Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries

Christian Gloeckner1, Ramon Kranaster1, Andreas Marx1

1 Department of Chemistry, University of Konstanz, Konstanz, Germany
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090183
Online Posting Date:  May, 2010
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Abstract

The protocols in this article describe the construction of a mutant DNA polymerase library using error‐prone PCR (epPCR) as a method for gene randomization, followed by screening of the library using two different approaches. The examples described use an N‐terminally truncated form of the thermostable DNA polymerase I of Thermus aquaticus (Taq DNA polymerase), namely Klentaq (KTQ), and protocols are included for the identification of variants with (1) increased DNA lesion‐bypass ability and (2) enhanced selectivity for DNA match/mismatch recognition. The screening assays are based on double‐stranded DNA detection (using SYBR Green I) which can be carried out using standard laboratory equipment. The described assays are designed for use in a 384‐well plate format to increase screening throughput and reduce material costs. For improved accuracy and ease of liquid handling, the use of an automated liquid handling device is recommended. Curr. Protoc. Chem Biol. 2:89‐109. © 2010 by John Wiley & Sons, Inc.

Keywords: DNA polymerase; directed evolution; screening; PCR; primer extension

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Error‐Prone PCR Library Construction
  • Basic Protocol 2: Expression of DNA Polymerase Variants and Lysate Preparation
  • Basic Protocol 3: Distinguishing Between PCR‐Active and ‐Inactive Variants by Screening DNA Polymerase Mutant Libraries in a PCR‐Based Assay
  • Basic Protocol 4: Screening DNA Polymerase Mutants for Enhanced Single‐Nucleotide Discrimination
  • Basic Protocol 5: Screening Mutant DNA Polymerases for Lesion‐Bypass Ability
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Error‐Prone PCR Library Construction

  Materials
  • 5 U/µl Taq DNA polymerase (Roche)
  • 10× Taq buffer (see recipe)
  • 25 mM MgCl 2
  • 1 mM MnCl 2
  • 10 µM forward cloning primer KTQ fw (5′‐ATG GTA CGT CTC AGC GCG CCC TGG AGG AGG CCC CCT‐3′)
  • 10 µM reverse cloning primer KTQ rev (5′‐ATG GTA CGT CTC ATA TCA CTC CTT GGC GGA GAG CCA GTC‐3′)
  • 400 pM template (pASK‐IBA37plus‐KTQ‐wt) (pASK‐IBA37plus from IBA GmbH)
  • 1× loading buffer for agarose gel electrophoresis (Voytas, )
  • 0.8% agarose gel (Voytas, )
  • 1‐kb DNA ladder (New England Biolabs)
  • 2 mM dNTP mix (2 mM dATP, dCTP, dGTP, dTTP)
  • Gel elution kit (Qiagen)
  • PCR reaction clean‐up kit (Qiagen)
  • Restriction endonucleases (New England Biolabs):
    • 20 U/µl DpnI
    • 10 U/µl BsmBI
    • 10 U/µl BsaI
  • 10× reaction buffer in which both DpnI and BsmBI are active (e.g., Buffer 4; New England Biolabs)
  • 1 U/µl T4 DNA ligase and 10× ligase buffer
  • pASK‐IBA37‐plus (BsaI‐digested and dephosphorylated; see recipe)
  • SOC medium (see recipe)
  • Electrocompetent E. coli BL21 (DE3) Gold (Stratagene)
  • LB liquid medium and plates containing 100 µg/ml carbenicillin (see recipe)
  • Plasmid miniprep kit (e.g., Qiagen, Invitrogen)
  • Sequencing primers:
    • Forward: 5′‐GAG TTA TTT TAC CAC TCC CT‐ 3′
    • Reverse: 5′‐CGC AGT AGC GGT AAA CG‐3′)
  • 60% (v/v) glycerol in LB medium
  • Thermal cycler
  • Nanodrop ND‐1000 UV/Vis spectrophotometer (http://www.nanodrop.com/)
  • Cuvettes for electroporation, 1 mm electrode distance (BioRad)
  • Electroporator Gene Pulser Xcell (BioRad)
  • Incubation shaker, Titramax 1000 (Heidolph)
  • Autoclaved wooden toothpicks
  • 384‐well deep‐well plates (ABgene)
  • Gas‐permeable adhesive seals (ABgene)
  • Self‐adhesive aluminum/paper seals (ABgene)
  • Adhesive tape (Tesa or Scotch)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and replica plating (Elbing and Brent, )

Basic Protocol 2: Expression of DNA Polymerase Variants and Lysate Preparation

  Materials
  • 384‐well plate containing E coli glycerol cultures ( protocol 1)
  • LB liquid medium containing 100 µg/ml carbenicillin (see recipe)
  • 4 mg/liter anhydrotetracycline (AHT; IBA GmbH) in LB medium
  • 1× KTQ lysis buffer (with lysozyme; see recipe)
  • Automated liquid handling device (Hamilton Microlab Star)
  • 96‐well deep‐well plates (2.2 ml well volume; VWR)
  • Thermostatic incubators and shakers
  • Gas‐permeable adhesive seal (ABgene)
  • Self‐adhesive aluminum/paper seal (ABgene)
  • Spectrophotometer
  • Refrigerated floor‐model centrifuge with multiwell‐plate carrier
  • 75°C water bath

Basic Protocol 3: Distinguishing Between PCR‐Active and ‐Inactive Variants by Screening DNA Polymerase Mutant Libraries in a PCR‐Based Assay

  Materials
  • 10× KTQ reaction buffer (see recipe)
  • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
  • 0.1 µM DNA template F90A: 5′‐d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC CGC CGT GGA CAG AGG ACT GCA GAA AAT CAA CCT A TC CTC CTT CAG GAC CAA CGT ACA GAG)‐3′ (custom synthesis)
  • 10,000× SYBR Green I (Invitrogen, cat. no. S‐7563)
  • 100 µM forward primer F20+: 5′‐d(CGT TGG TCC TGA AGG AGG AT)‐3′ (custom synthesis)
  • 100 µM reverse primer F20: 5′‐d(CGC GCA GCA CGC GCC GCC GT)‐3′ (custom synthesis)
  • Cell lysates in 96‐well plates ( protocol 2)
  • 96‐well PCR plate (VWR)
  • Automated liquid handling device (Hamilton Microlab Star)
  • Reagent dispenser (Multidrop, Thermo Scientific)
  • Optically clear heat‐sealing film (ABgene)
  • Refrigerated floor‐model centrifuge with multiwell‐plate carrier
  • Thermo‐sealer or electric iron
  • Real‐time PCR thermal cycler
  • 384‐well deep‐well plates (ABgene)
  • Autoclaved wooden toothpicks

Basic Protocol 4: Screening DNA Polymerase Mutants for Enhanced Single‐Nucleotide Discrimination

  Materials
  • 10× KTQ reaction buffer (see recipe)
  • 10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP)
  • 10,000× SYBR Green I (Invitrogen, cat. no. S‐7563)
  • 100 µM forward primer F20+: 5′‐d(CGT TGG TCC TGA AGG AGG AT)‐3′ (custom synthesis)
  • 100 µM reverse primer F20: 5′‐d(CGC GCA GCA CGC GCC GCC GT)‐3′ (custom synthesis)
  • 0.1 µM DNA template F90A (match): 5′‐d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC CGC CGT GGA CAG AGG ACT GCA GAA AAT CAA CCT A TC CTC CTT CAG GAC CAA CGT ACA GAG)‐3′ (custom synthesis)
  • 0.1 µM DNA template F90G (mismatch): 5′‐d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC CGC CGT GGA CAG AGG ACT GCA GAA AAT CAA CCT G TC CTC CTT CAG GAC CAA CGT ACA GAG)‐3′ (custom synthesis)
  • Cell lysates in 96‐well plates ( protocol 2)
  • 96‐well PCR plate, semi skirted (VWR)
  • Automated liquid handling device (Hamilton Microlab Star)
  • Reagent dispenser (Multidrop, Thermo Scientific)
  • Refrigerated floor‐model centrifuge with multiwell‐plate carrier
  • Optically clear heat‐sealing film (ABgene)
  • Thermo‐sealer or electric iron
  • Real‐time PCR thermal cycler

Basic Protocol 5: Screening Mutant DNA Polymerases for Lesion‐Bypass Ability

  Materials
  • 10 µM primer F20+: 5′‐d(CGT TGG TCC TGA AGG AGG AT)‐3′ (custom synthesis)
  • 1 µM DNA template F90A: 5′‐d(CCG TCA GCT GTG CCG TCG CGC AGC ACG CGC CGC CGT GGA CAG AGG ACT GCA GAA AAT CAA CCT A TC CTC CTT CAG GAC CAA CGT ACA GAG)‐3′ (custom synthesis)
  • 10× KTQ buffer (see recipe)
  • 10 mM dNTP mix (only three dNTPs; 10 mM each dCTP, dGTP, and dTTP)
  • 10 mM dNTP mix (all four dNTPs; 10 mM dATP, dCTP, dGTP, and dTTP)
  • 500 mM Tris⋅Cl, pH 9.2
  • 500 mM EDTA
  • 20× SYBR Green I (prepare from 10,000× stock, as purchased from Invitrogen, cat. no. S‐7563)
  • Heat‐inactivated E. coli lysates (see protocol 2)
  • 384‐well plates, black, flat bottom, square shaped wells (VWR)
  • Automated liquid handling device (Hamilton Microlab Star)
  • Reagent dispenser (Multidrop, Thermo Scientific)
  • Self‐adhesive aluminum/paper seal (ABgene)
  • Thermal cycler
  • Floor‐model centrifuge with multiwell‐plate carrier
  • Plate reader for 384‐well plates, heatable (POLARStar optima, BMG Labtech; http://www.bmglabtech.com)
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Figures

Videos

Literature Cited

   d'Abbadie, M., Hofreiter, M., Vaisman, A., Loakes, D., Gasparutto, D., Cadet, J., Woodgate, R., Pääbo, S., and Holliger, P. 2007. Molecular breeding of polymerases for amplification of ancient DNA. Nat. Biotechnol. 25:939‐943.
   Barnes, W.M. 1992. The fidelity of Taq polymerase catalyzing PCR is improved by an N‐terminal deletion. Gene 112:29‐35.
   Brogan, A.P., Eubanks, L.M., Koob, G.F., Dickerson, T.J., and Janda, K.D. 2007. Antibody‐catalyzed oxidation of delta(9)‐tetrahydrocannabinol. J. Am. Chem. Soc. 129:3698‐3702.
   Cadwell, R.C. and Joyce, G.F. 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2:28‐33.
   Cirino, P.C., Mayer, K.M., and Umeno, D. 2003. Generating mutant libraries using error‐prone PCR Methods Mol. Biol. 231:3‐9.
   Elbing, K. and Brent, R. 2002. Growth on solid media. Curr. Protoc. Mol. Biol. 59:1.3.1‐1.3.6.
   Glieder, A., Farinas, E.T., and Arnold, F.H. 2002. Laboratory evolution of a soluble, self‐sufficient, highly active alkane hydroxylase. Nat. Biotechnol. 20:1135‐1139.
   Kranaster, R. and Marx, A. 2009. Taking fingerprints of DNA polymerases: Multiplex enzyme profiling on DNA arrays. Angew. Chem. Int. Ed. Engl. 48:4625‐4628.
   Lee, Y.‐F., Tawfik, D.S., and Griffiths, A.D. 2002. Investigating the target recognition of DNA cytosine‐5 methyltransferase HhaI by library selection using in vitro compartmentalisation. Nucleic Acids Res. 30:4937‐4944.
   Loakes, D., Gallego, J., Pinheiro, V.B., Kool, E.T., and Holliger, P. 2009. Evolving a polymerase for hydrophobic base analogues. J. Am. Chem. Soc. 131:14827‐14837.
   Loh, E., Choe, J., and Loeb, L.A. 2007. Highly tolerated amino acid substitutions increase the fidelity of Escherichia coli DNA polymerase I. J. Biol. Chem. 282:12201‐12209.
   Reetz, M.T. and Wu, S. 2009. Laboratory evolution of robust and enantioselective Baeyer‐Villiger monooxygenases for asymmetric catalysis. J. Am. Chem. Soc. 131:15424‐15432.
   Sauter, K.B.M. and Marx, A. 2006. Evolving thermostable reverse transcriptase activity in a DNA polymerase scaffold. Angew. Chem. Int. Ed. 45:7633‐7635.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 2.5A.1‐2.5A.9.
   Wilhelm, J. and Pingoud, A. 2003. Real‐time polymerase chain reaction. ChemBioChem 4:1120‐1128.
Key References
   Sauter and Marx, 2000. See above.
  Construction of the DNA polymerase library (KTQ) by epPCR as described in .
   Gloeckner, C., Sauter, K.B.M., and Marx, A. 2007. Evolving a thermostable DNA polymerase that amplifies from highly damaged templates. Angew. Chem. Int. Ed. 46:3115‐3117.
  Screening for lesion‐bypass ability as described in .
   Strerath, M., Gloeckner, C., Liu, D., Schnur, A., and Marx, A. 2007. Mutations in motif C on the mismatch‐extension selectivity of Thermus aquaticus DNA polymerase. ChemBioChem 8:395‐401.
  Screening for PCR activity and increased selectivity–single nucleotide discrimination as described in Basic Protocols 3 and 4.
   Summerer, D., Rudinger, N.Z., Detmer, I., and Marx, A. 2005. Enhanced fidelity in mismatch extension by DNA polymerase through directed combinatorial enzyme design. Angew. Chem. Int. Ed. 44:4712‐4715.
  Screening for increased selectivity–single nucleotide discrimination as described in .
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