Using a Single-Nucleotide Polymorphism to Predict Bitter Tasting Ability

1. Using a Single-Nucleotide Polymorphism to Predict Bitter Tasting Ability

2. 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.

3. 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

5. Day 1 Procedure • Label a 1.5 mL tube with your initials. • Rinse your mouth with saline solution for 30 seconds. • Spit solution into paper cup. • Transfer 1000 ml of your saliva to your 1.5 mL tube. • Spin tube in a microcentrifuge at full speed for 90 seconds.

6. Carefully pour off supernatant into the paper cup. • Set a micropipet to 30 ml. Resuspend cells by pipetting in and out. • 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. • Place your tube in a thermal cycler programmed at 99oC for 10 minutes. • After boiling, vigorously shake the PCR tube for 5 seconds.

7. Spin tube for 90 seconds at full speed. • 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. • Label the cap and side of the tube with your initials. • Place your sample into the freezer.

8. Day 2 Procedure • Obtain a PCR tube containing a Ready-to-go PCR bead. Label tube with your initials. • Add 22.5 ml of PTC primer/loading dye mix to the tube. Allow the bead to dissolve for a minute or so. • Add 2.5 ml of your cheek cell DNA directly into the primer/loading dye mix.

9. Place your tube in a thermal cycler. Program 30 cycles Denaturing: 94oC 30 sec Annealing: 64oC 45 sec Extending: 72oC 45 sec

10. Day 3 Procedure • Label a 1.5 mL tube with your initials and with a U. • Transfer 10 ml of your PCR product to the U tube. Store this on ice until Day 4. • Add 1 ml of restriction enzyme HaeIII directly into the PCR product remaining in the PCR tube. Label this tube D. • Place your PCR tube in a thermal cycler for 30 minutes at 37oC. • Store sample in freezer until day 4. • Make a 2% agarose gel.

11. Day 4 Procedure • Load 20 ml of marker into the far left lane of the gel. • Load 10 ml of the U and 16 ml of the D into different wells. • Make a diagram of your gel with the lanes labeled. • Run gel until dye has moved at least 50 mm from the wells. • Mrs. Swenson will stain gels with EtBr.

12. Conclusion • Does your actual genotype match your predicted genotype? Your predicted genotype is based on the results of the PTC taster taste. • How does HaeIII discriminate between tasters and nontasters? • Why do heterozygous tasters show 3 bands?