DNA sequencing using the enzymatic extension reaction makes use of the chemical difference between normal deoxyribonucleotides and dideoxyribonucleotides. Deoxyribonucleotides contain a hydroxyl group in position 3 of the sugar ring, and DNA polymerase can join the phosphate of the next nucleotide to that hydroxyl end. A dideoxynucleotide can be added to a growing chain but does not contain a hydroxyl group at position 3, so no further nucleotides can be added. Addition of a dideoxynucleotide stops DNA synthesis. The DNA template to be sequenced is denatured in a tube.
The reaction mix contains a primer complementary to the template, DNA polymerase, the four deoxyribonucleotides and one labeled dideoxyribonucleotide, in this case, dideoxy-A. The dideoxy-A is present at lower concentration than the analogous deoxy-A. The primer base pairs with a complementary sequence and DNA synthesis begins.
When the dideoxyribonucleotide is inserted, DNA synthesis on that molecule stops. With the optimum concentrations for the deoxy and dideoxyribonucleotides, there will be molecules synthesized that stop at each of the A's in the new sequence. The fragments generated are separated by size using gel electrophoresis, and the tag on the dideoxyribonucleotide allows the fragments to be visualized. DNA sequencing reactions are done using each of the four dideoxyrival nucleotides, and if each dideoxy has a unique fluorescent label, all four reactions can be carried out in the same tube. When the reaction products are separated, there should be labeled fragments corresponding to each base along the template, with the four bases represented by different recognizable tags.