Size-dependent, chromatographic separation of double-stranded DNA which is not based on gel permeation mode

A new procedure for size-dependent fractionation of DNA was investigated. DNA fragments ranging from 10 to 40 kbp were separated by using columns for high-performance gel permeation chromatography. However, the order of elution was opposite to that which would be expected for gel permeation chromatography, i.e., smaller fragments were eluted faster than larger fragments, though separation based on normal gel permeation chromatography was observed when smaller DNA fragments (less than 5 kbp) were applied. The size range of DNA which can be resolved by this new procedure was found to depend on both particle size and flow rate; the use of a column packed with smaller particles or the application of a faster flow rate enabled us to resolve smaller DNA fragments, but the pore size or chemical nature of the column packing had scarcely any effect on the resolution. This mode of separation was attained by using both silica and polymer packings. The results suggest that the separation is based on a hydrodynamic phenomenon.