UNIT 8.2A Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Hill, D. E. 2001. Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence. Current Protocols in Molecular Biology. 46:8.2A:8.2.1–8.2.7.
- Published Online: 1 MAY 2001
- Published Print: APR 1999
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.