Linking phenotype to genotype in the classroomminipcr
Guest post by Vivek Bajaj, PhD, MA, Science Instructor and Director of Professional Development, The Innovation Institute.
In our molecular biology and genetics class, we are currently studying the concept of phenotype (the outwardly expressed trait) and how this relates to a genotype (the information in the genetic code as determined by DNA sequence). Nucleotide changes can alter the genes, resulting in non-functional proteins or indeed different proteins that result in different phenotypes. Single nucleotide polymorphisms or SNPs are variations at single positions among individuals. They are the most common form of variation in the human genome and account for much of our genetic and phenotypic diversity.
In this lab we explored a SNP associated with the ability to taste the chemical phenylthiocarbamide (PTC) which tastes bitter. We assessed this phenotype and how it correlated with the students’ genotype at the TAS2R38 locus, which codes a gene for a taste receptor expressed in gustatory papillae (taste buds). The two alleles for the TAS2R38 gene are a “taster” and a “non-taster allele.” The difference in these alleles are the result of three SNPs within the gene coding for this receptor protein.
In order to assess their phenotypes, the students tasted a strip of PTC paper and classified their phenotype as either strong taster, moderate taster, or non-taster. These predicted the following respective genotypes—TT (homozygous taster) for strong, Tt (heterozygous) for moderate, and tt (homozygous non-taster) for a non-taster. A control strip of plain paper that did not contain any PTC was also tasted.
To assess genotypes, cheek cells were scraped from each student and DNA was isolated from the cells using an extraction buffer. The DNA from this preparation was used to amplify a 250 base pair region of the TAS2R38 gene that contains one of the SNPs. In the taster allele, the SNP in this region generates a specific restriction site for the restriction enzyme Fnu4H1. In the non-taster allele, this restriction site is absent. Thus the taster vs non-taster genotypes can be distinguished by treating the PCR products with the restriction enzyme and analyzing the results using gel electrophoresis. Gel electrophoresis is a method of analyzing DNA based on its size (base pair length). As a phenotypic taster, the genotype will be represented as two distinct bands on the gel (150bp and 100bp) since the restriction site is present. In a non-taster, only one 250bp band will appear. Since we carry two copies of every gene in our cells, an intermediate phenotype (moderate taster, Tt) is also possible. This genotype is represented as the appearance of all three bands. One copy of the gene has the restriction site and the other does not.
Please refer to the gel picture for the following analysis of the data. Lane number is from left to right, with lane 1 being a control set of DNA size standards. Correlation between phenotypes and genotypes was relatively good. As can be seen from the gel, in lane 2 (going from left to right) the phenotype was moderate taster. Indeed 3 bands can be seen indicating a probable Tt genotype. The lane 3 student was a strong taster, consistent with the presence of two bands. The lane 7 student was a non-taster, consistent with the presence of one band. Lanes 6 and 9 students identified themselves as non-tasters, however the gel seems to indicate they are heterozygous (Tt) tasters. As an explanation for this apparent discrepancy, polymorphisms in TAS2R38 can account for ~85% of the variance in PTC tasting ability. The other 15% may be due to the involvement of other genes and/or simply a broad spectrum of tasting sensitivity. The expression of the phenotype is influenced by other factors, such as gender (women are more sensitive to PTC and are more likely to be super-tasters than men). Other factors besides genetics can influence the sense of taste, such as temperature, aging, colds, flu, and allergies.
The Innovation Institute would like to thank our friends at miniPCR for providing us with the reagents and equipment to do this lab. We were particularly excited by our results as this is the first time we have amplified our own DNA for genomic analysis.