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

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許台宜(Tai-Yi Hsu) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

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
(Designing of phosphate–methylated DNA( nDNA) as probe/primer for in situ hybridization and improving single nucleotide polymorphism(SNP) discrimination by PCR/qPCR)

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摘要(中)

miRNA是一種單股的非編碼微小RNA片段，長度18-23個核甘苷酸，近幾年的研究已證實miRNA的表達與癌症疾病有很強的關聯性，且可被視為診斷和預後的生物標誌。為了要更準確判斷miRNA表達，有幾種常見方法用來檢測miRNA，例如，即時聚合酶連鎖反應、微型核糖核酸晶片、次世代定序或是原位雜交。然而，由於miRNA其序列長度較短，家族成員之間的序列相似性，造成檢測上的困難。近年來，科學家已提出許多新方法，例如鎖核酸（LNA）和肽核酸（PNA）以獲得更好的靈敏度和特異性信號。在本研究中，我們使用本實驗室開發研究的核酸類似物，其磷酸骨幹上的氧可以經由甲基化的改質，使帶負電的磷酸根轉變成甲基磷酸三酯鍵methyl phosphotriester (MPTE)而成為電中性，使用MPTE修飾的DNA序列可以降低靜電排斥效應，同時增強雙股雜交時的親和力，因此可以提高其microRNA檢測的靈敏性和專一性，並將其應用於ISH和qPCR的方法。
在上述幾種檢測miRNA的方法中，原位雜交技術(ISH)是唯一可以對單個細胞中miRNA表達和定位的方法。我們使用MPTE修飾的探針檢測HCT116細胞系（人結腸癌細胞系）中轉染的外源miR-524-5p和內源miR-21和miR-29a的表達。實驗結果顯示，在MPTE修飾的探針都有相較於未修飾的探針較強的染色訊號，且分別使用一個錯配和三個錯配的探針在不同的雜交溫度下皆可顯示出高的信噪比。
及時定量聚合酶鏈反應（qPCR）是一種已被廣泛用於分析miRNA表達的方法。然而，因同一家族中的miRNA常具有相似的序列或單一核苷酸多態性（SNP）。先前的研究已證實許多疾病的產生與單一核苷酸多態性有關，因不易被一般 DNA 引子或探針所辨識，而導致錯誤的治療與診斷。在本研究中，我們使用miR-29a和miR-29c作為目標序列。使用含有MPTE修飾的DNA作為引物，透過調整不同MPTE修飾數的引物和退火溫度，來增加其單核苷酸多態性的辨識能力。從實驗結果得知，經由MPTE修飾的引子在單一鹼基錯誤配對辨識能力優於一般DNA引子，且我們能透過調控退火溫度來得到MPTE修飾的引子更好的專一性。
基於成功的將MPTE修飾的序列應用在ISH和qPCR方法中並改善引子和探針的專一性及靈敏度，未來可進一步將其應用在GC rich的序列中，預期MPTE修飾的寡核苷酸具有在不同的生物分子檢測平台和疾病治療用藥的潛在能力。

摘要(英)

MicroRNA (miRNA) is a small non-coding RNA molecule, playing an essential role in the expression and regulation of genes. The expression patterns of miRNAs are important to the verification of their predicted function. miRNA levels have been demonstrated to have a strong correlation with disease progression in cancer, which can be considered as diagnostic and prognostic biomarkers. Currently, there are many ways for analyzing miRNA, such as qRT-PCR assays, microarray assays, in situ hybridization(ISH) and the next-generation sequencing(NGS). However, the challenge of detecting specific miRNA and remains
because of its small size, sequence similarity among the miRNA family members. In recent years, many novel nucleotide derivatives have been proposed to gain better signals of sensitivity and specificity such as Locked Nucleic Acid (LNA) and Peptide nucleic acid (PNA).
In this study, we developed DNA containing neutral methyl phosphotriester internucleotide (MPTE) linkages apply in ISH and qPCR.
The use of MPTE modified DNA sequence could reduce electrostatic repulsion effect, and as a consequance, could enhance the duplex formation which can specific and sensitive detection of microRNAs.
In situ hybridization is the only method which can provides insight into both the level and localization in single cell. We used MPTE modified probe for the detection of mimic exogenous miR-524-5p that transfected into HCT116 cell lines (human colon cancer cell lines) and the expression of endogenous miR-21 and miR-29a. We successfully demonstrated improved hybridization efficiency and show the high mismatch discrimination with high signal noise ratio in different temperature.
Quantitative polymerase chain reaction (qPCR) is a powerful method, which has been widely used to quantify the miRNA expression. However, miRNAs in the same family often have overlapping targets or single nucleotide polymorphism(SNP) that are not easy to be discriminated by unmodified DNA primers. we use miR-29a and miR-29c as targets which are both miRNA-29 (miR-29) family share the same mature sequence in vitro, while differ in one nucleotide. Using DNA containing MPTE linkages as primers to enhance the specificity of the SNP discrimination. By adjusting modification number of MPTE modified primer and annealing temperature, we achieve the optimum operating conditions for precise detection of miRNAs.
Depend on the success of applying DNA containing MPTE linkages in ISH and qPCR methods, it could be expected the potential ability of MPTE modified oligonucleotides developing into different biomolecular detection platform and possibly theoretic agent in the future.

關鍵字(中)

★ 微核醣核酸
★ 原位雜交
★ 即時定量聚合酶鏈鎖反應

關鍵字(英)

★ miRNA
★ in situ hybridization
★ qPCR

論文目次

摘要 i
Abstract iii
誌謝 v
圖目錄 x
表目錄 xiii
第一章緒論 1
第二章文獻回顧 3
2.1核酸分子 3
2.1.1 核酸分子介紹 3
2.1.2 核糖核酸 (Ribonucleic acid, RNA) 5
2.1.3 微小核糖核酸 (microRNA) 6
2.1.4 鹼基含量之研究 (GC-content) 7
2.2 核酸類似物 9
2.2.1 鎖核酸 (Locked Nucleic Acid, LNATM) 9
2.2.2 肽核酸 (Peptide Nucleic Acid, PNA) 10
2.3.3 中性去氧核醣核酸 (Phosphate–methylated DNA, nDNA) 12
2.3 單一核苷酸多態性 16
2.4 分子生物檢測平台 17
2.4.1 聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 17
2.4.2 即時定量聚合酶鏈鎖反應 18
2.5 原位雜交技術 22
2.5.1 原位雜交 (In Situ Hybridization, ISH) 22
2.5.2 原位雜交技術訊號放大技術 25
第三章實驗方法與儀器設備 27
3.1 實驗藥品 27
3.1.1 細胞培養 27
3.1.2 序列設計 27
3.1.3 核酸萃取 30
3.1.4 即時聚合酶鏈式反應 30
3.1.5 原位雜交 30
3.2 儀器設備 30
3.3 實驗方法 32
3.3.1 細胞解凍 32
3.3.2 微小核醣核酸萃取 32
3.3.3 即時聚合酶鏈式反應 33
3.3.4 原位雜交細胞培養 34
3.3.5 外源mimic miRNA轉染 34
3.3.6 原位雜交 35
3.3.7 電泳 36
第四章實驗結果與討論 39
4.1 nDNA合成探針檢測細胞中的miRNA之原位雜交方法 39
4.1.1 設計不同修飾數目的nDNA合成探針檢測細胞中的miRNA之原位雜交方法 39
4.1.2 使用nDNA合成探針檢測細胞中的內源性 miR-21、miR-29a原位雜交方法之探針修飾數目探討 43
4.1.3 使用nDNA合成探針檢測細胞中內源性的miRNA之原位雜交方法之專一性探討 47
4.1.4 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 54
4.2 nDNA 修飾引子應用於微小RNA (miRNA)檢測以提升專一性之實驗 57
4.2.1 設計nDNA引子以提升專一性之探討 58
4.2.2 以細胞內萃取之miRNA為模板設計nDNA引子以提升專一性之探討 65
4.3 修飾nDNA在不同鹽濃度下對於GC Rich 序列ΔTm之影響 69
第五章結論 72
第六章參考文獻 74

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指導教授

陳文逸(Wen-Yi Chen)

審核日期

2019-7-25

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