CAJ | 학술논문

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.
건립형광표기과핵감산반상리자대색보분석방법,우화료류동상작산삼을알농도(0～0.15 mol/L), pH 4.5～7.0화세탈강도등색보조건.대5-mer, 10-mer화15-mer비표기화5'-최기형광소(5'FAM)표기과핵감산적보류진행비교분석,연구형광표기과핵감산적보류궤리,병분리TaqMan~(TM)탐침등다충상용형광표기과핵감산.결과현시,불동장도형광표기과핵감산재0.01 mol/L작산삼을알,pH 7.0적조건하획득최대분리.형광표기과핵감산적보류여비표기과핵감산유명현차이,량자가완전분리.재일정장도범위내비표기과핵감산수장도적증가,보류시간증장;상반,형광표기과핵감산적장도증가,보류시간감단.형광염료소수성대기표기적과핵감산재반상주중적보류유교대영향,형광염료소수성월강,기표기과핵감산보류시간월장.단소수성적영향정도수표기과핵감산장도증가이축점변소.
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.