Linker‐Scanning Mutagenesis of DNA

John M. Greene1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.4
DOI:  10.1002/0471142727.mb0804s13
Online Posting Date:  May, 2001
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Abstract

Two protocols are described in which clusters of point mutations are introduced throughout a sequence of interest that has been cloned into a plasmid vector. The first protocol uses complementary oligonucleotides and requires a unique restriction site adjacent to the region that is to be mutagenized. A nested series of deletion mutations is first generated in the region. A pair of complementary oligonucleotides are synthesized to fill in the gap in the sequence of interest between the linker at the deletion endpoint and the nearby restriction site. The linker sequence actually provides the desired clusters of point mutations as it is moved or “scanned” across the region by its position at the varied endpoints of the deletion mutation series. An makes use of site‐directed mutagenesis procedures to introduce smaller clusters of point mutations throughout the target region.

     
 
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Table of Contents

  • Basic Protocol 1: Linker Scanning Using Nested Deletions and Complementary Oligonucleotides
  • Alternate Protocol 1: Linker Scanning Using Oligonucleotide‐Directed Mutagenesis
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Linker Scanning Using Nested Deletions and Complementary Oligonucleotides

  Materials
  • High‐copy‐number plasmid vector (unit 1.5)
  • Linkers (unit 3.16, Example 3.16.6)
  • Bal 31 nuclease (unit 3.12) or exonuclease III and S1 nuclease (units 3.11 & 3.12, respectively)
  • Restriction endonucleases and buffers (unit 3.1)
  • 0.5 M EDTA, pH 8 (optional; appendix 22)
  • 100% ethanol
  • Low gelling/melting temperature agarose (unit 2.6)
  • Competent E. coli cells (unit 1.8)
  • 29:1 acrylamide/bisacrylamide (unit 2.7) or sieving agarose (unit 2.8)
  • Synthetic oligonucleotides for the gene of interest
  • TE buffer ( appendix 22) containing 150 mM NaCl
  • T4 DNA ligase (measured in cohesive‐end units; unit 3.14)
  • 10× T4 DNA ligase buffer (unit 3.4)
  • Selective plates
  • Additional reagents and equipment for creating nested sets of deletions (unit 7.2), ethanol precipitation (unit 2.1), ligating DNA fragments (unit 3.16), isolating DNA fragments using low gelling/melting temperature agarose (unit 2.6), preparing and transforming competent E. coli cells (unit 1.8), minipreps of plasmid DNA (unit 1.6), nondenaturing polyacrylamide gel electrophoresis (unit 2.7) or sieving agarose gel electrophoresis (unit 2.8), and DNA sequencing (Chapter 7)

Alternate Protocol 1: Linker Scanning Using Oligonucleotide‐Directed Mutagenesis

  Additional Materials
  • M13 template (unit 1.15)
  • E. colidut ung strain (unit 8.1 and Table 97.80.47111.4.5)
  • T4 DNA polymerase (unit 3.5)
  • Additional reagents and equipment for oligonucleotide‐directed mutagenesis without phenotypic selection (unit 8.1)
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Figures

Videos

Literature Cited

Literature Cited
   Greene, J.M., Larin, Z., Taylor, I.C.A., Prentice, H., Gwinn, K.A., and Kingston, R.E. 1987. Multiple basal elements of a human hsp70 promoter function differently in human and rodent cell lines. Mol. Cell. Biol. 7:3646‐3655.
   McKnight, S.L. and Kingsbury, R. 1982. Transcriptional control signals of a eukaryotic protein‐coding gene. Science 217:316‐324.
Key Reference
  McKnight and Kingsbury, 1982. See above.
  Describes the basic concept of a linker scan.
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