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 by cutting up foreign DNA
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. RFLP Analysis 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 Case of the Missing Biology Teacher

14. The Disappearance of Ms. Mason
The Crime: Ms. Mason, an AP Biology teacher at FHS (Fictional High School), is missing. There were 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

15. 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.

16. Overview of Lab Pre-Lab: DNA cut with restriction enzymes
Day 1: Load and run gels
Day 2: Analyze and interpret results
Generate standard curve
Analyze results and weigh evidence

17. Pre-Lab
Samples of DNA were cut with restriction enzymes EcoRI and PstI
Handout: “Lesson 1: Restriction Digest of DNA Samples”.

18. Lab Day 1 Your lab group will be given 7 tubes:
“S” = Standard DNA Sample (for comparison)
“CS” = Crime Scene DNA
S1-S5 = Suspects’ DNA
Spin down the contents of the tubes using a centrifuge.
Handout: “Lesson 2: Agarose Gel Electrophoresis”.

19. Loading the Gel Add 5 μl of Loading Dye “LD” to each test tube sample.
Mix tubes by flicking with finger. Spin the tubes to bring contents to bottom.
Load the gel (from left  right):
Lane 1: DNA Standard (S), clear tube, 10 μl
Lane 2: CS, green tube, 20 μl
Lane 3: S1, blue tube, 20 μl
Lane 4: S2, orange tube, 20 μl
Lane 5: S3, purple tube, 20 μl
Lane 6: S4, pink tube, 20 μl
Lane 7: S5, yellow tube, 20 μl

20. Loading DNA samples into the wells

22. Running a Gel + _

23. Lab Day 2:
Using your phone or an iPad, take a good clear (straight-on) photo of your gel.
Make sure the wells and each band of DNA is visible
Handouts: “Electrophoresis Data Table” and “Semilog Graph Paper”.

24. Standard Curve 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

25. Standard HindIII Lambda DNA Sample

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

27. Data Analysis
Using a ruler, measure the distance (in mm) from the well to the center of each DNA band. Record the distances in the Electrophoresis Data table.
Using the data from the known Lambda/HindIII Standard DNA, plot distance vs. size for Bands 2-6 on the Semilog Graph Paper. Draw a best-fit line. This will be your standard curve.
Using the standard curve created in #2, determine the approximate size of each suspect or crime scene fragment. Fill in the data table for Suspects 1-5.

28. Post-Lab Questions
What were we trying to determine from this lab? Restate the problem.
Which of the DNA samples were fragmented? What would the gel look like if the DNA were not fragmented?
What caused the DNA to become fragmented?
What determines where a restriction endonuclease will "cut" a DNA molecule?

29. Post-Lab Questions
A restriction endonuclease "cuts" two DNA molecules at the same location. What can you assume is identical about the molecules at that location?
Do any of your suspect samples appear to have EcoRI or PstI recognition sites at the same location as the DNA from the crime scene?
Based on the above analysis, do any of the suspect samples of DNA seem to be from the same individual as the DNA from the crime scene? Describe the scientific evidence that supports your conclusion.

30. Conclusion Complete the Post-Lab Questions (#1-7) for analysis.
Attach into your lab notebook:
Lesson 1, Lesson 2, Photo of gel, Electrophoresis Data Table, Semilog Graph, Post-Lab Questions