Nucleotide clusters in deoxyribonucleic acids: sequence analysis of DNA using pyrimidine oligonucleotides as primers in the DNA polymerase I repair reaction

• Published in: Biochemistry (Easton) 1978-03, Vol.17 (5), p.841-850
• Main Authors: Kaptein, John S, Spencer, John H
• Format: Article
• Language: English
• Subjects: Bacteriophages; Base Sequence; DNA Polymerase I - metabolism; DNA Repair; DNA, Viral - metabolism; DNA-Directed DNA Polymerase - metabolism; Escherichia coli - enzymology; Oligodeoxyribonucleotides - metabolism; Oligonucleotides - metabolism; Pyrimidine Nucleotides - metabolism; Templates, Genetic
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00598a015

Pyrimidine oligonucleotides have been shown to prime the E. coli DNA polymerase I repair reaction, specifically and reproducibly. DNA molecules up to 30 nucleotides long have been obtained from the extension of oligopyrimidine primers, 9 to 11 nucleotides long isolated from the complementary (minus) strand of bacteriophage S13 RFDNA using S13 viral DNA as the template molecule. The sequences of the extended primers were determined from mobility shift following separation of partially extended primers by ionophoresis and homochromatography, and by a modification of the "plus" system of Sanger and Coulson (1975). The 3' leads to 5' exonuclease activity of E. coli DNA polymerase was utilized for the "plus" system in the presence of single dNTPs and also with two dNTPs in the reaction, to give a nearest neighbor type of analysis for sequence confirmation. The ready availability of oligopyrimidine primers from any DNA and the simplification of the "plus" method broaden the range of applicability of the primed DNA polymerase I repair reaction for DNA sequence analysis.