SEPARATION AND MEASUREMENT OF DIFFUSION-COEFFICIENTS OF LINEAR AND CIRCULAR DNAS BY FLOW FIELD-FLOW FRACTIONATION

In this paper flow field-flow fractionation (flow FFF), an elution separation method, is utilized to separate and to measure the translational diffusion coefficients D of a variety of linear and both single- and double-stranded circular DNA chains in the molecular weight range M = (0.4-4.8) X 10(6) Da. Equations for component retention times, band broadening, and resolution are given and compared with experimental results. The tradeoff between resolution and separation speed is discussed and experimentally realized. Overloading studies show that approximately 1 mug of individual DNAs can be isolated per 10-20-min run; the procedure can be readily automated for repetitive runs. Values of D obtained from retention time measurements are tabulated, and these as well as literature D values (for M = (0.058-38) X 10(6) Da) are compared with expressions of Kirkwood-Riseman (KR), Mandelkern-Flory (MF), Tirado-Garcia de la Torre (TG), and Yamakawa-Fujii (YF). The predicted Ds of MF agree well with data over the high M range ((0.4-38) X 10(6) Da), while the rigid-rod equation of TG fits data quite well up to M = 2 x 10(6) Da, an M for which the DNA chains is approximately 70 times longer than that displaying rigid-rod behavior. We find also that D is reasonably described by the simple form D = AM(-b) over the 3-decade M range examined. Factors involved in the application of FFF to DNAs with M > 10(7) Da are discussed including shear degradation, transition to a steric mechanism of FFF, and use of condensed DNA. Severe overloading effects induced by chain entanglement rendered preliminary attempts unsuccessful, but future prospects for applying FFF to high-M DNA are found favorable.