DNA Electrophoresis in Polymer Solutions. Separation Mechanisms studied by Fluorescence and Linear Dichroism Spectroscopy
Capillary electrophoresis of DNA in polymer solutions is a promising technique for separation of DNA molecules according to size. The primary aim of the work presented in this thesis was to get a better understanding of the electrophoretic separation mechanism in these media by studying the DNA migration behavior during electrophoresis in both entangled and non-entangled polymer solutions using linear dichroism spectroscopy and fluorescence video microscopy.
Since the fluorescence microscopy technique requires optical probing, the DNA was stained with the cyanine dye YOYO, a dimer of oxazole yellow (YO) with excellent fluorescent properties. The first part of the work deals with photophysical properties of YO and YOYO, their binding to DNA and the influence of probing with YOYO on the electrophoretic behavior of DNA. We found that YO and YOYO intercalates and bisintercalates, respectively, in DNA. Furthermore, binding of YOYO to DNA has quite a small effect on the electrophoretic behavior of DNA; it slows down the orientation dynamics and lowers the mobility but does not seem to affect the global migration mode. Thus, YOYO is suitable as a DNA probe in electrophoresis context.
Fluorescence microscopy observations at low electric fields (<25 V/cm) and linear dichroism measurements at fields up to 150 V/cm on YOYO-stained T2 DNA (164 kbp) in polyacrylamide solutions have shown that, in a highly entangled polymer solution (0.4% (w/w) polyacrylamide, MW 18-106), DNA reptates with oscillations in a similar way as in agarose gel. The degree of reptation-like motion and the degree of orientation decreases gradually as the concentration and/or molecular weight of the polymer is decreased. However, even at polymer concentrations well below the overlap concentration, the polymer has a strong influence on the DNA migration behavior, resulting in, for example, a hundredfold increase in orientation compared to in free buffer solution.
polarized light spectroscopy