CONSTANT DENATURANT CAPILLARY ELECTROPHORESIS (CDCE) - A HIGH-RESOLUTION APPROACH TO MUTATIONAL ANALYSIS

Using a zone of constant temperature and denaturant concentration in c apillary electrophoresis, we have devised a simple, rapid, and reprodu cible system for separating mutant from wild type DNA sequences with h igh resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the us e of linear polyacrylamide at viscosity levels that permit facile repl acement of the matrix after each run. For a typical 100 bp fragment, p oint mutation-containing heteroduplexes are separated from wild type h omoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 10(4) molecules with a linear dynamic range of six or ders of magnitude. The relative limit of detection at present is 3 x 1 0(-4) i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences. The new approach should be applicable to the identific ation of low frequency mutations, to mutational spectrometry and to ge netic screening of pooled samples for detection of rare variants.