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Biotechnology: Restriction Enzyme Analysis of DNA

Biotechnology: Restriction Enzyme Analysis of DNA. AP Biology Investigation 9 Gel Electrophoresis Lab. Introduction. Genetic Engineering : process of manipulating genes and genomes Biotechnology : process of manipulating organisms or their components for the purpose of making useful products.

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Biotechnology: Restriction Enzyme Analysis of DNA

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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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