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Dideoxy sequencing

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Dideoxy sequencing

The most popular method of dna sequence determination (c.f. Maxam gilbert sequencing). 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.