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 50x4.6-mm column packed with porous, 2.5 micrometer 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 (Cy3) dyes, as well as dually-labeled TaqMan probes. Purification of a 0.1-micromole oligonucleotide synthesis in a single injection was demonstrated.
Fountain
Kenneth J
KJ
Waters Corporation, 34 Maple Street, 01757, Milford, MA, USA.
Gilar
Martin
M
Budman
Yeva
Y
Gebler
John C
JC
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D005456:Fluorescent Dyes / Q000737:chemistry*; D007477:Ions; D013058:Mass Spectrometry; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification*
D017931:DNA Primers; D005456:Fluorescent Dyes; D007477:Ions; D009841:Oligonucleotides