Mutagenesis with Degenerate Oligonucleotides: Creating Numerous Mutations in a Small DNA Sequence

David E. Hill1

1 Applied Biotechnology, Cambridge, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.2A
DOI:  10.1002/0471142727.mb0802as46
Online Posting Date:  May, 2001
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Abstract

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.

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 10× E. coli DNA polymerase I buffer (unit 3.4)
  • 10 mM dNTP mix of 2.5 mM 4dNTP mix (unit 3.4)
  • [α‐32P]dNTP (400 to 800 Ci/mmol; see unit 3.4)
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • TE buffer, pH 7.5 ( appendix 22)
  • 3 M sodium acetate, pH 5.2
  • Buffered phenol (unit 2.1)
  • Ethanol
  • Gel elution buffer: 0.5 M ammonium acetate/1 M EDTA (store protected from light up to several months at room temperature)
  • T4 DNA ligase (measured in cohesive‐end units; unit 3.14)
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1), denaturing polyacrylamide gel electrophoresis (unit 7.4), phenol extraction and ethanol precipitation (unit 2.1), nondenaturing polyacrylamide gel electrophoresis (unit 2.7), transformation of E. coli (unit 1.8), and DNA sequence analysis (Chapter 7)
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Figures

Videos

Literature Cited

Literature Cited
   Hill, D.E., Hope, I.A., Macke, J.P., and Struhl, K. 1986. Saturation mutagenesis of the yeast His3 regulatory site: Requirements for transcriptional induction and binding by GCN4 protein. Science 234:451‐457.
   Hill, D.E., Oliphant, A.R., and Struhl, K. 1987. Mutagenesis with degenerate oligonucleotides: An efficient method for saturating a defined DNA region with base pair substitutions. Meth. Enzymol. 155:558‐568.
   Matteucci, M.D. and Heyneker, H.L. 1983. Targeted random mutagenesis: The use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5′ of an ATG initiation codon. Nucl. Acids Res. 11:3113‐3121.
   Oliphant, A.R., Nussbaum, A.L., and Struhl, K. 1986. Cloning of random‐sequence oligodeoxynucleotides. Gene 44:177‐183.
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