Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores.
A dual labeled oligonucleotide used as TaqMan® or 5' nuclease probe for in vitro diagnostic has been purified through orthogonal ion-pairing reversed phase chromatography, using polymeric semi-preparative and preparative PRP-1 column. We studied the mechanism of separation of oligonucleotides using ion-pairing reversed phase chromatography. We found that elution profiles of dye labeled oligonucleotides can be controlled by use of specific ion-pairing reagents. Here, we report a method for purification of an oligonucleotide containing an internally positioned rhodamine dye using two orthogonal chromatographic steps, in which the primary step resolves mostly by differences in hydrophobicity by using a weak ion-pairing reagent, and a secondary step uses a strong ion-pairing reagent for separation of length variants. Purification is demonstrated for both 1 and 15μmol scale syntheses, and amenable to further scale up for commercial lot production.
Roche Molecular Diagnostics, 4300 Hacienda Drive, Pleasanton, CA 94588, USA. Electronic address: concordio.anacleto@roche.com.
Concordio
C
Anacleto
Roche Molecular Diagnostics, 4300 Hacienda Drive, Pleasanton, CA 94588, USA. Electronic address: randall.ouye@roche.com.
Randall
R
Ouye
Roche Molecular Diagnostics, 4300 Hacienda Drive, Pleasanton, CA 94588, USA. Electronic address: nancy.schoenbrunner@roche.com.
Nancy
N
Schoenbrunner
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D007477:Ions / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification*
D005456:Fluorescent Dyes; D007477:Ions; D009841:Oligonucleotides
Dual labeled probes; Ion pairing reversed phase; Oligonucleotides; PCR; Polymeric reversed phase column; Preparative scale up