Analysis of Fluorescent Dye-labeled Oligonucleotides by Ion Pair Reversed Phase High Performance Liquid Chromatography
- VernacularTitle:荧光标记寡核苷酸的反相离子对色谱分析
- Author:
Peng LI
;
Jing WANG
;
Yunhua GAO
;
Liqing WU
;
Linghui SHENG
;
Boqiang FU
- Publication Type:Journal Article
- Keywords:
Fluorescent dye-labeled oligonucleotides;
fluorescent dyes;
ion pair reversed phase high performance liquid chromatography;
retention
- From:
Chinese Journal of Analytical Chemistry
2009;37(12):1722-1726
- CountryChina
- Language:Chinese
-
Abstract:
An analytical method of fluorescent dye-labeled oligonucleotides was established by ion pair reversed phase high performance liquid chromatography(IP-RP-HPLC) which was improved by optimizing the effects of triethylamine-acetic acid(TEAA)(0-0.15 mol/L), pH(4.5-7.0) and gradient. Comparing the retention of 5, 10 and 15-mer unlabeled oligonucleotides with that of 5'-carboxyfluorescein(5'FAM) labeled oligonucleotides, the mechanism of fluorescent dye-labeled oligonucleotides retention was studied. In addition, TaqMan~(TM) probes as wellas other common fluorescent dye-labeled oligonucleotides were concerned. The results showed that the best resolution of different length fluorescent dye-labeled oligonucleotides was observed under the condition of 0.01 mol/L TEAA and pH 7.0. The retention behavior of fluorescent dye-labeled oligonucleotides was different from that of unlabeled oligonucleotides significantly, and therefore they can be separated completely. The results indicated that the retention of unlabeled oligonucleotides enhanced with the increase of the length of molecule. In contrast, the retention of fluorescent dye-labeled oligonucleotides was reduced with the increase of the length of molecule. For the hydrophobicity of fluorescent dyes made a great impact on the retention, a longer retention time the labeled oligonucleotides would take while the hydrophobicity of fluorescent dyes was higher. However, the effect of the hydrophobicity was limited as the length was increased to a certain level.
