Microsoft PowerPoint - BCX-N2.ppt

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1 BST 生化科技系生物化學實驗 Polymerase Chain Reaction

2 細胞中 DNA 的複製模式 : Semiconservative replication 圖引用自 :Nelson, D. L. and Cox, M. M. (2000) Lehninger Principles of Biochemistry. 3rd Ed., Worth Publishers. Fig

3 DNA polymerase 催化細胞中 DNA 的複製 : 5 3 DNA-directed DNA polymerases (dnmp) n + dntp (dnmp) n+1 + PPi 圖引用自 :Nelson, D. L. and Cox, M. M. (2000) Lehninger Principles of Biochemistry. 3rd Ed., Worth Publishers. Fig. 25-5

4 DNA-directed DNA polymerase 反應需求 : dntps Primer Template Mg 2+ 圖引用自 :Nelson, D. L. and Cox, M. M. (2000) Lehninger Principles of Biochemistry. 3rd Ed., Worth Publishers. Fig. 25-5

5 DNA polymerase I of E. coli : 第一個被發現的 DNA polymerase 103 kd, 928 amino acid residues N 5 3 Exonuclease 3 5 Exonuclease Polymerase C Primer removal Proofreading DNA synthesis (Primer extension)

6 Large fragment of DNA polymerase I: Proteolytic enzyme 可將 DNA polymerase I 切成兩段 N 5 3 Exonuclease 3 5 Exonuclease Polymerase C Small fragment Large fragment (Klenow fragment) Klenow fragment 在分子生物實驗中的應用極多, 例如, 修補 DNA 末端放射線核酸探針標幟等等最初的 polymerase chain reaction 反應即是利用 Klenow enzyme 進行

7 Polymerase chain reaction: 1 copy P2 Target DNA Denaturation P1 1st cycle Annealing Polymerization 2 copies

8 Polymerase chain reaction: 2 copies Denaturation Annealing 2nd cycle Polymerization 4 copies

9 Polymerase chain reaction: 3rd cycle 4th cycle 5th cycle 8 copies 16 copies 32 copies 第 n 次反應後, 理論上目標 DNA 有 2 n copies, 但實際情況下會受到限制, 為什麼? 2 n copies

10 Experimental approach: Standard PCR Primer design, Preparation of template DNA Determination of experimental parameters PCR Analysis of products Purification of products Subcloning

11 Primer design: Primer length: nt Melting temperature (T m ): 兩引子之差異小於 5 C G/C content: ~ 40%-60% PolyN stretches Complementary sequences Specificity 3 -end sequence

12 T m & annealing temperature T m = H [ S + R ln(c/4) C log 10 [K + ] H: the enthalpy for helix formation S: the entropy for helix formation R: the molar gas constant c: the concentration of primer T m = 2(A+T) +4(G+C) C Annealing tempreature = (T m - 5 ) C

13 Primer design: Primer length: nt Melting temperature (T m ): 兩引子之差異小於 5 C G/C content: ~ 40%-60% PolyN stretches Complementary sequences Specificity 3 -end sequence

14 Experimental approach: Standard PCR Primer design, Preparation of template DNA Determination of experimental parameters PCR Analysis of products Purification of products Subcloning

15 Determination of experimental parameters: Reaction buffer Enzyme Denaturation temperature and time Annealing temperature and time Elongation temperature and time Cycle number

16 Reaction buffer General components: Tris-Cl, mm, ph 8.3 ~8.8 KCl, < 50 mm MgCl 2, mm over total [dntp] dntps, ph 7.0, M each Primers, M each Additives, e.g., BSA, gelatin, NP-40 etc.

17 Determination of experimental parameters: Reaction buffer Enzyme Denaturation temperature and time Annealing temperature and time Elongation temperature and time Cycle number

18 Enzyme Taq polymerase (from Thermus aquaticus) T. Thermophilus DNA polymerase Vent polymerase (from Thermococcus litoralis). 是否具 proofreading 功能? 目標基因的長度? 是否在產物末段多加上一個 A?

19 Determination of experimental parameters: Reaction buffer Enzyme Denaturation temperature and time Annealing temperature and time Elongation temperature and time Cycle number

20 Experimental parameters For Taq polymerase: Concentration: units/100 l Denaturation: C, 30 sec - 1 min Elongation: C, min (2 4 kb/min) Cycle number: 25-35

21 為什麼你的 PCR 失敗? MgCl 2 濃度不對 : 沒有試最適濃度? 使用前沒有混合均勻? dntp 濃度太高? 已經降解了? 反應液中存在 inhibitors? 例如 :EDTA, chloroform, phenol, ethanol, SDS, sarkosyl, tracking dyes,.? Enzyme 太多? Template 過多或過少? Primers 濃度不對? Primers 設計不良? Annealing temperature 不對? Reagents 有雜質?

22 Always remember Work clean. Titrate MgCl 2. Do not use too much template DNA. Do not use PCR products in PCR preparation areas. Always include negative and positive control in every experiment. Wear gloves.

23 10 分鐘添加反應組成份循環30 次N2 以聚合連鎖反應增殖 DNA 準備反應液連鎖反應產物檢定 N1 反應液 72 E. coli DNA 加熱變性高溫變性引子黏合 PCR 產物 N3 限制分析冰浴聚合反應膠體電泳

24 本次實驗反應程式 : , 2 min 95, 45 sec 55, 30 sec 72, 1.5 min 30 cycles 5 72, 10 min 6 4 貯存於冰箱, 下週進行分析

25 本週實驗進行要點 : 製備膠片電泳膠片分析準備電泳樣品定量準備 PCR TAs: 示範鑄膠說明儀器操作 1,3 組 : 進行 DNA 稀釋測定吸光值準備電泳樣品 2,4 組 : TAs: 示範 sample loading 進行 DNA 稀釋測定吸光值 1,3 組 : 進行 DNA 稀釋準備 PCR 反應液 (DNA 及酵素除外 ) 2,4 組 : 準備 PCR 反應液 (DNA 及酵素除外 ) 進行 DNA 稀釋 TAs: 說明 PCR 程式設定 TAs: 協助判讀結果, 決定 DNA 是否可用於 PCR PCR 各組添加 PCR 反應之 DNA 及酵素

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)