臺灣博碩士論文加值系統

中文摘要
本研究係利用商業陰離子交換薄膜與樹脂純化質體核酸。藉由純化前處理將高分子量的核醣核酸移除，再藉由改變清洗液中醇類濃度探討其對分離質體核酸的影響。
在批次研究中發現，提高醇類濃度有助於質體核酸回收率的提高，且增加異丙醇濃度至45%時有最佳質體核酸回收率與純度。
以陰離子交換薄膜純化質體核酸，在利用鈣離子作純化前處理之後，以1.5M氯化鈉40%異丙醇當清洗液，可以移除核醣核酸與蛋白質，得到純度100%的質體核酸，回收率約在43±3%。
以陰離子交換樹脂純化質體核酸，則利用1.0M胍基鹽酸與50%異丙醇作純化前處理。在批次小量純化程序，利用1.0M氯化鈉與40%異丙醇當清洗液，可純化得到純度100%的質體核酸，其回收率近乎100%。而以重力流方式純化質體核酸，固定1.5M氯化鈉並以降低異丙醇濃度階梯梯度來作為純化條件，可純化不同大小質體核酸，得到純度100%質體核酸。重力流純化7.5毫升菌液時，4.7kb質體核酸與8.0kb質體核酸回收率分別為68.5±3%與72±5%。在純化300毫升菌液時，4.7kb質體核酸與8.0kb質體核酸回收率則分別為75±6%與50±3%。在純化3公升菌液時，4.7kb質體核酸與8.0kb質體核酸回收率則分別為70%與20%。
本研究利用商業薄膜與樹脂純化質體核酸，在清洗液中加入異丙醇可得到高純度高回收率質體核酸。且回收再生的陰離子交換樹脂，可以再用於純化質體核酸且不會影響其純度。

英文摘要
In this study, commercial anion exchange membrane and resin were used to purify plasmid DNA. After the removal of high molecular RNA in a pretreatment procedure, various alcohol concentrations in the washing buffer were examined for purification of plasmid DNA.
In batch experiments, we found that the increase of alcohol concentrations can enhance plasmid recovery and that the buffer containing 45% isopropanol had optimal purity and recovery.
When anion exchange membrane was used for purification of plasmid DNA, calcium was added in the pretreatment to remove high molecular weight RNA. The remaining RNA and protein can be removed by using a washing buffer containing 40% isopropanol and 1.5M NaCl. The purified plasmid DNA has a purity 100% and a recovery of 43±3%.
When anion exchange resin was used for purification of plasmid DNA, 1M guanidine-Cl and 50% isopropanol were added in the pretreatment to remove high molecular weight RNA. In small scale of purification process, a washing buffer containing 1M NaCl and 40% isopropanol was used. The obtained plasmid DNA has 100% purity and a recovery close to 100%. For plasmid purification by gravity-flow, the purification condition employed buffers containing 1.5M NaCl with step-down isopropanol concentrations. The plasmid DNA of different sizes obtained by this condition had 100% purity. When the gravity-flow method was used to purify 7.5 milliliter of fermentation broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 68.5±3% and 72±5%, respectively. When purification process was scaled up to 300 milliliter broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 75±6% and 50±3%, respectively. When purification process was scaled up to 3 liter broth, the recovery of 4.7kb and 8.0kb plasmid DNAs were 70% and 20%, respectively.
In conclusion, the use of commercial anion exchange membrane and resin coupled with washing buffer of various isopropanol concentrations can be used to obtain plasmid DNA of high purity and high recovery. Additionally, the use of regenerated anion exchange resin will not affect the purity of the purified plasmid DNA.

目 錄
中文摘要 I
英文摘要 II
目 錄 IV
圖目錄 VIII
表目錄 XI
第一章 緒論 1
1-1前言 1
1-2 研究動機與目的 2
第二章 文獻回顧與原理 3
2-1基因治療與核酸疫苗 3
2-2質體核酸純化的要求 3
2-3純化質體核酸的步驟 4
2-3-1微生物培養 4
2-3-2細胞的回收 4
2-3-3細胞溶離 5
2-3-4 上清液的收集與純化前處理 5
2-3-5 質體核酸的純化 6
2-3-5-1陰離子交換層析法 6
2-3-5-2膠體層析法 7
2-3-5-3疏水性層析法 8
2-3-5-4組合式純化法 8
2-3-6 測量方法 9
2-3-6-1質體核酸、核醣核酸定量 9
2-3-6-2蛋白質定量 10
第三章 實 驗 11
3-1 實驗物質 11
3-1-1欲純化質體核酸(pEPFG-C1、pCMV SPORT-βgal) 11
3-2實驗藥品與儀器 12
3-3 實驗方法 14
3-3-1藥品的配法 14
3-3-2大腸桿菌轉形 16
3-3-3微生物培養 16
3-3-3-1搖瓶培養 16
3-3-3-2發酵槽培養 17
3-3-4細胞溶離 18
3-3-5純化前處理 18
3-3-5-1薄膜純化前處理 18
3-3-5-2離子交換樹脂純化前處理 19
3-3-6離子交換薄膜純化 19
3-3-6-1批次（batch） 19
3-3-6-2流動（flow） 20
3-3-7陰離子交換樹脂純化 20
3-3-7-1批次（batch） 20
3-3-7-2流動（flow） 21
3-3-7-3重力流動（gravity flow） 22
3-3-8 Q Sepharose再生 22
3-3-9樣品處理 23
3-3-10瓊脂凝膠分析法 23
3-3-11分析方法 23
3-3-11-1質體核酸測量 23
3-3-11-2核醣核酸測量 24
3-3-11-3蛋白質測量 24
第四章 結果與討論 25
4-1不純物對量測標準曲線的影響 25
4-1-1質體核酸的測量 25
4-1-2蛋白質的量測 25
4-2純化前處理的探討 26
4-2-1陰離子交換薄膜純化前處理 26
4-2-2陰離子交換樹脂純化前處理 26
4-3陰離子交換薄膜的探討 27
4-3-1探討陰離子交換薄膜純化質體核酸的條件 27
4-3-2以批次方式探討不同濃度異丙醇對質體核酸與核醣核酸分離的影響(固定氯化鈉濃度為1M) 28
4-3-3不同濃度的醇類在批次操作對質體核酸回收率的影響 29
4-3-3-1清洗液中氯化鈉濃度為1.0M 29
4-3-3-2清洗液Gu-Cl濃度為2.0M 30
4-3-4流動純化質體核酸條件的探討 31
4-3-5薄膜質量平衡的探討 32
4-3-5-1溫度與轉速的探討 32
4-3-5-2洗脫液pH對脫附的影響 33
4-3-6純化質體核酸 33
4-4陰離子交換樹脂對質體核酸純化的探討 34
4-4-1以批次條件探討陰離子交換樹脂純化質體核酸 34
4-4-1-1以批次方式探討不同濃度的異丙醇對質體核酸與核醣核酸分離的影響(固定氯化鈉濃度為1.0M) 34
4-4-1-2不同醇類濃度對Q Sepharose純化質體核酸的影響 35
4-4-1-3不同溶劑濃度對DEAE Sepharose純化質體核酸的影響 36
4-4-2以批次最佳條件利用流動純化質體核酸 38
4-4-3探討流動階梯梯度純化質體核酸條件 39
4-4-4重力流純化不同的質體核酸 40
第五章 結 論 43
參 考 文 獻 45
圖目錄
圖 2.1 製備質體核酸步驟 4
圖 3.1 β-gal質體圖譜 11
圖 3.2 pEPFG-C1質體圖譜 11
圖3.3本研究所用圓片型薄膜分離器之正視及側視圖 51
圖4.1質體核酸校正曲線 52
圖4.2含蛋白質與核醣核酸時質體核酸測量校正曲線 53
圖4.3蛋白質標準校正曲線 54
圖4.4含質體核酸與核醣核酸時測量蛋白質校正曲線 55
圖4.5薄膜對核酸吸附與脫附曲線 56
圖4.6以階梯梯度流動方式利用陰離子交換薄膜純化質體核酸 57
圖4.7以批次方式探討薄膜在不同濃度的異丙醇對質體核酸與核醣核酸分離的影響 58
圖4.8以批次探討薄膜在不同濃度的溶劑對質體核酸回收率的影響(固定氯化鈉濃度為1.0M) 59
圖4.9在氯化鈉為1.0M情況下，以批次方式探討薄膜在不同濃度的溶劑對質體核酸與核醣核酸分離的影響 60
圖4.10以批次探討薄膜在不同濃度的溶劑對質體核酸回收率的影響(固定Gu-Cl濃度為2.0M) 61
圖4.11在Gu-Cl 2.0M情況下，以批次方式探討薄膜在不同濃度的溶劑對質體核酸與核醣核酸分離的影響 62
圖4.12利用薄膜在批次最佳條件以流動方式來純化質體核酸 63
圖4.13薄膜在流動方式提昇NaCl濃度(固定1.5M NaCl)並改變不同濃度異丙醇對質體核酸純化的影響 64
圖4.14不同pH值洗脫（Elute）液對質體核酸純化的影響 65
圖4.15流動方式利用薄膜純化質體核酸 66
圖4.16M以批次方式探討不同濃度的異丙醇對質體核酸與核醣核酸分離的影響 67
圖4.17 Q Sepharose以批次探討不同濃度的溶劑對質體核酸回收率的影響(固定氯化鈉濃度為1.0M) 68
圖4.18 Q Sepharose以批次探討不同濃度的溶劑對質體核酸純度的影響(固定氯化鈉濃度為1.0M) 69
圖4.19 Q Sepharose以批次探討不同濃度的甲醇對質體核酸純化的影響(固定氯化鈉濃度為1.0M) 70
圖4.20 Q Sepharose以批次探討不同濃度的乙醇對質體核酸純化的影響(固定氯化鈉濃度為1.0M) 71
圖4.21 Q Sepharose以批次探討不同濃度的異丙醇對質體核酸純化的影響(固定氯化鈉濃度為1.0M) 72
圖4.22 DEAE Sepharose以批次探討不同濃度的溶劑對質體核酸回收率的影響(固定氯化鈉濃度為1.0M) 73
圖4.23 DEAE Sepharose以批次探討不同濃度的溶劑對質體核酸純度的影響(固定氯化鈉濃度為1.0M) 74
圖4.24 DEAE Sepharose以批次探討不同濃度的甲醇對質體核酸純化的影響(固定氯化鈉濃度為1.0M) 75
圖4.25 DEAE Sepharose以批次探討不同濃度的乙醇對質體核酸純化的影響(固定氯化鈉濃度為1.0M) 76
圖4.26 DEAE Sepharose以批次探討不同濃度的異丙醇對質體核酸純化的影響(固定氯化鈉濃度為1.0M) 77
圖4.27以批次最佳條件利用流動與重力流純化質體核酸 78
圖4.28固定鹽類濃度、降低iPrOH濃度利用重力流純化質體核酸 79
圖4.29重力流最佳條件純化小量質體核酸pEGFP-C1與β-gal 80
圖4.30重力流純化中量pEGFP-C1質體核酸 81
圖4.31重力流純化中量β-gal質體核酸 82
圖4.32重力流純化大量pEGFP-C1質體核酸 83
圖4.33重力流純化大量β-gal質體核酸 84
表目錄
表2.1 質體核酸的純度 3
表3.1 使用之藥品 12
表3.2 使用之儀器 13
表3.3各種清洗液的配製 85
表4.1 本研究使用薄膜與樹脂之性質與價格 86
表4.2不同溶劑濃度在水相中的dielectric constant 87
表4.3薄膜流動純化結果 88
表4.4重力流純化4.7kb質體核酸 89
表4.5重力流純化8.0kb質體核酸 90
表5.1各種純化質體核酸方法的比較 91

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