單一分子生技研究的新領域近年來在國際間發展迅速，也為生物醫學提供了一個創新的舞台。而利用單分子的研究來發展DNA定序技術，預計將會是下一個具有很大潛力且可行的方法，它提供了一個作為基因體定序研究的利器。不過現行的技術研究仍有許多的問題及瓶頸亟待克服。本核心實驗室將以對DNA分子的操縱能力為基礎，配合已著手進行架設之光鑷子系統，在現今的國際研究中提供DNA CD-ROM定序的創新想法，並且初步進行利用酵素配合雷射光之激發控制研究，以期研發出具前瞻性的研究方法與成果。我們希望憑著巨觀的初步觀察，來作為接下來單分子微觀技術的基礎。
本論文主要架構可分成三部分：第一部份將對現行DNA定序技術做個概述與探討，並且整理出現行國際研究單分子定序所遭遇到的問題；第二部分則提出DNA CD-ROM 定序技術的初步構想與策略；最後則著重於實驗構想中，前段之“雷射光輔助外切核酸分解酶之控制”的研究。在研究中，初步完成了生物實驗流程的建立，並且著重於尋找出調控酵素活性的相關參數，以利後續研究之參考。結果顯示溫度因子在15℃~25℃之間，以每5℃切除速率增加一倍的關係影響酵素活性，並在37℃時達到最佳反應溫度；依實驗結果計算出ExoIII反應之活化能為19.1Kcal/mole；當利用添加NaCl來調控反應離子濃度時，酵素反應速率會隨NaCl濃度增加而線性的減少；當NaCl濃度接近270mM時，酵素的活性完全受到抑制反應速率接近0。至於實驗的再現性好壞尚無法評估，還需要有更多的重複實驗來增加統計量。
另外，針對cDNA微陣列晶片系統所研究的優化實驗，也整理於附錄4，內容主要為晶片雜合反應條件的優化研究。

Recently, the research of single molecule techniques has developed rapidly and now provides an innovative stage for bio-medical researches. Single molecule approach may provide a potential way and novel solution to developing DNA sequencing techniques that may probably be a powerful tool for genome research in the present day. However, at the present stage there still exist many obstacles that deserve to overcome. In this study, based on the capabilities of the DNA manipulation, together with the optical tweezers system, established in our core Lab, we will propose a new sequencing method with the help of exonuclease and laser excitation such that one can clip the single nuclide of a DNA and put them on the CD-ROM for ultra-fast and precision sequencing. Hope that this approach via macro and preliminary observation as a first step may be formed the basis of the following single molecule detection studies.
This thesis contains three major sections, the brief introduction of DNA sequencing and the list of the difficulties of the current single molecule technique, the fundamental idea and the preliminary strategy of DNA CD-ROM sequencing, and the study of “Laser Assisted Exonuclease Activities”, respectively. In this thesis, the key point is to find out the important related parameters in an effort to control the activity of exonuclease included the assist of laser. Finally, the results of the experiences concerning an “Optimization of DNA Hybridization Protocol for Microarray System” are clearly described in the appendix 4.

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