Directed Mutagenesis Using the Polymerase Chain Reaction

Brendan Cormack1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.5
DOI:  10.1002/0471142727.mb0805s37
Online Posting Date:  May, 2001
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Abstract

This unit contains two basic protocols for introducing base changes into specific DNA sequences. The first describes the incorporation of a restriction site and the second details the generation of specific point mutations. An describes generating point mutations by sequential PCR steps. Although the general procedure is the same in all three protocols, there are differences in the design of the synthetic oligonucleotide primers and in the subsequent cloning and analyses of the amplified fragments.

     
 
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Table of Contents

  • Basic Protocol 1: Introduction of Restriction Endonuclease Sites by PCR
  • Basic Protocol 2: Introduction of Point Mutations by PCR
  • Alternate Protocol 1: Introduction of a Point Mutation by Sequential PCR Steps
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Introduction of Restriction Endonuclease Sites by PCR

  Materials
  • DNA sample to be mutagenized
  • pUC19 plasmid vector ( Figure 1.5.2) or similar high‐copy‐number plasmid having M13 flanking primer sequences
  • TE buffer ( appendix 22)
  • 10× MgCl 2‐free PCR amplification buffer (unit 15.1) supplemented with MgCl 2 as appropriate (see step )
  • 2 mM 4dNTP mix (unit 15.1)
  • 500 ng/µl (100 pmol/µl) M13 forward and reverse flanking sequence primers (New England Biolabs)
  • 5 U/µl Taq DNA polymerase (units 15.1 & 3.5)
  • Mineral oil
  • Chloroform (unit 2.1)
  • Buffered phenol (unit 2.1)
  • 100% ethanol
  • Appropriate restriction endonucleases (Table 8.5.1)
  • 500‐µl microcentrifuge tube
  • Automated thermal cycler
  • Additional reagents and equipment for subcloning DNA (unit 3.16), plasmid DNA miniprep (unit 1.6), synthesis and purification of oligonucleotides (units 2.11 & 2.12), PCR amplification (unit 15.1), DNA extraction and precipitation (unit 2.1), quantitation of DNA by absorbance spectrometry ( 3.NaN), restriction endonuclease digestion (unit 3.1), agarose and polyacrylamide gel electrophoresis of DNA (units 2.5 & 2.7), purification of DNA from low gelling/ melting agarose gels (unit 2.6), ligation of DNA fragments (unit 3.16), transformation of E. coli (unit 1.8), and DNA sequence analysis (unit 7.4)
    Table 8.5.1   Materials   Relative Efficiencies of Restriction Enzyme Cleavage When Restriction Site is Near End of DNA Fragment a   Relative Efficiencies of Restriction Enzyme Cleavage When Restriction Site is Near End of DNA Fragment

    Enzyme Oligo sequence   % cleavage
    2 hr 20 hr
    AflIII CCACATGTGG >90 >90
    AscI GGCGCGCC >90 >90
    AvaI CCCCCGGGGG >90 >90
    BamHI CGGGATCCCG >90 >90
    BglII GAAGATCTTC 75 >90
    BssHII TTGGCGCGCCAA 50 >90
    BstEII GGGT(A/T)ACCC 0 10
    BstXI CTGCAGAACCAATGCATTGGATGCAT 25 >90
    ClaI CCATCGATGG >90 >90
    EcoRI GGAATTCC >90 >90
    HaeIII GGGGCCCC >90 >90
    HindIII CCCAAGCTTGGG 10 75
    KpnI GGGGTACCCC >90 >90
    MluI CGACGCGTCG 25 50
    NcoI CATGCCATGGCATG 50 75
    NdeI GGAATTCCATATGGAATTCC 75 90
    NheI CTAGCTAGCTAG 10 50
    NotI AAGGAAAAAAGCGGCCGCAAAAGGAAAA 25 >90
    NsiI CCAATGCATTGGTTCTGCAGTT >90 >90
    PacI CCTTAATTAAGG 0 >90
    PmeI AGCTTTGTTTAAACGGCGCGCCGG 75 >90
    PstI AAAACTGCAGCCAATGCATTGGAA >90 >90
    PvuI ATCGATCGAT 10 25
    SacI CGAGCTCG 10 10
    SacII TCCCCGCGGGGA 50 90
    SalI ACGCGTCGACGTCGGCCATAGCGGCCGCGGAA 10 75
    ScaI AAAAGTACTTTT 75 75
    SmaI TCCCCCGGGGGA >90 >90
    SpeI GACTAGTC 10 >90
    SphI ACATGCATGCATGT 10 50
    StuI AAGGCCTT >90 >90
    XbaI GCTCTAGAGC >90 >90
    XhoI CCGCTCGAGCGG 10 75
    XmaI TCCCCCCGGGGGGA >90 >90

     aReprinted with permission from New England Biolabs.

Basic Protocol 2: Introduction of Point Mutations by PCR

  Materials
  • DNA sample to be mutagenized
  • Klenow fragment of E. coli DNA polymerase I (unit 3.5)
  • Appropriate restriction endonuclease (Table 8.5.1)
  • Additional reagents and equipment for synthesis and purification of oligonucleotides (units 2.11 & 2.12), phosphorylation of oligonucleotides (unit 3.10), electrophoresis of DNA on nondenaturing agarose and low gelling/melting agarose gels (units 2.5 & 2.6), restriction endonuclease digestion (unit 3.1), ligation of DNA fragments (unit 3.16), transformation of E. coli (unit 1.8), plasmid DNA miniprep (unit 1.6), and DNA sequence analysis (unit 7.4)
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Figures

Videos

Literature Cited

Literature Cited
   Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Erlich, H.A. 1988. Primer‐directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487‐491.
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