Dideoxy Sequencing of DNA

Abstract The enzymic sequencing approach uses a DNA polymerase to synthesize transcripts that have been terminated at specific bases. A primer anneals to a complementary region on the template strand and acts as a starting point for DNA synthesis, which occurs in the presence of a mixture of deoxyribonucleotides (building blocks) and specific dideoxyribonucleotides (chain‐terminators). Originally four separate reactions were set up, each with a base‐specific chain‐terminator. Separation of these reaction products on a DNA sequencing gel will generate a ladder of bands indicating the position of each terminated base relative to the end of the sequencing primer. A homogeneous sequencing template should generate a single band in one of the four lanes where a band in the ‘A stop’ lane indicates an ‘A’, a band in the ‘C stop’ lane represents a ‘C’ and so on. In this manner it is possible to deduce the DNA sequence of the target template by identifying consecutive bands in one of the four lanes of the ladder. Since DNA synthesis occurs in the 5′ to 3′ direction, the sequence is read 5′ to 3′ from the bottom to the top (origin) of the gel. Although it was originally suited to the sequencing of single‐stranded DNA templates, there have been many advances in the chemistry of enzymatic sequencing, including the application of thermostable DNA polymerases and fluorescent dye‐terminators as used in automated DNA sequencers.