標題: | 發展蛋白質電晶體作為第三代去氧核醣核酸定序平台 Developing third generation DNA sequencing platform based on protein transistor |
作者: | 黃國華 Huang Gue-Wha 國立交通大學材料科學與工程學系 |
關鍵字: | 去氧核醣核酸定序;蛋白質電晶體;單分子;DNA sequence;protein transistor;single molecule |
公開日期: | 2013 |
摘要: | 本計畫提出的第三代去氧核醣核酸序列解碼平台,是將其與單分子蛋白質電晶體相結合。上一世代的去氧核醣核酸定序技術的主要缺點是 : 過短的解序長度以及過高的錯誤判讀機率。就基因體定序成果而言,上一代基因體定序技術的效率仍差強人意。第三代的基因體定序技術(或稱為”單分子定序”技術),可解讀較長的基因序列並大幅降低判讀錯誤機率,這將在個人基因體計畫解讀及藥物客製化領域帶來革命性的突破。我們將去氧核醣核酸聚合酶鍵結在蛋白質電晶體上,並藉由觀測電子傳遞效應,進而即時監控定序反應的進行。本研究團隊已將單分子酵素架接在蛋白質電晶體上的技術開發完成。藉由此接合技術,可將蛋白質電晶體轉化成為一個極度敏感的感測器,對於酵素進行催化反應時產生的酵素轉型現象具有高度感測性。我們具有即時解析催化反應中分子變化的能力。本計畫提出的單分子酵素平台具有下列優勢 : 能夠在等同於原生去氧核醣核酸聚合酶催化速率,並於去氧核醣核酸合成反應中區分出4種不同的含氮鹼基,而錯誤判讀的機率等同於去氧核醣核酸複製時的機率,並且能夠解讀長度將近106個鹼基對的序列。另一方面,核酸外切酶也會被架接到本蛋白質電晶體系統。去氧核醣核酸的降解,可以提供來自前向合成反應中與聚合酶合成反應不同的序列資訊。本蛋白質電晶體系統將能提供一個真正的第三代基因體定序平台。 A third generation DNA sequencing platform based on the single molecule protein transistor (proT) is proposed. The main shortage of the next generation DNA sequencing is short reading length and high error rate. For de novo genomic sequencing, the next generation sequencing techniques are far from satisfactory. The third generation sequencing, or single molecule sequencing, promises long reading length with low error rate will revolutionize personal genomic project and personal medicine. We will conjugate DNA polymerase onto proT and perform sequencing reaction monitored by the electric conductivity. Conjugating single molecule enzyme onto proT has been performed in the lab. The conjugation has transformed proT into a sensitive detector sensing the enzymatic trajectory of single molecule enzyme during catalysis. We were capable of dissecting the molecular detail of catalysis in real-time. The current proposal taking advantage of single molecule enzyme platform will be able to distinguish 4 different nucleotides during the synthesis of second DNA strand with the speed equaling the native DNA polymerase catalysis, the error rate equaling the DNA replication, and reading length approaching megabase. Alternatively, exonuclease will be conjugated to the system. Instead of polymerization, DNA degradation will provide sequencing information complimentary to the forward synthesis. This system will represent a true third generation sequencing platform. |
官方說明文件#: | NHRI-EX102-10249EI |
URI: | http://hdl.handle.net/11536/93113 https://www.grb.gov.tw/search/planDetail?id=2751473&docId=402245 |
顯示於類別: | 研究計畫 |