Exact Method for Sequencing Procedure____________________________________

Restriction enzyme fragments obtained from ΦX174 replicative form.

Fragments from 5µg of ΦX174 in 5 µl of water mixed with 1 µl of viral ΦX174 DNA and 1 µl of H x 10 buffer,
    heated to 100 °C for 3 minutes, and then incubated at 67°C for 30 minutes.

Solution diluted to 20 µl with H buffer.
Samples of 2µl taken for each incubation and mixed with 2 µl DNA polymerase and 2 µl of “mix”
    (composed of 1 µCi [α-³²P]dATP, 1µl 1.5 X H buffer and the appropriate dNTPs and ddNTPs).

Incubation at room temperature for 15 minutes.

1 µl of 0.5mM dATP added, incubation continued for 15 minutes.
    [If omitted, some termination at A residues occurred due to low concentration of the [α-³²P]dATP. ]

Small primers were left attached, long primers were removed with 1 µl of restriction enzyme and incubation at 37°C.

Reaction mixtures denatured.

Applied to acrylamide gel for electrophoresis next to one another in the following order of inhibitors:
ddGTP, ddATP, ddTTP, ddCTP/araCTP.