Nucleic Acids Research · 1976 · 15 citations · 5 references
Life SciencesBiochemistryMolecular BiologyDna ReplicationEnzymologyNucleic Acid BiochemistryDna LigaseDna StructureNucleic Acid SynthesisPolymerase Chain ReactionBackbone LabilityDna Backbone ChemistryDna Duplex StabilityDna Polymerase IEnzymatic Dna SynthesisEnzymatic SynthesisSucrose Gradient CentrifugationT4 Dna Ligase
Duplex circular phiX174 DNA (RF I) containing some phosphoramidate links in the backbone chain of the (-) strand was synthesized by reaction of 5'-amino-5'-deoxythymidine 5'-triphosphate, dCTP, dGTP, and 3H-dATP with DNA polymerase I and DNA ligase (T4) on a (+) strand phiX174 amber 3 DNA template. The yield of duplex DNA was higher when dTTP was included along with the amino analog in the initial reaction system or was added late in the synthesis. RF I DNA was observed as a rapidly sedimenting species in an alkaline sucrose gradient, and the presence of phosphoramidate linkages was demonstrated by the unusual lability of the duplex DNA in a weakly acidic solution.
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