Fractionation and molecular weight determination of deoxyribonucleic acid fragments using agarose column chromatography

DNA fragments of several sizes have been produced by shearing E. coli DNA under different pressures. These fragments have been used to demonstrate that column chromatography on agarose Bio-Gel A-15M can provide a rapid, inexpensive fractionation and sizing method for single-stranded nucleic acids having masses between 10⁵ and 10⁶ daltons. Both chromatographic and electrophoretic analysis of the sheared DNA indicated that discrete fragment populations were produced at each shearing pressure and that these fragments were distributed essentially symmetrically around a mean piece size. The average molecular weight of the several DNA fragment distributions was determined electrophoretically by comparison with standard DNA fragments obtained from restriction endonuclease cleavage of SV40 viral DNA. The molecular weights of the denatured, sheared fragments (single-stranded) ranged from 1.25 × 10⁵ to 7.4 × 10⁵. The single-stranded DNA fragments were chromatographed over agarose Bio-Gel A-15M and a linear relationship was found to exist between the mobilities and logarithms of the molecular weights. Readily available tRNA, 5s RNA, and φX174 single-stranded circular DNA chromatographed at the extremes of the linear relationship and could be used to calibrate the column chromatography.