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PCR ( P olymerase C hain R eaction)

PCR ( P olymerase C hain R eaction). 聚合酶 链 式 反应. 生化教研室： 牛永东. What is PCR ?. P CR is to make a huge number of copies of a gene in vitro. HISTORY. lives in a small apt. near the beach.

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PCR ( P olymerase C hain R eaction)

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  1. PCR (Polymerase Chain Reaction) 聚合酶链式 反应 生化教研室： 牛永东

  2. What is PCR ? PCR is to make a huge number of copies of a gene in vitro

  3. HISTORY lives in a small apt. near the beach • In April, 1983, Kary Mullis took a drive on a moonlit California mountain road. During that drive, he conceived the Polymerase Chain Reaction (PCR) 2. He received a $10K bonus from Cetus, Cetus later sold the technolgy to La Roche for $300 million

  4. 1993 Nobel prize “I do my best thinking while driving”

  5. OUTLINE • 3 concepts • 5 Essential Components • 3 steps • One sentence

  6. 3 Concepts of the PCR • Denaturation :变性 • Renaturation：复性 3. Semiconservative replication：半保留复制 the double strandDNA open to single stranded DNA two single stranded DNA form double strandDNA replication, one make two

  7. Old new Semi-conservative replication

  8. salts (ions) DNA Polymerase Template DNA • dNTPs Primers 5Essential Components of PCR Reaction

  9. TaqDNA Polymerase • Taq : Thermus aquaticus 2. 95℃,T1/2(half life)40min 3. Polymerization

  10. dT dG dA dC Polymerization聚合反应 TaqPolymerase dT 5 ’ 3 ’ 3 ’ 5 ’ A Taqpolymerase: 72℃, 2,000～4,000 bases/min

  11. How PCR works • three steps: • Denaturation (变性): 90～97℃ • Annealing(退火): 45～55℃ • Extension(延伸): around72℃

  12. Step 1 : Denaturation变性 94 ℃ TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATGACATCGATTTGATCGAT ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA

  13. Step 2 : Annealing退火 T℃ primer-dependent TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATGACATCGATTTGATCGAT GCTGCTACGTAG CGTAGTAGCA ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA

  14. Taq Polymerase Taq Polymerase Step 3 : Extension延伸 72 ℃ TGAACGTAGTAGCATAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATGACATCGATTTGATCGAT TACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGA GCTGCTACGTAG CGTAGTAGCA TAGCAATGTACATCATCAGATAGACGATTGACAGTGATCTCGACGATGCATCATCGCGATAT ACTTGCATCATCGTATCGTTACATGTAGTAGTCTATCTGCTAACTGTCACTAGAGCTGCTACGTAGTACTGTAGCTAAACTAGCTA

  15. 1. Denaturation 2. Annealing 3. Extension 聚合酶链式反应 PCR.EXE

  16. After 25~30 cycles Last Step : 72℃2 min ?

  17. One sentence One makes two, two make four, four make anything • 一生二，二生四，四生万物

  18. Thank you for your tolerance！

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