磷酸根甲基化去氧核醣核酸：合成、定性及其應用之研究

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姓名

林耀能(Yao-neng Lin) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

磷酸根甲基化去氧核醣核酸：合成、定性及其應用之研究
(Phosphate-Methylated DNA as Neutralized DNA (nDNA)：Synthesis, Properties and Potential Applications)

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摘要(中)

近年來，由於生物技術的快速進展，人體中基因所表現、調控的功能，與其所提供人體當下的資訊也逐漸明朗。基因晶片是瞭解人體內的基因表現資訊很有用的工具，以非侵入性、大量、快速且低成本之檢測方式得到樣品中的DNA、RNA的表現資訊，可應用於早期的病症診斷，也可以針對治療效果進行監控及甚至提出進一步的治療方向。
　　為了增加基因晶片的檢測能力，基因晶片上的核酸探針對於特定序列的目標核酸雜交辨識性、專一性、靈敏度是關鍵因素，而專一性的問題取決於使用的核酸探針與其目標物的雜交性質，以及周遭水溶液環境中鹽離子的影響，因此有許多研究團隊使用了多種核酸類似物作為探針進行測試，並針對其應用範圍進行探討。
　　本研究主要開發一種核酸類似物，其磷酸骨幹的負電由甲基化所遮蔽，使核酸分子不帶電，與互補股序列的DNA雜交時會有更好的穩定性，並可以在低鹽濃度溶液環境下進行雜交，期待其對於序列的辨識性質更佳，且應用於電訊號敏感的感測平台上具有其優勢。
　　因此研究中以固相化學法合成不帶電DNA並初步探討其應用於基因晶片之性質優勢。實驗中以質譜及NMR對特殊改質後的核苷單體原料定性、確認化學反應完成並探討合成條件，合成為oligo後以HPLC進行成分分析，純化後以質譜測量分子量確認合成成功，再以CD及電泳證明了其核酸雜交、辨識序列的能力，最終我們將不帶電DNA應用於FET生物感測器，並調整感測水溶液環境之條件，其實驗結果證明了中性DNA於基因檢測平台中的優越能力。

摘要(英)

Intensive efforts have been achieved on the sequencing of whole human genome, which revealed the blueprint of what we are made of, but we do not yet know how it work without a guideline. DNA microarray is a useful tool for early diagnosis and prompt therapeutic intervention, however, low DNA probe recognition efficiency are usually affected by some specific sequences. To improve the sensitivity and specificity of DNA probe, we synthesized a novel neutral DNA (nDNA), which is a DNA analogue with the backbone phosphate groups changed by methylposphate groups. nDNA showed high affinity to hybridize with its complementary regular DNA and nDNA, even at low ionic strength buffer conditions. In particular, nDNA also improve the recognition efficiency for mismatch DNA sequences. In this study, we synthesized and characterized four nDNA nucleosides for nDNA synthesis. nDNAs were synthesized through the solid-phase synthesis and purified by reversed-phase chromatography. From the circular dichroism spectrum and gel electrophoresis we found that nDNA and its complementary DNA can form duplex, and this duplex shows higher Tm than regular DNA duplex. Finally, we took nDNA as probe on FET (field-effect transistor) sensor for gene detection, the results revealed that nDNA can enhance the recognition efficiency under low ionic strength condition.

關鍵字(中)

★ 去氧核醣核酸
★ 場效電晶體

關鍵字(英)

論文目次

目錄
摘要 iii
Abstract iv
誌謝 v
目錄 vi
圖目錄 ix
表目錄 xiii
第一章緒論 1
1.1 研究背景 1
1.2 研究動機 2
1.3 論文架構 3
第二章文獻回顧 4
2.1 生物標記Biomarker 4
2.2 基因晶片Gene chip 5
2.3 核酸類似物 5
2.3.1 肽核酸PNA 5
2.3.2 鎖核酸LNA 6
2.3.3 嗎啉基寡核苷酸MOs 7
2.3.4 中性DNA，n DNA 8
2.4 nDNA合成 10
第三章實驗藥品、儀器設備與方法 13
3.1 實驗藥品 13
3.1.1 化學品 13
3.1.2 實驗耗材 16
3.2 儀器設備 16
3.3 實驗方法 17
3.3.1 核苷單體改質與管柱純化 17
3.3.2 nDNA原料準備、合成 19
3.3.3 nDNA保護基步驟 20
3.3.4 nDNA濃度定量 21
3.3.5 nDNA HPLC分析純化 23
3.3.6 nDNA Agarose電泳 24
3.3.7 nDNA PAGE電泳 25
3.3.8 nDNA圓偏光二色光譜儀測定構型 26
3.3.9 nDNA UV-vis分光光譜儀 27
第四章結果與討論 29
4.1 nDNA nucleosides 保護基的選擇 29
4.2 原料單體amidite改質成果定性 33
4.2.1 以NMR針對化學結構鑑定 33
4.2.2 以質譜儀確認改質後分子量 34
4.2.3 產物之存放以及使用觀察 36
4.3 nDNA以HPLC進行成分分析 37
4.4 nDNA以質譜定性 43
4.5 nDNA去保護基條件 49
4.5.1 去保護基時間 49
4.5.2 不同酸鹼中和方式 51
4.6 nDNA 穩定性 52
4.7 nDNA電泳 57
4.7.1 Agarese電泳 58
4.7.2 PAGE電泳 61
4.8 nDNA 以圓二色光譜儀測定構型 63
4.9 nDNA應用於FET生物晶片初步成果 67
第五章結論與未來展望 69
5.1 結論 69
5.2 未來展望 69
第六章參考文獻 72
附錄Appendixes 75

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指導教授

陳文逸

審核日期

2015-8-21

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