Genotyping PTC Allele Study Notes

Genotyping PTC Allele Study Notes

Introduction to PTC Tasting

• PTC (Phenylthiocarbamide) is a compound that produces a bitter flavor.
• Observations from the early 1930s:
  • Some individuals can taste PTC (called "tasters").
  • Others cannot taste it (called "non-tasters").
• Importance: Sensitivity to bitter flavors is advantageous as many toxic plants have a bitter taste, aiding in the avoidance of harmful ingestion.
• Prevalence: Approximately 75% of individuals tested demonstrate sensitivity to PTC.

Genetics of PTC Sensitivity

• The gene responsible for tasting PTC is termed TAS2R38.
  • Location: Long arm of chromosome 7, specifically at positions 7q35-q36.
  • Function: Encodes a membrane-bound receptor protein expressed in gustatory cells of taste buds.
• Allelic Variants:
  • Two primary alleles exist:
  • T (taster allele)
  • t (non-taster allele)
• Inheritance Patterns:
  • Homozygous dominant (TT) and heterozygous (Tt) individuals are categorized as "tasters."
  • Only homozygous recessive (tt) individuals are classified as "non-tasters."
• Gene Sequencing:
  • Completed in 2003, revealing two haplotypes (taster and non-taster), differing at three nucleotide positions (SNPs), and resulting in three amino acid differences between the alleles.
  • The taster haplotype is ancestral, as proven by the presence of the same amino acid sequence in primates such as chimpanzees and gorillas.

SNP and Genotype Identification

• Important SNP: Located at position 785; a mutation results in an Fnu 4HI restriction site.
  • Taster allele has C785, while the non-taster allele has T785.
• Restriction site sequences:
  • Taster Fnu 4HI sequence: 5’-GCNGC-3’
  • Non-taster sequence: 5’-GTNGC-3’ (prevents enzyme digestion).

Laboratory Exercise Overview

Determining Phenotype via Tasting Experiment

• Method: Use a PTC test strip to taste for bitterness and record observations.

Estimating Allele Frequencies in the Classroom Population

• Hardy-Weinberg Principle: Describes conditions for allele/genotype frequencies in a population at genetic equilibrium:
  1. No mutations.
  2. No migration in/out of the population.
  3. Infinitely large population.
  4. Equal survivorship and reproduction for all genotypes.
  5. Random mating.
• Implications: Real classroom conditions won't meet these criteria, but can estimate allele frequencies using Hardy-Weinberg equations.

Mathematical Foundations

• Definitions:
  • Let p be the frequency of the dominant allele (T) and q be the frequency of the recessive allele (t).
  • Equation: $p + q = 1$
• Genotype Frequencies:
  • $p^2$ = frequency of TT (homozygous tasters)
  • $2pq$ = frequency of Tt (heterozygous tasters)
  • $q^2$ = frequency of tt (non-tasters)
  • Formula: $p^2 + 2pq + q^2 = 1$

Method for Frequency Calculations

1. Collect phenotype responses from the class and record them.
2. Calculate frequency of “non-tasters” using the square root of their frequency (i.e., $q = ext{sqrt}(q^2)$).
3. Calculate frequency of the taster allele from $p = 1 - q$.

Genotyping Process

DNA Extraction via Chelex Method

• Chelex is a resin used to preserve DNA during extraction by chelating Mg2+ ions, which are cofactors for degrading enzymes.
  • Procedure:
  1. Swish 10 ml of 0.9% NaCl in your mouth and spit into a cup.
  2. Pipette 1 ml of saline into a microcentrifuge tube and centrifuge to collect cells.
  3. Add 30 µl of 0.9% NaCl to resuspend the pellet.
  4. Transfer into a tube containing Chelex solution and incubate at 95°C for 10 minutes.
  5. Centrifuge again and collect the supernatant as the DNA extract.

PCR Amplification

• Use Polymerase Chain Reaction (PCR) to amplify a 303 bp segment of the PTC gene.
  • Taq DNA polymerase used for heat stability.
  • PCR Process includes denaturation, annealing, and extension phases, resulting in billions of DNA copies within a few cycles.
• PCR Protocol:
  • Incubate at 95°C for 10 mins followed by 30 cycles of:
  • Denaturation at 95°C for 30 seconds
  • Annealing at 58°C for 30 seconds
  • Extension at 72°C for 60 seconds
  • Final extension at 72°C for 7 minutes.

Restriction Enzyme Digestion for Genotyping

• Use Fnu 4HI enzyme to digest the PCR products and distinguish between taster and non-taster alleles.
  • Experimental Setup:
  1. Prepare digestion tubes and add DNA amplicons and buffer according to protocols.
  2. Incubate at 37°C for one hour.
  3. Complete analysis via agarose gel electrophoresis.
• Band patterns observed will differentiate allele types (homozygous tasters vs. non-tasters).

Agarose Gel Electrophoresis

• Prepare a 3% agarose gel and load it with samples including a DNA ladder for size reference.
• Run the gel, stain with ethidium bromide, and visualize the bands under UV light to determine genotype based on their migration and size.

Determining Genotype

• Using band patterns from the gel:
  • Homozygous taster: 238 bp and 64 bp bands.
  • Non-taster: one band at 303 bp.
  • Heterozygous taster: all three bands 303 bp, 238 bp, and 64 bp visible.

Data Analysis and Interpretation

Calculating Chi-square Values

• To compare observed genotypes to expected frequencies from Hardy-Weinberg calculations:
  • $ext{Chi-square} = \frac{(S<em>{observed} - S</em>{expected})^2}{S_{expected}}$
• Determine if the observed frequencies fit the Hardy-Weinberg model.

Importance of Findings

• Discuss implications if Hardy-Weinberg equilibrium is not supported; conditions that disrupted assumptions should be noted.