无胶筛分毛细管电泳分离相差单个碱基的脱氧寡核苷酸方法初探  

Preliminary researches for the method of separation of oligonucleotides with difference of a single base in sequence from each other by non-gel sieving capillary electrophoresis

作　　者：尚明美[1] 刘秀文[1] 汤仲明[1] 宋海峰[1] 陈惠鹏[2] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《军事医学科学院院刊》2005年第3期268-271,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金资助项目(39870878和39930180)

摘　　要：目的:建立和优化分离不同碱基数目硫代脱氧寡核苷酸的有效方法。方法:用去离子水溶解硫代脱氧寡核苷酸样品并采用核酸蛋白分析仪定量。在无胶筛分毛细管电泳分离分析之前超声波和高速离心脱气。无胶筛分毛细管电泳以线性大分子ssDNA100RGel作为分离介质。为得到最佳效果,对毛细管温度、分离电压、进样方式和进样时间进行了优化。结果:灵敏度、迁移时间和重现性的最佳实验条件为:毛细管温度25℃,电动进样10kV10s,DAD检测器波长为255nm,18kV恒压分离模式。在此条件下,成功分离了依次相差一个碱基的21种脱氧寡核苷酸[pd(A)4060]。结论:建立并优化了一种简单快速分离相差单个碱基的脱氧寡核苷酸的毛细管电泳方法。Objective:To establish and optimize a method for effective separation of the oligodeoxynucleotides with difference of a single base. Methods:Oligodeoxynucleotides were dissolved in de-ionized water and quantitated by (DU640) Nucleic Acid and Protein Analyzer at wavelength 260 nm. The aqueous solution of the oligodeoxynucleotides was degassed by ultrasonic device and centrifugation before analyzing by non-gel sieving capillary electrophoresis (NGCE). The separating packing was the linear macromolecule ss-DNA 100-R gel. Experimental factors including the capillary temperature, separating electric voltage, the injection mode and the injection time were optimized to obtain the optimal experimental conditions. Results: Optimized separating conditions for sensitivity, migration time and reproducibility were obtained: capillary temperature at 25 ℃, electric injection at 10 kV for 10 s, constant voltage with - + operating mode at separation voltage of 18 kV and DAD detection at 255 nm. A mixture of 21 oligonucleotides with the sequence difference of a single base was separated with high solution. Conclusion:An optimal simple and fast method for separation of oligonucleotides with difference of a single base by capillary electrophoresis(CE) was established and validated as feasible.

关 键 词：反义 寡核苷酸类 硫代修饰 电泳 毛细管 无胶筛分 

分 类 号：O629[理学—有机化学]