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dc.contributorJung-Yie Kaoen_US
dc.contributor.authorTseng, Kuo-Tungen_US
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dc.description.abstract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。zh_TW
dc.description.abstractRolling 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.en_US
dc.description.tableofcontents中文摘要 i ABSTRACT ii 目錄 iii 表目錄 vi 圖目錄 vii 附錄目錄 viii 第一章 緒論 1 第一節 滾動循環增幅(Rolling circle amplification,RCA) 1 第二節 單核苷酸多型性(Single nucleotide polymorphism,SNP) 3 第三節 甲基四氫葉酸還原酶 (Methylenetetrahydrofolate reductase,MTHFR) 基因 5 第四節 點突變檢測法簡介 8 一、Single-strand conformation polymorphism (SSCP) 8 二、Real-time PCR 8 三、Denaturing high performance liquid chromatography (dHPLC) 9 四、Restriction fragment length polymorphism(RFLP) 9 五、Direct DNA sequencing 10 第五節 研究動機 12 第二章 材料與方法 14 第一節 材料 14 一、Genomice DNA 14 二、寡核苷酸(Oligo nucleotides) 14 三、儀器 15 第二節 方法 15 一、PCR 15 二、不對稱PCR (Asymmetric PCR,As-PCR) 16 三、連接酶鏈反應(ligase chain reaction, LCR) 17 四、Exonuclease I digestion 18 五、超分支滾動循環擴增(hyperbranched rolling circle amplification,HRCA) 18 六、瓊脂凝膠電泳(agarose gel electrophoresis) 20 第三章 結果 21 一、比較PCR與不對稱PCR(Asymmetric PCR)差異 21 二、扣鎖探針設計(Padlock probe design) 21 三、連接酶鏈反應(ligation chain reaction,LCR) 23 四、滾動循環增幅(Rolling circle amplification,RCA)與超分支滾動循環增幅(hyperbranched RCA,HRCA) 23 五、Bst DNA聚合酶RCA放大 24 六、利用本法做基因分型驗證 25 第四章 結論 26 第五章 討論 27 參考文獻 31 表 33 圖 36 附錄 43zh_TW
dc.subjectSingle Nucleotide Polymorphismen_US
dc.subjectLigation Chain Reactionen_US
dc.subjectRolling Circle Amplificationen_US
dc.titleLigation Chain Reaction-Rolling Circle Amplification for Single Nucleotide Polymorphism Genotypingen_US
dc.typeThesis and Dissertationzh_TW
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item.openairetypeThesis and Dissertation-
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