Our group really enjoyed learning about the PTC Taster/Nontaster that took place over multiple lab sessions. This is an excellent experiment to follow in order to gain a better understanding and prepare for using restriction enzymes to genotype SNPs.
The diagram below shows the Tas2R38 gene. Underlined are 3 SNPs that are associated with the taster allele. The blue shows the bases associated with the non-taster allele. The green and red represent the primers used by Wellesley College. (Our primers are a bit different to create greater differences between the bands.)
Fnu4H1 Restriction Site
3’-CGNCG-5” N can be any of the 4 bases
Here are the phenotypes we observed for the class:
Taylor – light bitterness
Carson – bitter
Regina – bitter
Anna – no taste
Mike – no taste
Cesar – no taste?
Take a further look and check out the NCBI gene info here. Then browse their database to see if you can find the multiple SNPs in the TAS2R38 gene!
Due to exciting future Citizen Salmon events we’ve been a bit slow on the Bioinformatics front. We are currently focused on the concept of using restriction enzymes to identify what alleles a sample may have for a specific SNP. Remember that SNPs are single nucleotide polymorphisms, and can present themselves as single base replacements (‘T’ or ‘G’) or as deletions. Restriction enzymes allow us to cut DNA at particular sites:
NEB provides a large range of restriction enzymes with different recognition sites. https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes
To practice this concept we’ll soon be running the PTC Tasting Lab. PTC is a chemical that tastes sour to some people while others may taste nothing. This is a common trait typically talked about in high school because it strongly follows Mendelian inheritance. It is also useful for us because much of the variation in bitterness perception is caused by three single nucleotide polymorphisms found on the TAS2R38 gene.
In this lab, we will isolate our DNA, run PCR on TAS2R38, and perform a DNA restriction digest using Fnu4HI. This restriction enzyme’s recognition site is
(N representing any of the 4 bases). One of the TAS2R38 SNPs reads GCTGC (taster) or GTTGC (non-taster), resulting in a cut for one allele but not the other.
For a further look, click here for a great explanation of the lab we’ll be running.