JBC, Vol. 250, Issue 12, 4607-4618, Jun, 1975
DNA sequence analysis. Terminal sequences of bacteriophage phi80
R. Bambara and R. Wu
Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA
have been determined. Sequences of the cohesive ends were obtained through
the use of two standard methods. The first method involved the
incorporation of all four labeled deoxyribonucleotides into the phi80
cohesive ends using DNA polymerase I. The DNA was then partially digested
with micrococcal nuclease or pancreatic DNase. The products were separated
by two-dimensional electrophoresis and characterized by composition,
3'-terminal, and nearest neighbor analyses. The second method involved
partial incorporation using one, two, or three labeled deoxyribonucleotides
followed by similar analyses. Sequences of the double-stranded regions
adjacent to the cohesive ends were determined by three new methods. These
methods were: (a) the DNA was specifically labeled at the 3' terminus and
then partially degraded. Labeled oligonucleotide products were sequenced by
their mobilities on various separation systems. (b) The cohesive ends were
enlarged by limited degradation with exonuclease III. After this treatment,
the DNA was partially repaired with labeled nucleotides, digested, and the
products were analyzed. (c) A synthetic ologonucleotide primer was bound to
phi80 DNA which had been repaired with DNA polymerase I, and then partially
digested with lambda-exonuclease. The primer was extended into the region
of interest by partial repair with labeled nucleotides. The extended primer
was isolated and analyzed.
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