Simultaneous measurements of mobility, dispersion, and orientation of DNA during steady-field gel electrophoresis coupling a fluorescence recovery after photobleaching apparatus with a fluorescence detected linear dichroism setup.
Orientation of molecules is responsible for the loss of separability during steady-field gel electrophoresis. In this work we develop a technique to measure simultaneously the relevant parameters involved in the separation mechanism: electrophoretic mobility, band broadening, and molecular orientation. To do that we have associated a fluorescence recovery after photobleaching (FRAP) apparatus with a fluorescence detected linear dichroism setup. This coupling allows one to follow the buildup of orientation during the FRAP experiment. Because orientation involves a change in the angular distribution of fluorescence, we have added a fluorescence polarization setup which can be used in parallel with the FRAP and gives an exact value of the steady-state orientation factor. We illustrate the possibilities of these combined experiments by analyzing the coupling of electrophoretic transport and orientation of lambda DNA in 1% agarose gels.
Tinland
B
B
Institut Charles Sadron, CNRS, 6 rue Boussingault, 67083 Strasbourg, France.
Meistermann
L
L
Weill
G
G
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry*; D004058:Diffusion; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D008432:Mathematical Computing; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D013057:Spectrum Analysis
D004279:DNA, Viral