Optimized detection of DNA point mutations by double gradient denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis displays the highest detection rate among mutation scanning methods. In classical denaturing gradient gel elec trophoresis the denaturant gradient range and migration times vary for ever y amplicon to be scanned, greatly affecting the routine application of the method. As an alternative, we developed double gradient denaturing gradient gel electrophoresis where a gradient of pore size is superimposed over the denaturing one, allowing maintenance of the zone-sharpening effect even ov er prolonged time runs, and adoption of identical run time conditions for a ll fragments analyzed. Here double gradient denaturing gradient gel electro phoresis has been applied to the analysis of a number of point mutations an d polymorphisms located in several exons of three different genes, the cyst ic fibrosis transmembrane conductance regulator, the beta-globin and the p5 3 genes.