Random Mutagenesis by PCR

David S. Wilson1, Anthony D. Keefe2

1 Zyomyx, Hayward, California, 2 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.3
DOI:  10.1002/0471142727.mb0803s51
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Error‐prone PCR (EP‐PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence. Using EP‐PCR, the 5′ and 3′ boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly, it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled as a function of the number of EP‐PCR doublings performed. The EP‐PCR technique described here is for a 400‐bp sequence, and an is for a library. EP‐PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn2+, increasing the Mg2+ concentration, and using unequal dNTP concentrations.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Mutagenesis of a DNA Sequence
  • Alternate Protocol 1: Mutagenizing a Library of Sequences
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Mutagenesis of a DNA Sequence

  Materials
  • 100 mM Tris⋅Cl, pH 8.3 ( appendix 22)
  • 2 M KCl
  • 200 mM MgCl 2
  • 25 mM dCTP, pH ∼7
  • 25 mM dTTP, pH ∼7
  • 5 mM dATP, pH ∼7
  • 5 mM dGTP, pH ∼7
  • 100 µM each 5′ and 3′ PCR primers
  • 200 pg/µl DNA template (400 bp in length)
  • 25 mM MnCl 2
  • 5 U/µl Taq DNA polymerase
  • 100‐µl PCR tubes (Sarstedt)
  • Thermal cycler (see unit 15.1)
  • TOPO T/A cloning kit (Invitrogen)
  • QIAprep kit (Qiagen)
  • Additional reagents and equipment for PCR amplification (unit 15.1) and agarose gel electrophoresis (unit 2.7)

Alternate Protocol 1: Mutagenizing a Library of Sequences

  • 30 ng/µl DNA template (library)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Cadwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Meth. Appl. 2:28‐33.
   Eckert, K.A. and Kunkel, T.A. 1991. DNA polymerase fidelity and the polymerase chain reaction. PCR Meth. Appl. 1:17‐24.
   Fromant, M., Blanquet, S., and Plateau, P. 1995. Direct random mutagenesis of gene‐sized DNA fragments using polymerase chain reaction. Anal. Biochem. 224:347‐353.
   Leung, D.W., Chen, E., and Goeddel, D.V. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1:11‐15.
   Spee, J.H., de Vos, W.M., and Kuipers, O.P. 1993. Efficient random mutagenesis method with adjustable mutation frequency by use of PCR and dITP. Nucleic Acids Res. 21:777‐778.
   Vartanian, J.‐P., Henry, M., and Wain‐Hobson, S. 1996. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res. 24:2627‐2631.
   Xu, H., Petersen, E.I., Petersen, S.B., and El‐Gewely, M.R. 1999. Random mutagenesis libraries: Optimization and simplification by PCR. BioTechniques 27:1102‐1108.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library