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Mullis KB. The unusual origin of the polymerase chain reaction.

Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990 Apr;262(4):56-61, 64-5. What is the Polymerase Chain Reaction?. Template: Base-paired dsDNA. 3’. 5’. 3’. 5’. What is the Polymerase Chain Reaction?. 1. Heat Denature. What is the Polymerase Chain Reaction?.

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Mullis KB. The unusual origin of the polymerase chain reaction.

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  1. Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990 Apr;262(4):56-61, 64-5

  2. What is the Polymerase Chain Reaction? Template: Base-paired dsDNA 3’ 5’ 3’ 5’

  3. What is the Polymerase Chain Reaction? 1. Heat Denature

  4. What is the Polymerase Chain Reaction? 2. Anneal ssDNA Primers

  5. What is the Polymerase Chain Reaction? 3. Elongate ssDNA Primers on ssDNA template

  6. What is the Polymerase Chain Reaction? 4. Heat Denature

  7. What is the Polymerase Chain Reaction? 5. Anneal ssDNA Primers on ssDNA template

  8. What is the Polymerase Chain Reaction? 6. Elongate ssDNA Primers on ssDNA templates

  9. Products:1 Cycle +

  10. Products: 2 Cycles

  11. Products: 3 Cycles

  12. Products: 4 Cycles

  13. Products: 5 Cycles

  14. Products: 2n Cycles

  15. How much DNA is that? • Given: 1000 bp of dsDNA has a MW of 630 000 • Question: how much would you have after amplifying • 1 molecule through 30 cycles • 10 molecules through 40 cycles? • Answer (A): (6.3 x 105/6.0221415 x 1023) x 230 = 1.123 x 10-9 g = 1.123 ng • Answer (B): 11 500 x 10-9 g = 11.5 µg How would you detect that? Ethidium bromide Down to ~ 50 ng / band Down to ~ 50 pg / band

  16. denature heat 94 rapidlycool extend 70 heat heat anneal 45 Thermal Cycling Temp Time 180 120 60

  17. Factors Affecting PCR:

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