cDNA芯片表面核酸固定化的优化

Surface Optimization of Glass Slide Used for The Immobilization of cDNA Probes

cDNA芯片技术表面核酸固定化影响因素众多 ,其中涉及选择载体、固定于玻片的DNA片段浓度、玻片DNA片段的固定方法、玻片预处理方法、DNA片段的变性、溶解DNA片段的点样液等等 .针对这些因素进行了优化筛选实验 ,以便于提高cDNA芯片技术检测基因表达的效率 .

Microarray hybridization is a powerful technique in life science since it allows a relatively straightforward determination of gene expression, in respect to distribution as well as quantification. Large-scale hybridization-based mRNA expression mapping projects call for robust and high efficient microarray hybridization techniques that are amenable to automation. However, a major drawback of microarray is the fact that all standard protocols available include time consuming optimization steps of several critical parameters such as DNA fixation to surface of the slides, hybridization conditions and washing procedures. The aim of this investigation was a rational design of DNA probes which were adapted to the standard protocol and could therefore be used without changing any parameter. A rapid microarray technique for quantitative analysis has been recently introduced. For highly repetitive DNA probes the hybridization time and the number of washing steps were reduced considerably by formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes showed visible hybridization signals well suited for quantitative image-screening. The discrimination of minor and major binding sites to surface of glass slide was possible by automated image-processing. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature, spotting buffer) of the given microarray protocol. By comparation of several candidate conditions, the optimized standard protocol was constructed.