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. Supplies needed per lab group
Supplies needed per student
2 – 1.5 ml tubes (label w/ class id #)
1 – dixie cup
1 – disposable 1 ml pipette
1 – µl micropipette
1 – box of pipette tips
1 – sharpie (fine point)
Supplies needed per lab group

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

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

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

8. Lab Journal
Draw a flowchart that traces the DNA through part I of the lab

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

10. 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?

11. 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

12. 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

13. Materials needed per student:
1 – PCR .2 ml tube w/ white bead
1 – .5 – 10 µl micropipette
1 – box pipette tips
Materials needed per lab group:

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

15. 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:

16. 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

17. 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

18. 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

19. 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

20. 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

22. 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

23. 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

24. 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

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

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

27. Example Gel Results

28. 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

29. 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

30. 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?

31. 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.