Manufacturing DNA microarrays of high spot homogeneity and reduced background signal
Manufacturing DNA microarrays of high spot homogeneity and reduced background signal
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DOI：
10.1093/nar/29.7.e38
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发表时间：
2001-04-01
影响因子：
14.9
通讯作者：
Hoheisel, Joerg D.
中科院分区：
文献类型：
--
作者：
Diehl, Frank;Grahlmann, Susanne;Hoheisel, Joerg D.
DNA 微阵列分析在很大程度上取决于斑点质量和玻璃支持物的低背景信号。通过使用甜菜碱作为柠檬酸钠盐点样溶液的添加剂，在胺化表面（例如涂有广泛使用的聚-L-赖氨酸或氨基硅烷的载玻片）上，点样 PCR 产物的结合效率和 DNA 点的均匀性均得到显着提高。此外，非特异性背景信号显着减弱。同时，在排列过程中，甜菜碱减少了保存 PCR 产物的微量滴定盘孔中的蒸发。在非极性非水溶剂 1,2-二氯乙烷和酰化催化剂 N-甲基咪唑的存在下，随后用琥珀酸酐对芯片表面的封闭得到显着改善。此过程可防止在硼酸盐缓冲液中经常使用的水性溶剂 1-甲基-2-吡咯烷酮中出现的整体背景信号，因为 DNA 在封闭过程中会从斑点中重新溶解，只会再次结合在整个玻璃表面上。
Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.