次世代定序關鍵酵素與樣品庫製備流程最佳化研究

Abstract

次世代定序技術(Next-generation Sequencing, NGS)是一個新穎的定序技術，相較於傳統定序方法，不僅快速經濟，並且具有高輸出量、高解析度與低錯誤率的特性，而樣品庫製備(sample preparation)流程則是決定次世代定序結果優劣的重要實驗步驟。雖然市售已有許多樣品庫製備的試劑套組可供選擇，然而大多仍具有實驗操作步驟繁瑣、耗時長及成本高等狀況。 本研究運用酵素工程的技術，使用大腸桿菌(Escherichia coli)表達樣品庫製備實驗中所需要的五種關鍵酵素(T4 DNA polymerase, Klenow fragment, T4 polynucleotide kinase, Klenow fragment (3’ to 5’ exo-)和T4 DNA ligase)。我們運用基因合成的方式構築質體(plasmid)，並優化基因序列(codon)使其更適合使用大腸桿菌進行表達，以增加酵素之表現量。 此外，不同的定序平台或不同種類的樣品，均需要搭配其專屬的試劑套組，在實驗操作上有諸多限制且無法彈性運用。 本研究使用實驗室自行生產之酵素，進行樣品庫製備流程最佳化之研究，以建立一套實驗操作更簡便、節省時間及成本並且效率更好的樣品庫製備流程。

Next-generation Sequencing (NGS) technology is the state-of-the-art sequencing technology in many biological research and clinical applications. NGS is more sensitive, efficient and economical than traditional Sanger sequencing method. It also offers high-throughput, high-resolution and low failure rate. Therefore, applications of NGS technology are important and essential to current developments of biological research. Sample preparation is the critical experimental step of NGS, but the commercial NGS sample preparation kits are costly, time-consuming, and are complicated in workflow. Moreover, different NGS platforms and various samples require specific kits for different purposes. This restricts the flexibility between each library preparation kit and dramatically increases the cost while switching among experiments. To reduce the cost and increase the workflow of sample preparation more flexible, this study is to optimize the DNA library preparation protocol for NGS with self-produced enzymes, and to create a streamlined workflow which is more cost-effective, flexible, and efficient. The aims of this thesis is to overexpress the five key enzymes, T4 DNA polymerase, Klenow fragment, T4 polynucleotide kinase, Klenow fragment (3’ to 5’ exo-) and T4 DNA ligase of NGS sequencing library preparation by Escherichia coli with enzyme engineering. We construct the plasmid with gene synthesis, and optimize the codon for E. coli overexpression. This work reduces the costs of the DNA library preparation. We will overexpress these enzymes with large-scale, and make NGS popular to popularize universal, promote developments and persistence of biological research.