利用恆溫滴定微卡計於核酸分子雜交反應之熱力學與機制的研究

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劉安振(An-Chen Liu) 查詢紙本館藏

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論文名稱

利用恆溫滴定微卡計於核酸分子雜交反應之熱力學與機制的研究
(Studies of Thermodynamics and Mechanism of DNA Hybridization by Isothermal Titration Calorimetry)

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摘要(中)

中文摘要
DNA分子雜交反應基本上包含氫鍵作用力、鹼基間堆疊作用力與水合去水合作用。在單股DNA分子主幹上帶負電的磷酸根對DNA分子雜交反應有相當程度的影響，故親疏水作用力及靜電作用力在DNA分子雜交反應上皆扮演著重要角色。在雜交反應過程中兩互補之單股DNA分子在進行雜交前，在單股DNA分子周圍先有一去水合步驟發生，且此步驟可能伴隨著單股DNA分子的結構轉變，由unstructured single strand state轉變到helical(stacked) single strand state，再藉由氫鍵產生與鹼基對堆疊作用形成雙螺旋結構，最後在雙螺旋DNA分子外圍之水合作用主要作用在穩定雙螺旋結構。在整個雜交反應中，單股DNA分子的結構轉變、氫鍵之形成、鹼基對間之堆疊作用與水合作用等均為放熱反應；而去水合步驟則為吸熱反應。各個作用均會對不同DNA雜交條件，如DNA分子長度、雜交反應之環境溫度及鹽濃度等之雜交反應熱造成不同程度的貢獻。
本實驗利用恆溫滴定微卡計(ITC)量測DNA分子在不同溫度、不同DNA分子長度、不同GC%及不同鹽濃度溶液下之雜交交互作用的雜交反應熱(△H，hybridization enthalpy)，藉此可得知DNA分子間交互作用力的大小及溫度鹽濃度等實驗條件對各個作用力的影響，然後再進一步探討雜交反應之反應機制。並期望從雜交熱力學中推導出雜交反應之可能機制與熱力學經驗關係式，以提供DNA生物晶片反應較佳之條件。並檢討可否利用DNA雜交熱力學之量測以偵測SNP(Single Nucleotide Polymorphism)問題之可能性！
文獻中的實驗大都利用微分掃描卡計及紫外線光譜儀掃描一溫度範圍(大都在283K~383K)來測量雜交反應熱，其結果均為放熱；而本研究之恆溫滴定微卡計的實驗均在一恆溫條件下進行，實驗結果大多為吸熱反應，造成此不同之實驗結果可能是由於文獻中之實驗溫度不斷改變可能造成DNA分子之結構改變，此由heat capacity的改變即可證明，說明了單股DNA分子之結構於不同溫度下之改變及去水合作用影響之重要性。另外造成不同實驗結果原因可能在於使用的緩衝溶液不同。在DNA分子雜交過程中若只簡單地視為去水合(dehyhration)及雜交(hybridization)兩大步驟，而由能量觀點來說明，去水合步驟牽涉到水分子的亂度改變，為吸熱反應；雜交步驟主要為氫鍵的鍵結，為放熱反應，但另外鹼基堆疊作用則主要為entropy之貢獻為主。本研究之結果應可推測去水合步驟對雜交反應有決定性的影響，此結果可提供DNA晶片業者於雜交反應之環境條件上之理論基礎。此外，本實驗也對DNA分子長度、溶液鹽濃度、實驗溫度與GC%對雜交反應熱會造成不同程度的貢獻做討論。

摘要(英)

Abstract
DNA hybridization reaction includes ssDNA structural arrangement hydrogen bond interaction, stacking interaction between base pairs, hydration step and dehydration step. The DNA hybridization reaction is mainly affected by the negatively changed phosphate group in DNA backbone. Hydrophobic interaction between the base pairs intermolecule and intramolecule of ssDNA and electrostatic interaction play important roles in DNA hybridization reaction. Specifically, before DNA hybridization, single strand DNA molecules proceeded conformational transform from unstructured single state to helical single strand state and dehydration step. Then, double helical structure was form due to hydrogen bond interaction, stacking interaction between base pairs and hydration step. Conformational transform of single strand DNA, hydrogen bond, stacking between base pairs and hydration step are exothermic and dehydration step is endothermic, basically. Therefore, DNA hybridization enthalpy is affected to different extent by the above mentioned steps.
In this study, we measured the DNA hybridization enthalpy by isothermal titration calorimetry at different temperature, DNA length, GC% and salt concentration to understand DNA molecules interaction and effects of different experiment conditions and to discuss the DNA hybridization mechanism. An experimental equation of hybridization enthalpy as function of the reaction parameters was proposed in our study.
Our results demonstrated that the hybridization enthalpy is exothermic. The dehydration step plays a profound role in DNA hybridization process. It also provided theoretical basis that to select hybridization conditions for application of biochip. Furthermore we indicated the difference of hybridization enthalpy in various DNA length and between perfect match and mismatch can be differentiated in a proper reaction condition. Therefore, we may possibly detect the SNP problem by isothermal titration calorimetry measurement.

關鍵字(中)

★ 恆溫滴定微卡計
★ 核酸

關鍵字(英)

★ isothermal titration calorimetry
★ DNA hybridization

論文目次

目錄
中文摘要.....................................................................................................I
Abstract.....................................................................................................III
目錄..........................................................................................................IV
圖目錄.....................................................................................................VII
表目錄......................................................................................................XI
第一章緒論(introduction)......................................................................1
第二章實驗原理與文獻回顧................................................................3
2.1 核酸介紹...........................................................................................3
2.1.1 核酸之研究歷史....................................................................3
2.1.2 去氧核醣核酸在遺傳上的作用............................................7
2.1.3 聚核苷酸的共價結構............................................................8
2.1.4 去氧核醣核酸(DNA)的結構...............................................13
2.1.5 去氧核醣核酸(DNA)的變性...............................................20
2.2 去氧核醣核酸分子的水合、去水合作用與雜交反應...................24
2.3 單一核酸多型性(single nucleotide polymorphism).......................29
2.4 恆溫滴定微卡計.............................................................................32
2.4.1 卡計之基本介紹..................................................................32
2.4.2 微分熱掃描卡計與恆溫滴定微卡計之差異......................32
2.4.3 恆溫滴定微卡計之基本介紹..............................................33
2.5 卡計與紫外線光譜儀於DNA雜交反應之研究...........................38
第三章實驗藥品與步驟......................................................................41
3.1 實驗藥品.........................................................................................41
3.2 實驗儀器設備.................................................................................43
3.3 實驗步驟.........................................................................................43
3.3.1 溶液配製..............................................................................43
3.3.1.1 SSC緩衝溶液................................................................43
3.3.1.2 SDS溶液.......................................................................44
3.3.1.3 互補之單股DNA溶液.................................................44
3.3.2 恆溫滴定微卡計之操作步驟..............................................44
3.4 實驗量測.........................................................................................46
3.4.1 雜交反應熱實驗..................................................................46
3.4.2 單股DNA分子稀釋熱實驗................................................47
3.4.2.1 Single Target(ST) DNA分子稀釋熱實驗....................47
3.4.2.2 Single Probe(SP) DNA分子稀釋熱實驗.....................47
第四章結果與討論..............................................................................48
4.1 改變不同鹽濃度與溫度實驗.........................................................48
4.2 改變不同GC %實驗.......................................................................63
4.3 改變不同DNA分子長度實驗.......................................................67
4.4 One-mismatch及Two-mismatch實驗............................................74
4.5 熱力學經驗式推導.........................................................................79
第五章結論與建議..............................................................................85
5.1 結論.................................................................................................85
5.2 建議.................................................................................................86
第六章參考文獻..................................................................................87
附錄..........................................................................................................94

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指導教授

陳文逸(Wen-Yih Chen)

審核日期

2003-7-9

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