These dideoxynucleotides are used as triphosphate (ddl, TP) and can be incorporated in a growing chain, but they terminate synthesis, since they can not form a phosphodiester bond with next incoming deoxynucleotide triphosphate (dNTP).

Following steps are involved in Sanger's dideoxy method for DNA sequencing.

(i) Four reaction tubes are set up, each containing single stranded DNA sample (cloned in M13 phage) to be sequenced, all four dNTPs (radioactively labelled) and an enzyme for DNA synthesis (DNA polymerase I). Each tube also coritains a small amount (much smaller amount relative to four dNTPs) of one of the four ddNTP, so that four tubes have each a different ddNTP, bringing about termination at a specific base adenine (A), cytosine (C), guanine (G) and thymine (T).

In this new approach, of a different fluorescent dye is tagged to the oligonucleotide primer in each of the four reaction tubes (blue for: A, red for C, etc.). The four reaction mixtures are pooled and electrophoreses together in a single Polyacrylamide of the tube.

A high sensitivity fluorescence detector, placed near the bottom of the tube, measures the amount, of each fluorophore as a function of time. The sequence is determined from the temporal order of peaks corresponding to four different dyes.