運用nDNA 修飾引子於PCR及qPCR平台以提升專一性之研究

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姓名

阮翰賢(Han-Xian Ruan) 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

運用nDNA 修飾引子於PCR及qPCR平台以提升專一性之研究
(Enhancing Specificity by nDNA modified primers on PCR and qPCR)

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摘要(中)

本研究經由在引子上修飾nDNA並進行PCR及qPCR實驗，應用nDNA在與互補的DNA雜交時產生靜電排斥力較低之特性，以期能夠達成提升檢測專一性與準確性之結果。
有些疾病的產生，僅是緣於染色體序列的單一鹼基的突變，且時常因含量過低而不易被偵測。因此如何正確的檢驗出突變的序列就變得日益重要。但是在量測單核?酸多態性（Single Nucleotide Polymor- phism，SNP）、微核糖核酸（microRNA，miRNA）表現量等性質時，易有一般之去氧核醣核酸(DNA)常有分辨性不足，而容易產生誤判之性質。本研究以一種DNA之類似物— nDNA並加入引子序列中，以期能達成提升檢測準確性與專一性之效果。
在本文中，我們首先應用nDNA修飾引子於實驗上，以實驗證明nDNA修飾於引子上不會造成酵素無法辨識，接著分別以完全互補及不完全互補的引子，比較當使用一般DNA及nDNA修飾引子進行PCR及qPCR實驗之結果差異。本研究選用之研究對象分別為plasmid (pUC19)、rs4646-c/-a(基因片段)與miRNA反轉錄之cDNA(let-7b/7e)。實驗結果顯示nDNA修飾於引子可以讓完全互補及不完全互補的引子之間的差異為以DNA引子進行同樣實驗的千倍以上。經由上述實驗，我們證明nDNA能夠提升其檢測專一性，並且於較高之接合溫度時能夠提升檢測準確性，我們希望未來能夠運用此特性於更高精確度的疾病檢測或是治療上。

摘要(英)

This research is about using modified DNA (nDNA) primers on polymerase chain reaction (PCR) and quantification polymerase chain reaction (qPCR) experiments. Taking advantage of the property that nDNA-DNA duplex can modify the electrostatic repulsion during the hybridization of two strands, proving nDNA primers can provide higher specificity on detection. Some diseases are caused by single nucleotide mutantion on the sequences, difficult to be discriminated due to the low content. It becomes an important issue to detect these mutants precisely around the world. Technology that can improve the accuracy of detection is what people desired. In this study, nDNA modified primers compare with regular DNA primers, hoping to enhance specificity in detection.
In this research, first, we use nDNA modification around ′ end of primers to verify that these nDNA designs wouldn’t interfere the amplification of templates during PCR. And then we use different kinds of polymerase and different sequences to prove that the results are generally accepted. The result indicated that nDNA modified primers can be amplified generally. Second part, DNA and nDNA applied in enhancing specificity experiments. Comparing the different amplification results between fully complementary (perfect match) primers and partial complementary (mismatched) primers by either nDNA or DNA primers on PCR and qPCR experiments. By annealing temperature gradient method, we found out that nDNA primers have better discrimination between perfect match and mismatched than DNA primers. After ensuring the provement of specificity when using the nDNA primers, we applied nDNA primers on microRNA (miRNA) amplification. miRNA are ~bp RNAs and some of them have only one nucleotide difference, which make them hard to be discriminated. In this part, we choose let-b and let-e as target, comparing the amplification results between perfect match and mismatched primers by either nDNA and DNA primers. The results also show that nDNA primers have better discrimination than DNA primers.
This research proved that nDNA modified primers can improve specificity in PCR and qPCR experiments and have better discrimination in higher annealing temperature. These results can be applied in medical aspect for more credible and for more precise detection.

關鍵字(中)

★ 中性DNA
★ 聚合?鏈鎖反應
★ 即時聚合?鏈鎖反應
★ 專一性

關鍵字(英)

★ nDNA
★ PCR
★ qPCR
★ specificity

論文目次

中文摘要 i
Abstract vii
目錄 viii
圖目錄 xi
表目錄 xiii
1、緒論 1
2、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 去氧核醣核酸結構 3
2.1.3 核醣核酸結構 6
2.1.4 核酸雜交反應 8
2.2 DNA類似物 8
2.2.1 ?核酸 9
2.2.2 鎖核酸(LNA) 10
2.2.3 嗎?基寡核?酸(Morpholino oligomers，MOs) 13
2.2.4 磷酸甲基化核酸 (phosphate-methylated DNA) 13
2.3 生物感測器 16
2.3.1 紫外光/可見光光譜儀 16
2.3.2 聚合?鏈鎖反應 18
2-3-2 逆轉錄聚合?鏈鎖反應 23
2.4 單核?酸多態性 24
2.4.1 錯誤配對辨識能力之研究 25
3、實驗藥品、儀器設備與方法 26
3.1 實驗藥品 26
3.2 儀器設備 28
3.3 實驗方法 28
3.3.1 熔點量測實驗 28
3.3.2 雜交實驗 29
3.3.3 PCR 30
3.3.4 qPCR 33
4、結果與討論 35
4.1 nDNA修飾引子是否能被酵素辨識之測試 35
4.2 nDNA修飾引子提升專一性之實驗 40
4.2.1 Mismatch on primer 40
4.2.2 Mismatch on template 45
4.3 nDNA修飾引子應用於微小RNA(miRNA)檢測以提升專一性之實驗 50
5、結論與未來展望 59
未來展望 59
參考文獻 61
附錄 67

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2017-1-24

推文