developed to test oligonucleotide-directed mutations: T----C transition, T and C deletions (delta T and delta C), C insertion, double mutations (A----G, delta T), (T----C, A----G), and large oligonucleotide deletions (36 or 44 nucleotides). The system includes 9 variants of the phage M13 DNA carrying fragment of beta-galactosidase gene, and oligodeoxyribonucleotides partially noncomplementary to DNA sequence of this gene. Six variants are obtained by the site-localized mutagenesis, the other were described earlier. Induced mutations are easily tested by phenotype change of transformed bacteria (Lac+----Lac-); by formation or loss of the sites for BamHI and EcoRI restrictases; by DNA hybridization with 32P-labeled oligonucleotides; and by DNA sequencing by the Sanger method. The system is used to study the role of some factors, such as completeness of RF DNA synthesis, thermal stability of the oligonucleotide: DNA complex, quality of enzymes and substrates used in polymerase reaction, mutation type or the efficiency of mutagenesis. A number of unexpected mutations were observed in the course of oligonucleotide-directed mutagenesis. Lower yields of some mutants induced by oligonucleotides are shown to be due to the action of repair systems of bacteria. |
