HLA-DRB1-12寡核苷酸芯片的制备及优化(英文)

Fabrication and Optimization of HLA-DRB1-12 Oligonucleotide Microarray

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 。

Oligonucleotide microarray is developed on the basis of hybridization on the solid substrate. The pre activated glass substrates and the terminal modification of the oligonucleotides are the two important factors in the process of fabrication for microarray. In order to compare the hybridization signal intensity of the different terminal modified oligonucleotide probes, the eight kinds of oligonucleotides were designed according to the sequence of HLA DRB1 12, including the amino modified oligonucleotides with PEG spacer and the one without spacer, the phosphorothioate modified oligonucleotides with PEG spacer and the one without spacer. They were modified on 5′ terminal and 3′ terminal, respectively. In addition, the oligonuclotides probes with the internal spacer of different number of PEG were designed to observe the relationship between the spacer of PEG and the hybridization efficiency. These probes were respectively fixed on the bromoacetylation activated and glutaraldehyde activated slides to manufacture the two kinds of microarray which hybridized with the fluorescence labeled PCR product of HLA DRB1 12 gene. The results from the study demonstrated that the signal intensity of 3′ amino modified probes with the internal spacer of different number of PEG on the bromoacetylation activated slides was stronger than the others. It is concluded that the 3′ amino modified oligonucleotide with an internal PEG spacer and the bromoacetylation activated slide enhanced the hybridization efficiency and were worthy to be proposed for the fabrication of HLA microarray or other kinds of microarrays for detecting fluorescence labeled PCR product in the future study.