臺灣博碩士論文加值系統

實現個人化醫療是現代醫療的新趨勢。配合基因資訊的分析，醫療人員能夠更精準地給予不同病患合適的療程。倘若倚靠基因晶片並配合大型基因資料庫，在臨床上便能快速且精確地得到病患特定表現的基因，可以在診斷時，可以更快地提供相關的基因資訊。目前的基因晶片仍舊有相當多問題，螢光標定的不穩定性、價格昂貴，基因晶片靈敏度及準確度不夠……等需要解決。透過矽奈米線場效電晶體(SiNWFET)其高靈敏度、免標定、及時偵測等特性作為新一代檢測DNA序列雜交的平台具有相當大的潛力。然而，場效電晶體在檢測DNA序列雜交的電訊號時，易受到環境鹽離子濃度影響造成檢測上的困難。根據本實驗室過去的研究，將DNA序列磷酸骨幹上的氧經由甲基化的改質，使帶負電的磷酸根轉變成甲基磷酸三酯鍵methyl phosphotriester (MPTE)而成為電中性，可以降低序列本身負電量，使得DNA序列得以於低鹽離子濃度下進行雜交，進而使場效電晶體檢測靈敏度上升。
現今的基因晶片主要是倚靠點陣法(spotting method)所製作出來，其晶片上探針密度相較於Affymetrix公司利用類似半導體製程的原位合成法(in situ synthesis)所生產之晶片低，若在臨床上需要更精準地得知病患的基因資訊，勢必需要更高密度的探針以檢測差異表現的基因。因此，本研究為了開發能夠在場效電晶體上進行原位合成nDNA(phosphate-methylated DNA)序列的原料，我們利用(R,S)-1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate (MeNPOC-Cl)作為原料，以兩步親核反應合成出5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite(光敏化中性核苷酸)，並藉由本校的600MHz核磁共振儀及質譜儀得到定性分析，然而其本身的不穩定性，導致我們目前尚無法得到乾淨的產物。此外，我們也利用紫外光光譜儀及質譜儀去分析目標產物照光後的特性及其產物分子量，並嘗試在溶液相中合成出二聚體(dimer)。
最終，5’-MeNPOC-2’-nucleoside p-methoxy phosphoramidite是可以利用本研究的合成途徑被合成出來，但仍需改善純化方法以得到乾淨產物。倘若未來能夠找到一個合適目標產物的純化方法，便能利用5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以原位合成的方式在矽奈米線場效電晶體上合成出相當高密度的寡核苷酸探針，以得到高通量的基因晶片，在臨床上為精準醫療提供更準確的基因資訊。

Personal treatment has been a new medical trend recently. Based on the analysis of gene information, professional healthcare could give every individual a precise medical treatment. From the perspective of clinical diagnosis, if we could combine gene chip technology and a huge gene database, we would obtain the gene information immediately. However, there are still numerous problems in gene chip technology, such as the instability and the cost of fluorescent label, the sensitivity of gene chip. Silicon nanowire field effect transistor (SiNWFET) could be a huge potential for next generation detection platform due to its high sensitivity, label-free technique, and high response time…etc. However, the environmental ionic concentration would influence the sensitivity of SiNWFET when we detect the electronic signals of duplex formation. According to the previous studies in our lab, by neutralizing site-specific phosphate backbone with methyl phosphotriester (MPTE) inter-nucleoside linkage(s), we improved the electronic signals of SiNWFET when DNA/DNA hybridization under low ionic concentration.
The probe density on gene chip which fabricated with spotting method is lower than those fabricated with in situ synthesis that developed by Affymetrix. In order to have a more precise gene information on clinical diagnosis, there must be higher dense probes on microarray, which is called high throughput microarray. Therefore, in this study, we use (R,S)-1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate (MeNPOC-Cl) as the raw material to develop novel deoxynucleosides for in situ synthesized oligonucleotides, which contain site-specific methyl phosphotriester (MPTE) inter-nucleoside linkage(s), on SiNWFET. Using 2-step nucleophilic reactions, 5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite can be synthesized and can be qualitative analyzed with 600MHz NMR and ESI-QTOF-MS, but so far, we cannot get the pure products due to their instability. In addition, we characterized these deoxynucleosides by UV spectrum and MASS spectrum after solution phase illumination test, and try to synthesize dinucleotide in solution phase.
Eventually, 5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite can be synthesized. In the future, if we could improve the purification method for the desired products, we aim to fabricate a high-throughput FET-based DNA microarray with these novel deoxynucleosides by in situ synthesis method and provide a more precise gene information on clinical diagnosis.

摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 x
表目錄 xv
第一章 緒論 1
第二章 文獻回顧 4
2.1基因檢測 4
2.2核酸及核酸類似物 5
2.2.1 去氧核醣核酸及其結構 5
2.2.2 肽核酸(Peptide Nucleic Acid, PNA) 8
2.2.3 鎖核酸(Locked Nucleic Acid, LNA) 9
2.2.4 中性去氧核醣核酸(oligonucleotides containing site-specific methyl phosphotriester(MPTE) inter-nucleoside linkage(s), nDNA) 10
2.3原位合成(in situ synthesis) 11
2.4 光敏化基團(Photolabile Protecting Group, PPG) 17
第三章 實驗藥品、方法與儀器設備 20
3.1 實驗藥品 20
3.1.1 化學品 20
3.2.2 實驗耗材 23
3.2 儀器設備 24
3.3 合成方法 25
3.3.1 MeNPOC-thymidine與MeNPOC-guanosine(ibu)合成 25
3.3.2MeNPOC-thymidine p-methoxy phosphoramidite 與MeNPOC- guanosine(ibu) p-methoxy phosphoramidite 合成 36
3.4 照光反應 41
3.5 溶液相二聚體合成方法 43
第四章　結果與討論 44
4.1 MeNPOC-nucleoside 定性結果 44
4.1.1 MeNPOC-thymidine定性結果 44
4.1.2 MeNPOC- guanosine(ibu) 定性結果 48
4.2 MeNPOC-nucleoside p-methoxy phosphoramidite 定性結果 52
4.2.1 MeNPOC-thymidine p-methoxy phosphoramidite定性結果 52
4.2.2 MeNPOC-guanosine(ibu) p-methoxy phosphoramidite定性結果
55
4.3 MeNPOC-nucleoside p-methoxy phosphoramidite於萃取與純化時產物損壞討論 57
4.3.1 MeNPOC-thymidine p-methoxy phosphoramidite萃取比例的討論
58
4.3.2 MeNPOC-thymidine p-methoxy phosphoramidite不同純化方式結果 60
4.3.2.1 MeNPOC-thymidine p-methoxy phosphoramidite以前處理過的矽膠純化結果 62
4.3.2.2 MeNPOC-thymidine p-methoxy phosphoramidite以LH20中性樹脂純化結果 64
4.3.2.3 MeNPOC-thymidine p-methoxy phosphoramidite以再結晶純化結果 66
4.4 MeNPOC光敏化基團光化學動力學探討 67
4.5溶液相二聚體合成 76
第五章　結論與未來展望 78
第六章　參考文獻 80
第七章 補充資料 85

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