The process relies restriction enzymes to produce DNA of varying lengths. On cloned pieces of DNA the enzymes stop DNA replication at one of the four bases. The lengths of the DNA strands can be worked out using gel electrophoresis.
Process
A primer is added to a single-strand length of DNA to be sequenced. To this template DNA is added a mixture of the four normal deoxy-nucleotides (dATP, dGTP, dCTP and dTTP). Also added are the dideoxy-nucleotides (ddATP, ddGTP, ddCTP and ddTTP) in limited quantities. Each of these floresces a different colour when illuminated by a laser beam. The enzyme [DNA polymerase]? is then added.
The DNA polymerase catalyses the joining of deoxy nucleotides to the correspondng bases. However if by chance a dideoxy nucleotide is joined to a base, then that fragment of DNA will stop further pairing. Fragments of all sizes should be obtained due to the randomness of when a dideoxy nucleotide is added.
When incubation is complete, the fragments are separated (with a resolution of just one nucleotide) by gel electrophoresis, from longest to shortest. As each dideoxy nucleotide floresces a different colour under laser light, it is possible to read off which base occurs at each diplacement from the original primer.
Uses
The dideoxy method is now highly automated as a result of development occuring under the Human Genome Project, where it is used to sequence fragments of our genomes.
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