臺灣博碩士論文加值系統

Thermus thermophilus 的DNA 聚合酶屬於一種可以DNA 為模板複製DNA 的熱穩定聚合酶，
當Mn2+取代Mg2+的存在之下，Tth DNA 聚合酶則同時具有反轉錄酶的活性。正因為Tth DNA 聚合酶有熱穩定的特性以及兩種活性功能，因此常常被用在PCR 及RT-PCR 之中。但是其在PCR 的應用中並不像Taq DNA 聚合酶來得多，且在cDNA 的反轉錄中也不較MMLV RT 來得廣，最主要的原因在於目前對於Tth DNA 聚合酶尚未完全了解其活性功能，以及反應的環境也尚未最佳化。RecA 在重組作用（單股DNA 鍵結、鍊交換、ATP 水解活性）及DNA 修補作用（單股DNA
鍵結、輔蛋白水解酶活性）中扮演重要的角色。SSB 因其具單股DNA 鍵結活性，因此在DNA 的
複製、RNA 的反轉錄以及DNA 的修補上是非常重要的。DnaA 是一種複製起始因子，在原核生物的DNA 複製時，可以在OriC（複製起始點）上驅動DNA 的解螺旋或變性。DnaB 與UvrD 因各具有解螺旋酶的活性，在DNA 複製的起始與修補中是很重要的存在。在本研究中這些基因都是由Thermus thermophilus HB8 的基因體中以PCR 擴增而得。將polA 基因克隆至pUC18 載體並轉型至大腸桿菌XL10-gold 中；另外，dnaA、dnaB、recA、ssb、uvrD 基因則是克隆至pET-22b 載體並轉型至大腸桿菌Rosetta 2 中。各自以1 公升培養量以IPTG 誘導蛋白質表達，並通過鈷離子樹脂後以3-30 mM imidazole 作純化。純化後的Tth DNA 聚合酶共有240,000 U 並稀釋成5 U/μl，而純化後的DnaA、DnaB、RecA、SSB、UvrD 則分別得到3.85 mg、1.6 mg、4.49 mg、3.47 mg、3.7 mg 並具有50,000 U/mg、100,000 U/mg、50,000 U/mg、50,000 U/mg、100,000 U/mg 的活性。我們運用兩種解螺旋酶（DnaB 與UvrD）、SSB 與Tth DNA 聚合酶進行等溫HDA 反應以擴增人類基因體DNA及質體DNA。另外也發展一種等溫RT-HDA 反應以擴增人類RNA。並以real-time PCR 分析的方式用以證明HDA 與RT-HDA 的專一性產物生成。目前在HDA 的敏感度為105 個DNA，而RT-HDA則是106 個RNA。當DnaA 加入質體DNA 的HDA 反應時，其產率可增加1.2 倍，對照RecA 則在DnaB 所進行的HDA 反應可增加產率約2.2 倍，但在UvrD 中則沒有任何效果。綜合以上結果，我們目前已成功利用Tth 聚合酶及其核酸相關蛋白，發展出一項等溫核酸擴增技術。

The thermostable DNA polymerase from Thermus thermophilus (Tth) is a DNA-dependent DNA polymerase. Tth DNA polymerase also possesses reverse transcriptase activity while Mn2+ instead of Mg2+ is present. Because of its thermal stable property and dual activity functions, Tth DNA polymerase has been used in PCR and RT-PCR. Nonetheless, the amplification yield of Tth DNA polymerase in PCR is not as high as Taq DNA polymerase and the utility in cDNA synthesis is not as well accepted as MMLV RT. The reasons are partly due to not fully understand its activities and lacking of conditions optimization. Recombinase A (RecA) is known important in recombination (single-stranded DNA binding, strand exchange, ATP hydrolysis activities) and in DNA repair (single-stranded DNA binding and coprotease activities). SSB with the single-stranded DNA binding activity is important in DNA replication, RNA transcription, and DNA repair. DnaA is a replication initiation factor which promotes the unwinding or denaturation of DNA at oriC, during DNA replication in prokaryotes. DnaB and UvrD with helicase activity are important in initiation of DNA replication. In this study, these genes are amplified from Thermus thermophilus HB8 genome by PCR. The polA gene was cloned into the pUC18 vector and transformed into the E. coli XL10-gold, whereas the dnaA, dnaB, recA, ssb, uvrD genes were cloned into the pET-22b vector and transformed into the E. coli Rosetta 2. The Tth DNA polymerase was expressed in XL10-gold and DnaA, DnaB, RecA, SSB, UvrD were expressed in Rosetta 2 with the induction of IPTG by a scale of 1 liter. These proteins were purified by the cobalt resin column in the presence of 3-30 mM imidazole. The purified Tth DNA polymerase with a total activity of 240,000 U was finally diluted to 5 U/μl. These purified DnaA, DnaB, RecA, SSB, UvrD have total amounts of 3.85 mg, 1.6 mg, 4.49 mg, 3.47 mg, and 3.7 mg with the activities of 50,000 U/mg, 100,000 U/mg, 50,000 U/mg, 50,000 U/mg, and 100,000 U/mg, respectively. We used two helicases (DnaB and UvrD), SSB, and Tth DNA polymerase to carry out an isothermal helicase-dependent amplification (HDA) reaction to amplify both the human genomic DNA and plasmid DNA. In addition, an isothermal RT-HDA reaction was also developed to amplify the human RNA. The specific products generated by HDA or RT-HDA can be demonstrated by real-time PCR assay. Currently, the sensitivities of HDA and RT-HDA were 105 copies on DNA template and 106 copies on RNA template respectively. When DnaA was added into the reaction of HDA on plasmid DNA template, the yield was increased by 1.2-fold. In HDA, the RecA increased the yield of the DnaB mediated HDA by 2.2-fold, but had no effect on the UvrD mediated HDA. Taken together, we have successfully developed an isothermal nucleic acid amplification by using Tth DNA polymerase and related nucleic acid interaction proteins.

Abstract….………………………………………………………………………………………………...Ⅰ
中文摘要………………………………………………………...…………………………………...….. Ⅲ
目錄………………………………………………………………………………………………….……Ⅳ
圖表目錄………………………………………………………………………………………………….Ⅶ
中英文名詞對照………………………………………………………………………………………….Ⅷ
前言………………………………………………………………………………………………………1
實驗材料…………………………………………………………………………………………………..5
一、藥品與試劑……………………………………………………………………………………….5
二、儀器設備…………………………………………………………………………………………..8
實驗方法………………………………………………………………………………………………..9
一、目標基因（target gene）的克隆…………………………..…………………………………….9
二、蛋白質的表達與純化………..………………………………………………………………….11
三、蛋白質濃度的測定………………………………………………………………………………14
四、蛋白質活性分析…………………………………..…………………………………………….14
五、RecA 與SSB 對PCR 的影響…………………………………………………………..………16
六、等溫擴增（Isothermal amplification）………………………………………………………….17
結果……………………………………………………………………………………………………..20
一、目標基因（target gene）的克隆結果……………………………………………………………20
二、蛋白的表達與純化結果…………………………………………………………………………20
三、Tth DNA polymerase 活性分析結果………..…………………………………………………..20
四、RecA 活性分析結果………………………………………………………………………………21
五、SSB 活性分析結果……………………………………………….……….…………………….21
六、解螺旋�﹛]DnaB、UvrD）活性分析結果……………………………………………………..21
七、RecA 與SSB 對於PCR 所造成的影響結果…………………………………………………..22
八、HDA（helicase-dependent amplification）的結果…….…..………….………….…..………..22
九、RT-HDA（reverse transcription helicase-dependent amplification）的結果.………………….22
十、DnaA 參與HDA 擴增質體DNA 的結果.………………………………………..…………….23
十一、HDA 與RT-HDA 反應敏感度結果......….….…..….….……..…...…...........................….…..23
結論……………………………………………………………………………………………………..24
討論……………………………………………………………………………………………………..25
參考文獻…………………………………………………………………………………………………28
實驗圖表………………………………………………………………………………………………….31
附錄……………………………………………………………………………………………………..55
附錄一、NASBA（nucleic acid sequence-based amplification）反應示意圖.……………………..55
附錄二、SDA（strand displacement amplification）反應示意圖…………………………………..56
附錄三、RCA（rolling circle amplification）反應示意圖…………………………………………57
附錄四、LAMP（loop-mediated isothermal amplification of DNA）反應示意圖…………………58
附錄五、HDA（helicase-dependent amplification）反應示意圖…………………………….……59
附錄六、SPIA（single primer isothermal amplification）………………………………………….60
附錄七、Qβ RNA amplification 偵測反應示意圖………………………………………….………..61
附錄八、Branched DNA signal amplification 偵測病毒反應示意圖.…………………….………..62
附錄九、pET 系統下的調控機制…….…………………………………………………….………..63
附錄十、pRARE 質體的圖譜…………………….………………………………………………….64
附錄十一、pyT&A 載體的圖譜……….……….…………….…………………………………..…65
附錄十二、Taq DNA 聚合�﹛BE. coli DnaA 結構示意圖…………………..……..……………….66
附錄十三、Taq DnaB、E. coli SSB 結構示意圖…………………………………………………….67
附錄十四、E. coli RecA、E. coli UvrD 結構示意圖………………………….……………………68
附錄十五、Tth DNA 聚合�※穧]及胺基酸序列……………………………………………………69
附錄十六、Tth DnaA 基因及胺基酸序列…………….……………………………………….……70
附錄十七、Tth DnaB 基因及胺基酸序列…………....………………………….………………….71
附錄十八、Tth RecA 基因及胺基酸序列..………………………..……………………….………..72
附錄十九、Tth SSB 基因及胺基酸序列…………………………………………………………….73
附錄二十、Tth UvrD 基因及胺基酸序列…………………………………..……………………….74
附錄二十一、Tth MutS 基因及胺基酸序列…………………………………..…………………….75
附錄二十二、Taq DNA 聚合�※穧]序列及胺基酸序列…………………..……………………….76
附錄二十三、pUC18 載體OriC 序列……………………………………………………………….77
附錄二十四、RT-HDA 所使用引子於DNA 及RNA 模板擴增的序列…………………………….77
附錄二十五、所有clones 的列表………………………………………….………...……………….78

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