以缺口標記法於脂雙層伸展之DNA上快速建立DNA圖譜

Abstract

自從華生、克立克共同發表DNA的雙股螺旋結構，人類展開了一連串相關研究，企圖一窺生物遺傳之全貌，隨之發展出的DNA定序與DNA圖譜幫助使得人類解開遺傳的密碼，例如準確地找出製造胰島素的基因，某些遺傳疾病反映在異常的染色體結構。 本研究著重於快速建立DNA圖譜，期待能應用於病原體篩檢等具有時效急迫性的領域。傳統DNA圖譜的建立，需要透過限制酶酵素將樣本DNA剪切成數片段，隨後還需搭配聚合酶鏈鎖反應增幅片段的數目，以及凝膠電泳的交叉比對分析，經過上述等繁複步驟才能得到樣本DNA之圖譜。近年來許多研究提出「DNA單分子圖譜」，藉由直觀地觀察「一根」線性DNA上的標記點的相對位置與數目，學者將能得到樣本DNA之圖譜。 單分子圖譜牽涉DNA拉伸與DNA序列標記等兩大技術。關於DNA拉伸，奈米通道侷限拉伸法為近十年來之顯學，然而奈米通道的製程所費不貲，操作上也相當複雜，這對於實際應用是很大的阻力。我們實驗室所用之脂雙層拉伸法則克服了這些挑戰，我們選用正電脂雙層與負電DNA具有靜電吸引力而使得DNA得以吸附於其上，由於支托脂雙層的二維流動性，DNA得以逐漸展開，最後在表面上的溝槽之側壁達到最低自由能，因此能夠自發拉伸呈一直線。本研究將使用-DNA(48502 bp)以及T4GT7-DNA(165647 bp)作為研究樣本，此拉伸法皆能夠使兩者達到80%的拉伸率，已能夠媲美次50奈米級的奈米通道中的DNA拉伸率。 關於DNA序列標記，我們過去嘗試過限制酵素以及雙肽核酸標定的方法，雖然皆能成功建立正確的DNA圖譜，然而兩種方法皆有各自的限制，限制酵素法雖然耗時較少，但須全程拍攝剪切過程，受限於CCD視野大小而難以一次收集足夠數據，此為採樣效率不佳；雙肽核酸法雖然改善了採樣效率差的問題，但讓雙肽核酸標記於DNA上所花的反應時間過長，因此與「快速建立DNA圖譜」的目標相違背。 因此，我們使用另一種同時滿足採樣效率與反應時間快速的方法－缺口標記法，我們分別使用Nb.BbvCI與Nt.BspQI缺口內切酵素在l-DNA與T4 GT7 DNA上建立圖譜。缺口標定法乃是利用缺口內切酵素在雙股DNA的其中一股特定序列上製造出缺口，再由DNA聚合酶將具有螢光修飾的核苷酸合成於DNA骨幹上，缺口標記反應時間只需2~3小時，而且反應後的DNA能夠保持其完整性與拉伸率。為了正確判讀的標記位置，我們將擷取的光強度分布圖加以擬合，因此我們將能有系統地得出的標記位置。 我們發現脂雙層系統與缺口標記法能夠彼此相容，由兩者所建立的DNA圖譜具有低成本、操作容易、高準確、高精確以及快速建立等優勢，因此本研究非常有潛力應用於快速疾病檢測、病原體篩檢等醫療檢測技術。

Our research is to establish rapid DNA mapping, which has the great potential to apply to medical diagnostics and pathogen identification. In tradition, DNA mapping needs PCR, gel electrophoresis and analysis for de novo assembly, the whole process is complicated and time-consuming. Recently, the field of single-molecule biophysics has produced new techniques that characterize individual DNA molecules, allowing for establishing the DNA mapping without a great deal of DNA samples. Using the direct linear analysis (DLA), we can intuitively obtain DNA optical mapping from the sequence-specific optical signal on linear DNA. The two main techniques in employing this concept are DNA stretching and DNA sequence-specific labeling. We have managed to make DNA adopt an extended conformation on the patterned lipid bilayer, where DNA spontaneously gathers and unravels along the root of the trench walls. For both T4GT7(166kbp) and lambda DNA(48.5kbp), the relative DNA extension can reach as high as 85% of their contour lengths, comparable to the highest degree of DNA extension obtained in sub-50nm nanochannels. About DNA sequence-specific labeling, we have performed the restriction enzyme method and bis-PNA labeling. However, there are some limitations in the both mapping. Restriction enzyme provides a rapid mapping, but we can’t capture enough data at one time due to the field of view. On the other hand, bis-PNA labeling takes a lot of time, contradicting our goal, 「rapid DNA mapping」. To overcome the above challenges－sampling efficiency and time-consuming, we performed another DNA mapping technique－nick-labeling. We used nicking enzyme to cut specific sequence on one strand of dsDNA, and then DNA polymerase incorporated the fluorescent nucleotide in the nicked site. In addition, the image analysis was conducted by fitting the optical intensity profile of the DNA, such that we can read out the location of the optical signals on the DNA. We found that the patterned lipid bilayer is compatible to nick-labeling and other labeling techniques we tried. The nick-labeling DNA mapping on lipid bilayer has strength in low-cost, easy-to-use, high accuracy and precision, rapid mapping. Therefore, our research has a great potential to apply to the medical diagnostics and pathogen identification.