单链脱氧核糖核酸探针在羧基聚苯乙烯小珠表面的微阵列条件及固液杂交特性  

Optimal Condition of the Microarray of Single-Stranded Deoxyribonucleic Acid Probe on Carboxylate Polystyrene Bead Surface and the Hybridization of Microarrayed Probe at Solid Liquid Interface

作　　者：黄承志[1] 李原芳[1] 黄新华[1] 范美坤[1] 

机构地区：[1]西南师范大学环境化学研究所,重庆400715

出　　处：《分析化学》2000年第11期1344-1349,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.20045002);教育部高等学校骨干教师资助计划(No.200065)资助项目

摘　　要：优化在 1 乙基 3 ( 3 二甲基氨丙基 ) 碳化二亚胺 (EDC)存在下 ,5′ NH2 单末端和 5′ TR 3′ NH2 双末端修饰单链脱氧核糖核酸 (DNA)探针在 10 μm羧基聚苯乙烯小珠表面进行微阵列的条件 ,研究 5′ NH2 单末端修饰DNA探针与其碱基互补序列进行的固 液杂交特性。结果表明 ,在pH 4 .6～ 5.6,EDC浓度为 0 .5～ 0 .7g L时 ,DNA探针在小珠表面具有最大的微阵列能力。微阵列在小珠表面的 5′ NH2 单末端修饰DNA探针 ,与其碱基互补序列的杂交反应遵守二级反应动力学 ,杂交优化条件除取决于溶液介质的pH值、离子强度和碱基互补序列在溶液介质中的浓度外 ,还取决于环境温度和杂交时间。测定了 2 5℃下微阵列在小珠表面的 2 0 merDNA探针与其溶液中互补序列的杂交反应常数。This paper reports the optimal condition for the microarray of 5′ TR/3′ NH 2 double terminus modified single stranded deoxyribonucleic acid probe on 10 μm carboxylate polystyrene bead surface in the presence of 1 Ethyl 3 (3 dimethylaminopropyl) carbodiimide (EDC). It was found that a 20 mer DNA probe has its most strong microarray ability on the bead surface when pH was 4.6～5.6 and EDC concentration 0.5～0.7×10 -3 g/mL. We made micorarray of 5′ NH 2 single stranded DNA probe at the same microarray condition as the double terminus modified probe to test the hybridization properties of 5′ NH 2 single stranded probe on the bead surface. It has proved that the hybridization follows second order kinetics, and depends on the pH, ionic strength and the sample concentration of DNA in aqueous solution, and surrounding temperature and the reaction time. The hybridization constant was determined at room temperature for a microarrayed 20 mer DNA on bead surface with its complementary DNA sequence in solution.

关 键 词：脱氧核糖核酸探针 羧基聚苯乙烯小珠 优化 杂交 

分 类 号：Q523[生物学—生物化学] O632.13[理学—高分子化学]