Denaturing HPLC purification of tritylated oligonucleotides using tetraethylammonium hydroxide.
Purification of oligonucleotides by HPLC is limited by association between failure sequences and full-length oligonucleotide. We describe a protocol for denaturing purification of 5'-dimethoxytritylated oligonucleotides that ensures that separation of tritylated and non-tritylated species will not be complicated by strand association. Fully denaturing conditions are produced by the use of tetraethylammonium hydroxide, which is a basic reagent with ion-pairing properties similar to triethylammonium acetate. The method also includes two other convenient features: a) the option of loading the crude oligonucleotide without removing concentrated ammonium hydroxide and b) detritylation on the column with separation of dimethoxytrityl alcohol.
Hobbs
F W
FW
Dupont Merck Pharmaceutical Company, Wilmington, DE 19880.
Yarem
J A
JA
D001483:Base Sequence; D001709:Biotechnology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds
D009838:Oligodeoxyribonucleotides; D011089:Polydeoxyribonucleotides; D013757:Tetraethylammonium Compounds; D019789:Tetraethylammonium