Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA

We present a study aimed at understanding the factors affecting the separat ion of large DNA molecules by capillary electrophoresis in polymer solution s In a first series of experiments, a systematic study of the effect of ope rational parameters an the development of an electrohydrodynamic instabilit y resulting in DNA aggregation and spurious peaks in the electropherograms is presented. The results are discussed in regard to a recent theory of ele ctrohydrodynamic instabilities in, macroion suspensions, recently proposed by Isambert et al. Overall, the results provide strong support to the theor y. Some situations of interest for applications, and not explicitly conside red in the theory, such as asymmetric field poising and the use of polymer polymer additives in the buffer, were also considered. Furthermore, robust optimized protocols for high resolution separation of DNA in the range of 1 00 base pairs to 160 kilobase pairs, are proposed. As predicted by the mode l, it is shown that using a concentrated isoelectric buffer (histidine) str ongly reduces aggregation as compared to the use of a conventional buffer a t the same concentration, and allows separation of DNA from 100 bp to 160 k bp in less than 10 min. We also present a systematic study of the dependenc e of the mobility vs DNA size, pulse frequency and field strength. The resu lts are discussed with respect to the Biased Reptation with Fluctuations mo del and a good agreement is obtained. (C) 1999 John Wiley & Sons, Inc.