Open AccessConstant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational anaylsis
Author(s) -
Konstantin Khrapko,
John S. Hanekamp,
William G. Thilly,
A. B. Belen'kii,
František Foret,
Barry L. Karger
Publication year - 1994
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/22.3.364
Subject(s) - capillary electrophoresis , detection limit , biology , dna , microbiology and biotechnology , fluorescence , point mutation , mutant , electrophoresis , gel electrophoresis , chromatography , genetics , chemistry , gene , physics , optics
Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes 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 orders of magnitude. The relative limit of detection at present is 3 x 10(-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 identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.