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研究生:李采璘
研究生(外文):Tsai-Ling Li
論文名稱:使用不帶電中性核酸探針於原位雜交技術檢測微核醣核酸之研究
論文名稱(外文):MicroRNA In Situ Hybridization with Phosphate-methylated oligonucleotides (nDNA) Probe
指導教授:陳文逸陳文逸引用關係
指導教授(外文):Wen-Yih Chen
學位類別:碩士
校院名稱:國立中央大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:85
中文關鍵詞:原位雜交技術微核醣核酸核酸分子專一性
外文關鍵詞:In Situ HybridizationmicroRNAnucleic acidspecificity
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微核醣核酸(microRNA)是一群非編碼的小核醣核酸分子,序列長度約為18-22個核苷酸,在基因表現的調控上扮演重要的角色;藉由和其目標信使核糖核酸(messanger RNA)的互補序列結合誘發mRNA的降解以調節基因的表現。在先前的許多研究皆指出了不正常的微核糖核酸表現量和一些人類的疾病有相當大的關聯性。因此,特定的核糖核酸可以做為疾病檢測或癒後預測的生物標記分子(biomarkers)。發展具有專高靈敏度及高專一性的檢測平台因而變成一項重要的議題;現今的檢測平台及方法包括原位雜交技術(in situ hybridization) ,逆轉錄聚合酶鏈式反應(qRT-PCR),北方點墨法(northern blotting),微型核糖核酸晶片定序(miRNA microarray profiling);在上述這些技術中,僅有原位雜交技術可以達到同時檢測核醣核酸表現量及觀察在細胞或組織中的分布情形,且保有細胞或組織原有的形態。然而此技術的最大限制在於因微核糖核酸的序列長度太過於短小且核醣核酸本身較脆弱易被降解的特性,若使用一般去氧核醣核酸分子(DNA)做為檢測探針的情況下,檢測結果缺乏專一性。
在本研究中,我們在原位雜交技術中使用的檢測探針是一種由本實驗室開發研究的核酸類似物,一種不帶電中性核酸,是將單股的去氧核醣核酸上的核酸骨幹上帶負電的磷酸根基團修飾上甲基,形成甲基磷酸三酯鍵 (MPTE),使其不帶電;因此與其互補的序列做雜交時,靜電排斥的作用力下降形成較穩定的MPTEDNA/DNA雙股螺旋結構,因而具有較高的檢測專一性。且其疏水性的性質使得中性DNA更容易穿進細胞膜。我們利用此種探針於檢測HCT116人類結腸癌細胞株的外源微核糖核酸(miR-524-5p)及在結腸癌細胞株內oncomiR,內源核醣核酸(miR-21)。實驗結果皆顯示在相同的實驗條件下,修飾過MPTE的探針比起一般DNA探針具有較高的訊號強度,我們可以看到使用優化設計的不帶電中性核酸探針於原位雜交技術檢測微核醣核酸的可行性且可成功的提高檢測訊號且維持檢測專一性。期望藉由建立使用中性DNA於ISH的方法,將此種核甘酸發展成有潛力的疾病治療用藥。
MicroRNAs (miRNAs) are a cluster of small, non-coding RNA molecules, generally 18-22 nucleotides in length, that play important roles in regulating gene expression by binding to the target messenger RNAs (mRNAs) and inducing mRNAs degradation. Previous studies have shown several correlations between aberrant miRNAs expression and a variety of human diseases. Hence, typical miRNAs are considered as diagnostic and prognostic biomarkers. That is to say, the need of developing highly sensitive and specific detection methods is necessary. Current detecting methods include In Situ Hybridization (ISH), Real time PCR, northern blotting, and miRNA microarray profiling technology. Above these methods, only ISH provide miRNA information about both expression level and localization in a single cell level. However, the main challenge of using DNA oligonucleotides as detecting probe is that the results lack of specificity since the small size and the nature fragile characterization of the target miRNA. In this study, we applied an alternative DNA analogue, which contains site-specific neutral methyl phosphotriester internucleotide linkages (MPTE), shows improved the hybridization properties due to the reduction of electrostatic repulsion between the double strands MPTEDNA/RNA duplex. And the lipophilic character allow the probe transport through cell membrane easily. In Situ Hybridization methods were performed to visualize mimic exogenous miR-524-5p we transfected into HCT116 cell lines (human colon cancer cell lines) and the well-known oncomiR in colon cancer cell lines, endogenous miR-21, by 3’-digoxigenin (DIG) labelled MPTE modified probe. Through optimal design of the modification of MPTE, the results demonstrated improved hybridization efficiency while remaining detecting specificity. Based on the success of applying MPTE probe on detecting miRNA through ISH, we expected the potential ability of MPTE modified oligonucleotides developing into theoretic agent.
摘要 i
Abstract iii
誌謝 v
目錄 vi
圖目錄 x
表目錄 xiv
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 - 2 -
第二章 文獻回顧 - 4 -
2.1 核酸分子介紹 - 4 -
2.1.1 去氧核醣核酸(deoxyribonucleic acid,DNA) - 4 -
2.1.2 核糖核酸(Ribonucleic acid,RNA) - 6 -
2.1.3 微小核糖核酸(microRNA) - 7 -
2.2 原位雜交技術的發展與原理 - 8 -
2.3 核酸類似物 - 10 -
2.3.1 鎖核酸(Lock nucleic acid, LNA) - 10 -
2.3.2 肽核酸(Peptide nucleic acid, PNA) - 12 -
2.3.3 中性去氧核醣核酸( Neutralized DNA, nDNA) - 13 -
2.4 原位雜交技術訊號放大探針/試劑 - 19 -
2.5 反義核酸 (antisense) 調控基因表現 - 22 -
第三章 實驗方法與儀器設備 - 24 -
3.1 實驗藥品 - 24 -
3.2 儀器設備 - 26 -
3.3 實驗方法 - 27 -
3.3.1 細胞培養 - 27 -
3.3.2 外源mimic miRNA轉染 - 28 -
3.3.3 原位雜交技術(In Situ Hybridization, ISH) - 29 -
3.3.3 逆轉錄聚合酶鏈式反應(qRT-PCR) - 30 -
3.3.4 聚丙烯醯胺凝膠電泳 (Polyacrylamide gel electrophoresis, PAGE) - 30 -
第四章 實驗結果與討論 - 31 -
4.1 建立用nDNA合成探針檢測細胞中的miRNA之原位雜合方法-外源性mimic miR-524-5p - 31 -
4.1.1 設計不同修飾數目的nDNA合成探針檢測細胞中的miRNA之原位雜合方法 - 36 -
4.1.2 nDNA合成探針檢測細胞中的miRNA之原位雜合方法之專一性探討 - 38 -
4.2 用nDNA合成探針檢測細胞中的miRNA之原位雜合方法-內源性 miR-21 - 42 -
4.2.1 設計不同修飾位置的nDNA合成探針檢測細胞中的miRNA之原位雜合方法 - 44 -
4.2.2 nDNA合成探針檢測細胞中內源性的miRNA之原位雜合方法之專一性探討 - 47 -
4.2.3 調整細胞透化時間來探討nDNA合成探針對於細胞膜穿透之能力。 - 50 -
4.3 用螢光標定nDNA合成探針檢測細胞中的miRNA之原位雜合方法-內源性 miRNA - 52 -
第五章 結論 - 54 -
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1. 甲基磷酸三酯鍵中性核酸引子/探針的設計應用於微核醣核酸原位雜交及改善PCR /qPCR單一核酸多態性檢測
2. 改良二氧化矽纖維膜分離程序於培養的細胞中微核醣核酸之純化
3. 運用nDNA 修飾引子於PCR及qPCR平台以提升專一性之研究
4. 設計不帶電中性核酸探針於矽奈米線場效電晶體來改善富含GC鹼基核醣核酸之檢測專一性
5. 以熱力學的觀點探討鈉離子與錯誤鹼基配對在一般核酸和中性DNA雜交形成雙 股螺旋之機制
6. 探討中性DNA與一般DNA雜交反應熱力學與結合機制之研究
7. 利用兩性離子表面塗層探討抗石蓴孢子及藤壺腺介幼蟲附著之研究
8. 使用中性不帶電去氧核醣核酸探針於矽奈米線場效電晶體檢測微核醣核酸之研究
9. 設計中性DNA引子及探針以提升PCR與qPCR專一性之研究
10. 使用不帶電中性核酸探針於矽奈米線場效電晶體檢測去氧核醣核酸與微核醣核酸之研究
11. 矽奈米線場效電晶體多點之核酸檢測研究
12. 利用SPRi探討中性DNA探針相較於一般DNA探針在低鹽雜交環境下之優勢
13. 探討聚乙烯二醇化對於聚乙烯二醇化人類副甲狀腺素 (1-34) 與疏水介面間交互作用之影響
14. 於矽奈米線場效電晶體進行不同化學表面改質並利用核酸適體探針檢測肌鈣蛋白I之研究
15. 利用恆溫吸附以及恆溫滴定卡計探討二氧化矽表面吸附DNA之吸附機制