Non-isocratic capillary electrophoresis for detection of DNA point mutations

Abstract

Capillary electrophoresis under non-isocratic conditions is reviewed here. In particular, these conditions are elicited by programming the temperature over the run, just as in temperature-programmed gas chromatography. There is at least one major instance in which this technique is particularly useful: detection of DNA point mutations in PCR-amplified DNA fragments. In this case, migration of DNA homo- and hetero-duplexes against a denaturing gradient (either thermal or chemical) is the only technique capable of developing a pattern of four zones, indicative of the presence of a point mutation in genomic DNA, and due to the formation of two homo- (W t/W t, M/M, where W t = wild type and M = mutant) and two hetero- (W t/M and W t/M) duplexes, formed by fully denaturing and then re-annealing the W t and M filaments in solution. It is demonstrated that it is possible to obtain such gradients (which do not exist in space, i.e., along the length of the capillary, but in time), simply by producing voltage ramps, which in turn generate temperature ramps from within the capillary lumen, rather than from without, by circulating liquids and thermostats. This technique can be applied to any possible type of mutation, from low- to intermediate to high-melters, i.e., from 40°C up to 60–65°C. Examples of separations of point mutations in the cystic fibrosis transmembrane regulator gene and in the β-globin chain gene are shown.