Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template.

Abstract

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.