Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores.
J Chromatogr A
; 1329: 78-82, 2014 Feb 14.
Article
em En
| MEDLINE
| ID: mdl-24462467
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Oligonucleotídeos
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Cromatografia Líquida de Alta Pressão
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Cromatografia de Fase Reversa
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Corantes Fluorescentes
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article