PCR Genotyping the TAS2R38 PTC receptor

• 5’-CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG-3’ (Forward Primer)
• 5′-AGGTTGGCTTGGTTTGCAATCATC-3′ (Reverse Primer)

1. PCR the DNA samples extracted from cheek cells using the PCR Beads
2. Pour 2% agarose into casting apparatus in refrigerator
   • 2 gels per class need to be made → 100ml of TBE with 2g agarose
   • add 5μl SYBR safe solution into the molten agarose before casting
   • place 2 sets of combs into the gel → at one end and in the middle
3. Digest PCR product with HaeIII
   1. remove 10μl of PCR product into a fresh tube
   2. add 1μl of HaeIII enzyme into tube
   3. incubate for 10 minutes at 37°C
4. load gel with DNA ladder, Digested and Undigested
   • Undigested sample is from the original PCR
5. Run gel at 120V for 20 minutes
6. Visualize on UV transilluminator

SNP detection

The longer primer ends with the sequence “GG”. Both alleles at this locus will amplify equally well with this primer set, however, one allele will have the sequence “GGGC” and another “GGCC”. “GGCC” is the restriction site for the enzyme HaeIII. The digestion of this amplified DNA will be digestible for one allele and yield a DNA fragment the size of the large primer (44 bp) as well as the remainder of the amplicon. Because of this difference in digestion profile of the amplicon, we can identify the 2 alleles at this locus.

Analysis questions

1. What is the size of the PCR product?
   • Perform an in silico PCR on the Tas2R38 gene and identify the size of the amplicon.
2. The long primer is 44bp. If the amplicon of the allele digests, what are the sizes of fragments expected following HaeIII?
3. Which allele is the one that can be identified through HaeIII digestion?
   • use the results of the PTC paper test
4. Some lanes contain 3 bands instead of 1 or 2. Can you explain this?