MODULE 4
GENOTYPE TO PHENOTYPE
PTC Analysis
,LEARNING OUTCOMES - The student will be able to:
• Comprehend how DNA encodes traits that are transmitted across generations
• Understand that PCR is a technique for amplifying specific parts of the genome
• Understand the link between genotype and phenotype
• Purify your own genomic DNA and amplify your PTC locus using PCR
• Perform a restriction digest on the PCR product followed by gel electrophoresis to visualize your
PTC allelic variability
• Perform Hardy-Weinberg analysis on your genotypic and phenotypic data
OBJECTIVES
The ultimate goal of this experiment is to determine your
personal allelic genotype and predict your ability to taste
phenylthiocarbamide (PTC). You will compare your results
with your classmates’ results in a population study using
Hardy-Weinberg analysis. By completing these objectives,
you will determine if you carry the genetic allele that confers
the ability to taste PTC using CAPS markers. You will examine
the allelic variation of the PTC allele of your entire class as a
population. You will also observe whether the determined
genotype correctly predicts the PTC sensitivity phenotype, or if
there might be other genetic traits that you are not detecting that
could also affect PTC sensitivity.
BACKGROUND
Our ability to taste is mediated by GPCRs (G-protein-coupled
receptors). Taste receptor genes (surface proteins) of the
TAS1R and TAS2R families encode GPCRs expressed in taste
receptor cells of the gustatory papillae in the tongue. Various
taste receptors mediate specific taste modalities (bitter, sweet,
savory, etc.). Humans have 43 different receptors for bitter taste
within the TAS2R gene family. One of the best characterized
taste receptors is encoded by the TAS2R38 gene. This receptor
binds ligands such as 6-npropylthiouracil (PROP6) and
phenylthiocarbamide (PTC), two chemicals similar to those
found in bitter tasting vegetables. When ligands bind to the
receptor, they cause a conformational change in the TAS2R38
receptor protein, which activates a signal transduction pathway Figure 4.1. How does the PTC gene work?
that ultimately transmits a nerve impulse to the brain letting it https://learn.genetics.utah.edu/content/basics/ptc/
know “that’s bitter” (Fig. 4.1). The gene is 1143 nucleotide
base pairs (bp) long and located on the long arm of chromosome
7 along with nine other genes for bitter taste receptors.
The Genetics of PTC tasting
A genotype is the genetic basis of a trait, the genetic information that codes for the phenotype. The ability
to taste PTC is inherited as a simple Mendelian trait. As with other simple Mendelian traits, such as cleft
chin or widow’s peak, the gene associated with the ability to taste PTC exists in two allelic forms: the
dominant allele (T), which confers the ability to taste PTC, and the recessive non-taster allele (t). The
combination of these different alleles within an individual is referred to as a genotype, which in turn dictates
phenotype: in this case whether an individual is a “taster” or “non-taster”. The genotype of a taster can be
either homozygous dominant (TT) or heterozygous (Tt). If a person is a non-taster then their genotype is
homozygous recessive (tt).
2
, Within the general population, about 70% of the people tested can taste PTC, whereas the other 30% cannot.
The ability to discern bitter tastes evolved as a mechanism to prevent early humans from eating poisonous
plants. If the ability to taste bitter compounds conveys a selective advantage, then shouldn't non-tasters have
died off long ago? Why do so many people still carry the non-tasting PTC variant. Even though PTC is
considered a dominant trait with simple Mendelian inheritance, tasters vary greatly in their sensitivity to
PTC. There are many other factors that affect PTC tasting ability. Having a dry mouth inhibit your PTC
sensibility, what you ate or drank before sampling PTC paper may also affect your tasting ability. Also, an
individual's sensitivity may change over time.
Different Haplotypes of the TAS238 Gene
By comparing DNA sequences between tasters and non-tasters, scientists determined that there are three
single nucleotide polymorphisms (SNPs) found on the TAS2R38 gene that differentiate the taster allele (T)
from the non-taster allele (t) resulting in two alleles referred to as PAV and AVI, as detailed in Table 4.1.
Table 4.1 – Polymorphisms within the TAS2R38 gene
The PAV allele is associated with high sensitivity to the bitter taste of PTC (‘Taster’ allele), while AVI is associated
with little or no sensitivity (‘Non-taster’ allele). In human populations we find individuals that are homozygous for
PAV (‘strong tasters’), homozygous for AVI (‘non-tasters’), or heterozygous (‘moderate tasters’).
This polymorphism is a genetic marker known as a cleaved amplified polymorphic sequence – or a “CAPS”
marker. If you are a taster, then you have a single nucleotide polymorphism (SNP) located at position 785.
This SNP creates a cleavage site for the restriction enzyme Fnu4H1. SNPs found on the same chromosome
or a combination of alleles are referred to as a haplotype. These genes are so close together on the
chromosome that they’re almost always inherited as a block.
• The common non-taster allele has a G at nucleotide position 145 (G145), T at position 785 (T785) and
A at position 886 (A886). This non-taster allele produces a polypeptide with alanine, valine and
isoleucine at these sites and therefore is referred to as the AVI allele (Alanine-Valine-Isoleucine)
• The common taster allele has C145, C785, and G886, produces a polypeptide with proline, alanine,
and valine at these sites, and is referred to as the PAV allele (for Proline-Alanine-Valine) Table 4.1
In this module you will have an opportunity to observe whether you can taste the compound PTC. You will
then analyze your DNA to determine whether you are a homozygous non-taster (tt: AVI/AVI), homozygous
taster (TT: PAV/PAV), or heterozygous taster (Tt: PAV/AVI).
Figure 4.2 is an overview of the methods you will use to determine your genotype. The first step is to extract
genomic DNA from your cheek cells. Since this extract contains complete chromosomes and far too few
copies of the gene to analyze, you will use polymerase chain reaction (PCR) to make billions of copies of
a 303 bp region of the TAS2R38 gene that contains the SNP at nucleotide position 785. The T and t alleles
can be distinguished with a restriction digest assay using Fnu4H1 enzyme which only cleaves the taster
allele. Finally, to view the results of the digest, you will use gel electrophoresis to separate and view the
DNA fragments of different sizes. At the same time, you will test your ability to taste the bitter compound
PTC and correlate it with your genotypes.
3
GENOTYPE TO PHENOTYPE
PTC Analysis
,LEARNING OUTCOMES - The student will be able to:
• Comprehend how DNA encodes traits that are transmitted across generations
• Understand that PCR is a technique for amplifying specific parts of the genome
• Understand the link between genotype and phenotype
• Purify your own genomic DNA and amplify your PTC locus using PCR
• Perform a restriction digest on the PCR product followed by gel electrophoresis to visualize your
PTC allelic variability
• Perform Hardy-Weinberg analysis on your genotypic and phenotypic data
OBJECTIVES
The ultimate goal of this experiment is to determine your
personal allelic genotype and predict your ability to taste
phenylthiocarbamide (PTC). You will compare your results
with your classmates’ results in a population study using
Hardy-Weinberg analysis. By completing these objectives,
you will determine if you carry the genetic allele that confers
the ability to taste PTC using CAPS markers. You will examine
the allelic variation of the PTC allele of your entire class as a
population. You will also observe whether the determined
genotype correctly predicts the PTC sensitivity phenotype, or if
there might be other genetic traits that you are not detecting that
could also affect PTC sensitivity.
BACKGROUND
Our ability to taste is mediated by GPCRs (G-protein-coupled
receptors). Taste receptor genes (surface proteins) of the
TAS1R and TAS2R families encode GPCRs expressed in taste
receptor cells of the gustatory papillae in the tongue. Various
taste receptors mediate specific taste modalities (bitter, sweet,
savory, etc.). Humans have 43 different receptors for bitter taste
within the TAS2R gene family. One of the best characterized
taste receptors is encoded by the TAS2R38 gene. This receptor
binds ligands such as 6-npropylthiouracil (PROP6) and
phenylthiocarbamide (PTC), two chemicals similar to those
found in bitter tasting vegetables. When ligands bind to the
receptor, they cause a conformational change in the TAS2R38
receptor protein, which activates a signal transduction pathway Figure 4.1. How does the PTC gene work?
that ultimately transmits a nerve impulse to the brain letting it https://learn.genetics.utah.edu/content/basics/ptc/
know “that’s bitter” (Fig. 4.1). The gene is 1143 nucleotide
base pairs (bp) long and located on the long arm of chromosome
7 along with nine other genes for bitter taste receptors.
The Genetics of PTC tasting
A genotype is the genetic basis of a trait, the genetic information that codes for the phenotype. The ability
to taste PTC is inherited as a simple Mendelian trait. As with other simple Mendelian traits, such as cleft
chin or widow’s peak, the gene associated with the ability to taste PTC exists in two allelic forms: the
dominant allele (T), which confers the ability to taste PTC, and the recessive non-taster allele (t). The
combination of these different alleles within an individual is referred to as a genotype, which in turn dictates
phenotype: in this case whether an individual is a “taster” or “non-taster”. The genotype of a taster can be
either homozygous dominant (TT) or heterozygous (Tt). If a person is a non-taster then their genotype is
homozygous recessive (tt).
2
, Within the general population, about 70% of the people tested can taste PTC, whereas the other 30% cannot.
The ability to discern bitter tastes evolved as a mechanism to prevent early humans from eating poisonous
plants. If the ability to taste bitter compounds conveys a selective advantage, then shouldn't non-tasters have
died off long ago? Why do so many people still carry the non-tasting PTC variant. Even though PTC is
considered a dominant trait with simple Mendelian inheritance, tasters vary greatly in their sensitivity to
PTC. There are many other factors that affect PTC tasting ability. Having a dry mouth inhibit your PTC
sensibility, what you ate or drank before sampling PTC paper may also affect your tasting ability. Also, an
individual's sensitivity may change over time.
Different Haplotypes of the TAS238 Gene
By comparing DNA sequences between tasters and non-tasters, scientists determined that there are three
single nucleotide polymorphisms (SNPs) found on the TAS2R38 gene that differentiate the taster allele (T)
from the non-taster allele (t) resulting in two alleles referred to as PAV and AVI, as detailed in Table 4.1.
Table 4.1 – Polymorphisms within the TAS2R38 gene
The PAV allele is associated with high sensitivity to the bitter taste of PTC (‘Taster’ allele), while AVI is associated
with little or no sensitivity (‘Non-taster’ allele). In human populations we find individuals that are homozygous for
PAV (‘strong tasters’), homozygous for AVI (‘non-tasters’), or heterozygous (‘moderate tasters’).
This polymorphism is a genetic marker known as a cleaved amplified polymorphic sequence – or a “CAPS”
marker. If you are a taster, then you have a single nucleotide polymorphism (SNP) located at position 785.
This SNP creates a cleavage site for the restriction enzyme Fnu4H1. SNPs found on the same chromosome
or a combination of alleles are referred to as a haplotype. These genes are so close together on the
chromosome that they’re almost always inherited as a block.
• The common non-taster allele has a G at nucleotide position 145 (G145), T at position 785 (T785) and
A at position 886 (A886). This non-taster allele produces a polypeptide with alanine, valine and
isoleucine at these sites and therefore is referred to as the AVI allele (Alanine-Valine-Isoleucine)
• The common taster allele has C145, C785, and G886, produces a polypeptide with proline, alanine,
and valine at these sites, and is referred to as the PAV allele (for Proline-Alanine-Valine) Table 4.1
In this module you will have an opportunity to observe whether you can taste the compound PTC. You will
then analyze your DNA to determine whether you are a homozygous non-taster (tt: AVI/AVI), homozygous
taster (TT: PAV/PAV), or heterozygous taster (Tt: PAV/AVI).
Figure 4.2 is an overview of the methods you will use to determine your genotype. The first step is to extract
genomic DNA from your cheek cells. Since this extract contains complete chromosomes and far too few
copies of the gene to analyze, you will use polymerase chain reaction (PCR) to make billions of copies of
a 303 bp region of the TAS2R38 gene that contains the SNP at nucleotide position 785. The T and t alleles
can be distinguished with a restriction digest assay using Fnu4H1 enzyme which only cleaves the taster
allele. Finally, to view the results of the digest, you will use gel electrophoresis to separate and view the
DNA fragments of different sizes. At the same time, you will test your ability to taste the bitter compound
PTC and correlate it with your genotypes.
3
