Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans

Abstract

Constant denaturant electrophoresis is a DNA separation technique based on the principle of cooperative melting equilibrium. DNA sequences with distinct high and low melting domains can be utilized to separate and identify molecules differing by only one base pair in the lower melting domain. Combined with capillary gel electrophoresis and when coupled with high fidelity DNA amplification, this approach can detect mutants at a fraction of 10 −6. Modifications to the capillary elecctrophoretic system have also increased DNA loading capacity which allows for analysis of rare mutations in a large, heterogeneous population such as DNA samples derived from human tissues. Employment of this technology has determined the first mutational spectrum in human cells and tissues in a mitochondrial sequence without phenotypic selection of mutants.