A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides.
A method is described for the efficient saturation mutagenesis of a segment of DNA. A pool of mixed oligodeoxynucleotides (oligos) is generated in a single synthesis by deliberately contaminating each nucleotide reservoir with a low concentration of the other three monomers. The concentration at which a particular level of substitution (i.e. single, double, etc.) is favored can be calculated from simple probability. The pool of single-stranded, mixed oligos is then cloned directly into a double-stranded vector. This is achieved by designing the oligo such that it has ends which are complementary to those left by restriction enzymes generating 5' and 3' overhanging ends. The efficiency of the cloning procedure that we describe is greater than 90%. As a result, direct sequence analysis of transformant clones is justified. In the example described, 2/3 of M13 plaques containing an inserted oligo carried one or more mutations. Mutations generated by this method are randomly distributed throughout the inserted DNA and include all types of substitution. The ease and efficiency of the procedure eliminate any requirement for genetic selection of mutants and thus allow phenotypically silent mutations to be obtained.
D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant; D005822:Genetic Vectors; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides