DIDEOXY SEQUENCING

dideoxy sequencing: translation

(= Sanger dideoxy sequencing)

The most popular method of DNA sequence determination (cf. Maxam-Gilbert method). Starting with single-stranded template DNA, a short complementary primer is annealed, and extended by a DNA polymerase. The reaction is split into 4 tubes (called "A, C, G or T") each containing a low concentration of the indicated dideoxy-nucleotide, in addition to the normal deoxynucleotides. Dideoxynucleotides, once incorporated, block further chain extension, and so each tube accumulates a mixture of chains of lengths determined by the template sequence. The 4 reactions are denatured and run out on an acrylamide sequencing gel in neighbouring lanes, and the sequence read up the gel according to the order of the bands.