PCR Sequencing Protocols

Volume 65 of the series Methods in Molecular Biology™ pp 133-136

DNA Sequencing by the Chemical Method

  • Eran PicherskyAffiliated withDepartment of Biology, University of Michigan

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The chemical method of sequencing DNA (1) has some advantages and some disadvantages compared with the enzymatic method (2). The major disadvantage is that it takes more time to produce the same amount of sequence. This is so for two main reasons. First, the DNA has to be end-labeled and then reisolated prior to the actual chemical sequencing reactions, a process that usually requires an additional day. Also, because more DNA is used in the reaction and because the lower specific activity of the sequenced DNA requires the use of an intensifying screen in the autoradiography, bands are not as sharp as in the enzymatic method and therefore it is difficult to obtain reliable sequence past about nucleotide 250 (unless very long gels are run).