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PCR 理論

PCR 理論. 1970 Khorana 等人提出其理論 DNA 尚不能定序 寡核苷酸合成不易 1983 Kary Mullis 等人提出實際操作方法 獲得 1993 年諾貝爾化學獎. 進行 PCR 所需的材料與過程. 熱穩定 DNA 聚合酶 (Thermostable DNA polymerase) Taq - Thermus aquaticus 合成 DNA 片段長度有限 無校正功能 (proofreading) Pfu - Pyrococcus furiosis 引子 (primers) 人工合成的寡核苷酸 (18 ~ 32 鹼基 )

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PCR 理論

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  1. PCR理論 • 1970 Khorana等人提出其理論 • DNA尚不能定序 • 寡核苷酸合成不易 • 1983 Kary Mullis等人提出實際操作方法 • 獲得1993年諾貝爾化學獎

  2. 進行PCR所需的材料與過程 • 熱穩定DNA聚合酶(Thermostable DNA polymerase) • Taq- Thermus aquaticus • 合成DNA片段長度有限 • 無校正功能(proofreading) • Pfu- Pyrococcus furiosis • 引子(primers) • 人工合成的寡核苷酸 (18 ~ 32 鹼基) • 三磷酸去氧核苷酸(Deoxynucleoside Triphosphates，簡稱dNTPs) • 模版DNA (template DNA) • 取樣時避免污染

  3. PCR的原理與方法一 • 變性(Denaturation) • 加熱（95℃）將模版DNA雙股分開 • 引子黏合(primer annealing) • 降低溫度使特異性引子黏合至單股模版DNA的互補序列 （溫度40 ～ 72℃） • 延展(extension) • DNA聚合酶開始合成新股DNA （最佳反應溫度72℃） • 此三步驟為一個循環，一般進行25 ～30個循環

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