寡核苷酸探针制备的优化

Optimization of Oligonucleotide Probe Fabrication

目的:探讨寡核苷酸微阵列制备中适合使用的探针浓度、探针缓冲液pH值、离子强度、优化杂交条件。方法:选取人白细胞抗原DQA1位点,针对多态性集中的外显子2设计一对保守引物及16条特异性分型探针;分别用ddH2O和0.1、0.2mol/L碳酸盐缓冲液(pH=7)稀释探针至100μmol/L;选取合适的缓冲液浓度后,调碳酸盐缓冲液为5.0、6.0、7.0、8.0、9.0、10.0等6种pH值,选取最优pH值及离子强度,分别溶解探针至20、50、100、200μmol/L,比较上述不同条件的杂交结果。结果:用0.1mol/L、pH9的碳酸盐缓冲液溶解探针杂交效果最佳;探针浓度为20μmol/L时信号弱,其他浓度下无显著差别。结论:通过探针制备的优化可以提高杂交效率,探针浓度与杂交信号强度无明显正相关。

Objective： To explore the optimal probe concentration, appropriate pH and ion intensity of probe buffer, as well as optimal hybridization conditions. Methods： A pair of conservative primers and a set of 16 specific probes were designed according to the polymorphism of the HLA-DQA1 exon 2. The probes were resuspended to 100 μmol/L by pH7.0 carbonate buffer. And then carbonate buffer was adjust to pH5.0, 6.0, 7.0, 8.0, 9.0, 10.0 in optimal ion intensity respectively. Then, with optimal pH and ion intensity, probes were resuspended to 20, 50, 100, 200 μmol/L. Hybridization results were evaluated respectively. Results： The optimal hybridization result comes from 0.1 mol/L, pH=9 carbonate buffer. Hybridization signal is weak when probe concentration is 20 μmol/L, while hybridization signal is no much different in other concentration. Conclusion： Hybridization signal could be improved by optimization of probe fabrication. The strength of signal does not relevant with probe concentration obviously.