Molecular Techniques and Methods

Random Mutagenesis by Taq DNA Polymerase

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The error rate of Taq DNA polymerase is the highest of the known thermostable DNA polymerases, in the range of 0.1-2 x 10-5 per nucleotide per pass of the polymerase. Over the course of PCR, in which the polymerase makes an average of 20-25 passes, the cumulative error rate is roughly 10-3 per nucleotide. This is insufficient to generate a diverse library of variant sequences, especially over a region shorter than 1,000 nucleotides. A further drawback is that the errors made by Taq DNA polymerase under standard PCR conditions are heavily biased toward AT to GC changes.

This protocol devised a mutagenic PCR that has an overall error rate of about 7 x 10-3 per nucleotide and does not exhibit substantial sequence bias.

  • 7 mM MgCl2 is used to stabilize non-complementary base pairs.

  • 0.5 mM MnCl2 is added to diminish the template specificity of the Taq DNA polymerase.

  • High concentration (1 mM) dCTP and dTTP are used to promote misincorporation.

  • Two times execess (5 units/ 100 ul reaction) Taq DNA polymerase is added to promote chain-extension beyond positions of base mismatch.

  • This protocol introduces errors at a frequency of 0.66-0.13% per position over the course of the PCR. Nearly all of these changes are base substitutions.




    MATERIALS AND SOLUTIONS

    Native Taq Polymerase (or AmpliTaq DNA Polymerase, Perkin-Elmer)


    10 x Reaction Buffer (1 ml)
    70 mM MgCl2 --------------------------------- 7 ul of 1 M MgCl2
    500 mM KCl ---------------------------------- 500 ul of 1 M KCl
    100 mM Tris-HCl (pH 8.3) -------------------- 100 ul of 1 M Tris-HCl
    0.1% (w/v) Gelatin -----------------------------100 ul of 1% Gelatin
    Distilled H2O ---------------------------------- 293 ul


    10 x dNTPs (1 ml)
    2 mM dATP ----------------------------------- 20 ul of 100 mM dATP
    2 mM dGTP ----------------------------------- 20 ul of 100 mM dGTP
    10 mM dCTP --------------------------------- 100 ul of 100 mM dCTP
    10 mM dTTP --------------------------------- 100 ul of 100 mM dTTP
    Distilled H2O ---------------------------------- 760 ul


    5 mM MnCl2 (1 ml)
    5 mM MnCl2 --------------------------------- 5 ul of 1 M MnCl2
    Distilled H2O --------------------------------- 995 ul
  • Do not combine with the 10 x Reaction Buffer, which would result in the formation of a precipitate that disrupts PCR amplification.




    PROCEDURES

    1. Add the following in the PCR tube in a pre-PCR area.

    Components
    Volume Added
    Final Concentration
    10 x Reaction Buffer
    10 ul
    1 x
    10 x dNTPs
    10 ul
    -
    0.3 nM PCR Primer 1
    10 ul
    30 pmole
    0.3 nM PCR Primer 2
    10 ul
    30 pmole
    Template DNA
    -
    20 fmole
    5 mM MnCl2
    10 ul
    0.5 mM
    Distilled H2O to make a final volume of
    100 ul
    -
    Native Taq DNA Polymerase
    2 ul
    5 U


    2. Mix well and confirm that a precipitate has not formed.

    3. Add 5 units of Taq DNA polymerase.

    4. Do PCR reaction as follow:

    1 cycle
    Denaturation
    95oC
    3 min
    30 cycles
    Denaturation
    95oC
    1 min
    Annealing
    Tm-5oC
    1 min
    Synthesis
    72oC
    1 min
    No extension cycle


    5. Do phenol: chloroform extraction of the reaction products.
    Do chloroform: IAA extraction.

    6. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2.5 vol Ethanol. to precipitate.

    7. Centrifuge 15 min at 13,000 rpm to pellet DNA.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Beckman, BA, Mildvan, AS, Loeb, LA (1985) On the fidelity of DNA replication: Manganese mutagenesis in vitro. Biochemistry 24: 5810-5817.

  • Cadwell, C, Joyce, GF (1992) Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2: 28-33.

  • Cha, RS, Thilly, WG (1993) Specificity, efficiency, and fidelity of PCR. PCR Methods Appl. 3: 518-529.

  • Hubner, P, Lida, S, Aiber, W (1988) Random mutagenesis using degenerate oligodeoxyribonucleotides. Gene 73: 319-325.

  • Leung, DW, Chen, E, Goeddel, DV (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1: 11-15.

  • Matteucci, MD, Heyneker, HL (1983) Targeted random mutagenesis: The use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5' of an ATG initiation codon. Nucleic Acids Res. 11: 3113-3121.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".
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