【作者】 刘全俊；

【导师】 陆祖宏；

【作者基本信息】 东南大学 ， 生物医学工程， 2006， 博士

【摘要】 基因芯片技术是一种高通量的现代生物检测技术,在功能基因研究、新药筛选、疾病的基因诊断等领域具有广泛的应用。基因芯片检测是一个复杂的过程,包括:靶基因的扩增、扩增产物的标记、芯片杂交与清洗、杂交信号检测及分析等。在这样的一个复杂检测流程中,容易产生由于操作过程的复杂性而带来的检测结果不稳定,从而影响了基因芯片的推广应用。我们研制基因扩增杂交一体化检测仪的目的是为了实现从基因扩增、荧光标记、杂交与清洗、芯片信号检测的一系列流程的自动化操作,以减少检测结果的人为因素的干扰,提高基因芯片在基因多态性分析及疾病诊断等方面的应用水平。为了进一步简化基因扩增杂交检测一体化芯片结构及其仪器的复杂性,我们又提出了管盖基因芯片。为能够对多个靶基因实现定量检测,我们在本实验室提出的TaqMan微阵列芯片的基础上,设计和研制了定量基因芯片检测系统,并设计了一种新的FRET定量检测基因芯片,可用于同一样品中不同基因的高通量定量检测。1.基因扩增杂交一体化芯片及检测系统针对基因芯片检测过程的复杂性,我们设计了集成化的操作平台—基因扩增杂交检测一体化芯片,用于实现基因扩增、杂交、清洗、检测的自动化以减少复杂操作过程中的人为因素影响。基因扩增杂交检测一体化芯片的结构包括二个功能区域:十四个通道的玻璃毛细管作为基因扩增区域和由十四个在玻片上固定了核酸探针阵列的基因芯片组成的杂交检测区。使用了硅胶作为密封件,分别将二个区域相连。该芯片可实现十四个通道的基因扩增与基因芯片检测一体化,检测样品流程所需要的时间为3~4小时。基于上述生物芯片,我们设计和研制了基因扩增杂交一体化检测仪,能够进行PCR、生物芯片杂交和荧光检测的一体化操作。基因扩增杂交一体化检测仪主要由温度精确控制系统、样品自动注入系统和荧光检测几部分组成,包括:计算机、加热炉、荧光显微镜、温度控制器、试样装夹及送进机构、运动控制箱、清洗机构等。与南京依科机电有限公司合作开发出相应的产品,通过了江苏省医疗器械检测所的检验,建立了医疗器械注册产品标准。该仪器有如下的结构特点:(1)利用蠕动泵,电磁开关等进行液体的控制。通过软件控制,实现了反应体系在基因扩增区域与杂交区域的密封与流动。通过液体的输送,实现了基因扩增产物与杂交液的混合及与芯片的杂交反应。(2)杂交过程的温度控制通过空气介导的温控体系来实现。具有变温快、温度场均匀等优点。此仪器能够对PCR反应区的温度进行精确控制,其温度上升的速率可达到20 oC/s,下降的速率可达到10oC/s。(3)芯片的荧光杂交信号检测是通过荧光显微镜与CCD的图像采集技术实现的。芯片被固定在二维移动平台上在显微镜下进行荧光信号的扫描阅读,在PC机上显示,存入计算机。基于上述芯片和仪器,我们发展了用于乙型肝炎基因突变检测的试剂,可以对乙型肝炎YMDD突变基因进行检测,并在南京市第二医院进行了临床检测,检测准确率达到100%。通过与在普通更多还原

【Abstract】 The microarray technique, a high-throughput detection method, has been widely applied in the fields of the gene expression research, the SNP studies and the diagnosis development, and etc. The microarray progress is a very complex process including the gene fragment amplification, gene fragment labeling, hybridization and washing, microarray signal capture, data analysis and etc. Because of its complexity and multi-step, it is quite easy to cause unstable results. So that it has a negative impact on practical application of the microarray technique. Now, an integrated instrument of polymerase chain reaction, gene chip hybridization and fluorescence detection has been developed to perform an automatic process. The instrument is able to decrease potential disturbance of operators and increase the stability of results of the SNP detection or the disease diagnosis. Again, a microarray-in-a-tube system, which can perform the microarray progress in a sealed tube, has been developed to simplify the complexity and decrease the cross contamination in microarray progress. Furthermore, in order to solve the multi-gene quantitative problem, a quantitative microarray scanner has been designed and developed based on our lab quantitative PCR microarray technique. The scanner can be used for the multi-gene quantitative detection. And a new type of quantitative microarray was designed. The detection probes were the immobilized FRET probes. The high-throughput quantitative method would increase the efficiency of gene expression research, disease research, drug development, and drug screen and disease diagnosis.1. The integrated instrument of polymerase chain reaction and microarray hybridization and fluorescence detectionTo overcome the complexity of progress of the microarray technique, an integrated microarray, which integrates polymerase chain reactions, biochip hybridization and fluorescence detection, has been designed, developed and built. The instrument is capable of executing the whole three-step process automatically. It is good for decreasing the interfering of operators. The PCR Hybridization chips have two functional areas. The polymerase chain reaction area, made of a glass capillary, has xxx tubes with fourteen-channels. The hybridization area, covered with a glass fixed onto the chip with a rubber ring, consists of fourteen chips with detectors. The linkers between them are rubber tubers, which can be pressed by the machine to hold the liquid in the glass capillary when temperature cycles. The typical running time is about 3~4 hours for an experiment with the fourteen-unit bis-function microarray.Based on the chips above, an integrated instrument of polymerase chain reaction and microarray hybridization and fluorescence detection has been designed and constructed. The integrated instrument makes all steps of the microarray be performed automatically. It consists of a precision temperature controlling system, an automatic sample injection system and a fluorescence detection system. The hardware includes a computer, an oven, a fluorescence microscope, a temperature controller, a 2-dimension stage and a washing system. The features of the system are listed as follows.更多还原