【作者】 李文；

【导师】 何继善； 汤建新；

【作者基本信息】 中南大学 ， 生物信息物理学， 2010， 博士

【摘要】 基因芯片已被广泛运用到生物诊断、生物表达、生物组、疾病诊断、药物筛选、发现新基因及各种病原体的诊断等生物科学领域中的重大应用价值,其光芒已渗透到生命科学的各个领域,成为后基因组时代科学研究强有力的工具。近年来,国外以Illumina、Affymetrix、Agilent、Roche-Nimble Gen等公司推出了各种基因芯片合成系统平台,虽然能够完成基因芯片的自动化操作,但由于其均需要了芯片及相关试剂和试剂盒、工具数据库及芯片分析软件工具、芯片制备系列平台仪器及其零配件、扫描检测仪器、杂交反应设备,使得价格普遍在数百万人民币以上,且其合成芯片技术都存在着专利保护,合成的基因芯片价格高昂,目前国内生物芯片产品主要用户为规模较大的科学研究单位,市场空间相对单一狭小,规模较小科研院所都负担不起,造成了基因芯片难以临床应用和推广的瓶颈。因此,研制和开发一种具有自主知识产权,又能快速、准确、低成本的基因芯片合成系统平台非常重要。本研究基于自主创新的活版印刷基因芯片(DNA微阵列)原位合成的技术,研制了一种成本低廉、质量可靠、工艺简单、特别适合于产业化的基因芯片合成仪,构建活版印刷原位合成基因芯片系统平台,制备了甲基化特异性寡核苷酸芯片。具体内容包括：1.活版印刷原位合成基因芯片合成仪根据活版印刷法合成DNA微阵列的原理,本文设计和加工活版印刷原位合成基因芯片合成仪,并采用三坐标仪对模板、反应槽底座精度、模板和底座组装后各位置公差以及二维定位系统的精度进行检测,结果表明：模板精度和反应槽底座精度均为3μm,模板和底座组装后各位置公差小于8μm,X轴的重复精度为8.8μm,定位精度为14.4μm；Y轴的重复精度为9μm,定位精度为15μm,能符合活版印刷法制备基因芯片的要求。2.构建活版印刷基因芯片合成系统平台本文以活版印刷DNA合成仪器为核心,结合无水无氧专用手套箱、分子杂交仪、荧光扫描仪构建了活版印刷基因芯片合成系统平台,并采用完全正交回归实验研究了反应槽中碱基单体的体积,压印平台的压印时间,机械手压印时的气体压力对活版印刷基因芯片原位合成系统芯片合成效率的影响。研究表明：压印时间为45s,碱基单体体积为1200ul,气体压力为0.12MP为系统平台最优的合成条件,基因芯片压印效果为100%。本文运用上述合成条件和活版印刷基因芯片合成系统平台,成功研制出了p53基因第四外显子72密码子单碱基突变寡核酸微阵列,各阵点上同一探针寡核苷酸的杂交荧光强度均匀一致,实现了单个碱基正错配的检测。3.肺癌甲基化特异性寡核苷酸芯片制备及验证本文采用活版印刷基因芯片合成系统平台,研制了RARB、FHIT、RASSF1A基因启动子甲基化特异性寡核苷酸芯片,在肺癌患者和正常人群进行高通量的平行检测,并采用MSP方法扩增FHIT、RARB FASS1A基因启动子,验证甲基化芯片的正确性。研究结果表明：芯片检测结果与MSP方法检测结果在统计学上差异不显著(p>0.05),甲基化特异性寡核苷酸芯片是一种低成本、质量可靠、高灵敏度、高特异性的基因芯片,能应用于临床分析；其活版印刷基因芯片原位合成系统平台是一种成本低廉、质量可靠、工艺简单、特别适合于产业化的基因芯片制备系统平台。4. FHIT、RARB和RASSF1A基因启动子甲基化与肺癌易感性的研究本文通过采用活版印刷基因芯片原位合成系统平台合成FHIT、RARB、FASS1A基因启动子甲基化芯片,并对肺癌患者和正常人群进行研究,研究结果表明：RARB、RASSF1A基因启动子甲基化是高水平甲基化,有望成为肺癌早期诊断侯选的分子标志物；FHIT基因启动子甲基化是低水平甲基化,有望成为肺癌早期诊断侯选的分子标志物。更多还原

【Abstract】 Gene chip has been widely applied to all kinds of biological science such as biological diagnosis, biological expression, biological group, disease diagnosis, drug screening, discovery of new genes and the diagnosis of various pathogens, penetrated into all fields of life sciences, and become a powerful tool for the research post-genomic era. In recent years, some gene-chip synthesis platform were yielded by Illumina, Affymetrix, Agilent, Roche-Nimble Gen and others foreign company, including related eagents and kits, database, microarray analysis software tools, chip preparation series platform equipment and spare parts, scanning instrumentation and hybridization equipment. Moreover, the platform was difficultly applied to most of the molecular biology laboratory in our country because of the price of platform more than several million RMB, gene-chip synthesis technology protecting by patents and the high price of synthetic gene chip. So, Research and development of a proprietary gene-chip synthesis platform should be important, which was fast, accurate and low cost platform.This research was developed a low-cost, reliable, simple process, particularly suitable for industrial synthesis of the gene chip platform which used in-situ synthesis of biochip basing on the principle of typography and synthesize methylation-specific oligonucleotide microarray to detect lung cancer. The major content was as follows:1. The synthesizer for preparing gene chip of typography techniqueThe DNA synthesizer was designed and manufactured according to preparing gene chip of typography technique. The template, the reaction tank base, two-dimensional positioning system and the tolerance of assemble between the template and the reaction tank was detected the accuracy by using three-coordinated measurement instrument. The accuracy of printing template and the reaction tank base were 3μn, the position tolerance of assembly between the template and the reaction tank was less than 8μm, X axis repeatability was 8.8μm, X-axis positioning accuracy was 14.4μm, Y axis repeatability was 9μm, y-axis positioning accuracy was 15μm.The testing results showed that the accuracy of the machine was good enough to meet the demand of in-situ synthesis of biochip based on the principle of typography printing.2. Building the microarray platform for typography techniqueThe microarray platform included glove box without anhydrous and oxygen, hybridization instrument, fluorescence scanner and DNA synthesis equipment as to core. The synthesis efficiency of gene chip synthesis platform was affected on the time of imprinting, the volume of the reactor base monomer and the gas pressure of manipulator with imprint. And the completely orthogonal regression experiment was adopted to research the synthesis efficiency. The results showed that the synthetic efficiency of system was 100 percent when imprinted time was 45 second, the volume of the reactor base monomer was 1200 ul and the gas pressure of manipulator was 0.12 MP.Moreover, the oligonucleotide microarray of the fourth exon mutation in p53 gene was produced. The hybridization results showed that the fluorescence intensity is uniform in same oligonucleotide probe of microarray and oligonucleotide microarray could detect single base mismatch and match.3. Methylation-specific PCR method identifying the result of the methylation specific oligonucleotide microarrayMethylation-specific oligonucleotide microarray of RARB, FHIT and RASSF1A gene was synthesized by the gene chip synthesis platform, further, the Methylation-specific oligonucleotide microarray was detected between lung cancer and normal groups. The results of methylation-specific PCR were verified in that of methylation chip. The results indicated there were no association between methylation-specific PCR and methylation-specific oligonucleotide microarray (p>0.05). Methylation specific oligonucleotide chip was a low-cost, reliable, high sensitivity and high specificity of gene chips. And which could be used in clinical research. The in-situ synthesis microarray platform was a cheap, reliable and simple process, particularly suitable for industrial preparation of the gene chip synthesis platform.4. Analysis of lung cancer susceptibility by methylation-specific oligonucleotide microarray Methylation-specific oligonucleotide microarray of RARB, FHIT and RASSF1A gene was synthesized by the gene chip synthesis platform, detecting the lung cancer susceptibility between lung cancer patients and normal people. The results showed that the promoter methylation of RARB and RASSF1A gene was high level and the promoter methylation of FHIT gene was low level.The promoter methylation of FHIT, RARB and RASSF1A gene were candidate molecular markers for early diagnosis of lung cancer.更多还原