Chain termination method

The polymerase chain reaction is used to produce millions of cloned pieces of DNA. Substances are added to randomly stop the creation of DNA at each of the four bases (depending on the substance), producing pieces of DNA of almost every length. The lengths of the DNA strands can be worked out using gel electrophoresis. Markers on each strand show which base each strand ends with. When the results from the strands are combined, it is possible to work out the sequence of bases at any point.

Detailed 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. These have a fluorescent tagging such that each of these fluoresces 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 tagged dideoxy nucleotide fluoresces a different colour under laser light, it is possible to read off which base occurs at each displacement from the original primer.

Uses

The dideoxy method is now highly automated as a result of development occurring under the Human Genome Project, where it is used to sequence fragments of our genome.