Difference between revisions of "TAS2R38 PCR amplification"
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! Reagent !! Volume | ! Reagent !! Volume | ||
|- | |- | ||
| − | | '''extracted | + | | '''extracted gDNA''' || 10 µL |
|- | |- | ||
| − | | ''' | + | | '''Green GoTaq Mix + primers added''' || 20 µL |
|- | |- | ||
| − | + | | '''Final Volume''' || 30.0 µL | |
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | | '''Final Volume''' || | ||
|} | |} | ||
<br> | <br> | ||
| Line 22: | Line 16: | ||
'''PCR conditions:''' | '''PCR conditions:''' | ||
# 5 minutes at 95˚C. | # 5 minutes at 95˚C. | ||
| − | # | + | # 30 seconds at 95˚C. |
| − | # | + | # 30 seconds at 55˚C. |
| − | # | + | # 30 seconds at 72˚C. |
# repeat steps 2 - 5 29 more times. | # repeat steps 2 - 5 29 more times. | ||
# hold at room temperature. | # hold at room temperature. | ||
| Line 32: | Line 26: | ||
'''4.''' Before you can send your DNA off for sequencing, you will need to [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html clean up your PCR products] | '''4.''' Before you can send your DNA off for sequencing, you will need to [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html clean up your PCR products] | ||
| + | |||
| + | '''This protocol was developed by Dr. Mark Barsoum and the 2012 RISE students.''' | ||
| + | |||
| + | <center>'''References:'''<br></center> | ||
| + | * [http://www.ncbi.nlm.nih.gov/gene/5726 NCBI Gene entry] | ||
| + | * [http://www.ncbi.nlm.nih.gov/nuccore/NG_016141.1?from=5001&to=6143&report=fasta FASTA sequence] | ||
| + | * [http://omim.org/entry/607751 OMIM entry (brief review and some gene structure, including SNP markers)] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/12595690 Original cloning and identification of SNP markers] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/12584440 First identification of the TAS2R family] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/21036327 Overview of genetics of the chemical senses] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/17108952 Molecular cell biology of taste] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/22792271 Recent cell biology] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/18524836 Association with nicotine dependence and alcohol consumption in African Americans 1] | ||
| + | * [http://www.ncbi.nlm.nih.gov/pubmed/17250611 Association with nicotine dependence and alcohol consumption in African Americans 2] | ||
| − | ''' | + | '''Excellent review:'''<br> |
| + | Tepper Beverly J. 2008. Nutritional implications of genetic taste variation. Annual Review of Nutrition. 28: 367-88.<br> | ||
| + | <br> | ||
| + | '''Evolution and diversity:'''<br> | ||
| + | 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.<br> | ||
| + | <br> | ||
| + | '''More on nutrition and food selection:'''<br> | ||
| + | * 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. | ||
| + | <br> | ||
| + | '''Diseases:'''<br> | ||
| + | * 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. | ||
Latest revision as of 12:03, 3 October 2013
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:
- 5 minutes at 95˚C.
- 30 seconds at 95˚C.
- 30 seconds at 55˚C.
- 30 seconds at 72˚C.
- repeat steps 2 - 5 29 more times.
- hold at room temperature.
- 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.
- NCBI Gene entry
- FASTA sequence
- OMIM entry (brief review and some gene structure, including SNP markers)
- Original cloning and identification of SNP markers
- First identification of the TAS2R family
- Overview of genetics of the chemical senses
- Molecular cell biology of taste
- Recent cell biology
- Association with nicotine dependence and alcohol consumption in African Americans 1
- Association with nicotine dependence and alcohol consumption in African Americans 2
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.
