Imaging and dynamic studies of DNA during capillary electrophoresis: Implications for biopolymer electrophoretic resolution.

Abstract

Linear double-stranded (ds) DNA (5 kbp or larger) exhibits noticeable band broadening and asymmetry during capillary electrophoresis (CE) in dilute linear polymers. The band broadening mechanism has been termed dispersion and theorized to result from a distribution in entanglement lifetimes between polymer and DNA. To assess the effect of DNA electrophoretic dynamics on separation resolution, high-speed video (222 frames/s) fluorescence microscopy was used to visualize DNA during electrophoresis in dilute hydroxyethyl cellulose (HEC). Pulsed electric fields sharpen DNA bands during CE in dilute polymer solutions, enhancing resolution. To investigate band sharpening, linear lambda dsDNA (48.5 kbp) was imaged in dilute HEC using pulsed electric fields. With field inversion frequencies close to 50 Hz and modulation depths (AC voltage/DC voltage) of 180% (DC bias 100 V/cm) the dsDNA extension-contraction periodicity due to entanglement with HEC increased dramatically compared to DC conditions. However, the DNA extension-contraction cycle in oscillating electric fields showed little change compared to DC fields. It was hypothesized weak polymer entanglements were removed, increasing the periodicity of the DNA extension-contraction cycle and retarding mobility dispersion. Visualization studies showed 40 and 90 kbp supercoiled (sc) plasmids exhibited an elastic rod electrophoretic motion, completely different than the familiar U or J shapes seen with linear dsDNA. The elastic-rod motion was due to the nature of the entanglement between scDNA and polymer. Supercoiled plasmids migrated with a higher degree of periodicity in the polymer entanglement-disentanglement cycle than linear dsDNA. The scDNA visualization studies helped explain the CE mobility dependence of scDNA and small linear dsDNA (<1 kbp) on DNA size. DNA aggregation, which can severely limit CE resolution, was also visualized during electrophoresis in dilute HEC solutions. Time fractals were used to measure the rates of lambda dsDNA aggregation. Aggregation was severe with field inversion because of the high driving electric fields. FRET (fluorescence resonance energy transfer) microscopy was used to probe the structure of DNA aggregates. A random entanglement of DNA was observed within the aggregates as expected.