Biology

5Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability

Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

STUDENT LAB INSTRUCTIONS

INTRODUCTION

Mammals are believed to distinguish only five basic tastes: sweet, sour, bitter, salty, and umami (the taste of monosodium glutamate). Taste recognition is mediated by specialized taste cells that communicate with several brain regions through direct connections to sensory neurons. Taste perception is a two-step process. First, a taste molecule binds to a specific receptor on the surface of a taste cell. Then, the taste cell generates a nervous impulse, which is interpreted by the brain. For example, stimulation of “sweet cells” generates a perception of sweetness in the brain. Recent research has shown that taste sensation ultimately is determined by the wiring of a taste cell to the cortex, rather than the type of molecule bound by a receptor. So, for example, if a bitter taste receptor is expressed on the surface of a “sweet cell,” a bitter molecule is perceived as tasting sweet.

A serendipitous observation at DuPont, in the early 1930s, first showed a genetic basis to taste. Arthur Fox had synthesized some phenylthiocarbamide (PTC), and some of the PTC dust escaped into the air as he was transferring it into a bottle. Lab-mate C.R. Noller complained that the dust had a bitter taste, but Fox tasted nothing—even when he directly sampled the crystals. Subsequent studies by Albert Blakeslee, at the Carnegie Department of Genetics (the forerunner of Cold Spring Harbor Laboratory), showed that the inability to taste PTC is a recessive trait that varies in the human population.

Bitter-tasting compounds are recognized by receptor proteins on the surface of taste cells. There are approximately 30 genes for different bitter taste receptors in mammals. The gene for the PTC taste receptor, TAS2R38, was identified in 2003. Sequencing identified three nucleotide

Albert Blakeslee using a voting machine to tabulate results of

taste tests at the AAAS Convention, 1938. (Courtesy Cold

Spring Harbor Laboratory Research Archives)

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positions that vary within the human population—each variable position is termed a single nucleotide polymorphism (SNP). One specific combination of the three SNPs, termed a haplotype, correlates most strongly with tasting ability.

Analogous changes in other cell-surface molecules influence the activity of many drugs. For example, SNPs in serotonin transporter and receptor genes predict adverse responses to anti-depression drugs, including PROZAC® and Paxil®.

In this experiment, a sample of human cells is obtained by saline mouthwash. DNA is extracted by boiling with Chelex resin, which binds contaminating metal ions. Polymerase chain reaction (PCR) is then used to amplify a short region of the TAS2R38 gene. The amplified PCR product is digested with the restriction enzyme HaeIII, whose recognition sequence includes one of the SNPs. One allele is cut by the enzyme, and one is not—producing a restriction fragment length polymorphism (RFLP) that can be separated on a 2% agarose gel.

Each student scores his or her genotype, predicts their tasting ability, and then tastes PTC paper. Class results show how well PTC tasting actually conforms to classical Mendelian inheritance, and illustrates the modern concept of pharmacogenetics—where a SNP genotype is used to predict drug response.

Blakeslee, A.F. (1932). Genetics of Sensory Thresholds: Taste for Phenyl Thio Carbamide. Proc. Natl. Acad. Sci. U.S.A. 18:120-130.

Fox, A.L. (1932). The Relationship Between Chemical Constitution and Taste. Proc. Natl. Acad. Sci. U.S.A. 18:115-120.

Kim, U., Jorgenson, E., Coon, H., Leppert, M., Risch, N., and Drayna, D. (2003). Positional Cloning of the Human Quantitative Trait Locus Underlying Taste Sensitivity to Phenylthiocarbamide. Science 299:1221-1225.

Mueller, K.L., Hoon, M.A., Erlenbach, I., Chandrashekar, J., Zuker, C.S., and Ryba, N.J.P. (2005). The Receptors and Coding Logic for Bitter Taste. Nature 434:225-229.

Scott, K. (2004). The Sweet and the Bitter of Mammalian Taste. Current Opin. Neurobiol. 14:423-427.

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

Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

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7Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability

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LAB FLOW

I. ISOLATE DNA BY SALINE MOUTHWASH

II. AMPLIFY DNA BY PCR

III. DIGEST PCR PRODUCTS WITH HaeIII

IV. ANALYZE PCR PRODUCTS BY GEL ELECTROPHORESIS

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

Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

8

Your teacher may instruct you to collect a small sample of cells to observe under a microscope.

Before pouring off supernatant, check to see that pellet is firmly attached to tube. If pellet is loose or unconsolidated, carefully use micropipet to remove as much saline solution as possible.

METHODS

I. ISOLATE DNA BY SALINE MOUTHWASH

1. Use a permanent marker to label a 1.5-mL tube and paper cup with your assigned number.

2. Pour saline solution into your mouth, and vigorously rinse your cheek pockets for 30 seconds.

3. Expel saline solution into the paper cup.

4. Swirl the cup gently to mix cells that may have settled to the bottom. Use a micropipet with a fresh tip to transfer 1000 µL of the solution into your labeled 1.5-mL microcentrifuge tube.

5. Place your sample tube, along with other student samples, in a balanced configuration in a microcentrifuge, and spin for 90 seconds at full speed.

6. Carefully pour off supernatant into the paper cup. Try to remove most of the supernatant, but be careful not to disturb the cell pellet at the bottom of the tube. (The remaining volume will reach approximately the 0.1 mark of a graduated tube.)

7. Set a micropipet to 30 µL. Resuspend cells in the remaining saline by pipetting in and out. Work carefully to minimize bubbles.

8. Withdraw 30 µL of cell suspension, and add it to a PCR tube containing 100 µL of Chelex®. Label the cap and side of the tube with your assigned number.

9. Place your PCR tube, along with other student samples, in a thermal cycler that has been programmed for one cycle of the following profile. The profile may be linked to a 4°C hold program. If you are using a 1.5-mL tube, use a heat block or boiling water bath.

Boiling step: 99°C 10 minutes

10. After boiling, vigorously shake the PCR tube for 5 seconds.

Reagents (at each student station)

0.9% saline solution, 10 mL 10% Chelex®, 100 µL (in 0.2- or 0.5-mL PCR

tube)

Supplies and Equipment

Permanent marker Paper cup Micropipets and tips (10–1000 µL) 1.5-mL microcentrifuge tubes Microcentrifuge tube rack Microcentrifuge adapters Microcentrifuge Thermal cycler (or water bath or heat

block) Container with cracked or crushed ice Vortexer (optional)

Food particles will not resuspend.

The near-boiling temperature lyses the cell membrane, releasing DNA and other cell contents.

Alternatively, you may add the cell suspension to Chelex in a 1.5-mL tube and incubate in a boiling water bath or heat block.

9Using a Single-Nucleotide Polymorphism to Predict Bitter-Tasting Ability

Copyright © 2006, Dolan DNA Learning Center, Cold Spring Harbor Laboratory. All rights reserved.

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