| Summary: | 碩士 === 中原大學 === 化學工程研究所 === 100 === Two sequence-inversed probes were microarrayed on glass slides to study the steric effect on the hybridization efficiency with their DNA targets. A fluorescence laser scanner and an atomic force microscope (AFM) were utilized to investigate the hybridization efficiency in different hybridization orders and their corresponding depth changes on the chips. The sequences of two targets were designed to be fully complementary to their shared DNA probe in a coaxial stacking configuration. In other words, after the first DNA target is pre-hybridized onto the probe, the second one is stacked onto the non-hybridized region of the same probe. The pre-hybridizing and the second DNA targets were distinguished by two distinct fluorescent dyes.
The two probes (77mer) were microarrayed into ten 10X10 matrixes in five different concentrations, with the top row as the positive control and the bottom as the negative control. The pre-hybridization was verified by recognizing the fluorescence signal of the short-strand DNA target, the Arabidopsis thaliana gene fragments. After the measurement of the fluorescence intensity, the pre-hybridized probe was measured its corresponding thickness by AFM. Finally, the pre-hybridized probe was further hybridized the long-chain DNA target, the Human beta actin gene fragment, stacking with the previous Arabidopsis target. And again, the corresponding thickness changes were measured by AFM.
The hybridization efficiency was investigated through the comparison between the stacking and individual hybridization configurations. AFM was used to measure the depths of two probes at different steps of hybridizations. The results indicated that the depths increased as the hybridization proceeded. Probe#1, pre-hybridizing closer to the chip surface, received a thicker depth than Probe#2, pre-hybridizing farther from the chip surface. The AFM-measured depths was concluded to have a qualitative correlation with the fluorescent intensity.
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