【摘要】 应用SpotBotTM芯片点样仪对4种不同的芯片点样液(3×SSC、50%DMSO、3×SSC+1.5mol/L甜菜碱和MSP)和5个不同公司生产的7种芯片片基(UltraGAPSTM、GAPSⅡ、SuperAmine、SuperAmineBarcoded、Baio氨基载玻片、PL-100C多聚-L-赖氨酸片基和POLY-PREPTM片基)进行了比较分析,发现UltraGAPSTM、GAPSⅡ、SuperAmine和SuperAmineBarcoded片基用3×SSC+1.5mol/L甜菜碱能得到最佳的点样和杂交结果。同时对克隆PCR产物的扩增和纯化方法、用于点样的DNA样品的浓度、点样环境条件(温度、相对湿度等)、点样后芯片的处理方法、探针的标记方法、芯片的杂交及杂交后的处理等进行了比较、分析和优化。应用优化后的条件成功地在每张芯片上点制27648个点,并杂交、扫描得到高质量的芯片结果。更多还原

【Abstract】 Four types of printing solvent (3×SSC, 50%的DMSO, 3×SSC+1.5 mol/L betaine and MSP) and seven printing substrates (UltraGAPSTM substrates, GAPS Ⅱ substrates, SuperAmine Barcoded substrates, SuperAmine substrates, Baio Amine slides, PL-100C Poly-L-Lysine slides and POLY-PREPTM slides), came from five different companies, were compared and analyzed with the spotting robot of SpotBotTM. The results showed that the combination of four slides, UltraGAPSTM, GAPS Ⅱ, SuperAmine and SuperA-mine Barcoded with 3 × SSC+1.5 mol/L betaine could produce the best printing and hybridization results, the optimized parameters. At the same time, a comprehensive comparison, analysis and optimization of the amplification methods of clone DNA, the purification methods of PCR products, the concentrations of arrayed DNA in the printing solutions, the printing environment conditions (including environmental temperature, relative humidity and so on), the treatment methods of substrates after printing, the labeling methods of probe, the conditions of hybridization and post-hybridization washing were carried out. The microarrays with 27 648 spots per substrate were successfully produced, and the high quality hybridization results were subsequently achieved using these optimized conditions.更多还原