The 3′-terminal nucleotide sequences of T7 DNA

Paul T. Englund

Research output: Contribution to journalArticle

Abstract

A simple procedure has been developed for determining short nucleotide sequences at 3′ termini of duplex DNA molecules. The DNA is incubated at 11 °C with the T4 DNA polymerase and a single α-32P-labeled deoxynucleoside triphosphate. The enzyme sequentially degrades each strand from the 3′ terminus until a mononucleotide is released which can be replaced by transfer from the 32P-labeled triphosphate. Subsequent reaction consists of alternating removal and replacement of the 32P-labeled nucleotide, and the enzyme never penetrates deeper into the strand. These reactions result in the steady-state incorporation of about one mole of radioactive nucleotide per mole of strand at a unique position in the sequence at or near the original 3′ terminus. In separate experiments, the DNA is labeled with each of the four 32P-labeled nucleotides and the strands of the DNA are separated to permit independent analysis of the two termini. Sequence information is obtained from the 32P-labeled strands by nearest-neighbor analyses and from a related method for identifying the terminal nucleotide of each strand. Using this method on phage T7 DNA, a trinucleotide sequence at the terminus of each strand was determined. The r strand terminates with the sequence pApGpA and the l strand terminates with the sequence pCpCpT.

Original languageEnglish (US)
Pages (from-to)209-224
Number of pages16
JournalJournal of molecular biology
Volume66
Issue number2
DOIs
StatePublished - May 14 1972

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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