Polymer Dynamics in Electrophoresis of DNA

Abstract

Gel electrophoresis of nucleic acids with a constant electric field cannot resolve DNA fragments much above 50 kilobase pairs (Kbp) (1). However, properly implemented pulsed electric fields extend the range of resolution into megabases, as shown in Figure I (2). For this technique to be effective, a strategy for choosing the shapes and durations of the electric field pulses must be developed to optimize the resolution in a given size range. In this article, we describe the theoretical ideas behind the development of such a strategy (3). Some basic facts about gel electrophoresis are as follows: electric fields typically used are 1-10 V/cm; DNA fragments separated by current protocols arc up to 10 mega base pairs (Mbp); and pore sizes of typically used agarose gels are � 1/10 J.lm (4). The velocities of the DNA molecules are very slow, approximately I mm/hour. With pulsed fields, the shapes of the voltage pulses are usually rectangular, and pulse times vary from seconds to hours (5); high-frequency secondary pulses of 10100 ms are sometimes inserted into the primary low-frequency pulses (6). The pulse times and voltages are generally changed many times during a separation, because a given pulse shape and duration usually affects only a narrow segment of the distribution of molecular sizes, and the mixtures to be separated often contain a wide distribution of sizes.