Efficiency of separation of DNA mutations by constant denaturant capillary electrophoresis is controlled by the kinetics of DNA melting equilibrium

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Abstract
Constant denaturant capillary electrophoresis (CDCE) separation takes place in the heated portion of the capillary where faster‐moving, unmelted DNA fragments are in equilibrium with slower‐moving, partially melted forms. Within a certain temperature range, the position of the melting equilibrium and thus the average electrophoretic mobility of each mutant is different. The resulting difference in mobility allow sequences containing single base pair point mutations to be separated from each other. We report the results of experiments in which we explored the rules defining separation efficiency by varying the parameters of CDCE. We discovered an unusual peak broadening mechanism. In contrast to most other DNA electrophoresis systems, peak width in CDCE steadily decreases with the square root of the separation speed. Moreover, the peak width displays a sharp maximum at a specific temperature. To account for these observations, we use a model which describes CDCE separation as a random walk. According to this model, peaks in CDCE are broad because the kinetics of the melting equilibrium are slow and there‐fore the number of random walk steps represented by melting/renaturation transitions is relatively small. In addition to providing a satisfactory interpretation of the data, the model also predicts that separation efficiency will increase as the ionic strength of the running buffer is increased and as the concentration of denaturant in the buffer is decreased. These predictions were verified and were used to establish conditions for high‐resolution CDCE suitable for separating complex mixtures of single base pair mutants.