Oligodeoxyribonucleotides as Site-Specific Mutagens
①Classical genetic analysis of cells and viruses involves spontaneous or induced mutation together with screening or selection for a changed phenotype followed by assignment of the changed DNA to a specific location in the genome. This approach has been very useful in defining the various functions of DNA both in coding and regulatory regions ②The advent of rapid DNA sequence determination methods has created a new situation where extensive regions of DNA sequences can be known wit out a precise knowledge of the functions of that DNA. ③Deliberate, defined modification of a DNA at a specific site followed by examination of the biological properti of the modified DNA offers a way out of this dilemma. ④A number of methods have recently been developed for in vitro modification of the genomic DNA and recombinant plasmids (1,2). These methods in general operate by introduction, using chemical or enzymatic means, of a limited number of changes in the vicinity of a restriction endonuclease cleavage site. The procedure has been called site-directed mutagenesis reversed genetics or the production of constructed mutants (1,2). Whilst the general locality of the introduced changes are known, their nature or exact position are not defined until the sequence of the mutant DNA is known. Usually more than one type of changed DNA is produced. in this article an alternate strategy for the production of defined point mutations, site-specific mutagenesis, will be described. This method uses a synthetic oligodeoxyribonucleotide, different at only one nucleotide from wild-type, as a specific mutagen, and involves in vitro integration of the oligodeoxyribonucleotide into genomic DNA. The experiments described here were performed with bacteriophage ØX174 DNA; in principle the method is applicable to any circular genome of a similar size. Thus the method is appropriate not only for small circular bacteriophage and viral DNAs but also for parts of larger genomes cloned as recombinants in bacterial plasmid vectors.
KeywordsMismatched Nucleotide Mutant Production Site Specific Mutagen Transversion Mutation Phage Plaque
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