- 學位論文
以雙肽核酸標定染色體DNA於脂雙層上快速建立基因圖譜
Using bisPNA-Labeling Technique to Mark Chromosomal DNA for Rapid Gene Mapping on Lipid Bilayers
摘要
近年來交通便利性提升使傳染病更容易傳播，在醫療上需分析致病原才能達到精準治療，利用直接線性分析法建立DNA基因圖譜即為有潛力應用於疾病檢測的技術。其概念是先將DNA特定序列處以螢光標記後再將DNA拉伸呈線性，則DNA上的螢光標記點數量與相對位置可代表此樣本DNA的專一條碼，與基因資料庫比對後即可辨識病原體。相較於傳統基因檢測的方法，直接線性分析法不需要PCR步驟增量DNA也不需以膠體電泳做分析，因此具有操作簡單且省時的優勢。 直接線性分析法主要包含DNA標定及DNA拉伸的技術。本研究團隊先前已成功展示如何在脂雙層上拉伸DNA，並利用bisPNA標定λ-DNA以建立其基因圖譜。此基因圖譜分析平台除了具有低成本、簡易操作、圖譜準確性高等優點，也因使用bisPNA標定法而具有較高的序列選擇彈性，於疾病檢測技術上將更具潛力。然而本研究團隊先前以bisPNA標定DNA的時間需要約21小時，其中19小時是利用透析法將反應後多餘的bisPNA移除以避免錯誤標記點，使得此標定方法在實際醫療應用上受到限制，因此加速bisPNA標定的過程是本研究的主要目標。此外由於先前研究主要是使用實驗上常見的標準短鏈λ-DNA(48502bp)，故為了展示此基因圖譜平台可用於更廣泛的DNA，我們不但自行合成長鏈λ-DNA多倍體並建立其基因圖譜，也進一步檢驗以此技術拉伸大腸桿菌DNA的可行性。 於本研究中為了加速移除反應後多餘bisPNA的步驟，我們提出利用奈米磁珠捕獲法取代先前使用的透析法。奈米磁珠捕獲法的概念是利用表面帶負電的奈米磁珠捕獲帶正電的bisPNA，並以強力磁鐵快速移除帶有bisPNA的奈米磁珠。在實驗上我們先對此方法進行可行性及操作參數測試，並透過此捕獲法成功建立高準確性的短鏈λ-DNA基因圖譜。實驗結果顯示我們建立的奈米磁珠捕獲bisPNA流程，可以取代原本相當耗時的透析法，將此分離步驟縮短至1小時內，整體加速近19倍。 由於長鏈λ-DNA多倍體上標記點的理論位置與數量即為單一λ-DNA的倍數，因此較容易驗證我們所開發基因圖譜平台對長鏈DNA的標定結果。我們自行合成長鏈λ-DNA多倍體(≈300kbp)並配合改良後的bisPNA標定法進行標記，實驗結果顯示隨著標定過程進行，長鏈λ-DNA多倍體的長度會逐漸減小，且雖然我們可以在約3倍λ-DNA(150kbp)上產生標記點，但其數量以及間隔皆與理論不符。造成上述實驗結果的原因我們認為與利用接合酵素合成λ-DNA多倍體有關，因為接合酵素可能會影響標定反應之結果，也可能引發使DNA長度減小的未知反應。 最後為了測試真實細菌DNA與本團隊所開發脂雙層拉伸技術之相容性。我們透過市售DNA萃取套組取得大腸桿菌的DNA，並成功以脂雙層裝置拉伸約350kbp的大腸桿菌片段，此片段大小已接近某些致病原之DNA大小如摩氏摩根氏菌(538kbp)及生殖道黴漿菌(580kbp)，故本研究證明真實細菌DNA與脂雙層拉伸系統具相容性，也展現了本研究團隊所開發之基因圖譜平台應用於快速篩檢致病原之潛力。
並列摘要
DNA optical gene mapping by direct linear analysis (DLA) is an efficient method to extract low-resolution genetic information from DNA for genomic study and pathogen identification. The concept of DLA is first to label specific sequences on DNA with fluorescent markers and then to linearize DNA to read the genetic information from the number and the positions of the markers. In brief, applying DLA for gene mapping mainly includes two key techniques: DNA labeling and DNA linearization. In our previous studies, we have developed a low-cost DLA platform based on bisPNA-labeling technique and the method of DNA linearization on patterned lipid bilayers. The bisPNA-labeling technique has high flexibility on the choice of target sequences and therefore bears more potential in detecting growing number of pathogens. However, removing redundant bisPNA during the labeling process to decrease false markers is too slow to make the bisPNA-labeling technique efficient for medical diagnostics. Therefore, accelerating the process of removing redundant bisPNA is the primary goal of this research. In addition, since our previous studies mainly used relative short λ-DNA (48502bp), we have also tested the potential of our DLA platform to more general long-chain DNA including the synthesized multi-λ-DNA and E. coli DNA extracted directly from E. coli bacterials. In order to accelerate the removal of redundant bisPNA, we proposed to use negatively charged magnetic-nanoparticles (MNPs) to capture positively charged bisPNA after the labeling reaction. The MNPs can later be quickly removed by a strong magnet. After testing the feasibility and operating parameters of this method, we have successfully constructed an accurate λ-DNA gene map. Using MNPs allowed us to remove redundant bisPNA within 1 hour, a 19 times acceleration in comparison with the dialysis method used in the past. We have also performed a bisPNA mapping of multi-λ-DNA using the new capturing process by MNPs. Experimental results showed that we can label and linearize multi-λ-DNA, but the number of markers and the length between markers were not in line with the expectation. Besides, we found that the length of multi-λ-DNA decreases during the process of bisPNA-labeling. We speculate that the ligase enzyme and other cofactors used in the synthesis of multi-λ-DNA may be the cause of DNA fragmentation. To test our platform with more general DNA, we have extracted E. coli DNA and successfully linearized E. coli DNA fragment of the size about 350 kbp, already close to the full length of the DNA of some common pathogens such as Morganella (538kbp) and Mycoplasma (580kbp). Hence, we have demonstrated the compatibility between bacterial DNA and our lipid bilayers based DNA linearizing system. Moreover, we demonstrated our DNA mapping platform has great potential in rapid pathogen identification.