High-performance affinity chromatography of DNA. II. Porosity effects.

A DNA-silica, (dT)18-silica, was prepared and used in a study of the chromatography of the oligonucleotide, (dA)18, based upon base pairing. It was shown that hybridization efficiency did not depend upon flow-rates up to 2 ml/min for the small columns (22 x 2 mm) used. As increasing amounts of (dA)18 were loaded onto the columns, the columns were found to saturate at a well defined capacity that was always less than the amount that theoretically could have been bound. Maximum capacity was achieved whenever the loading temperature was at least 20-25 degrees C below the temperature at which the loaded oligonucleotide would elute. The effects of porosity on both coupling efficiency and capacity were measured and suggest that pore sizes in the 300-500 A range are most appropriate for this form of chromatography.