1. Four reaction tubes (A, G, C, T) are set, each containing:
a. the purified DNA of interest.
b. a primer sequence.
c. DNA polymerase (Taq).
d. the four deoxynucleotide phosphates
(dNTPs- dATP, dGTP, dCTP, dTTP).
e. one radioactively labeled dNTP, usually
f. one dideoxynucleotide (didNTP) per tube-
didATP in tube A, didGTP in G, didCTP
in C, didTTP in T.
The ratio of dNTP to didNTP is adjusted so that one didNTP is incorporated into the growing DNA chain for every one hundred nucleotides. Each time a didNTP is incorporated, synthesis stops at a single nucleotide, generating a fragment of distinct size. Since billions of DNA fragments are produced in each tube, sufficient DNA is present to make a visible band in each lane of a polyacrylamide gel containing urea to prevent the DNA from renaturing during electrophoresis. The gel is dried and sandwiched with x-ray film, which when developed shows the banding pattern.
A modified protocol uses fluorescent dyes of four different colors, each bonded to a different dNTP per tube. The gel is passed through an automated reader, which displays a graph of peaks for each color, representing each of the nucleotide bases. This automated process increases the speed of sequencing manyfold over the autoradiograph protocol.