The 3′-terminal nucleotide sequences of T7 DNA

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.