Analyzing the PTC Taster Gene (tas2r38) through PCR Amplification The human taste process *Food is recognized by a taste receptor where the protein binds to the receptor most closely related to the 5 tastes: Sweet, Bitter, Sour, Salty, and Umami *The shapes of the protein closely matches the shape of the related receptor. *The receptor sends a nerve impulse to your brain which interprets it as one of those tastes. *The receptors, neuron messages and interpretation are all determined by your genetics, though can be altered by environment or injury. The human taste process Bitter Tasting Chemical PTC ( ) *Arther Fox in the late 20’s was using this chemical in a lab at Du Pont. *His collegue complained that he could taste the chemical in the air, but Fox was not experiencing the same taste sensation. *This was tested with many co-workers and friends and genetics was thought to play a role. *It is said that paternity was even tested by this early on. Bitter Tasting Chemical PTC *Albert Blakeslee, in 1932, determined that the ability to taste this chemical must be a dominant trait when most test subjects could taste the chemical.* *In 2004, the gene responsible was located on chromosome 7. We get one allele from our mother and one from our father. The gene is called TAST2R38 *The gene is just over 1000bp in length *There are three areas of variance that causes the taster/nontaster forms or 3 SNPS-Single Nucleotide polymorphism. Postition Taster Nontaster 145 C (proline) G (alanine) 785 C (alanine) T (valine) 886 G (valine) A (isoleucine) In this lab, you will: *Extract your own DNA using Chelex *Use Polymerase Chain Reaction (PCR) to amplify a portion of your own TAS2R38 gene *Use a restriction enzyme to potentially cut your TAS2R38 genes *Use gel electrophoresis to separate any fragments produced by the restriction enzyme activity Amplifying TAST2R38 with PCR *Primers used in the experiment: CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG AGGTTGGCTTGGTTTGCAATCATC Amplified Region is 221 bp with the 145 position SNP Predicting Alleles and Trait *If PCR was done correctly, everyone will have a very large amount of 221 bp PCR product *To predict the alleles, you have to separate the dominant from recessive *This is where the 145 SNP comes in Predicting Alleles and Trait *Using HAEiii enzyme, a restriction digest can be done at this SNP *HAEiii restriction site is GGCC • The pcr product that has GGCC will be cut into two pieces • The pcr product that has GGGC will not cut • Those that are heterozygous will have a mixture of both. Some product with GGCC and some product with GGGC RDigest of PCR Product PCR PRODUCT = 221 BP length 145 SNP ...GGCGGGCACT... ... CCGCCCGTGA... ...GGCGGCCACT... ...CCGCCGGTGA... Non-taster allele taster allele Add HAEiii for RD ...GGCGG ...CCGCC CCACT... GGTGA... 2 pieces 44bp 177 bp ...GGCGGGCACT... ...CCGCCCGTGA... 1 piece 221 bp Visualization of DNA results using gel electrophoresis tt TT Tt U D U D U D M Non-Taster Taster Taster Hetero Day 1: Isolating your DNA 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Label two microtubes and cup of saline solution with your identity number given to you by your teacher. Pour saline solution in your mouth and vigorously rinse your cheeks for 1 minute. Expel solution back into cup. Carefully, over garbage or sink pour spit into your microtube. Close tube tightly and put into spectrafuge (high powered). It will spin for 5 minutes. Look for cell pellet. Carefully pour of supernatant into waste and keep cell pellet in tube. Pipette 250 ul of 10% chelex into your microtube and resuspend cell pellet by vortexing or mixing solution up and down in pipette. Once resuspended fully, close lid tightly and poke a hole using a tack into the top of the lid. Place into 99o hot block for 10 minutes. Resuspend solution one more time and then spin in a minicentrifuge for 30 seconds so the Chelex beads fall to bottom of tube. Without disturbing the beads at the bottom, pipette about 50 ul of solution into second tube. This is your extracted DNA. Your teacher will freeze your tube for next class. Day 2: Performing PCR 1. PCR tubes should be on ice. Label PCR tube with your identity number then put back on ice. 2. Obtain your DNA tube from your teacher and carefully thaw it on countertop. 3. Pipette 5 ul of your DNA into your PCR tube. 4. Come up to front of room with your 20 ul pipette set at 20 ul and a fresh tip. Pipette 20 ul of master mix from the teachers stock 5. Return to your seat and quickly vortex pcr tube, then spin it down in minicentrifuge. 6. Bring it to the pcr machine, put on ice if teacher is waiting for other student tubes or put directly in PCR machine. 7. Your teacher will run the program, once all tubes are finished. ** Hint: Work carefully and quickly to get this done. The master mix is time sensitive. Day 3: Restriction Digest of PCR Product 1. Label two 1.5 ml microtube with your number and one with “U” for Undigested and one with “D” for digested. 2. Obtain your PCR product. 3. Pipette 10ul of PCR product into “U” tube. 4. Pipette 12ul of PCR product into your “D” tube. 5. With new tip, pipette 2ul of HAEIII enzyme into your “D” Tube. 6. Mix by tapping tube, give it a quick spin down in microcentrifuge and put into waterbath at 37o for 1 hour. 7. Bring your “U” tube to the front and your teacher will pull your “D” tube and put both into the refrigerator for tomorrow. Day 4: Running your PCR Products “U” and “D” on a 2% PCR Gel 1. Obtain your “U” and “D” tube. 2. Set up your electrophoresis box with 1x TAE gel and a 2% PCR gel. 3. Label two new microtubes with “U” and “D” 4. Pipette 2ul of loading dye into your new “U” tube and “D” tube. 5. Pipette 10 ul sample from your original “U” into your new “U” tube. 6. Pipette 10 ul of sample from your original “D” tube into your new “D” tube. 7. Add 3 ul of dH2O to each one of the new tubes. 8. Load 10ul of PCR Marker into the first lane of gel. 9. Load 12 ul of “U” tube into next open lane. 10. Load 12 ul of “D” tube into next open lane. 11. Record which lanes your samples are in. 12. When all lanes are loaded, close the electrophoresis box and run gel at 75V for 35-40 minutes. 13. Carefully remove your gel from box when finished running and take a picture of your gel using the UV camera. 14. Analyze your results.
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