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Polymerase Chain Reaction

PCR. Polymerase Chain Reaction. (PCR). PCR. PCR produces billions of copies of a specific piece of DNA from trace amounts of starting material. (i.e. blood, skin cells, bone) Allows scientists to isolate pure quantities of specific DNA sequences

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Polymerase Chain Reaction

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  1. PCR Polymerase Chain Reaction (PCR)

  2. PCR • PCR produces billions of copies of a specific piece of DNA from trace amounts of starting material. (i.e. blood, skin cells, bone) • Allows scientists to isolate pure quantities of specific DNA sequences • 230 = over 1 billion copies of a specific DNA fragment; large enough quantity to be analyzed • Used in: • Criminal Investigations to identify suspects • Sequencing the Human genome

  3. How does DNA from the Crime Scene help in the lab?

  4. What DNA is Used? • 46 Chromosomes code for 30,000 to 50,000 genes; only 5% of your DNA • Exons = DNA that is coded or expressed into proteins • Noncoding DNA has more diversity; since this DNA rarely leaves the DNA to head to ribosomes • Introns = DNA that is rarely expressed • Increased number of mutations

  5. PCR: How do we get started?

  6. What ingredients are needed? • Target DNA – the DNA that needs to be copied • Primers – short pieces of DNA that are designed to attach to each end of the DNA fragment that will be replicated • Taq polymerase – enzyme that reads the DNA • Comes from the bacteria Thermus aquaticus • Lives in the hot springs in Yellowstone; doesn’t fall apart in high temperatures • dNTPs – 4 nucleotides with the 4 different bases that are needed to replicate DNA • Buffer – gives the best environment for the enzymes to work • Mg Ions – needed by DNA polymerase to make DNA copies

  7. How Does PCR Work? • PCR machine is known as thermal cylcer • Machine changes to three different temperature changes during one cycle • Average number of cycles per run is 30 to 40 What happens at each temperature change?

  8. Temperature at 94ºC • Denaturing temperature • The target DNA falls apart • The H bonds holding the nitrogen bases together break • 2 individual strands of DNA are now present instead of a double helix.

  9. Temperature at 65ºC • Annealing Temperature • Primers attach to the ends of the Target DNA that needs to be copied • Annealing = attachment of the primers • Attach to complimentary bases of target DNA

  10. Temperature at 72ºC • Extension Temperature • Provides best temp for Taq polymerase to begin reading the DNA • Taq polymerase will synthesize a second strand of complimentary DNA • Taq polymerase always read target DNA from 3’ to 5’ end

  11. Repeat 30 times • The three temperature changes represents one cycle • Denature • Anneal • Extend • Repeat 30 times 230 = over 1 billion copies of the Target DNA • Once DNA is amplified (copied), it is visible on a gel

  12. One Cycle has all three different temperatures.

  13. Another Animation

  14. Cycle 1 Cycle 2 Cycle 3

  15. PCR Animation

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