荧光标记寡核苷酸的反相离子对色谱分析

建立荧光标记寡核苷酸反相离子对色谱分析方法,优化了流动相醋酸三乙胺浓度(0～0.15 mol/L), pH 4.5～7.0和洗脱强度等色谱条件.对5-mer, 10-mer和15-mer非标记和5'-羧基荧光素(5'FAM)标记寡核苷酸的保留进行比较分析,研究荧光标记寡核苷酸的保留机理,并分离TaqMan~(TM)探针等多种常用荧光标记寡核苷酸.结果显示,不同长度荧光标记寡核苷酸在0.01 mol/L醋酸三乙胺,pH 7.0的条件下获得最大分离.荧光标记寡核苷酸的保留与非标记寡核苷酸有明显差异,两者可完全分离.在一定长度范围内非标记寡核苷酸随长度的增加,保留时间增长;相反,荧光标记寡核苷酸的长度增加,保留时间减短.荧光染料疏水性对其标记的寡核苷酸在反相柱中的保留有较大影响,荧光染料疏水性越强,其标记寡核苷酸保留时间越长.但疏水性的影响程度随标记寡核苷酸长度增加而逐渐变小.

Analysis of Fluorescent Dye-labeled Oligonucleotides by Ion Pair Reversed Phase High Performance Liquid Chromatography

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.