RESUMEN
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.
Asunto(s)
ADN Viral/química , Electroforesis en Gel de Agar/métodos , Polarización de Fluorescencia/métodos , Bacteriófago lambda/genética , Difusión , Cómputos Matemáticos , Conformación de Ácido Nucleico , Fotoquímica , Análisis EspectralRESUMEN
Electrophoresis of DNA migrating in an ordered matrix is studied and compared with classical agarose gel electrophoresis. A well-defined migration medium is obtained by crystallization of monodisperse silica spheres. Electrophoretic mobility of DNA is measured with fluorescence recovery after photobleaching experiments. The main result is that, as it was the case for gel electrophoresis, diffusion and electrophoretic mobility of DNA in such a medium are well described with reptation theories.