Solid supported hydrolysis of apurinic sites in synthetic oligonucleotides for rapid and efficient purification on reverse-phase cartridges.
Rapid purification methods for synthetic oligonucleotides involving reverse-phase cartridges (RPC) and enzymatic hydrolysis have been introduced. These methods are based on a discrimination between the desired target fragment protected with a 5'-DMT group and incompletely elongated products possessing a 5'-hydroxyl function. For target products over 60 nucleotides, the rapid methods are of little use as reported to date. We have found that the problem is due to the presence of truncated 5'-DMT fragments generated from apurinic sites within the target product during NH4OH deprotection. These side products are co-purified with the target fragment when the rapid purification procedures are employed. If a step is included during deprotection to cleave the apurinic sites prior to removal of the crude product from the solid support (1 M lysine-HCl, pH 9 for 90 min at 60 degrees C), fragments up to 118 bases can be purified by RPC to near homogeneity.
Horn
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Chiron Corporation, Emeryville, CA 94608.
Urdea
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D001080:Apurinic Acid; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008239:Lysine; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000302:isolation & purification*; D011119:Polynucleotides; D014320:Triphenylmethyl Compounds
D015345:Oligonucleotide Probes; D011119:Polynucleotides; D014320:Triphenylmethyl Compounds; D001080:Apurinic Acid; D008239:Lysine