Fractionation of Native and Denatured Deoxyribonucleic Acid on Agarose Columns

Enzymatically prepared DNA fragments have been fractionated according to molecular weight on agarose columns. The upper limit of fractionation on the most porous gel used (Sepharose 2B) corresponds to a molecular weight of about 0.85·106 for double-stranded DNA. The relationships between kav and [η] or s as well as the [η] versus s relationship were determined for the 2B fractions. Sepharose 6B columns have been used to fractionate both native and denatured DNA fragments having a molecular weight lower than about 3.5·105. Sedimentation equilibrium studies using the short column technique (Van Holde, K. E., and Baldwin, R. L. (1958) J. Phys. Chem. 62, 734) at high speed (Yphantis, D. A. (1964) Biochemistry 3, 297), which only requires 0.5 µg of DNA, were done on the 6B chromatographic fractions. These were found to be practically monodisperse (Mz/Mω ≤ 1.1). Relationships between the sedimentation coefficients and the molecular weights of the 6B chromatographic fractions of native and denatured DNA and their elution volumes were determined. At the same time, the relationships between s and Mω were established in a molecular weight range where no data were previously available.