GFP质控芯片的初步研究

Preliminary Study of GFP Quality Control Microarray

目的:建立一种质量控制芯片来监测样品标记、杂交和检测过程中的失误。方法:针对GFP基因设计的4条60mer寡核苷酸探针和1条阳性对照探针poly(U)与流感寡核苷酸探针一起打印在DAKO玻片上,并构建了GFP基因的克隆载体和体外表达载体,将从这两种重组载体上获得的绿色荧光蛋白(GreenFluorescentProtein,GFP)基因的RNA、DNA片段和人的全血样品中的DNA用限制性显示技术(RestrictionDisplaytechnology,RD)扩增标记,将标记的样品和荧光标记的通用引物U分别与芯片杂交、检测,并对扫描的结果进行统计分析。结果:GFP探针与相应的样品杂交时出现阳性信号,阳性对照探针在所有的杂交中均出现阳性信号,而空白对照则未检测荧光信号。结论:建立的质控芯片具有较好的敏感性和特异性,可以用于基因芯片中的质量监控。

Objective： To establish a quality control microarray to monitor the process of labeling, hybridization and scanning of the microarray protocols. Method： Four oligonucleotide probes, one positive probe and influenza diagnostic probes were printed on DAKO slides. The gene encodhag the Green Fluorescence Protein （GFP） was subcloned into a cloning vector for in vitro expression of the GFP mRNAs. The RNA or DNA of GFP encoding sequence obtained from the recombined plasmids were respectively labeled by the restriction display （RD）. Control DNA from blood of healthy human volunteers were also labeled. The labeled DNA samples and cy3- universe primers U were hybridized with oligonucleotide probes arrayed on the slides. The fluorescent signals were scanned by C, enepix 4000B and analyzed with the statistics software Spss 11.0. Result： All the oligo probes complement to GFP gene and hybridized with the corresponding labeled samples gave strong signals , The signals of positive control probes were also strong in all hybridizations while the blank control was completely negative. Conclusion： The established quality control microarray have perfeet sensitivity and specificity and can be used to monitor the process of labeling, hybridization and scanning.