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Sanger method (dideoxynucleotide sequencing)

A technique for determination of the base sequence of a homogeneous polynucleotide that acts as a template in a replication system. A small amount of a specific dideoxynucleoside triphosphate is included with the four normal deoxynucleotide triphosphates during replication. Chain termination occurs whenever a dideoxynucleotide is incorporated that generates a labelled polynucleotide whose chain length, evaluated by polyacrylamide-gel electrophoresis, is indicative of the distance from the 5'-end of the primer to the position at which the normal nucleotide occurs. Detection of the electrophoresis bands may be by a label such as 32P or, more recently, by a fluorophore incorporated into the primer (dye primer sequencing) or the dideoxynucleotide (dye terminator sequencing). In fact, in a variation of the dye terminator method, the labelling of each of the four dideoxynucleotides with a fluorophore that emits at a different wavelength permits the identification of the base that terminates each electrophoresis band within a separated mixture of all four (single lane sequencing). (see also Maxam-Gilbert method (chemical cleavage method); plus and minus method)Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467


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