Download Using a Single-Nucleotide Polymorphism to Predict Bitter Tasting

Using a Single-Nucleotide
Polymorphism to Predict Bitter
Tasting Ability
Background
• Bitter Tasting compounds are recognized by
receptor proteins on the surface of taste cells.
• There are ~30 different genes for bitter taste
receptors in mammals.
• The gene for the PTC taste receptor, TAS2R38,
was identified in 2003.
• There are 3 nucleotide positions that vary within
the human population.
• One specific combination of the 3 SNPs,
correlates most strongly with tasting ability.
Genetic of PTC
• The inability to taste PTC is a recessive
trait (tt).
• Individuals with Homozygous dominant
(TT) or Heterozygous genotypes (Tt) have
the ability to taste PTC.
• ~75% of humans can taste PTC
Day 1 Procedure
1. Label a 1.5 mL tube with your initials.
2. Rinse your mouth with saline solution for
30 seconds.
3. Spit solution into paper cup.
4. Transfer 1000 ml of your saliva to your
1.5 mL tube.
5. Spin tube in a microcentrifuge at full
speed for 90 seconds.
6. Carefully pour off supernatant into the
paper cup.
7. Set a micropipet to 30 ml. Resuspend
cells by pipetting in and out.
8. Withdraw 30 ml and add it to a PCR tube
containing 100 ml of Chelex. Label the
cap and side of PCR tube with your
initials.
9. Place your tube in a thermal cycler
programmed at 99oC for 10 minutes.
10. After boiling, vigorously shake the PCR
tube for 5 seconds.
11. Spin tube for 90 seconds at full speed.
12. Use a pipet to transfer 30 ml of the clear
supernatant into a clean 1.5 mL tube. Be
careful to avoid pipetting any cell debris
and Chelex beads.
13. Label the cap and side of the tube with
your initials.
14. Place your sample into the freezer.
Day 2 Procedure
1. Obtain a PCR tube containing a Readyto-go PCR bead. Label tube with your
initials.
2. Add 22.5 ml of PTC primer/loading dye
mix to the tube. Allow the bead to
dissolve for a minute or so.
3. Add 2.5 ml of your cheek cell DNA
directly into the primer/loading dye mix.
4. Place your tube in a thermal cycler.
Program
30 cycles
Denaturing:
94oC
30 sec
Annealing:
64oC
45 sec
Extending:
72oC
45 sec
Day 3 Procedure
1. Label a 1.5 mL tube with your initials and with
a U.
2. Transfer 10 ml of your PCR product to the U
tube. Store this on ice until Day 4.
3. Add 1 ml of restriction enzyme HaeIII directly
into the PCR product remaining in the PCR
tube. Label this tube D.
4. Place your PCR tube in a thermal cycler for 30
minutes at 37oC.
5. Store sample in freezer until day 4.
6. Make a 2% agarose gel.
Day 4 Procedure
1. Load 20 ml of marker into the far left lane
of the gel.
2. Load 10 ml of the U and 16 ml of the D
into different wells.
3. Make a diagram of your gel with the
lanes labeled.
4. Run gel until dye has moved at least 50
mm from the wells.
5. Mrs. Swenson will stain gels with EtBr.
Conclusion
1. Does your actual genotype match your
predicted genotype? Your predicted
genotype is based on the results of the
PTC taster taste.
2. How does HaeIII discriminate between
tasters and nontasters?
3. Why do heterozygous tasters show 3
bands?