Materials Included In Kit

Additional Materials Required

Safety Precautions

Although the materials in this lab activity are considered nonhazardous at low concentrations, follow normal safety precautions. Wash hands thoroughly with soap and water before leaving the laboratory. Please consult current Safety Data Sheets for additional safety, handling and disposal information.

PTC paper is soaked with phenyl thiocarbamide (PTC). The LD₅₀ (Rat) for PTC is 3 mg/kg. Such a low LD₅₀ suggests that this is a very toxic substance. However, the solution used to make the taste test papers contains approximately 50 mg of PTC per liter of water. Using crude arithmetic we calculate that each strip of PTC paper would contain approximately 0.03 mg of PTC, and that a person would have to lick and ingest 5,000 strips of PTC paper to reach the LD₅₀ for a body weight of about 50 kilograms (110 pounds). A few sensitized individuals may have an allergic reaction to PTC.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Control and PTC Taste Test Papers may be disposed of according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 8 groups of students. Both parts of this laboratory activity can reasonably be completed in two or three 50-minute class periods. The laboratory can be read before coming to lab, and the data compilation and calculations can be completed after the lab.
• Any readily observable trait that is controlled by a single gene, such as attached ear lobes, can be substituted for PTC tasting.
• Data collection for Activity 2 is best done by asking for a show of hands of the genotype that each student assumed for each generation.
• In Activity 2, the number of each allele (p and q) must be obtained by counting the alleles directly.

Teacher Tips

• The gene that primarily affects the ability to taste PTC is called TAS2R38. It is part of a family of genes called TAS2R all of which are related to the ability to taste bitter substances. TAS2R38 is located on chromesome 7, location q35–q36. Three amino acid substitutions are the typical differences between tasters and non-tasters. Other TAS2R genes play a minor role in the ability to taste PTC. Certain TAS2R genes contribute to those individuals called “super-tasters.”
• One study found that many people who react strongly to PTC do not like to eat foods such as broccoli or coriander nor are they likely to smoke cigarettes or drink coffee or tea.
• The ability to taste PTC is present in about 70% of the overall human population, varying from 58% for Australians to 98% for Native American populations.
• Test the frequency of Thiourea tasters (Flinn No. AP7892) and Sodium Benzoate tasters (Flinn No. AP7891) and compare the data to that of the PTC tasters.

Sample Data

Genetics of PTC

Hardy-Weinberg Equilibrium

Table 1a. Group Frequency (Varies by group.)

Table 1b. Class Frequencies (Varies by class.) Table 1c. F₅ Calculations (Varies by class.) Selection

Table 2a. Group Frequency (Varies by class.)

Table 2b. Class Frequencies (Varies by class.) Table 2c. F₅ Calculations (Varies by class.) Heterozygous Advantage

Table 3a. Group Frequency (Varies by group.)

Table 3b. Class Frequencies Table 3c. Calculations Genetic Drift

Table 4a. Group Frequency (Varies by group.)

Table 4b. Class Frequencies Table 4c. Calculations

Answers to Questions

Genetics of PTC 

1. What was the percentage of heterozygous tasters in the class?

   Varies by class.

2. How do the classes’ phenotypes compare with the average in the North American population?

   Varies by class.

3. PTC is a very bitter chemical. What would be an evolutionary advantage to disliking very bitter foods?

   Many toxic chemicals are bitter so a strong distaste for bitter foods may have helped people avoid accidental poisoning.

Selection

1. How do the new frequencies of p and q compare to the initial frequencies in Case 1?

   The new p is much higher and the new q is much lower than that in Case 1. This is due to the selection against the homozygous recessive genotype and therefore against the allele.

2. Predict what would happen to the frequencies of p and q if you simulated another five generations.

   The frequency of q would continue to decline, but it would never reach zero because of the presence of the heterozygotes.

3. In a large population would it be possible to completely eliminate a deleterious recessive allele? Explain.

   No. It would always be present in the population in the unexpressed heterozygous condition.

Heterozygous Advantage

1. Explain how the changes in the frequencies of p and q in Case 2 compare with Case 1 and Case 3.

   q does not decrease as quickly in Case 3 as in Case 2, but it does decrease slightly less than Case 1.

2. Do you think the recessive allele will be completely eliminated in either Case 2 or Case 3?

   No. In Case 3 the recessive allele becomes favored in the heterozygous genotype. In Case 2 it was hidden in the heterozygous genotype.

3. What is the importance of heterozygotes in maintaining genetic variation in populations?

   The heterozygous genotype maintains genetic variation by not allowing either allele to be eliminated.

Genetic Drift 

1. Explain how the initial genotypic frequencies of the populations compare.

   They should be slightly different after five generations.

2. What do your results indicate about the importance of population size as an evolutionary force?

   Small populations are more likely to show variations in allele frequency.

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

How do populations evolve? What conditions would halt evolution? After doing this laboratory, you should be able to calculate the frequencies of alleles and genotypes in the gene pool of a population using the Hardy-Weinberg formula and discuss natural selection and other causes of microevolution as deviations from the conditions required to maintain Hardy-Weinberg equilibrium.

Concepts

• Evolution
• Hardy-Weinberg equilibrium model
• Allele frequency

Background

Population genetics is the study of allele frequency and its distribution and change under the influence of four evolutionary forces: natural selection, genetic drift, mutation, and migration. Evolution is generally defined as the total genetically inherited changes in individuals of a population’s gene pool. Allele frequency is a measure of the relative frequency of an allele in a population. Usually allele frequency is expressed as a proportion or a percentage. In population genetics, allele frequencies show the genetic diversity of a species population.

Individuals feel the effects of evolution, but it is the population as a whole that actually evolves. Godfrey Hardy, an English mathematician, and Wilhelm Weinberg, a German physician, developed this definition of evolution independently in 1908. They used mathematical modeling of probability to predict that gene pool frequencies are inherently stable but that continual evolution must be expected in all populations. Hardy and Weinberg concluded that evolution would not occur in a population if all of the following conditions were met:

1. Mutation is not occurring.
2. Natural selection is not occurring.
3. The population is infinitely large.
4. All members of the population breed.
5. All mating is totally random.
6. Everyone produces the same number of offspring.
7. There is no migration in or out of the population.

In other words, if no mechanisms of evolution are acting on a population, evolution will not occur—the gene pool frequencies will remain unchanged. However, since it is highly unlikely that any of these seven conditions, let alone all of them, will happen in the real world, evolution is the inevitable result.

Hardy and Weinberg developed a simple equation, now called the Hardy-Weinberg equilibrium equation or the Hardy-Weinberg principle or law. Scientists use the equation to determine the probable gene frequencies in a population and to track their changes from one generation to another. The equation is given in Equation 1. Where:

p is the frequency of the dominant allele for a trait
q is the frequency of the recessive allele for a trait

In other words, p is equal to the sum of all of the alleles in organisms with the homozygous dominant (AA) genotype plus one-half of the alleles in organisms who are heterozygous (Aa) for this trait in a population (see Equation 2). Similarly, q is equal to the sum of all of the alleles in individuals who are homozygous recessive (aa) plus one-half of the alleles in organisms who are heterozygous (Aa) for this trait in a population (see Equation 3). In the case where there are only two alleles for a trait (e.g., A and a), the frequency of p plus the frequency of q equals 100%. Stated mathematically as Equation 4. From these simple equations, Hardy and Weinberg realized that the chances of all possible combinations of alleles occurring randomly might be expressed mathematically (Equation 5). In this equation, p² is the predicted frequency of homozygous dominant (AA) genotype in a population, 2pq is the predicted frequency of heterozygous (Aa) genotype, and q² is the predicted frequency of homozygous recessive (aa) genotype.

The only observable genotype is that of a homozygous recessive (aa) organism because its phenotype is different from that seen in a homozygous dominant (AA) or a heterozygous (Aa) organism. In the Hardy-Weinberg Equilibrium Equation, therefore, q² is the frequency of the homozygous recessive (aa) genotype. Since it can be observed, the number of homozygous recessive (q²) organisms in a population can be counted. Using the equations to solve for p and q, it is possible to calculate the number of each type of allele.

Since the predicted frequencies of all three genotypes for the selected trait within the population can be calculated using the Hardy-Weinberg equation, it is possible to “see” evolution occur by comparing the mathematical results for successive number of alleles in each generation versus the first generation. In addition, the rate and direction of the evolution of a particular trait can be “seen.” However, the Hardy-Weinberg equation cannot determine which of the various possible causes of evolution are responsible for the changes in gene pool frequencies.

Remember that evolution occurs when mutation and natural selection causes the frequency of one allele to change. Before Hardy and Weinberg, it was thought that dominant alleles would eventually cause the extinction of the recessive alleles. This theory was called “genophagy” (literally “gene eating”). It was later shown to be incorrect. According to this once popular theory, dominant alleles always increase in frequency from generation to generation. Hardy and Weinberg were able to demonstrate with their equation that dominant alleles can just as easily decrease in frequency.

One way to test the Hardy-Weinberg Equilibrium Model is to use a simple, easy-to-test human trait. Using the class as a sample population, the allele frequency of a gene controlling the ability to taste phenyl thiocarbamide (PTC) will be determined. A bitter-taste reaction to PTC is evidence for the presence of the dominant allele (called “tasters”) in either the homozygous (AA) or heterozygous (Aa) genotype. The inability to taste the chemical (called “nontasters”) depends on the presence of homozygous recessive alleles (aa). In order to estimate the frequency of the PTC-tasting allele in the population, the number of nontasters, who are homozygous recessive (aa), must be counted. This is q². After determining q², p² can be calculated and finally the frequency of dominant alleles and the number of recessive alleles can be determined.

Experiment Overview

In Activity 1 the number of PTC tasters and nontasters will be determined.

In Activity 2, the entire class will represent a breeding population for four different cases, simulating an ideal population, a population undergoing natural selection, a population with heterozygous advantages, and a population undergoing genetic drift.

Materials

Safety Precautions

Handle the taste test papers as little as possible before use. Once any taste has been detected, immediately remove the test paper from the mouth and discard. Do not reuse any test papers; always use fresh test paper for every individual. Wash hands before touching the test papers and handle them as little as possible. Although the materials in Activity 2 are nonhazardous, follow normal safety precautions. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Activity 1. Estimating Allele Frequencies for PTC within a Sample Population

1. Place a piece of control taste test paper on your tongue. Move the control paper around until you have a taste sensation of the control paper (about 3–5 seconds). Note: Mentally note any taste you sense from the control paper and remember it as a negative taste test for the PTC paper.
2. Place a piece of PTC taste test paper on your tongue. If you sense a bitter taste (it will be obvious), then you are a taster of PTC. If the test paper tastes like the control paper, then you are a non-taster.
3. Report your status to the teacher so it can be recorded on the class overhead.
4. Record the class data on the Genetics of PTC Worksheet.
5. Calculate the decimal fraction corresponding to the frequency of tasters (p² + 2pq) by dividing the number of tasters in the class by the total number of students in the class. Record these on the Genetics of PTC Worksheet.
6. Calculate the decimal fraction corresponding to the frequency of nontasters (q²) by dividing the number of nontasters by the total number of students. Record these on the Genetics of PTC Worksheet.
7. Use the Hardy-Weinberg equation to determine the frequencies (p and q) of the two alleles. Record these on the Genetics of PTC Worksheet.
8. Calculate and record the values for p and q for the North American population given on the Genetics of PTC Worksheet.
9. Answer the questions on the Genetics of PTC Worksheet.

Activity 2. Simulating a Hardy-Weinberg Population

Case 1. The Ideal Hardy-Weinberg Population
The class will simulate a population of randomly mating heterozygous individuals with an initial gene frequency of 0.5 for the dominant allele A and 0.5 for the recessive allele a.

1. Each student collects two cards with an A and two with an a. Note: The four cards represent the products of meiosis. Each “parent” will contribute a haploid set of chromosomes (one allele) to the next generation.
2. All individuals will have the heterozygous genotype (Aa). This is recorded as F₀ in Table 1a of the Hardy-Weinberg Worksheet.
3. Turn the four cards over so that the letters do not show and shuffle them.
4. Turn the top card over (your partner should do the same).
5. Put the two cards together to represent the alleles for the first offspring. Example, Aa. Record the genotype of Offspring 1 in Table 1a of the Hardy-Weinberg Worksheet.
6. Replace the card into the stack of cards and reshuffle. Note: Each offspring begins with the same chance at each allele.
7. Turn the top card over (your partner should do the same).
8. Put the two cards together to represent the alleles for the second offspring. Example, aa. Record the genotype of Offspring 2 in Table 1a of the Hardy-Weinberg Worksheet.
9. Become the next generation (F₁) by assuming the genotypes of the two offspring. That is, one student assumes the genotype of the first offspring while the other student assumes the genotype of the second offspring.
10. Obtain, if necessary, new cards representing the alleles in the offspring (for a total of four cards). For example, Student 1 becomes genotype Aa and obtains cards A, A, a, a. While student 2 becomes aa and obtains cards a, a, a, a.
11. Randomly select another individual with whom to breed in order to produce the offspring of the next generation (F₂). Remember, the gender of your mate does not matter, nor does the genotype.
12. Repeat steps 4 through 11 until five generations (F₅) have been completed.
13. The teacher will collect the class data for each generation on the class overhead by asking you to raise your hand to report your genotype for each generation. Note: By only reporting your new genotype for each generation the entire class data will be collected.
14. Record the class data on Table 1b of the Hardy-Weinberg Worksheet.
15. Use the class data from Table 1b to calculate the number of dominant (A) alleles present in the fifth generation (F₅) using Equation 6.
    {10795_Procedure_Equation_6}
16. Record the value in Table 1c on the Hardy-Weinberg Worksheet.
17. Use the class data from Table 1b to calculate the number of recessive (a) alleles present in the fifth generation (F₅) using Equation 7.
    {10795_Procedure_Equation_7}
18. Record the value in Table 1c on the Hardy-Weinberg Worksheet.
19. Calculate the allele frequencies in the F₅ generation, p and q, using the data calculated in steps 16 and 17 and the Equations 8 and 9.
    {10795_Procedure_Equation_8}
    {10795_Procedure_Equation_9}
20. Record the results in Table 1c on the Hardy-Weinberg Worksheet.

Case 2. Selection
In this activity, the simulation will be more realistic because in the natural environment, not all genotypes have the same rate of survival. For this simulation, assume that the homozygous recessive (aa) individuals never reach reproductive age, while homozygous dominant and heterozygous individuals always do.

1. All individuals begin with the heterozygous genotype (Aa). This is recorded as F₀ in Table 2a of the Selection Worksheet.
2. Collect two cards with A and two with a.
3. Turn the four cards over so that the letters do not show and shuffle them.
4. Turn the top card over (your partner should do the same).
5. Put the two cards together to represent the alleles for the first offspring. Record the genotype of this offspring in Table 2a of the Selection Worksheet. Do not record the genotype for any homozygous recessive offspring. (Any time the offspring is homozygous recessive (aa), it dies before it can reproduce.)
6. In order to maintain a constant population size, the same two parents must continue mating until they produce two surviving offspring. To do this, replace your card into your stack of cards and reshuffle and repeat steps 4 and 5 until two offspring survive.
7. Become the next generation (F₁) by assuming the genotypes of the two surviving offspring. Obtain, if necessary, new cards representing the alleles in the offspring.
8. Randomly select another individual with whom to breed in order to produce the offspring of the next generation (F₂). Remember, the gender of your mate does not matter, nor does the genotype.
9. Repeat steps 3 through 8 until five generations (F₅) have been completed.
10. The teacher will collect the class data for each generation on the class overhead by asking you to raise your hand to report your genotype for each generation.
11. Record the class data on Table 2b of the Selection Worksheet.
12. Use the class data from Table 2b to calculate the number of dominant (A) alleles present in the fifth generation (F₅). Record the value in Table 2c on the Selection Worksheet.
13. Use the class data from Table 2b to calculate the number of recessive (a) alleles present in the fifth generation (F₅). Record the value in Table 2c on the Selection Worksheet.
14. Calculate the allele frequencies for the fifth generation, p and q, using the class data from Table 2c and the equations listed in Case 1.
15. Record the results in Table 2c on the Selection Worksheet.

Case 3. Heterozygous Advantage

Case 3 simulates a population in which the heterozygous genotype has an advantage over the homozygous dominant form while the homozygous recessive form is lethal (as in Case 2).

1. All individuals begin with the heterozygous genotype (Aa). This is recorded as F₀ in Table 3a of the Heterozygous Advantage Worksheet.
2. Collect two cards with A and two with a.
3. Turn the four cards over so that the letters do not show and shuffle them.
4. Turn the top card over (your partner should do the same).
5. Put the two cards together to represent the alleles for the first offspring. Record the genotype of this offspring in Table 3a of the Heterozygous Advantage Worksheet. As in Case 2, of the offspring is homozygous recessive (aa), it dies before it can reproduce. If the offspring is homozygous dominant (AA) flip a coin to determine if it lives or dies. If the coin lands heads up, then the offspring survives. If tails, the offspring does not survive.
6. Since a constant population size must be maintained, the same two parents must continue mating until they produce two surviving offspring. To do this, replace the allele card into the stack of cards and reshuffle. Repeat steps 3 through 5 until two offspring survive.
7. Become the next generation (F₁) by assuming the genotypes of the two offspring.
8. Obtain, if necessary, new cards representing the alleles in the offspring.
9. Randomly select another individual with whom to breed in order to produce the offspring of the next generation. Remember, the gender of your mate does not matter, nor does the genotype.
10. Repeat steps 3 through 10 until fifteen generations (F₁₅) have been completed.
11. The teacher will collect the class data for each generation on the Class Overhead by asking you to raise your hand to report your genotype for each generation.
12. Record the class data on Table 3b of the Heterozygous Advantage Worksheet.
13. Use the class data from Table 3b to calculate the number of dominant (A) alleles present in the fifth (F₅), tenth (F₁₀) and fifteenth (F₁₅) generations. Record the values in Table 3c on the Heterozygous Advantage Worksheet.
14. Use the class data from Table 3c to calculate the number of recessive (a) alleles present in the fifth (F₅), tenth (F₁₀) and fifteenth (F₁₅) generations. Record the values in Table 3c on the Heterozygous Advantage Worksheet.
15. Calculate the allele frequencies, p and q, using the equations given in Case 1. Record the results on the Heterozygous Advantage Worksheet.

Case 4. Genetic Drift
The activity will now examine the phenomenon of genetic drift. The class will be divided into smaller populations so that individuals from one isolated population do not interact with those from other populations.

1. Join the group to which you are assigned by the teacher.
2. All individuals will have the heterozygous genotype (Aa). This is recorded as F₀ in Table 4a of the Genetic Drift Worksheet.
3. Collect two cards with A and two with a.
4. Turn the four cards over so that the letters do not show and shuffle them.
5. Turn the top card over (your partner should do the same).
6. Put the two cards together to represent the alleles for the first offspring. Record the genotype of Offspring 1 in Table 4a of the Genetic Drift Worksheet.
7. Replace your card into your stack of cards and reshuffle.
8. Turn the top card over (your partner should do the same).
9. Put the two cards together to represent the alleles for the second offspring. Record the genotype of Offspring 2 in Table 4a of the Genetic Drift Worksheet.
10. Become the next generation (F₁) by assuming the genotypes of the two offspring.
11. Obtain, if necessary, new cards representing the alleles in the offspring.
12. Randomly select another individual within your assigned group with whom to breed in order to produce the offspring of the next generation (F₂). Remember, the gender of your mate does not matter, nor does the genotype.
13. Repeat steps 4 through 12 until five generations (F₅) have been completed.
14. The teacher will collect the class data for each generation on the class overhead by asking you to raise your hand to report your genotype for each generation.
15. Record the class data on Table 4b of the Genetic Drift Worksheet.
16. Use the class data from Table 4b to calculate the number of dominant (A) alleles present in the fifth generation (F₅). Record the value in Table 4c on the Genetic Drift Worksheet.
17. Use the class data from Table 4c to calculate the number of recessive (a) alleles present in the fifth generation (F₅). Record the value in Table 4c on the Genetic Drift Worksheet.
18. Calculate the allele frequencies, p and q, using the equations given in Case 1. Record the results on the Genetic Drift Worksheet.
19. PTC and Control taste test papers may be thrown in the regular trash.

Student Worksheet PDF

10795_Student1.pdf