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研究生:黃子芸
研究生(外文):Tzu-Yun Huang
論文名稱:利用單分子技術研究小腦失調症第31型特殊連續TGGAA重複序列結構動態學
論文名稱(外文):Single-molecule Study on the Structural Dynamics of TGGAA Tandem Repeats Associated with Spinocerebellar Ataxia Type 31 Disease
指導教授:侯明宏李以仁
指導教授(外文):Min-Hon HouI-Ren Lee
口試委員:孫英傑
口試委員(外文):Ying-Chieh Sun
口試日期:2016-07-25
學位類別:碩士
校院名稱:國立中興大學
系所名稱:基因體暨生物資訊學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:49
中文關鍵詞:重複序列小腦失調症單分子螢光共振能量轉移
外文關鍵詞:tandem repeatsTGGAASpinocerebellar Ataxiasingle-molecule spectroscopy
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TGGAA五核苷酸重複序列為造成神經退行性疾病脊髓小腦失調症第31型 (Spinocerebellar ataxia type 31, SCA31)的主要致病原因,患者16號染色體q22.1位置上的BEAN和TK2兩基因間會插入一段長度2.5~3.8kb、且包含至少110次重複 TGGAA序列的插入片段,並會於小腦內的浦肯亞神經細胞 (purkinje neurons)形成具細胞毒性的RNA凝聚體,在前人的研究中曾發現發病子代的插入片段比發病親代要來得更長,而隨著該片段長度越長、發病年齡亦越早,其中造成序列增長的可能原因為複製、修復或重組過程中,重複序列因非典型摺疊而滑動。本篇以單分子螢光共振能量轉移光譜觀察TGGAA二到八次重複序列如何摺疊,結果顯示三次以上重複者會形成類髮夾結構,而重複次數為奇數時,會形成頭對尾構型,重複次數為偶數時,會以少一個重複單位參與鍵結的方式形成類髮夾結構;最特別的是在6與8次重複序列中,有觀察到前述兩種構型間轉換的動態行為,分析所得結果後,本篇為TGGAA重複序列於合成時,因滑動而增長提出一可能機制。

Tandem pentanucleotide repeats of TGGAA is a specific sequence that has been shown to be responsible to the neurodegenerative disease Spinocerebellar Ataxia type 31. An insertion of TGGAA repeats with length of 2.5-3.8 kb is found between BEAN (brain expressed, associated with Nedd4) and TK2 (thymidine kinase 2 gene) on chromosome 16q22.1 exclusively in every patient. This insertion contains at least 110 TGGAA repeats and ultimately lead to the production of the toxic RNA foci in Purkinje neuron. The onset age is highly correlated to the length of the insertion and the expansion of this insertion was found in the inherited younger generation patients. This expansion is possibly due to the DNA slippage induced by a non-canonical folded structure during replication, repair, or recombination process. We use single-molecule spectroscopy to study the folding of 2 to 8 repeats of TGGAA sequences. Our result shows that the tandem repeats of TGGAA fold into a hairpin-like structure when the repeat number is greater than 2. Moreover, an end-to-end aligned hairpin-like structure was found in the molecules with odd repeats, while a single-repeat overhang that does not participate in the folding in addition to the hairpin-like structure is found in the case of even repeats. Interestingly, we also observed a dynamic behavior of interconversion of the abovementioned conformations in the molecules with 6 and 8 repeats. We propose a slippage mechanism that may lead to the expansion of the TGGAA repeats during DNA synthesis.

摘要 i
Abstract ii
目錄 iii
圖目錄 v
表目錄 vii
第一章、緒論 1
一、 文獻探討 1
(一) 核苷酸重複序列 1
(二) 脊髓小腦失調症第31型 2
(三) 已解出之TGGAA結構 10
二、 研究動機與目的 13
第二章、實驗儀器與方法 14
一、 實驗技術與儀器 14
(一) 單分子實驗技術 14
(二) 螢光共振能量轉移 15
(三) 全內反射螢光顯微鏡 17
二、 實驗樣品製備與流程 18
(一) 實驗玻片處理 18
(二) 實驗玻片組裝 20
(三) DNA序列 20
(四) 螢光染料Cy3標記 21
(五) DNA黏合反應 (DNA annealing) 23
(六) 令DNA附於微通道樣品槽 23
(七) 顯像緩衝溶液 (imaging buffer) 24
(八) 數據分析與處理 26
第三章、實驗結果 29
一、 實驗設計 29
二、 TGGAA重複序列頭對尾結構模擬 30
三、 TGGAA 2~8次重複序列摺疊情形 31
四、 利用模擬構型確認偶數組重複序列構型 34
五、 (TGGAA)8動態追蹤 35
六、 (TGGAA)6動態追蹤 36
七、 其餘(TGGAA)重複序列軌跡追蹤 39
第四章、討論 41
第五章、未來展望 44
第六章、參考資料 45



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