1. “Using a Single Nucleotide Polymorphism to Predict Bitter Tasting Ability” SNP Lab Instructions

2. Standard
Using lab techniques, such as DNA extraction, PCR, restriction enzymes and gel electrophoresis to determine the presence or absence of a SNP in a designated gene sequence

3. With this lab, I can… Extract my own DNA from my cheek cells
Amplify my DNA using PCR
Understand how HAEIII enzyme will identify a SNP in genotype
Determine my own genotype using gel electrophoresis

4. Lab Group Roles: Materials Manager Quality Control Manager
Time Manager
Task Manager
Everyone’s Responsibility Today:
Lab Technician
Recorder

5. 2 minutes
Hair pulled back
Goggles on
Supplies gathered

6. Supplies needed per lab group
Supplies needed per student
2 – yellow 1.5 ml tubes (label)
1 – dixie cup
1 – disposable 1 ml pipette
1 – toothpick
1 – purple (chelex) tube
1 – µl micropipette
1 – box of pipette tips
1 – test tube holder
1 – sharpie (fine point)
Supplies needed per lab group

7. Step 1: Obtain some cheek cells
30 seconds
1st - Swish vigorously
2nd - Gently scrape cheek with toothpick & mix into cup

8. Step 2: Separate the cheek cells from the Gatorade/spit mix
Think: Where is the DNA right now?

9. Step 3: Release the DNA from the cheek cells
In heat block
Detergent to break open cell membranes

10. Step 4: Extract only the DNA from the mix
Think: Where is the DNA right now?

11. Think-Pair-Share
Why did we discard the supernatant and keep the pellet after the first centrifuge step, and keep the supernatant and discard the pellet after the second centrifuge step?

12. Lab Journal
Draw a flowchart that traces the DNA through this part of the lab.
Begin with your cheek cells
End with the DNA on ice

13. Clean Up

14. Standard
Using lab techniques, such as DNA extraction, PCR, restriction enzymes and gel electrophoresis to determine the presence or absence of a SNP in a designated gene sequence

15. With this lab, I can… Extract my own DNA from my cheek cells
Amplify my DNA using PCR
Understand how HAEIII enzyme will identify a SNP in genotype
Determine my own genotype using gel electrophoresis

16. Materials needed per student:
1 – PCR (.2 ml) tube w/ white bead
1 – .5 – 10 µl micropipette
1 – box pipette tips
1 – sharpie
1 – tiny piece of colored tape
Materials needed per lab group:

17. Part II: PCR
2.5 μl
22.5 μl
Pulse only

18. Clean Up

19. Materials needed per student:
2 – 1.5 ml tubes
Label one tube “U” and the other tube “D”
Also label with your class ID #
1 – .5 – 10 µl micropipette
1 – box pipette tips
1 – sharpie
Materials needed per lab group:

20. Standard
Using lab techniques, such as DNA extraction, PCR, restriction enzymes and gel electrophoresis to determine the presence or absence of a SNP in a designated gene sequence

21. With this lab, I can… Extract my own DNA from my cheek cells
Amplify my DNA using PCR
Understand how HAEIII enzyme will identify a SNP in genotype
Determine my own genotype using gel electrophoresis

22. Part III: Digest DNA with HaeIII
Put Tube “U” back on ice.
10 μl
1 μl
Do this for both tubes
ONLY tube “D”
ONLY tube “D”
ONLY tube “D”
ONLY tube “D”
Pulse only
in heat block
For 2 hours

23. HaeIII and TAS2R38 gene HaeIII restriction enzyme
5’ GGCC 5’ ---GG CC--- 3’
3’ CCGG 3’ ---CC GG--- 5’
TAS2R38 gene variations
NONTASTER (t) TASTER (T)
GGCGGGCACT GGCGGCCACT
CCGCCCGTGA CCGCCGGTGA

24. HaeIII and TAS2R38 gene NONTASTER (t) TASTER (T) GGCGGGCACT GGCGGCCACT
CCGCCCGTGA CCGCCGGTGA
One band - Two bands
Full length of gene - One band 177 bp
220 bp - One band 44 bp

26. Lab Journal Assignment:
Write a formal hypothesis for your expected results
You already wrote an informal prediction based on your phenotype. Now let’s turn that into a formal hypothesis.
If, then statements help.
Example: If gender affects resting heart rate, then males will have a lower resting heart rate than females

27. Standard
Using lab techniques, such as DNA extraction, PCR, restriction enzymes and gel electrophoresis to determine the presence or absence of a SNP in a designated gene sequence

28. With this lab, I can… Extract my own DNA from my cheek cells
Amplify my DNA using PCR
Understand how HAEIII enzyme will identify a SNP in genotype
Determine my own genotype using gel electrophoresis

29. Step 4: Gel Electrophoresis
20 µl – DNA marker
10 µl – Undigested DNA
16 µl – Digested DNA

30. Interpreting Gel Results
(tt)Non-Taster Strong Taster (TT)
Think: What would three bands mean?

31. Example Gel Results

32. Did/Can you… Extract my own DNA from my cheek cells
Amplify my DNA using PCR
Understand how HAEIII enzyme will identify a SNP in genotype
Determine my own genotype using gel electrophoresis

33. Standard
Using lab techniques, such as DNA extraction, PCR, restriction enzymes and gel electrophoresis to determine the presence or absence of a SNP in a designated gene sequence

34. 2.1.3 Conclusion Questions
Explain how the HaeIII enzyme discriminates between the C-G polymorphism in the TAS2R38 gene.
Using what you know about genetics, SNPs, and the PTC gene, explain why it is possible for a person to be a “weak taster.”
Some studies have shown that PTC “tasters” are less likely to become smokers. Why do you think scientists are seeing this correlation?
How can the techniques described in this lab be used to test for human disease genes? Would this type of testing work on every disease with a genetic component?
What ethical issues are raised by human DNA typing experiments?

35. Reflection: In lab journal
How well does phenotype work to predict genotype?
Use not only your results, but those of the class to answer this question.
Provide evidence from the lab and your knowledge of the TAS2R38 gene and its inheritance pattern to support your answer.