Sanger “dideoxy” method

The Sanger dideoxy method of DNA sequencing allows large volumes of DNA to be sequenced so that it is possible to even determine whole genomes. This method uses deoxyribonucleoside triphosphates and dideoxyribonucleoside triphosphates. A DNA primer binds to the 5' end of the single-stranded DNA molecule to be sequenced (the template strand) to allow the DNA polymerase enzyme to attach and synthesize a new DNA chain against the template strand through Watson-Crick complementry base pairing. The new chain is built of deoxyribonucleoside triphosphates.

If a dideoxyribonucleotide triphosphate is incorporated into the growing newly synthesised chain, it termiantes synthesis at that point. There are 4 different dideoxyribonucleoside triphosphate chain-terminating molecules (ddCTP, ddGTP, ddATP, ddTTP) each containing one of the DNA bases, so ddCTP contains the base ‘C’ (Cytosine) etc. These 4 different molecules are used in 4 seperate DNA synthesis reactions on copies of the same single-stranded DNA template strand to be sequenced. Each reaction produces sequences of different lengths complementary to the template strand, which have terminated at different points.

These DNA fragments are then separated by gel electrophoresis which allows us to see how long each fragment is depending on how far through the gel it has travelled. By reading off each band on the gel the sequence of the newly synthesised strand as well as the template strand can be found out. So, the band which travelled furthest through the gel must have been the sequence which terminated at the first base. The band which was second furthest through the gel must have been terminated at the second base in the sequence. Etc. By looking at which dideoxyribonucleoside triphosphate bound to each of these DNA molecules you can determine the base on the template strand at the position it terminated because of complementary base pairing.