Genetics Techniques RFLP PCR AP Biology Unit 3

Genetics Techniques: RFLP & PCR AP Biology Unit 3

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

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.

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 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 • Animation #1 • Animation #2

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 • • 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) • Mg 2+ (cofactor that DNA Polymerase III needs to work) • Buffer • Thermocycler (machine that changes temperatures)

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 • 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 • animation