TAS2R38 PCR amplification

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1. When the DNA extraction cools (Isolate_genomic_DNA_from_a_single_hair_follicle), vortex the tubes for 30 seconds and then set up a new 500 µL microfuge tube by adding the following:

Reagent Volume
extracted gDNA 10 µL
Green GoTaq Mix + primers added 20 µL
Final Volume 30.0 µL


2. Run the thermocycler program called TAS2R38 with the heated lid enabled. PCR conditions:

  1. 5 minutes at 95˚C.
  2. 30 seconds at 95˚C.
  3. 30 seconds at 55˚C.
  4. 30 seconds at 72˚C.
  5. repeat steps 2 - 5 29 more times.
  6. hold at room temperature.
  7. END

3. When the PCR is completed (~2 hours), store DNA frozen until you are ready to clean up the DNA in order to have your two alleles sequenced.

4. Before you can send your DNA off for sequencing, you will need to clean up your PCR products

This protocol was developed by Dr. Mark Barsoum and the 2012 RISE students.

References:


Excellent review:
Tepper Beverly J. 2008. Nutritional implications of genetic taste variation. Annual Review of Nutrition. 28: 367-88.

Evolution and diversity:
Kim Unkyung, Wooding Stephen, Ricci Dante, Jorde Lynn B, and Drayna Dennis. 2005. Worldwide haplotype diversity and coding sequence variation at human bitter taste receptor loci. Human Mutation. 26: 199-204.

More on nutrition and food selection:

  • Duffy, V. B., Hayes, J. E., Davidson, A. C., Kidd, J. R., Kidd, K. K., & Bartoshuk, L. M. 2010. Vegetable intake in college-aged adults is explained by oral sensory phenotypes and TAS2R38 genotype. Chemosensory Perception, 3(3-4), 137-148.
  • Garcia-Bailo, B., Toguri, C., Eny, K. M., & El-Sohemy, A. 2009. Genetic variation in taste and its influence on food selection. Omics-a Journal of Integrative Biology, 13(1): 69-80.
  • Lipchock, S. V., Reed, D. R., & Mennella, J. A. 2012. Relationship between bitter-taste receptor genotype and solid medication formulation usage among young children: A retrospective analysis. Clinical Therapeutics, 34(3), 728-733.
  • Mennella, J., Pepino, Y., & Reed, D. 2005. Genetic and environmental determinants of bitter perception and sweet preferences. Pediatrics, 115(2), E216-E222.


Diseases:

  • Carrai, M., Steinke, V., Vodicka, P., Pardini, B., Rahner, N., Holinski-Feder, E., . . . Campa, D. 2011. Association between TAS2R38 gene polymorphisms and colorectal cancer risk: A case-control study in two independent populations of caucasian origin. Plos One, 6(6), e20464.
  • Basson MD,Bartoshuk LM,Dichello SZ,Panzini L,Weiffenbach JM,et al. 2005. Association between 6-n-propylthiouracil (PROP) bitterness and colonic neoplasms. Dig. Dis. Sci. 50:483–89.
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