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生物芯片技术是基于杂交原理发展而来的 ,是将固相反应的原理和形式应用于大分子识别反应 (核酸杂交、抗原抗体结合或酶促的模板依赖性的连接、延伸等反应 )中 ,以达到对大量的目标分子进行快速、平行的特异识别。寡核苷酸芯片的制备过程中关键部分是基片的表面化学处理和探针末端的不同修饰。为了比较不同末端修饰探针在不同化学处理的载玻片上的杂交信号的强弱 ,本研究根据HLA DRB1 12的序列设计 8种不同类型的探针 ,即 4种 5′末端修饰探针 ,包括末端氨基修饰探针 (N) ,氨基加四聚乙二醇间隔臂探针 (NL) ,硫代探针 (S) ,硫代加四聚乙二醇间隔臂探针 (SL)和 4种 3′末端修饰探针 ,同样包括末端氨基修饰探针 ,氨基加四聚乙二醇间隔臂探针 ,硫代探针 ,硫代加四聚乙二醇间隔臂探针。将这 8种探针分别固定在溴化芯片和醛基芯片上 ,与末端标记荧光的不对称的PCR产物进行杂交 ,通过比较杂交结果荧光信号的强弱 ,筛选出探针同活化基片的最佳组合 ,从而达到优化寡核苷酸芯片制备的目的。另外 ,为了进一步比较四聚乙二醇间隔臂对杂交信号的影响 ,设计末端连接不同数目四聚乙二醇的 3′氨基探针。结果显示 ,3′末端修饰探针杂交信号强于 5′末端修饰探针 ,探针在溴化芯片中的杂交信号强度高于在醛基芯片中杂交信
Biochip technology is based on the principle of hybridization developed from the principle and form of solid-phase reaction used in macromolecular recognition reaction (nucleic acid hybridization, antigen-antibody binding or enzymatic template-dependent ligation, extension and other reactions) In order to achieve a large number of target molecules for rapid, parallel specific recognition. A key part of the preparation of oligonucleotide chips is the chemical treatment of the surface of the substrate and the different modifications at the ends of the probes. In order to compare the intensity of the hybridization signals of different chemically modified slides with different terminal modification probes, we designed eight different types of probes according to the sequence of HLA DRB12, that is, four kinds of 5 ’end modification probes, including Amino-modified probe (N), Amino group plus polyethylene glycol spacer probe (NL), thio probe (S), thio- and tetraethylene glycol spacer probe (SL) The 3’-end modified probe also includes a terminal amino-modified probe, an amino-added tetraethylene glycol spacer probe, a thio probe, a thio-tetramethylene glycol spacer probe. The eight probes were immobilized on the brominated chip and the aldehyde-based chip, respectively, and the end-labeled fluorescent asymmetric PCR products were hybridized. The intensity of the fluorescent signal of the hybridization result was compared to screen the probes Optimal combination, so as to achieve the purpose of optimizing preparation of oligonucleotide chip. In addition, in order to further compare the effect of tetrapetaethylene spacer on the hybridization signal, a 3 ’amino-probe with different numbers of tetraethyleneglycol ends was designed. The results showed that the hybridization signal of the 3 ’terminal modified probe was stronger than that of the 5’ terminal modified probe, and the hybridization signal intensity of the probe in the brominated chip was higher than that in the hybrid chip