DNA Electrophoresis: Optical Probing and Microscopic Imaging to Understand Separation Mechanisms
The aim of this work was to combine spectroscopic, mobility, and fluorescence microscopy studies to elucidate the mechanisms behind DNA electrophoresis, a method to separate DNA molecules according to size.
The microscopy studies require staining of the DNA with a fluorescent dye. The newly synthesized cyanine dye YOYO, a dimer of oxazole yellow (YO), has been used for this purpose. An extensive part of the work has been to characterize the photophysical properties of YO and YOYO and how they interact with DNA, knowledge that is important in the interpretation of results from the electrophoretic studies. It was found that a single electronic transition roughly polarized along the long axis of the YO chromophore is responsible for the strong visible absorption band, and that the fluorescence quantum yield is related to the degree of rotation around the internuclear bond between the two ring systems in YO. Furthermore, the YO chromophores of YOYO were found to intercalate in both AT and GC sequences of DNA.
Fluorescence microscopy was used to study the orientation of the DNA molecules and the period time in the cyclic migration of long DNA in agarose gel. The distribution of period times was found to be broad and asymmetric. Comparing the observed orientation of the chain with the prediction of biased reptation theory, it was concluded that the theory underestimates the global orientation. In addition, local parts of the chain were found to be oriented by the electric field.
A study of the effect of the positively charged dye DAPI on the mobility and orientational behavior of DNA showed that DAPI decreases the mobility by 30 % and slows down the orientational dynamics of the DNA molecule by 60 %. This implies that DAPI causes an increase in the contour length, and a decrease of the electrokinetic charge and persistence length of the DNA. Given such implications neither the Ogston theory for short DNA nor the biased reptation theory for long DNA predicts correctly the reduction in mobility seen in the experiments. Therefore, these theories need to be developed further.
polarized light spectroscopy