Nucleotide Sequence Analysis of Deoxyribonucleic Acid
XIV. CONDITIONS FOR THE INCORPORATION OF RIBONUCLEOTIDES AND DEOXYRIBONUCLEOTIDES INTO SINGLE-STRANDED AREAS OF LONG DOUBLE-STRANDED DEOXYRIBONUCLEIC ACIDS
Richard T. Hamilton 1 and Ray Wu 1
From the
1 From the Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14850
At 5°, high salt concentration, and with Mn2+ as metal ion, Escherichia coli DNA polymerase I catalyzes the incorporation of ribo- and deoxyribonucleotides almost exclusively into the single-stranded cohesive ends of double-stranded 
DNA, using the 3'-hydroxyl ends as primers, and the 5' single-stranded regions as templates. The mixed ribodeoxyribo-oligomers are cleaved specifically at the points of ribonucleotide insertion, to give fragments with the expected sequence.
Partial inhibition of the insertion of ribonucleotides occurs under the above incubation conditions which confine the incorporation of ribo- and deoxyribonucleotides to the single-stranded cohesive ends. Calculations, based on the assumption that in a fixed percentage of cases no further ribonucleotide insertion occurs, give 10% blockage to ribocytidylate insertion, 30% blockage to riboguanylate insertion, and 60% blockage to riboadenylate insertion.
This system has been studied for possible use in DNA sequence analysis, commencing at the ends of a duplex DNA molecule.
Submitted on October 23, 1973
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