Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair

C. D. James*, I. M. Leffak

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

In vitro labeling of DNA molecules using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair has been examined as an alternative to existing methods of replacement synthesis labeling. It is shown that exonuclease III is able to act in a common restriction enzyme buffer [50 mm Tris (pH 8.0), 10 mm MgCl2, 50 mm NaCl] to produce a population of base-paired primer:template molecules which decrease uniformly in single-strand length with time. After heat inactivation of the exonuclease III and in the presence of radiolabeled deoxynucleotides the polymerase I reaction faithfully resynthesizes full-length molecules, asymmetrically labeled to high specific activity.

Original languageEnglish (US)
Pages (from-to)33-37
Number of pages5
JournalAnalytical Biochemistry
Volume141
Issue number1
DOIs
StatePublished - Aug 15 1984

Keywords

  • DNA
  • DNA polymerase I
  • exonuclease III
  • in vitro labeling

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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