Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis.
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.
Khrapko
K
K
Center for Environmental Health Sciences, MIT Cambridge 02139.
Hanekamp
J S
JS
Thilly
W G
WG
Belenkii
A
A
Foret
F
F
Karger
B L
BL
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation*; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation
D017931:DNA Primers