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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

Genetics Techniques: RFLP & PCR

AP Biology

Unit 3


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


  • 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.

Southern blotting
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

Southern blotting1
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

Rflp animations
RFLP Animations

  • Animation #1

  • Animation #2

Polymerase chain reaction
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

Materials needed for pcr
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)

Overview of pcr
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)

Differences between dna replication pcr
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

Pcr animation
PCR animation

  • animation