基因芯片—玻璃表面两种不同DNA探针固定化方法的比较研究

Genochips- comparative Studies on Two Different Methods for Immobilization of DNA Probes on Glass Surfaces

研究了基因芯片相关的DNA探针在芯片表面最佳固定化方法.用两种不同的双功能试剂1,4-苯二异硫氰酸酯和戊二醛分别把5＇-端氨基衍生的21-mer寡脱氧核苷酸探针直接共价固定到玻片表面,固定化的寡脱氧核苷酸探针与5＇-端FITC标记的互补靶序列进行分子杂交,杂交后用配有CCD的Ⅸ70型荧光倒置显微镜成像检测.结果表明,两种固定化方法的效果都比较好,能检测到靶序列的最低终浓度为1.5×10-9 mol/L优化了探针固定化时间、杂交时间、杂交温度等对DNA芯片分析性能的影响,为构建高灵敏度基因芯片打下良好基础.

Two different methods for immobilization of DNA probes on glass surfaces have been comparatively investigated. Glass slides were derivatized with 3-aminopropyltriethoxysilane. Then 5' - amino - modified 21 mer oligodeoxynucleotide probes were covalently immobilized onto the surface via 1,4-phenylene diisothiocyanate and glutaraldehyde, respectively. The surface molecular hybridization of the immobilized probes with FITC-labeled complementary target sequences was monitored with an inverted fluorescence microscope (Model IX70) coupled with a cooled - CCD. There was hardly any difference in the fluorescence intensity between the spots on the chips, obtained at high solution concentrations of DNA probes by the two immobilization methods respectively, indicating that these methods were satisfactory under the conditions used. However, the fluorescent signal for the chips obtained at low concentrations of DNA probes by the 1, 4-phenylene diisothiocyanate method was stronger than that by the glutaraldehyde method, indicating that the former method was better than the latter at low solution concentrations of DNA probes. The detection limit for the target sequence was 1.5 x 10(-9) mol/L. Some experimental conditions, including the time for DNA probe immobilization, hybridization time and temperature, were examined. The results obtained would be useful for the development of genochips.