Mutagenesis with degenerate oligonucleotides: creating numerous mutations in a small DNA sequence.
The procedure described in this unit is an efficient method for using the products of a single oligonucleotide synthesis to create numerous mutations in a small region of DNA (20 to 80 nucleotides). Mutant oligonucleotides are generated during the organic synthesis reaction by including low concentrations of the three non-wild-type nucleotide precursors with the wild-type precursor at each step of the synthesis. The product of such a DNA synthesis is a degenerate oligonucleotide, i.e., a complex mixture of related molecules, each of which has a defined probability of being altered from the wild-type sequence. The frequencies and types of zero, single, double, and higher order mutations can be set simply by choosing the appropriate amounts of non-wild-type precursors at each step of nucleotide addition during the synthesis.
Hill
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Applied Biotechnology, Cambridge, Massachusetts, USA.
D005821:Genetic Techniques; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D017354:Point Mutation
D009841:Oligonucleotides