Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair.
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.
D001482:Base Composition; D055598:Chemical Phenomena; D002621:Chemistry; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D010957:Plasmids
D004247:DNA; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III