不同探针在制备HLA-DRB1-12亚型点突变芯片中的比较

目的　探讨寡核苷酸芯片检测点突变过程中 ,不同类型探针在预先化学处理的玻片表面上固定效率的不同对于杂交信号的影响。方法　根据HLA DRB1 12亚型的基因序列设计四种 5′末端不同化学基团修饰的探针 ,通过与醛基化玻片和溴化玻片结合形成两种寡核苷酸微阵列芯片 ,与不对称荧光标记的目的片段杂交 ,通过荧光信号的强度判断最佳类型探针。在此基础上进一步设计两组四种碱基突变探针 ,根据杂交结果 ,判断最佳检测突变探针。结果　 5′末端氨基探针和硫代探针可以分别固定在两种类型的玻片上 ,溴化芯片中杂交信号强于醛基化芯片的杂交信号。其中 5′末端带有连接臂的氨基探针在检测点突变中阳性信号同阴性信号比值要高于 5′末端硫代探针 ,重复实验判型结果稳定一致。结论　溴化芯片中 5′末端带有连接臂的氨基探针在检测点突变中具有更明显的优势 ,为进一步的研究和制备点突变寡核苷酸芯片提供了条件

Comparison of different oligonucleotides in the fabrication of HLA-DRB1-12 mutation microarray

Objective To compare the different hybridization efficiencies of different probes and activated slides. Methods The four different 5' terminal modified probes were designed according to the sequence of HLA-DRB1-12 and the two kinds of different activated surfaces were made. The four modified oligonucleotides were immobilized respectively on the bromoacetylation activated slide surface and the aldehyde group coated slide to fabricate the two kinds of oligonucleotide microarray which could be hybridized with the fluorescence labeled as PCR product. Results The results of the study suggested that the bromoacertylation surface could give lower background and get stronger signals than aldehyde group coated surface. Moreover, the signal intensity of 5′amino-modified probes with the internal spacers of PEG were stronger than the signals intensity of the others and the ratio of the positive signal intensity to negative intensity of this kind of probes was higher than that of the others.Conclusion It can be concluded that 5'amino-modified probes with the internal spacers of PEG are more suitable for detecting the mutation in the microarray.