DNA‐Dependent DNA Polymerases

Rebecca B. Kucera1, Nicole M. Nichols1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.5
DOI:  10.1002/0471142727.mb0305s84
Online Posting Date:  October, 2008
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This unit presents characteristics and reaction conditions of the DNA‐dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.1), and the rapidly expanding class of lesion‐bypass DNA polymerases that play a role in DNA damage repair. Curr. Protoc. Mol. Biol. 84:3.5.1‐3.5.19. © 2008 by John Wiley & Sons, Inc.

Keywords: nick translation; 3′‐end labeling; blunting ends; random‐primer synthesis; whole genome amplification; PCR; lesion‐bypass

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Table of Contents

  • Introduction
  • General Notes on Polymerase Protocols
  • Enzyme: Escherichia coli DNA Polymerase I
  • Basic Protocol 1: Uniform Labeling of DNA by Nick Translation
  • Enzyme: Klenow Fragment of Escherichia coli DNA Polymerase I
  • Basic Protocol 2: Labeling of DNA by Random Oligonucleotide‐Primed Synthesis
  • Basic Protocol 3: Labeling the 3′ Ends of DNA
  • Basic Protocol 4: Repairing 3′ or 5′ Overhanging Ends to Generate Blunt Ends
  • Enzyme: T4 DNA Polymerase
  • Basic Protocol 5: Repairing 3′ or 5′ Overhanging Ends to Generate Blunt Ends
  • Enzyme: Native T7 DNA Polymerase
  • Enzyme: Modified T7 DNA Polymerase
  • Enzyme: Phi29 DNA Polymerase
  • Enzyme: Bst DNA Polymerase, Full Length and Large Fragment Forms
  • Enzyme: Taq DNA Polymerase and Its Stoffel Fragment
  • Other Thermophilic DNA Polymerases
  • Enzyme Class: Lesion Bypass DNA Polymerases
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

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Key Reference
   Abelson, J.N., Simon, M.I., and Campbell, J.L. (eds.) 1995. Methods in Enzymology, Vol. 262, DNA Replication, Academic Press, San Diego.
  Provides historical and practical information on various topics related to DNA polymerases.
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