Pulsed-field capillary electrophoresis on nucleic acids.

Abstract

Pulsed field capillary electrophoresis (PFCE) extends the high resolution range of nucleic acid separations from about 500-600 base pair (bp) to at least 2,000,000 bp (2 Mbp). With this technique separations are achieved in 4-15 min, compared to several hours by classical slab gel electrophoresis. In my work, PFCE is performed in buffers containing linear polymers, such as derivatized cellulose or linear polyethylene oxide. For field pulsing a computer-controlled high voltage amplifier is used in place of the customary high voltage power supply of capillary zone electrophoresis. Detection is by laser-induced fluorescence of intercalated ethidium ion. Under these working conditions, CE at constant field provides poor resolution for fragments in the kbp size range. To break the elongated field-induced conformations which are responsible for poor resolution, we impose periodic reversals of the electric field. The optimum field reversal time is a function of DNA length, and no fixed pulsed field protocol is satisfactory over a wide range of lengths. This problem is solved by employing several protocols. These include equal forward/reverse dwell times, with 0-40% amplitude in the reversal direction and equal forward/reverse amplitudes with unequal dwell times. The dwell-time limits are determined in advance, with the exact sequence calculated from random numbers generated throughout a run. More sophisticated protocol includes combinations of DC-only and pulsed field conditions. The DC/pulsed protocols are especially effective for samples containing very short and very long fragments. With these protocols I have been able to separate nucleic acid mixtures containing fragments as short as 75 bp and as long as 2 Mbp in 4 minutes.