利用螢光掃描儀與氣相原子力顯微鏡觀察DNA微陣列生物晶片上固定之共軸探針之偶合效率

Investigation of Hybridization Efficiency of A Sequence-Orientated Coaxial DNA Probe Microarryed on Biohips Using Atomic Force Microscope and Fluorescence Scanner

林彥旭(Yan-Syu Lin)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2012年)

https://doi.org/10.6840/cycu201200730

摘要

本研究針對兩序列相反，預偶合片段寡核苷酸（Oligo - nucleotide）之微陣列探針，藉由螢光掃描儀及原子動力顯微鏡(AFM)，探討不同偶合次序對偶合效率之影響及晶片表面之變化之關係。同軸堆疊之預偶合寡核苷酸片段和DNA標的物（Target）設計成與探針（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預偶合後，再將另一股標的物與同一探針序列上未偶合之部位偶合。

在表面已改質過的晶片上佈放十個10X10的矩陣，五種不同濃度之兩種探針(77mer)，矩陣內最上面一排為正控點，最下面一排為負控點。以螢光掃描確定DNA探針固定化後，進行阿拉伯芥短鏈基因片段標的物的單獨偶合，利用螢光掃描驗證單獨偶合成功之後，再使用原子動力顯微鏡量測厚度變化；最後進行人類β肌動蛋白長鏈基因片段標的物的堆疊偶合，利用螢光掃描驗證單獨偶合成功之後，再使用原子動力顯微鏡量測厚度變化。

在比較堆疊偶合與單獨偶合對偶合效率的研究中，原子力顯微鏡量測在不同程序後，兩個不同的DNA探針對應的厚度。結果顯示厚度會隨著偶合程序變大，預偶合於近晶片端之探針(Probe#1)，其厚度變化大於預偶合於遠晶片端之探針(Probe#2)，證明了厚度變化是與偶合效率有關之假設。

關鍵字

核苷酸 ； 偶合效率 ； 標的物 ； 原子力顯微鏡 ； 預偶合.微陣列.探針

並列摘要

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.

並列關鍵字

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

參考文獻

【16】 林俊耀，廖永剛，“我國動植物疫病檢測產品開發與應用現況”，第25-30 頁，農業生技產業季刊，台灣，2009

【74】 陳俊義、吳瑞璋，“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”，中原大學化工系，桃園，2010

【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).