以原子力顯微鏡觀察DNA生物晶片上固定之共軸探針之雜合效率

Investigation of DNA Hybridization Efficiency of Orientated Co-Axial Probes Immobolized on DNA-Arrayed Biochips Using Atomic Force Microscope

陳俊義(Jun-Yi Chen)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2010年)

https://doi.org/10.6840/cycu201000803

摘要

本研究於兩對調序列之微陣列探針，預先雜合片段寡核苷酸（Oligo - nucleotide），藉由螢光掃描儀及原子動力顯微鏡去探討不同雜合次序對雜合效率之影響及晶片表面之變化之關係。預先雜合之寡核苷酸片段和DNA標的物（Target）設計成與同軸堆疊之探針（Probe）序列完全互補。換而言之，先將一股DNA寡核苷酸片段與探針上完全互補序列雜合後，再與同一探針序列上未雜合之部位，偶雜合另一股完全互補的標的物。
預先雜合之寡核苷酸片段和DNA標的物，分別標定上明顯不同的螢光分子。藉由偵測螢光染色標定的訊號，證明晶片已經成功進行單獨雜合或堆疊雜合，可繼續使用AFM進行觀測。
使用AFM於不同DNA探針濃度下，作全面性的厚度探討，結果顯示厚度會隨著雜合作用變大，且近晶片端的Probe#1厚度增幅較遠晶片端的Probe#2大，推測DNA在Probe#1很可能是站立或傾斜在晶片表面；而遠晶片端的Probe#2則可能是傾斜或貼附晶片表面的情況。

關鍵字

核苷酸 ； 標的物 ； 雜合效率 ； 原子力顯微鏡 ； DNA預雜合 ； 微陣列 ； 探針

並列摘要

The research studies on two sequence-inversed microarrayed probes pre-hybridizing a oligo-nucleotide. The fluorescence scanner and atomic force microscope(AFM) were used to measure the hybridization efficiency in different hybridization order and the depth changes of the chip surface,respectively. The sequences of both pre-hybridizing oligo-nucleotide and target DNA were designed to be fully complementary to their shared DNA probe in a coaxial stacking configuration. In other words, the first DNA target is completely complementary with the probe, and then the second target stacked onto the non-hybridized site of the same probe.
The pre-hybridizing and the second DNA targets were distinguished by labeling with two distinct fluorescent dyes, and the hybridization efficiency was investigated through the comparison between the stacking and individual hybridization configurations based on the detection signals of the labeling dyes.
AFM was used to measure the thickness of two probes at various concentrations. The results indicated that the thickness will increase as the hybridization proceeded. Probe#1 immobilized close to the chip surface received more thickness then probe#2 chose to the solution. Hybridized target and probe#1 might partially stand on the surface, while probe#2 might lean on the surface. Conjecture that DNA probably stand or slope at the Probe#1 on the surface of the chip, and maybe slope or adhere to the surface of the chip.

並列關鍵字

AFM ； gene chips ； DNA pre-hybridization ； hybridization efficiency ； DNA microarray

參考文獻

【1】 Schaack, B., Reboud, J., Combe, S., Fouque, B., Berger, F., Boccard, S., Filhol-Cochet, O., Chatelain, F., "DropChip" cell array for DNA and siRNA transfection combined with drug screening,” NanoBiotechnology, Volume 1, Issue 2, Pages 183-189 (2005).

【2】 Wang, S. and Cheng, Q. “Microarray analysis in drug discovery and clinical applications,” Methods in Molecular Biology, Volume 316, Pages 49-65 (2005).

【3】 Douglas, C. and Ehlting, J. “Arabidopsis thaliana full genome longmer microarrays: A powerful gene discovery tool for agriculture and forestry,” Transgenic Research, Volume 14, Issue 5, Pages 551-561 (2005).

【5】 DJ Duggan, M Bittner, Y Chen, P Meltzer, JM Trent “Expression profiling using cDNA microarrays,” Nature Genetics, Volume 21, Issue 1, Pages 10-14 (1999).

【6】 Debouck C. and Goodfellow PN. “DNA microarrays in drug discovery and development,” Nature genetics, Volume 21, iSuppl 1., Pages 48-50 (1999).