Bridges 2014Using a Single Nucleotide Polymorphism to Predict
Bitter Tasting AbilityTasteTaste is mediated by specialized cells
that communicate with several brain regions through direct
connections to sensory neuronsTaste perception is a two-step
process: A taste molecule binds to a specific receptor on the
surface of a taste cellTaste cells generate a nervous impulse,
which is interpreted by the brainBitter Taste Discovery The
inability to taste phenylthiocarbamide (PTC) is a recessive trait
that varies in the human population.
PTC Taste Regulated by SNPThe gene for the PTC taste receptor,
TAS2R38, was identified in 2003Sequencing identified three
nucleotide positions that vary within the human populationeach
variable position is termed a single nucleotide polymorphism
(SNP)One specific combination of the three SNPs, termed a
haplotype, correlates most strongly with tasting abilitySNPs also
predict adverse responses to PROZAC and Paxil
Todays ActivitiesFigure out YOUR genotypeExtract DNA from your
cheek cells Boil Chelex resin, which binds contaminating metal
ionsPCR amplify a portion of the TAS2R38 geneDigest the PCR product
with restriction enzyme HaeIIIRecognition sequence includes one of
the SNPsOne allele is cut by the enzyme, and one is notProduces a
restriction fragment length polymorphism (RFLP)Gel visualize the
RFLPCompare genotype to phenotypeDNA Extraction
DNA ExtractionBoilingLyse cell (break apart/open up)Chelex Binds
contaminating metal ions-major inhibitor of PCRSpinPrecipitate out
debrisSupernatant
Supernatant
SupernatantPelletPolymerase Chain ReactionCreate many copies of
a SPECIFIC portion of DNARequires:Ready-To-Go PCR beadTaq
PolymeraseBufferMgCl2Nucleotides- A, G, C, TPrimer/Loading dye mix
(on ice!)Primers to target Portion of the TAS2R38 geneLoading dye
for gelTemplate DNA- cheek DNA from Part I
Thermocycling
Exponential Growth of Template DNA
PCR
Restriction Enzyme DigestEnzyme- protein that carries out
chemical reactionsRestriction enzymes cut DNA at a SPECIFIC
nucleotide sequence called a restriction siteThe HaeIII restriction
enzyme uses GGCC recognition siteIt only digests the TAS2R38 gene
at this siteRemember the TAS2R38 contains single nucleotide
polymorphismsYou may be GGCC and/or GGGCThe HaeIII may or may not
digest your DNA
