Neuroscience > Molecular Neurobiology

User Ratings

Your rating: None
Your rating: None
Your rating: None
 Add your comments

Directed Mutagenesis Using the Polymerase Chain Reaction

Brendan Cormack1

1Johns Hopkins University School of Medicine, Baltimore, Maryland

Publication Name: 
Current Protocols in Neuroscience
Unit Number: 
UNIT 4.11
DOI: 
10.1002/0471142301.ns0411s03
Online Posting Date: 
May, 2001
GO TO THE FULL TEXT:
PDF or HTML at Wiley Interscience
Are you the author of this protocol? Login or register and return to this page.

Abstract

The polymerase chain reaction (PCR) is most often used for the enzymatic amplification and direct sequencing of small quantities of nucleic acids. This technology can also be used as a quick and efficient method for introducing any desired sequence change into the DNA of interest. 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 alternate protocol 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.

     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Introduction of Restriction Endonuclease Sites by PCR
  • Basic Protocol 2: Introduction of Points Mutations by PCR
  • Alternate Protocol: Introduction of a Point Mutations by Sequential PCR Steps
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Materials

Basic Protocol 1: Introduction of Restriction Endonuclease Sites by PCR

 Materials
  • DNA sample to be mutagenized
  • pUC19 plasmid vector (Figure 4.11.2) or similar high-copy-number plasmid having M13 flanking primer sequences
  • TE buffer (appendix 2A)
  • 10× MgCl2-free PCR amplification buffer (see recipe) supplemented with MgCl2 as appropriate (see step )
  • 2 mM 4dNTP mix (see recipe)
  • 500 ng/µl (100 pmol/µl) M13 forward and reverse flanking sequence primers (New England Biolabs)
  • 5 U/µl Taq DNA polymerase
  • Mineral oil
  • Chloroform
  • Buffered phenol (appendix 2A)
  • 100% ethanol
  • Appropriate restriction endonucleases (Table 4.11.1)
  • Additional reagents and equipment for subcloning and DNA ligation (see cpmb unit 3.16 and appendix 1A in this manual), plasmid DNA miniprep (cpmb unit 1.6), synthesis and purification of oligonucleotides (cpmb units 2.11 & 2.12), PCR amplification (cpmb unit 15.1), DNA extraction and precipitation (cpmb unit 2.1A), quantitation of DNA by absorbance spectrometry (cpmb appendix 3D), restriction endonuclease digestion (cpmb unit 3.1), agarose and polyacrylamide gel electrophoresis of DNA (cpmb units 2.5A & 2.7), purification of DNA from low gelling/melting agarose gels (cpmb unit 2.6), transformation of E. coli (cpmb unit 1.8), and DNA sequence analysis (cpmb unit 7.4A)
     
    Table 4.11.1 Relative Efficiencies of Restriction Enzyme Cleavage When Restriction Site is Near End of DNA Fragment a
    EnzymeOligonucleotide sequence        % cleavage
    2 hr20 hr
    AflIIICCACATGTGG>90>90
    AscIGGCGCGCC>90>90
    AvaICCCCCGGGGG>90>90
    BamHICGGGATCCCG>90>90
    BglIIGAAGATCTTC75>90
    BssHIITTGGCGCGCCAA50>90
    BstEIIGGGT(A/T)ACCC010
    BstXICTGCAGAACCAATGCATTGGATGCAT25>90
    ClaICCATCGATGG>90>90
    EcoRIGGAATTCC>90>90
    HaeIIIGGGGCCCC>90>90
    HindIIICCCAAGCTTGGG1075
    KpnIGGGGTACCCC>90>90
    MluICGACGCGTCG2550
    NcoICATGCCATGGCATG5075
    NdeIGGAATTCCATATGGAATTCC7590
    NheICTAGCTAGCTAG1050
    NotIAAGGAAAAAAGCGGCCGCAAAAGGAAAA25>90
    NsiICCAATGCATTGGTTCTGCAGTT>90>90
    PacICCTTAATTAAGG0>90
    PmeIAGCTTTGTTTAAACGGCGCGCCGG75>90
    PstIAAAACTGCAGCCAATGCATTGGAA>90>90
    PvuIATCGATCGAT1025
    SacICGAGCTCG1010
    SacIITCCCCGCGGGGA5090
    SalIACGCGTCGACGTCGGCCATAGCGGCCGCGGAA1075
    ScaIAAAAGTACTTTT7575
    SmaITCCCCCGGGGGA>90>90
    SpeIGACTAGTC10>90
    SphIACATGCATGCATGT1050
    StuIAAGGCCTT>90>90
    XbaIGCTCTAGAGC>90>90
    XhoICCGCTCGAGCGG1075
    XmaITCCCCCCGGGGGGA>90>90
     a Reprinted with permission from New England Biolabs.

Basic Protocol 2: Introduction of Points Mutations by PCR

 Materials
  • DNA sample to be mutagenized
  • Klenow fragment of E. coli DNA polymerase I
  • Appropriate restriction endonuclease (Table 4.11.1)
  • Additional reagents and equipment for synthesis and purification of oligonucleotides (cpmb unit 2.11 &2.12), phosphorylation of oligonucleotides (cpmb unit 3.10), electrophoresis of DNA on nondenaturing agarose and low gelling/melting agarose gels (cpmb unit 2.5A & 2.6), restriction endonuclease digestion (cpmb unit 3.1), ligation of DNA fragments (cpmb unit 3.16), transformation of E. coli (cpmb unit 1.8), plasmid DNA miniprep (cpmb unit 1.6), and DNA sequence analysis (cpmb unit 7.4A also see appendix 1A in this manual).
Looking for materials?  Suppliers Guide
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience

Figures

  • Figure 4.11.1
    Introduction of a restriction site into a specific DNA fragment. (A) The fragment of interest is cloned into a high-copy-number vector. Sites for two oligonucleotide primers, such as the M13 forward and reverse primers (F and R), flank the cloning site. In two separate reactions, fragments upstream (A—E) and downstream (E—C) of the introduced site are PCR-amplified using the flanking primers and oligonucleotides containing the site to be introduced (primers F and 1, R and 2). The amplified fragments are digested with the appropriate restriction endonucleases, then ligated and subcloned into an appropriately cut vector and transformed into E. coli. The resultant plasmid contains an inserted fragment identical to the original DNA except for the introduced restriction site. (B) The oligonucleotides needed to change the primary sequence to an EcoRI restriction site are indicated. Note the four-base “clamp” sequence at the 5¢ end of the primers.

    View Image
  • Figure 4.11.2
    Introduction of a point mutation into a specific DNA fragment. (A) The fragment of interest is cloned into a high-copy-number plasmid vector. Sites for two oligonucleotide primers, such as the M13 forward and reverse primers (F and R), flank the cloning site. In two separate reactions, fragments upstream (A—M) and downstream (M—C) of the point mutation are PCR-amplified using the flanking primers and oligonucleotides containing the point mutation to be introduced (primers F and 3, R and 4). The fragments are digested with the appropriate restriction endonucleases and made blunt-ended. These fragments are ligated and subcloned into an appropriately cut vector and transformed into E. coli. The resultant plasmid contains an inserted fragment identical to the original DNA except for the introduced point mutation. (B) The oligonucleotides needed to generate a point mutation in the primary sequence are shown. Note that there are no “clamp” sequences at the 5¢ ends of the primers.

    View Image
  • Figure 4.11.3
    Introduction of a point mutation by sequential PCR steps. (A) The first steps of this protocol are as described in Figure 4.11.2A except primers 5 and 6 are used. The amplified fragments are then placed in the same tube and amplified in a second PCR step (using primers F and R only). This second PCR step obviates the need for the blunt-end ligation. The full-length fragment generated here is digested with the appropriate restriction endonucleases, subcloned into an appropriately cut vector, and transformed into E. coli. The resultant plasmid contains an inserted fragment identical to the original DNA except for the point mutation. (B) The oligonucleotide primers needed to generate a point mutation in this procedure are shown. Note that there are 12 bases of overlap between the two primers.

    View Image

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. P1rimer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.
     
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Interscience
Looking for Answers?
Do you have tips, tricks, or improvements to share?

Join the Conversation

Post new comment

The content of this field is kept private and will not be shown publicly.
CAPTCHA
This question is for testing whether you are a human visitor and to prevent automated spam submissions.