Recent methods for the preparative fractionation of trinucleotides from enzymic digests of RNA or DNA rely largely on substituted cellulose ion-exchange chromatography in 7M urea at neut.ral and acid pH (for a review, see Tener (1)). Fractionation is obtained according to chain length at pH 7, and according to net charge (base ratios) at pH 3. These two procedures suffice for the resolution of the 8 or 9 trinucleotides obtained from RNA by specific nucleases such as pancreatic RNase or RNase T,, respectively.
No nucleases of comparable specificity are known for DNA. Thus, isolation of trimers from enzymic hydrolyzates of DNA depends on the use of partial enzymic digests and enzymes with a preference, rather than a specificity, for linkages next to certain bases. The large number of trinucleotides so produced is difficult to separate by the two-step procedure described above. An additional step, employing column chromatography on DEAE-Sephadex in 50% MeOH instead of 7 M urea was recently found to separate trinucleotides from partial U&ago sphaerogena RNase digests of RNA according to Gp content (2). The mechanism of this separation is not known. However it was of interest to apply the procedure to partial micrococcal nuclease digests of calf t'hymus DNA (3-6). The results show that more than 23 trinucleotides may be so purified by a combination of the above three steps, and that further separation of sequence isomers such as TpGpAp-ApGpTp may be obtained by partition chromatography on cellulose columns with 30% ammonium sulfate at neutral pH.
Applications of the fractionation procedure to hydrolyzates of DNA and RNA with other nonspecific enzymes, as well as to hydrolyzates of RNA containing compounds with 2',3'-cyclic terminal phosphate residues, are discussed.