BME 210 Lecture 15 PCR - 15. Polymerase chain reaction...

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15. Polymerase chain reaction
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Polymerase chain reaction (PCR) PCR – a method to selectively multiply (amplify) a specific fragment of DNA molecule (e.g., a gene) PCR mimics DNA replication. PCR components: 1. DNA template – double-stranded DNA molecule containing the region to be multiplied 2. Thermostable DNA polymerase – enzyme for DNA synthesis (e.g., Taq polymeraze) 3. Two DNA primers single-stranded short DNAs that are complementary to regions on opposite strands of the copied DNA fragment (DNA–Polymerase requires primers to start synthesis) 4. Nucleotides - building blocks for the new DNA (dATP, dCTP, dGTP, dTTP) 5. Buffer - a solution suitable for the DNA polymerase
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PCR steps 1 PCR process is a series of cycles with each cycle consisting of 3 steps: (A) Denaturation • The temperature is increased to ≈95 C for 20-30 s • dsDNA is separated into two ssDNA
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PCR steps 2 (B) Annealing • The temperature is lowered to 50-65 C for 20-40 s • Two primers (red) attach to their complementary regions on opposite strands Primer 2 Primer 1
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PCR steps 3 (C) Elongation • The temperature is increased to ≈72 C (working temperature of Taq polymerase) • DNA polymerase binds and replicates DNA on both strands starting from the primers Steps 1-3 are repeated multiple times
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dsDNA Denature Anneal primers Taq Polymerase binds 96º 50º 72º Taq Taq 1 st PCR cycle Taq Taq Copy strands After the 1 st cycle - 2 dsDNA
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50º Anneal primers 2 Taq Taq Taq Taq 72º Polymerase binds 1 2 3 4 Taq Taq Taq Copy strands After 2 cycles: 4 dsDNA Taq 1 2 3 4 Denature 96º
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1 2 3 4 After 3 cycles: 8 double strands; 2 of them are short 3 rd PCR cycle
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1 2 3 4 After 4 cycles: 16 double strands; 8 of them are short
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1 2 3 4 After 5 cycles: 32 double strands; 22 have the same length
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This note was uploaded on 04/28/2010 for the course BME eng. biolo taught by Professor Fast during the Spring '10 term at University of Alabama at Birmingham.

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BME 210 Lecture 15 PCR - 15. Polymerase chain reaction...

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