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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生命科學院 > 依資料類型分類 > 碩博士論文 >  連結酶鏈反應-滾動循環擴增技術於單一核苷酸多型性分型之應用

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152191

標題: 連結酶鏈反應-滾動循環擴增技術於單一核苷酸多型性分型之應用
Ligation Chain Reaction-Rolling Circle Amplification for Single Nucleotide Polymorphism Genotyping
作者: 曾國棟
Tseng, Kuo-Tung
Contributors: 高振益
Jung-Yie Kao
生命科學院碩士在職專班
關鍵字: 單一核苷酸多型性;連接酶
Single Nucleotide Polymorphism;Ligation Chain Reaction;Rolling Circle Amplification
日期: 2012
Issue Date: 2013-11-07 13:15:04 (UTC+8)
Publisher: 生命科學院碩士在職專班
摘要: 連結-滾動循環擴增技術是一種運用在核酸分析上結合扣鎖式探針連結酶鏈反應及多分枝型滾動循環擴增的分析技術。一般設計的扣鎖式探針由於此偵測引子的存在而使得探針的長度大多在於100個核苷酸以上。本研究中利用重疊序列設計以縮減探針長度,設計出一段長64個核酸的探針用以分析MTHFR c677t多型性。在設計上是利用扣鎖式探針的兩端與MTHFR c677t序列互補故可辨識並與目標序列結合,當探針與目標序列結合後,連結酶可將探針頭尾連結形成一環狀探針,而當探針無法互補結合時,連接酶無法作用使探針依舊是線狀,存在環狀探針時,滾動放大的結果可使目標核酸序列的訊號在肉眼下簡單判讀。在結果上,利用SYBR Green I及即時偵測系統偵測可有效對MTHFR基因進行單一核苷酸分型。
Rolling circle amplification (RCA) of circularizing oligo nucleotide probes (padlock probes) is a sensitive method for short DNA fragment detection and amplification. Although oligonucleotide can be chemically synthesized, the longer probe sequences will have a lower synthesis yield and sequence accuracy. Here we designed a 64nt padlock probe for MTHFR c677t polymorphism detection. The shorter probe sequence makes it easy to obtain from chemical synthesis. In this study, the both ends of padlock probes are designed complementary to MTHFR c677t sequence. With a sensitive DNA ligase, the matched probe will be ligated and form a circular-form probe. Otherwise, the mismatched probe fails to be ligated and keeps in a liner form. The following hyper-branched rolling circle amplification of circular-form probe makes it easy to analyze the probe ligation result. RCA method detection sensitivity and specificity is based on probe ligation stage, which becomes a critical step in RCA detection. The ligase chain reaction method can help to increase ligation efficiency and more linked circle-form padlock probe yield for paired sequence. We amplified the circular-form padlock probe with detection primer and isothermal polymerase. The isothermal polymerase character makes RCA can be a ready-to-use detection method for study.
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