The use of 2-aminopurine fluorescence to study DNA polymerase function

Methods Mol Biol. 2009;521:381-96. doi: 10.1007/978-1-60327-815-7_21.

Abstract

The fluorescence of the base analog 2-aminopurine (2AP) is used in highly sensitive assays to determine kinetic parameters for DNA polymerase catalyzed reactions, including exonucleolytic proofreading and nucleotide binding and incorporation. Since 2AP fluorescence can also be used to probe DNA polymerase-induced conformational changes in 2AP-labeled DNA substrates, reaction steps that occur before product formation can be detected. Instruction is provided here in the use of 2AP fluorescence in steady-state and presteady-state assays to study DNA polymerase function and DNA replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 2-Aminopurine / chemistry*
  • Bacteriophage T4 / enzymology
  • Base Sequence
  • DNA Replication / physiology
  • DNA-Directed DNA Polymerase / metabolism*
  • Fluorescence
  • Kinetics
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / chemistry
  • Oligodeoxyribonucleotides / metabolism
  • Spectrometry, Fluorescence
  • Substrate Specificity

Substances

  • Oligodeoxyribonucleotides
  • 2-Aminopurine
  • DNA-Directed DNA Polymerase