Nucleotide sequence analysis of DNA. I. Partial sequence of the cohesive ends of bacteriophage λ and 186 DNA

Abstract

A general procedure has been developed for determining the deoxynucleotide sequence of the terminal region of a DNA molecule. When this terminal region is present as a single strand, as in bacteriophage lambda, Escherichia coli DNA polymerase can be used to repair the single-stranded region with the addition of radioactive nucleotides to the 3′-end copying the protruding 5′-terminated single strand. The partially labeled DNA can be degraded with nucleases, the radioactive oligonucleotides isolated, and their sequence determined. Using this procedure, partial sequences of deoxynucleotides in the protruding single strands of bacteriophage λ and 186 DNA have been determined. In the case of λ, the right cohesive end was analyzed. The first eight radioactive nucleotides added to the 3′-terminus are in the sequence dpGpGpGpCpGpGpCpG, reflecting a complementary sequence dpCpGpCpCpGpCpCpC for the inner eight nucleotides of the right-hand protruding strand which served as the template for the DNA polymerase repair synthesis. Since each of the two complementary cohesive ends of λ DNA is approximately 12 nucleotides in length, two-thirds of the sequence of nucleotide pairs in the cohesive region of λ has been determined by this analysis. These data refine and supersede the sequence reported previously for the ends of λ DNA (Wu & Kaiser, 1968). 186 DNA, which has cohesive ends of approximately 17 nucleotides in length, has been analyzed in the same way. The first four nucleotides added to the 3′-end of the right-hand cohesive end had a sequence dpGpGpCpG, differing from the sequence in λ. © 1970.