STRETCHING AND OVERSTRETCHING OF DNA IN PULSED-FIELD GEL-ELECTROPHORESIS .1. A QUANTITATIVE STUDY FROM THE STEADY-STATE BIREFRINGENCE DECAY

Using a sensitive birefringence instrument, the birefringence arising from the orientation of the DNA chain during electrophoretic transport has been recorded. This birefringence is shown to proceed both from t he alignment (stretching) of the molecule in the direction of the elec tric field and from the extension of the length of its primitive path (overstretching). The contribution of these two processes can be separ ated in the decay of the birefringence after the end of the applicatio n of the electric field. The fast relaxation of the overstretching occ urs first and is demonstrated to be the main contribution to the biref ringence. The orientation factor of the remaining stretched state and its decay can be quantitatively understood using the biased reptation model. It provides, in addition, a high value for the tube diameter or gel pore size a (4500 +/- 450 angstrom for a 0.7% agarose gel with a c(g)-0.6 dependence in the agarose concentration c(g)) and a low value for the effective charge per base pair (0.2e as compared to 0.5e usin g the condensation hypothesis). The contribution of overstretching to the birefringence is also quantitatively interpreted in term of the ch ange in the mean length 1 of DNA inside a pore size a. The dynamics of decay of this overstretching is well represented by a stretched expon ential with a stretching exponent alpha = 0.44. The mean decay time de creases slightly with increasing fields and scales with the overall DN A length close to N0(2). (C) 1993 John Wiley & Sons, Inc.