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論文中文名稱:複製中鏈黴菌DNA末端缺口序列之研究 [以論文名稱查詢館藏系統]
論文英文名稱:To identify the gap junction of replication intermediate of Streptomyces telomeres [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:99
出版年度:100
中文姓名:黃譯慧
英文姓名:Yi-Huei Huang
研究生學號:97688012
學位類別:碩士
語文別:中文
口試日期:2011-01-28
論文頁數:57
指導教授中文名:黃志宏
口試委員中文名:陳文盛;楊千金
中文關鍵詞:鏈黴菌複製中間物末端缺口序列S .lividansSCP1
英文關鍵詞:Streptomycesreplicating intermediatethe gap junction sequenceS.lividansSCP1
論文中文摘要:鏈黴菌線形染色體及線形質體的複製起始點都是由中間區域分別從左右兩邊朝末端進行。 複製過程中當最後一個引子被移除時造成延緩股(lagging strand)3’端突出(overhangs)形成單股間隙,單股DNA會折疊成二級結構,末端伴隨蛋白(TAP)會辨認特定二級結構與末端蛋白(TP)產生交互作用,將其帶往末端作為引子,鏈黴菌利用突出的單股DNA作為模版由DNA聚合酶及蛋白質補齊完整的長度。先前研究中發現,S. rochei菌株中pSLA2複製到末端所產生缺口大小為280個核乾酸。因此我們推測末端上有一特定序列可被引子酶辨認,使得每次複製缺口都是280個鹼基,若能確定此序列,便能與接下來的複製機制做連接更進一步了解鏈黴菌的複製機轉。
  我所使用的pLUS890L被構築帶有S. lividans及SCP1兩種不同類型的端粒。以此線型質體來檢測不同端粒的gap大小,由南方墨漬反應在同一個樣本下分析兩端端粒, 結果顯示,S. lividans端粒有出現複製中間產物的訊號;在SCP1端粒上似乎沒有疑似複製中間產物的訊號。並且我們也推論3’端上的單股間隙應是最後一個崗崎片段的位置,所以在此序列附近應會有引子酶辨認序列。所以我們想在活體內證實鏈黴菌中不同系統DNA端粒都有一特定序列能讓引子酶辨識,藉此線型質體來找尋缺口序列。我測試一個全新的方法,成功找出S. lividans染色體端粒的末端缺口序列。
論文英文摘要:Replication of the Streptomyces linear chromosome and linear plasmid is initiated from an internal origin. Bidirectionnally towards to the telomeres. The 3’end overhangs were formed by removal of the last primer at gap junction during replication, this single-strand DNA will fold into a secondary structure and recognized by terminal associated protein (TAP). TAP interact with terminal protein (TP), leading TP to the 3’ overhang ends as primer and patches single-strand gaps. In previous study, we had discovered that the gap size of S.rochei plasmid SLA2 is 280 base pairs. Thus, we hypothesized that there is a specific primase recognition site at 3’ end. The mechanism of Streptomyces replication can be further reveal if we could locate this specific sequence.
In this study, two different telomeres, S.lividans and SCP1 were constructed into a linear plasmid pLUS890. I used this plasmid to verify the gap junction size of different telomeres by southern blot detecting the telomeres of both ends. We found the signal of replication intermediate of S.lividans telomere but not SCP1 telomere. We also raised a hypothesis that the 3’ overhang should be the last okazaki fragment and the primase recognition sequence supposed to be somewhere near by. We want to prove that the primase recognition sequence is unique in different telomere systems in Streptomyces. By using linear plasmid, I had achieved a new approach and successfully identified the gap junction sequence of S. lividans telomere.
論文目次:目 錄

中文摘要……………………………………………….………………...……………….…ii
英文摘要…………………………………………………………………………iii
誌謝…………………………………………………………………………….….v
目錄………………………………………………………………………………...vi
圖目錄……………………………………………………………………………..viii
第一章 緒論…………………………………………………….....................…1
1.1 鏈黴菌簡介…………………………………………………………1
1.2 鏈黴菌線形DNA分子的末端複製.......……………….............……3
1.3 鏈黴菌的兩大類型端粒系統…………..…………………………….5
1.4 引子酶辨認序列 ……………………………………………….........7
1.5 本論文內容……………………………………….…………………..9
第二章 材料與方法………………………………………………………...……...10
2.1 菌種與質體…………………………………………………………..12
2.2 此篇論文所使用的引子序列 ….....….12
2.3培養基及緩衝液……………………………………………………….…12
2.4藥品酵素………………………………………………………….….12
2.5大腸桿菌質體之分離與純化…………………………………………12
2.6純化鏈黴菌total DNA…..………………………………………………12
2.7大腸桿菌的轉型………………………….……………………………13
2.8菌種儲存…………………………………………………….…..……13
2.9 南方墨漬法………………………………………………………….……13
2.10限制酶及連接酶的處理……………………………………...…...…….13
2.11聚合酶連鎖反應PCR(polymerase chain reaction)……….…………13
2.12純化正在複製之DNA…………………………………………………14
2.13檢測鏈黴菌生長曲線…………………………………………………16
2.14 End junction序列檢測方法.……………..………………………………17
第三章 實驗結果…………………………………………………..………………18
3.1 找尋引子酶辯識序列之策略…………...…………………...……18
3.2 端粒DNA複製中間產物之純化…………………………………20
3.3 Gap junction序列分析結果………………………..…..………………23
3.4檢測不同末端系統的Gap junction序列……………………………35
第四章 討論…………………………………………………………………..……39
參考文獻………………………………………………………………………...……43
附錄
Appendix A Media and buffer …….….45
Appendix B Isolation the total DNA of Streptomyces …………..48
Appendix C Plasmid isolation from E. coli ………………..49
Appendix D E.coli Competent cell preparation and transformation…………50
Appendix E Southern hybridization. …………………………………………53
Appendix F. Polymerase Chain Reaction…………………………………….57


圖目錄

圖1.1.1 數種鏈黴菌在固態培養基上生長情形…………...…………………………2
圖1.1.2 鏈黴菌生長過程……..………………………………………………………2
圖1.2 腺病毒複製機轉……...………………………………………………………4
圖 3.1.1端粒複製中間產物上gap junction的定序策略……………..……………19
圖 3.2.1 pLUS970L的端粒複製中間產物之偵測……………………...……………21
圖 3.2.2 TK64(pLUS970L)的生長曲線圖……………………………..……………22
圖 3.3.1 TK64(pLUS890L)的兩端端粒複製中間產物之偵測………..……………24
圖 3.3.2用pLUS970上的DNA片段測試PCR的實驗策略………...………………26
圖 3.3.3用pLUS970上DNA片段測試T4 DNA polymerase黏滑端切口修補作用…………………………………………………….……………………...………………28
圖 3.3.4將pLUS890L上的S. lividans染色體末端DNA複製中間產物,加入RNase H作用後的PCR結果……………………………………………………………………………29
圖 3.3.5以限制酶切割,確認TA cloning vector和第一次PCR的產物連接後所構築成的候選轉型質體…………………………………………………..…………………………....31
圖 3.3.6以限制酶切割,確認TA cloning vector和第二次PCR的產物連接後所構築成的候選轉型質體………………………………….………………….………………………...32
圖 3.3.7以限制酶切割,確認 TA cloning vector和第三次PCR的產物連接後所構築成的候選轉型質體…………………………………………………………..…….………………33
圖 3.3.8 Gap junction的序列分析…………………….……………….……………………34
圖3.4.1 3200(pLUS890L)的不同端粒系統的複製中間產物之偵測……………………………………………………………………...………..………..…37
圖 3.4.2 PI-VL的兩端端粒複製中間產物之偵測…………………….............…...……….38
圖 3.4.2 模擬DNA片段進行blunt ends ligation………………………….…………..……41
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論文全文使用權限:同意授權於2013-02-22起公開