Introduction: The Hardy-Weinburg theorem states that the gene pool of a nonevolving

population remains constant over generations. According to this theorem, the Mendelian

system has no tendency to alter allele frequencies and they should remain constant

forever. However, in reality, some other factor always intervenes and therefore Hardy-

Weinburg equilibrium cannot occur in nature. The system works similar to that of a deck

of cards: no matter how many times the deck is reshuffled, the deck itself remains the

same. The equation for Hardy-Weinburg equilibrium uses the letter p to represent the

frequency of one allele and the letter q to represent the frequency of the other allele.

Accordingly, p + q = 1, meaning that the combined frequencies of all possible alleles

must add up to 100% for that population locus. The equation calculating frequencies of

alleles in the gene pool if frequencies of genotypes are known is:

p2 + 2pq + q2 = 1.

This theory is important because it explains how Mendelian inheritance preserves genetic

variation from one generation to the next.

For a population to be in Hardy-Weinburg equilibrium, it must satisfy five

conditions. These conditions are, first, a very large population size. Second, no migration

can occur. Third, no net mutations will be accounted for. Fourth, random mating is

required. And finally, no natural selection can occur, meaning all genotypes must have an

equal chance of survival and reproduction. The Hardy-Weinburg equation describes the

expected norm. If all five conditions are met, no change should occur within the

allele/genotype frequencies of a population. The equation provides a norm by which

evolutionary change can be measured.

 The purpose of the following laboratory exercise was to not only

understand the aforementioned law of genetic equilibrium, but also to study the

relationship between evolution and changes in allele frequency using a sample

population. Other objectives include calculating allele and genotype frequencies using the

H-W theorem, discuss the effect of natural selection on allelic frequencies, and

explain/predict effect on allelic frequencies of selection against homozygous recessive.

The null hypotheses were assumed for the laboratory. These hypotheses

state that no relationship will be found between evolution and changes in allele frequency

and Hardy-Weinburg equilibrium theorem will not give an appropriate comparative

measure of evolution. Also, the ability to taste PTC cannot be used to determine allele

frequencies for a classroom or population of people.

Protocol: In part one of the laboratory exercise, allele frequencies for a specific trait

within a sample population (consisting, in this case, of a classroom of students) was

estimated. To do this, special PTC (phenylthiocarbamide) chemical papers were obtained.

Short strips of PTC paper were removed and handed out to students. The students placed

the strips on the tip of their tongues. If the student was a PTC taster, he/she would sense a

bitter taste. The number of tasters/nontasters was recorded, and then the decimal number

representing the frequency of tasters (p2 + 2pq) was calculated, as was the frequency of

nontasters (q2). The Hardy-Weinburg equation was used to determine the frequencies of

the two alleles, and the results were recorded.

In part two, each student was given four cards. Two of the four cards were

labeled with the capital letter A on one side of the card, and the remaining two cards were

labeled with a lowercase a. It was assumed that the class was a population of randomly
mating heterozygous individuals, each with the genotype Aa. Each student found a

partner and they both shuffled their cards face down so that the letter was not shown. The

top card of each of the partners’ pile was contributed to create Partner 1’s offspring

genotype. The cards were placed back in their respective piles, reshuffled, and the top

card was again contributed, this time to create Partner 2’s offspring genotype. Each of the

genotypes was recorded.

The two partners assumed the genotype of their offspring by creating a

hand of four cards of the appropriate genotype: for AA, a hand of four capital A’s, for aa,

a hand of all lowercase a’s, and Aa, two A’s and two a’s, just as before. The two

individuals then randomly selected a different (or possibly the same) partner to make

another generation of offspring with the same card-shuffling procedure. Class data was

collected. The process was repeated for five generations, each time randomly selecting a

mate, assuming offspring’s genotype, and recording class data.

The third part of the exercise was intended to demonstrate the probability

of recessive genotypes characterized by genetic diseases surviving to reproductive

maturity. The procedure followed was similar to that in the preceding part of the exercise.

The initial genotype (Aa) was assumed by all students. With a partner, each student

determined the genotype of his/her offspring and the offspring of his/her partner. This

time, however, every time an aa genotype was created, the offspring theoretically dies.

The set of partners who produced the fatal aa genotype tried again until they created a

surviving genotype. This process was repeated for five generations, each time selecting

against the recessive offspring. After each generation was created, the offspring of each

partner was recorded, as well as class data.

Data: The following table lists the percentages of phenylthiocarbamide tasters

based on the chemical paper test performed in part one of the exercise. The ability to taste

PTC is evidence of a presence of a dominant allele (either homo- or heterozygous). The

inability to taste PTC suggests a homozygous recessive allele. The results in Table 1 list

the percent of tasters and nontasters in the class and in the North American population

overall.
Table 1: Phenotypic Proportions of Tasters and Nontasters and Frequencies of the Determining Alleles

Phenotypes Allele Frequency

Based on the H-
W Equation
% Tasters % Nontasters p q
(p2 + 2 pq) (q2)

Class Population 60% 40% 0.3676 0.6325

North American
Population 55% 45% 0.3292 0.6708

According to the chart, and through calculations using the formulas p2 +

2pq + q2 and l - q = p, the percentage of heterozygous (2pq) tasters was approximately

46.5%. The percentage of heterozygous PTC tasters in North America was found to be

about 44.2%, so therefore the class average is relatively normal compared to the

population of the entire continent. The average of the class was only around 2% higher.

In the next table, Table 2, the class results are listed for Part 2 of the

exercise in which partners used genotype cards to create offspring for five generations.

These results are the total numbers of offspring with an AA, Aa, or aa genotype in the

class of sixteen students.

Table 2: Data of Part 2 of Laboratory Exercise Showing Class Totals for Each Genotype

Offspring’s Class Totals

Genotype for Each Genotype
Generation (AA, Aa, or AA Aa aa
aa)
1 Aa 4 6 6
2 Aa 2 8 6
3 Aa 3 7 6
4 Aa 5 8 3
5 Aa 6 5 5

The genotype frequencies for the sample class population can be

computed using the equation: Frequency of (AA/Aa/aa)= Total of (AA/Aa/aa)/Total AA +

Total Aa + Total aa. For the frequency of AA, the fraction divides 20/80. The frequency

of Aa is 34/80. The frequency of aa is 26/80. The theoretical genotype frequencies of the

beginning population where p and q = 0.5 are: p2 (AA) = .25, 2 pq (Aa) = .5, and q2 (aa) =

.25. However, after five generations of mating in the sample classroom population, the

frequencies were slightly different. For p2 (AA), the frequency was .25; for 2pq (Aa), the

frequency was .425; for q2 (aa), the frequency was .325.

Data 1: Calculations for A and a Alleles in Fifth Generation

Number of offspring with genotype AA =6x2= 12 A alleles

Number of offspring with genotype Aa = 5x1 = 5 A alleles
Total A alleles: 17
Total # alleles in population= 32
P= .53125

Number of offspring with genotype aa = 5x2 = 10 a alleles

Number of offspring with genotype Aa = 5x1 =5 a alleles
Total a alleles: 15
Total # alleles in population = 32
Q = .46875
 In the final table, Table 3, the survival rate of genetic diseases is

calculated. Both the hetero- and homozygous dominant genotypes will survive in a mock

environment, however a recessive genotype will not. There is no column for aa recessive

genotype because it did not survive into the first generation or to reproductive maturity.

Table 3: Chart of Genotypes Excluding that Which is Affected by Genetic Disease

Offspring’s Class Totals

Genotype
Generation (AA, Aa, or
aa)
1 Aa
2 AA
3 Aa
4 Aa
5 Aa
for Each Genotype
AA Aa
5 11
8 8
6 10
5 11
4 12

Data: Calculations for Alleles Present at the Fifth Generation for Table 3

Number of offspring with AA alleles 4x2= 8

Number of offspring with Aa alleles x1 = 12
Total A alleles = 20
Total # of alleles in population: 32
P = .625

Number of offspring with genotype aa x2= 0

Number of offspring with genotype Aa = 12
Total a alleles = 12
Total # of alleles in population: 32
Q = .375

In this part of the laboratory, it becomes apparent that through natural

selection, individuals with the genotype aa are eliminated, causing a decline in the

number of a alleles in this case. Therefore, the p frequencies in Part 3 are higher than

those in Part 2 of the lab exercise (in which the values were 0.5 for both p and q) and the
 q frequencies are lower than the q frequency in Part 2. This shows a trend toward A

dominance. If a similar process of selection proceeded for another five generations, the

frequency of q will continue to decrease, but it will not reach zero because the

heterozygous Aa will remain. Likewise, the frequency of A will continue to increase. The

difference in allele frequencies between Part 2 and Part 3 after five generations is

relatively apparent with the given data. In Part 2, the frequencies for A and a are very

closely related, with the a alleles at a slight loss. In Part 3, however, the frequencies are

quite different; for p, the frequency was .625 and q, a mere .375. This is a significant

difference. Q, or recessive a, had a much lower frequency in Part 3 and P, or A, had a

higher frequency.

Conclusion: In this lab, the relationship between evolution and changes in allele

frequencies were tested and observed. Using the Hardy-Weinberg equation, it was

determined how to calculate the frequencies of alleles and genotypes in the gene pool of a

population. For the Hardy-Weinberg theory to apply, the population must be in

equilibrium. This has five requirements; a large population, no migration, no selection, no

mutations, and random mating. Since only a classroom of students was used as a mock

population, the first of the five requirements listed was not met, therefore random mating

could not be ensured. In Part 2, all students started out as heterozygous, Aa. After five

“mating” simulations, the number of heterozygotes, dominant homozygotes, and

recessive homozygotes were counted. The Hardy-Weinberg theory predicts the p and q

frequencies to be .5 and .5. However, these results were not obtained. This is normal,

because Hardy-Weinberg Equilibrium does not occur in nature, nor was the experiment

performed under ideal conditions. Again, the small population size and not-so-random
 mating was probably the source of error here. In Part 3, the homozygous recessive

genotype assumed a deadly genetic disease and could not live to reproductive maturity.

This allowed for only homozygous dominant and heterozygous to survive. This created a

drastic difference in frequency; p was higher than q because the frequency of q in the

population would decrease due to natural selection.

In Part 1, it became apparent that PTC was tasted by a majority of the

population in not only the classroom, but also in North America. This, in combination

with the other data collected and discussed relating to Parts 2 and 3, leads to the rejection

of the null hypotheses. The Hardy-Weinberg Equilibrium indeed proved an appropriate

measure of evolutionary change in all parts of this experiment. Also, there was an evident

relationship between evolution and changes in allele frequency in, especially, Part 3 of

the exercise. The results of this lab were relatively accurate in the sense that the

procedure was performed as expected, but the population size of the classroom of

students was much too small. However, this was probably expected on the creation of the

lab, in order to show that Hardy-Weinberg equilibrium does not, and should not, occur in

nature. It is simply a norm by which evolutionary change can be measured. The results of

the lab exercises were conclusive and promoted a firm conviction on the rejection of the

null hypotheses.

Bibliography: Campbell, Neil. Reece, Jane. 2002. Biology: Sixth Edition. Addison and Wesley

Longman, Inc. California.

The College Board and ETS. Advanced Placement Biology Laboratory Manual

for Students. New York: 1991.

Measuring Allelic Frequency and Evolutionary Change Using the Hardy-Weinberg

Theorem and PTC Tasting

Tessa Rodes

April 7, 2009