Bridges 2014 Using a Single Nucleotide Polymorphism to Predict Bitter Tasting Ability

Taste Taste is mediated by specialized cells that communicate with several brain regions through direct connections to sensory neurons Taste perception is a two-step process: A taste molecule binds to a specific receptor on the surface of a taste cell Taste cells generate a nervous impulse, which is interpreted by the brain

Bitter Taste Discovery The inability to taste phenylthiocarbamide (PTC) is a recessive trait that varies in the human population.

PTC Taste Regulated by SNP The gene for the PTC taste receptor, TAS2R38, was identified in 2003 Sequencing identified three nucleotide positions that vary within the human population—each variable position is termed a single nucleotide polymorphism (SNP) One specific combination of the three SNPs, termed a haplotype, correlates most strongly with tasting ability SNPs also predict adverse responses to PROZAC® and Paxil®

Today’s Activities Figure out YOUR genotype Extract DNA from your cheek cells Boil Chelex resin, which binds contaminating metal ions PCR amplify a portion of the TAS2R38 gene Digest the PCR product with restriction enzyme HaeIII Recognition sequence includes one of the SNPs One allele is cut by the enzyme, and one is not Produces a restriction fragment length polymorphism (RFLP) Gel visualize the RFLP Compare genotype to phenotype

DNA Extraction

Boiling Lyse cell (break apart/open up) Chelex Binds contaminating metal ions-major inhibitor of PCR Spin Precipitate out debris

Supernatant

Pellet

Polymerase Chain Reaction Create many copies of a SPECIFIC portion of DNA Requires: Ready-To-Go PCR bead Taq Polymerase Buffer MgCl 2 Nucleotides- A, G, C, T Primer/Loading dye mix (on ice!) Primers to target Portion of the TAS2R38 gene Loading dye for gel Template DNA- cheek DNA from Part I

Thermocycling

Exponential Growth of Template DNA

PCR

Restriction Enzyme Digest Enzyme- protein that carries out chemical reactions Restriction enzymes cut DNA at a SPECIFIC nucleotide sequence called a restriction site The HaeIII restriction enzyme uses GGCC recognition site It only digests the TAS2R38 gene at this site Remember the TAS2R38 contains single nucleotide polymorphisms You may be GGCC and/or GGGC The HaeIII may or may not digest your DNA

Restriction Enzyme Digest

Gel Analysis