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Genetic Analysis through Restriction Enzyme Gel Electrophoresis

Explore the process of genetic engineering and biotechnology through the analysis of DNA using restriction enzymes and gel electrophoresis. Learn about restriction enzymes, RFLP analysis, and how DNA profiles are created. Solve the mystery of Ms. Mason's disappearance using AP Biology Lab 9 techniques.

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Genetic Analysis through Restriction Enzyme Gel Electrophoresis

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  1. Biotechnology: Restriction Enzyme Analysis of DNA AP Biology Investigation 9 Gel Electrophoresis Lab

  2. Introduction • Genetic Engineering: process of manipulating genes and genomes • Biotechnology: process of manipulating organisms or their components for the purpose of making useful products.

  3. Restriction Enzymes (RE) • Also called Restriction Endonucleases • Used to cut DNA strands at specific locations (restriction sites) • Discovered in bacteria used to repair DNA & defend against bacteriophages • Names: EcoRI (Escherichia coli), HindIII (Haemophilus influenzae) • Biologists use RE’s to manipulate & analyze DNA

  4. Palindrome = DNA sequence (4-10 base pairs) that reads same from both directions • EcoRI: 5’-GAATTC-3’  cuts G |AATTC • PstI: 5’-CTGCAG-3’  cuts CTGCA | G • Cuts in 2 places make restriction fragmentsthat have sticky ends(single-stranded overhanging ends) • Cuts exactly in center of restriction site makes blunt ends

  5. Blunt ends vs. “Sticky” ends

  6. AP Bio Lab 9 Briefing • RFLP = Restriction Fragment Length Polymorphism • RFLP Analysis: use RE’s to cut DNA so that a gel electrophoresis can be run to create a DNA profile

  7. Gel Electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current

  8. Gel Electrophoresis • DNA (- charged) will migrate towards (+) end • Shorter DNA  moves faster • Longer DNA  move slower

  9. How RE’s are used to create DNA profiles

  10. How RE’s are used to create DNA profiles

  11. The Disappearance of Ms. Mason The Crime: Ms. Mason, an AP Biology teacher at FHS (Fictional High School), is missing. There are blood drops found at the crime scene (CS). The Suspects: Five students who are failing Ms. Mason’s class • Suspect 1 (S1) – Amy • Suspect 2 (S2) – Bobby • Suspect 3 (S3) – Carla • Suspect 4 (S4) – Drew • Suspect 5 (S5) – Ellie

  12. What happened to Ms. Mason? Your Job: Determine whose DNA matches the blood drops found at the crime scene by analyzing the suspect DNA profiles created using gel electrophoresis.

  13. AP Bio Lab 9 Briefing • Standard Sample: Lambda DNA cut with HindIII • Sizes of thesefragments are known • Used as a basis for comparison to determine sizes of unknown DNA fragments • Create standard curve by graphing known data on semi-log paper

  14. Standard HindIII Lambda DNA Sample

  15. Standard Curve(plot of distance migrated vs. DNA size (base pairs)

  16. Data Collection:

  17. Data Analysis • Using a ruler, measure the distance from the well to the center of each DNA band. Record it in the data table. • Using the data from the known HindIII lambda DNA, plot distance vs. size for Bands 2-6 on the semilog graph paper. Draw a best-fit line. This will be your standard graph. • Using the standard graph created in #2, determine the approximate size of each suspect or crime scene fragment. Fill in the data table.

  18. Lab Write-Up

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