A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin.
Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
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van de Sande
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000138:chemical synthesis; D007202:Indicators and Reagents; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification*; D011137:Polystyrenes
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D011137:Polystyrenes; D004247:DNA; C003771:Amberlite XAD-2 resin