EFFICIENCY OF SEPARATION OF DNA MUTATIONS BY CONSTANT DENATURANT CAPILLARY ELECTROPHORESIS IS CONTROLLED BY THE KINETICS OF DNA MELTING EQUILIBRIUM

Constant denaturant capillary electrophoresis (CDCE) separation takes place in the heated portion of the capillary where faster-moving, unme lted DNA fragments are in equilibrium with slower-moving, partially me lted forms. Within a certain temperature range, the position of the me lting equilibrium and thus the average electrophoretic mobility of eac h mutant is different. The resulting differences in mobility allow seq uences containing single base pair point mutations to be separated fro m each other. We report the results of experiments in which we explore d the rules defining separation efficiency by varying the parameters o f CDCE. We discovered an unusual peak broadening mechanism. In contras t to most other DNA electrophoresis systems, peak width in CDCE steadi ly decreases with the square root of the separation speed. Moreover, t he peak width displays a sharp maximum at a specific temperature. To a ccount for these observations, we use a model which describes CDCE sep aration as a random walk. According to this model, peaks in CDCE are b road because the kinetics of the melting equilibrium are slow and ther efore the number of random walk steps represented by melting/renaturat ion transitions is relatively small. In addition to providing a satisf actory interpretation of the data, the model also predicts that separa tion efficiency will increase as the ionic strength of the running buf fer is increased and as the concentration of denaturant in the buffer is decreased. These predictions were verified and were used to establi sh conditions for high-resolution CDCE suitable for separating complex mixtures of single base pair mutants.