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Genetics Techniques: RFLP & PCR

Genetics Techniques: RFLP & PCR. AP Biology Unit 3. RFLP. How many fragments will result when each of these alleles are digested with DdeI?. Stands for Restriction Fragment Length Polymorphism

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Genetics Techniques: RFLP & PCR

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  1. Genetics Techniques: RFLP & PCR AP Biology Unit 3

  2. RFLP How many fragments will result when each of these alleles are digested with DdeI? • Stands for Restriction Fragment Length Polymorphism • Takes advantage of differences in DNA between individuals that result in different fragments when digested with restriction enzymes 3 fragments 2 fragments

  3. RFLP • To see RFLP, DNA is digested with the appropriate restriction enzymes and run on an agarose gel. • A Southern Blot is performed to complete the analysis.

  4. Southern Blotting • A method to visualize specific segments of DNA– usually a particular gene. • Uses radioactive probes that bind to the specific DNA segments • Ex. When testing for the hemoglobin alleles, the probe would bind to these regions

  5. Southern Blotting • Steps: • Soak gel in basic solution to separate DNA strands • Transfer DNA on to a nylon membrane (spacing of DNA is maintained) • Incubate with radioactive probe for specific segment • Wash away unbound probe • Detect probes using x-ray film autoradiograph

  6. RFLP Animations • Animation #1 • Animation #2

  7. Polymerase Chain Reaction • PCR allows scientists to amplify small, specific segments of DNA = make millions of copies of segment • Allows for amplification at an exponential rate • DNA Replication in a test tube

  8. Materials needed for PCR • Target DNA (the DNA you want to copy) • Free Nucleotides (A, T, C, G) • Primers (DNA primers, not RNA) • Taq Polymerase (heat stable DNA Polymerase III) • Mg2+ (cofactor that DNA Polymerase III needs to work) • Buffer • Thermocycler (machine that changes temperatures)

  9. Overview of PCR • Uses different temperatures to amplify DNA • Step 1: Separate existing DNA strands – 95ºC (Denaturation) • Step 2: Lower temperature to allow primers to bind to target DNA – 55ºC (Annealing) • Step 3: Raise temperature to allow Taq Polymerase to build DNA strand – 72ºC (Extension)

  10. Differences between DNA Replication & PCR • No Helicase or Topoisomerase – PCR uses the first heat step to completely separate the strands of DNA • No Primase – primers are already made • DNA primers (not RNA) – no need for DNA Polymerase I • No leading or lagging strands – DNA is completely unzipped, no Okazaki fragments

  11. PCR animation • animation

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