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 fragments that have sticky ends (single-stranded overhanging ends)
Cuts exactly in center of restriction site makes blunt ends

6. Blunt ends vs. “Sticky” ends

7. 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

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

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

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

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

13. 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

14. 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.

15. AP Bio Lab 9 Briefing Standard Sample: Lambda DNA cut with HindIII
Sizes of these fragments 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

16. Standard HindIII Lambda DNA Sample

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

18. Data Collection:

19. 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.

20. Lab Write-Up