臺灣博碩士論文加值系統

DNA 聚合酶被廣泛用於分子生物技術中，其中以溫泉中的嗜熱水生棲熱菌（Thermus aquaticus）所純化的熱穩定Taq DNA聚合酶為最廣泛應用極有價值。在Taq DNA聚合酶結構中，Motif A此為dNTPs結合區域，是影響酵素活性的關鍵位點。本論文研究針對Taq DNA聚合酶Motif A位點中第Ile614及Leu616殘基進行定點飽和突變，突變株的PCR活性與野生株相比，第Ile614殘基有8個突變株活性高於野生株，1個突變株活性相近於野生株，其餘10個突變株活性小於野生株；第Leu616殘基有3個突變株活性高於野生株，1個突變株活性相近於野生株，其餘15個突變株活性小於野生株。在18株較高PCR活性的突變株其複製DNA突變率與野生株相比，其中有10個突變株大於野生株，最大突變率是野生株的23.29倍；8個突變株小於野生株，最小突變率是趨近於零。在生物技術產業中，根據不同需求會使用不同得到各種特性的DNA聚合酶。本篇論文在第Ile614及Leu616殘基飽和突變不同特性突變株可以應用於不同DNA領域，PCR活性高的突變株可用於短時間複製長片段DNA、突變率低的突變株可用於複製出高正確性的DNA片段，突變率高的突變株可用於製備DNA隨機突變基因庫。

DNA polymerase is widely used in molecular biotechnology, among the thermostable Taq DNA polymerase purified by the Thermus aquaticus found in hot springs is extremely valuable for the widest application. In the Taq DNA polymerase structure, Motif A is a dNTPs binding region and the key site affecting enzyme activity. This studies the site-directed saturation mutation of Ile614 or Leu616 residue in the Motif A site of Taq DNA polymerase. The PCR activity of 8 mutants was higher than wild-type, 1 mutant was similar to wild-type, and the other 10 mutants were less than wild-type in the Ile614 residue mutation. The PCR activity of 3 mutants was higher than wild-type, 1 mutant was similar to wild-type, and the other 15 mutants were less than wild-type in the Leu616 residue mutation. The replication DNA mutation rate of the 18 mutants with higher activity compared with wild-type, among 10 mutants were higher than wild-type, and the maximum mutation rate was 23.29-fold higher than wild-type. The 8 mutants were lower than the wild-type, and the minimum mutation rate was close to zero. In the biotechnology industry, according to the different needs of the various features will be used to get different DNA polymerases. In this study, the different characteristics of saturation mutation in the I614 or L616 residue can be applied to different DNA fields. Mutants with high PCR activity can be used to replicate long-length DNA in a short time, mutants with low mutation rate can be used to replicate high correctness of DNA, and mutants with high mutation rates can be used to prepare DNA random mutation gene pools.

目錄
誌謝..........................................................I
目錄..........................................................II
表目錄........................................................IV
圖目錄........................................................V
中文摘要......................................................VI
英文摘要......................................................VII
前言..........................................................1
一、DNA聚合酶..................................................1
二、Taq DNA聚合酶..............................................2
三、DNA重組....................................................5
四、StEP (Staggered extension process，交錯延伸程序)............6
五、研究目的...................................................8
材料與方法.....................................................9
一、實驗材料...................................................9
二、培養基、緩衝液配製..........................................16
三、實驗儀器設備................................................21
四、實驗方法....................................................22
實驗結果.......................................................30
一、Taq DNA聚合酶第I614及L616殘基定點飽和突變質體構築、篩選與鑑定...30
二、Taq DNA聚合酶第I614及L616殘基各突變株之蛋白質表現、純化及定量...31
三、Taq DNA聚合酶第I614及L616殘基各突變株之PCR活性分析............32
四、Taq DNA聚合酶第I614及L616殘基突變株之DNA複製突變率比較.........34
五、最適化StEP方法..............................................36
六、StEP不同方法之置換率.........................................37
七、StEP方法與PCR增幅之突變率....................................37
討論及結論......................................................39
參考文獻........................................................45
表.............................................................49
圖.............................................................56

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29. 網路資料
胺基酸Wikipedia(http://0rz.tw/BiccC)
Taq DNA聚合酶Wikipedia(http://0rz.tw/dojPc)
QuikChange® Site-Directed Mutagenesis (http://0rz.tw/qpxhL)
PCR Wikipedia(http://0rz.tw/q8fKE)
RCS PDB(https://www.rcsb.org/structure/4DLG)
Staggered extension process(StEP) Wikipedia(http://0rz.tw/88key)