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 l 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 l. Resuspend cells by pipetting in and out.

8. Withdraw 30 l and add it to a PCR tube containing 100 l 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 l 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 Ready-to-go PCR bead. Label tube with your initials.

2. Add 22.5 l of PTC primer/loading dye mix to the tube. Allow the bead to dissolve for a minute or so.

3. Add 2.5 l 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 l of your PCR product to the U tube. Store this on ice until Day 4.

3. Add 1 l 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 l of marker into the far left lane of the gel.

2. Load 10 l of the U and 16 l 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?