Purification of dye-labeled oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography

Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50×4.6-mm column packed with porous, 2.5 μm C 18 sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of...

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Veröffentlicht in:	Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2003-01, Vol.783 (1), p.61-72
Hauptverfasser:	Fountain, Kenneth J., Gilar, Martin, Budman, Yeva, Gebler, John C.
Format:	Artikel
Sprache:	eng
Schlagworte:	Base Sequence Chromatography, High Pressure Liquid - methods DNA Primers Fluorescent Dyes - chemistry Ions Mass Spectrometry Oligonucleotides Oligonucleotides - chemistry Oligonucleotides - isolation & purification
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Zusammenfassung:	Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50×4.6-mm column packed with porous, 2.5 μm C 18 sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC–MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy™3) dyes, as well as dually-labeled TaqMan™ probes. Purification of a 0.1-μmole oligonucleotide synthesis in a single injection was demonstrated.
ISSN:	1570-0232 1873-376X
DOI:	10.1016/S1570-0232(02)00490-7