Download PTC Lab Instructions/Information

“Using a Single Nucleotide
Polymorphism to Predict Bitter
Tasting Ability”
SNP Lab Instructions
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
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
Supplies needed per student
• 2 – 1.5 ml tubes (label w/ class id #)
• 1 – dixie cup
• 1 – disposable 1 ml pipette
Supplies needed per lab group
• 1 – 20-200 µl micropipette
• 1 – box of pipette tips
• 1 – sharpie (fine point)
Step 1: Obtain some cheek cells
30
• 1st - Swish vigorously
• 2nd - Gently scrape
cheek with toothpick &
mix into cup
Step 2: Separate the cheek
cells from the Gatorade/spit mix
Think: Where is the DNA right now?
Step 3: Release the DNA from
the cheek cells
In heat
block
Detergent to break open
cell membranes
Lab Journal
• Draw a flowchart that traces the DNA
through part I of the lab
Step 4: Extract only the DNA
from the mix
Think: Where is the DNA right now?
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?
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
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
Materials needed per student:
• 1 – PCR .2 ml tube w/ white bead
Materials needed per lab group:
• 1 – .5 – 10 µl micropipette
• 1 – box pipette tips
Part II: PCR
22.5 μl
Pulse only
2.5 μl
Materials needed per student:
• 2 – 1.5 ml tubes
• Label one tube “U” and the other tube “D”
• Also label with your class ID #
Materials needed per lab group:
• 1 – .5 – 10 µl micropipette
• 1 – box pipette tips
• 1 – sharpie
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
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
Part III: Digest DNA with HaeIII
10 μl
Do this for both tubes
ONLY tube “D”
1 μl
Put Tube “U” back on
ice.
ONLY tube “D”
ONLY tube “D”
ONLY tube “D”
Pulse only
in heat
block
For 2 hours
HaeIII and TAS2R38 gene
• HaeIII restriction enzyme
– 5’ GGCC
– 3’ CCGG
5’ ---GG
3’ ---CC
CC--- 3’
GG--- 5’
• TAS2R38 gene variations
NONTASTER (t)
TASTER (T)
GGCGGGCACT
CCGCCCGTGA
GGCGGCCACT
CCGCCGGTGA
HaeIII and TAS2R38 gene
NONTASTER (t)
TASTER (T)
GGCGGGCACT
CCGCCCGTGA
GGCGGCCACT
CCGCCGGTGA
– One band
– Full length of gene
• 220 bp
- Two bands
- One band 177 bp
- One band 44 bp
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
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
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
Step 4: Gel Electrophoresis
20 µl – DNA marker
10 µl – Undigested DNA
16 µl – Digested DNA
Interpreting Gel Results
(tt)Non-Taster
Strong Taster (TT)
Think: What would three bands mean?
Example Gel Results
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
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
2.1.3 Conclusion Questions
1. Explain how the HaeIII enzyme discriminates between
the C-G polymorphism in the TAS2R38 gene.
2. Using what you know about genetics, SNPs, and the
PTC gene, explain why it is possible for a person to be a
“weak taster.”
3. Some studies have shown that PTC “tasters” are less
likely to become smokers. Why do you think scientists
are seeing this correlation?
4. 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?
5. What ethical issues are raised by human DNA typing
experiments?
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.