Non-isocratic capillary electrophoresis for detection of DNA point mutations.
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- (Wt/Wt, M/M, where Wt = wild type and M = mutant) and two hetero- (Wt/M and Wt/M) duplexes, formed by fully denaturing and then re-annealing the Wt 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 degrees C up to 60-65 degrees C. Examples of separations of point mutations in the cystic fibrosis transmembrane regulator gene and in the beta-globin chain gene are shown.
Righetti
P G
PG
University of Verona, Department of Agricultural and Industrial Biotechnologies, Italy.
Gelfi
C
C
D004247:DNA / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D017354:Point Mutation; D011108:Polymers
D011108:Polymers; D004247:DNA