Separation of Oligodeoxynucleotides by Reversed-Phase Chromatography1
We recently reported (1) that reversed-phase chromatography gives good resolution of oligonucleotides typically obtained by enzymatic digestion of RNAs. The present report describes the application of this technique for the separation of oligodeoxynucleotides.
Chromatographic methods and preparation of the chromatographic packing have been described previously (l-4). The column packing, designated RPC-5, consists of polychlorotrifluoroethylene support (Plaskon CTFE 2300 powder, Allied Chemical Company, Morristown, New Jersey) coated with methyltrialkylammonium chloride (Adogen 464 from Ashland Chemical Company, Columbus, Ohio). Columns (Chromatronix) were jacketed for temperature control. The column effluent was monitored simultaneously at 254 and 280 nm (Laboratory Data Control Duo-Monitor). Eluent solutions were made by diluting with water a stock ammonium acetate-acetic acid solution containing 1.86 M ammonium acetate and 4.14 M acetic acid with a resulting pH of 4.4. Oligodeoxynucleotides were obtained from Collaborative Research, Inc., Waltham, Massachusetts.
Figure 1 shows a chromatogram obtained from a mixture of oligodeoxynucleotides, ranging in size from dinucleotides to a hexanucleotide. Identification of the peaks was based on their spectral characteristics, and on the elution position as compared to the elution position of the individually chromatographed oligodeoxynucleotides. Conditions were chosen to give separation of dinucleotides as well as larger fragments of different sizes. Dinucleotides eluted in the order, d (PC-G), d (PA-G), d (pG-G) , i.e., in the order C, A, G, as expected at this pH. The series of deoxynucleotides, d(pA-G), d(pA-G),, and d(pA-G) 3 eluted in order of increasing size. The smaller, unlabeled peaks in Fig. 1 are presumably due to other contaminating oligodeoxynucleotides in the samples. Reversed-phase chromatography appears to have several advantages over other separation methods for separating oligodeoxynucleotides. Sam-' Research