Purification of crude DNA oligonucleotides by solid-phase extraction and reversed-phase high-performance liquid chromatography.
Purification of target oligodeoxyribonucleotides from failure sequence by-products of synthesis is often required for polymerase chain reaction primers, DNA sequencing and other oligonucleotide applications. We have developed purification protocols based on a reversed-phase mechanism ("trityl on" purification) using a 96-well Oasis HLB extraction plate. The Oasis HLB sorbent combines excellent pH stability with a high loading capacity allowing for single-step purification of 0.2 microM scale synthesis. After sample loading and washing, the oligonucleotide trityl group is cleaved on the plate with 2% trifluoroacetic acid. Target DNA is eluted with acetonitrile-0.36 mM triethylamine acetate, pH 11.3 (10:90, v/v). Typical yield of purified product is 60-95%. Final purity, measured by capillary gel electrophoresis, was found to be 90% or greater. Alternatively, highly pure oligonucleotides can be obtained by a RP-HPLC "trityl off" method using an XTerra C18 column. The use of volatile triethylamine acetate buffer as an ion-pair for RP-HPLC eliminates the need for further desalting.
Gilar
M
M
Waters Corporation, Milford, MA 01757, USA. martin_gilar@waters.com
Bouvier ESP
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*
D009838:Oligodeoxyribonucleotides