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FACULTY OF APPLIED SCIENCES AND COMPUTING

BABS2213 PRINCIPLES OF GENETICS


PRACTICAL 3
TITLE: HARDY WEINBERG EQUILIBRIUM
NAME: CHOONG MEL JUNE
GROUP: RBS 2 A1
STUDENT ID: 14WAR10521
DATE: 4 JUNE 20140
DEMONSTRATER: DR LOH KHYE ER











Objectives:
1. To derive the Hardy-Weinberg Equation.
2. To calculate the genotypic and allelic frequencies.
3. To discuss the conditions under which a population is in Hardy-Weinberg Equation.
4. To determine if a population is in Hardy-Weinberg Equation.
5. To design and perform a test of a disruption to Hardy-Weinberg Equation.
6. To identify why the Hardy-Weinberg Equation is difficult or impossible to demonstrate
with living organisms.

Hypothesis:
Hardy and Weinberg Equilibrium showed that if there was no migration, no mutation and no
selection in a large population, the frequencies of any pair of gene allele will tend to remain
constant from generation to generation.

Introduction:
A population is a group of organisms of the same species that live and breed in the same
area. Alleles are alternate forms of genes. In standard Mendelian genetics, two alleles - one from
each parent - control an inherited trait or characteristic (e.g. seed pod color). A gene pool is the
collective term representing all the genes, including all the different alleles, found in a population.

The written expression of the DNA code, called the genotype, is normally represented
using two letters, each letter representing one allele (e.g. AA). Dominant alleles are represented
by capital (uppercase) letters. Recessive alleles are represented by lowercase letters. Genotypes
with two dominant alleles (e.g. AA) are referred to as homozygous dominant. Genotypes with
two recessive alleles (e.g. aa) are referred to as homozygous recessive. Genotypes with one
dominant and one recessive allele (e.g. Aa) are referred to as heterozygous.

The physical expression of the genotype (e.g. brown hair, blue eyes) is called the
phenotype. In standard Mendelian genetics, the heterozygous condition (e.g. Aa) retains the
homozygous dominant phenotype because the dominant allele masks the phenotype of the
recessive allele. An example of this in humans would be a heterozygote for brown eye color. The
person would carry both a dominant brown allele A and a recessive blue allele a yet have
brown eyes. The dominant brown allele masks the recessive blue allele.

The population is considered the basic unit of evolution. The small scale changes
in the genetic structure of populations from generation to generation are called microevolution.
Microevolution is the study of the change in the frequency of an allele in a population from one
generation to the next. Mathematicians Hardy and Weinberg explained how an allele could
change in a population by first showing how it would not change, the Hardy-Weinberg principle.

Genetic equilibrium is the state in which allele frequencies remain constant. In 1908,
English mathematician G. H. Hardy and German physician W. Weinberg independently
developed models of population genetics that showed that the process of heredity by itself did
not affect the genetic structure of a population. It can be said the Hardy-Weinberg Principle is a
model used to help clarify evolutionary change by determining what happens if no change occurs.
When no change occurs and an environment is stable, genetic equilibrium is maintained. The
Hardy-Weinberg theorem states that the frequency of alleles in the population will remain the
same from generation to generation. Furthermore, the equilibrium genotypic frequencies will be
established after one generation of random mating. This theorem is valid only if certain
conditions are met:

- Random mating all individuals in a population have equal opportunity to pass on
their alleles to offspring.



- Large populations genetic drift, or random changes in allele frequency, has less
effect on large populations than small populations.







- No mutations - if genes randomly mutate, new alleles may be introduced into the
population and allele frequencies will change.



- No migration individuals cannot move into or out of the population.












- No natural selection - all genotypes must have equal probabilities of reproduction.



Two equations are used to model the Hardy-Weinberg Principle.
p + q = 1; where p represents the dominant allele, A, and q represents the recessive
allele, a. This equation, used to calculate allele frequency, equals 1 or 100% of the
population.
p
2
+ 2pq + q
2
= 1; where p
2
represents the homozygous dominant genotype, 2pq
represents the heterozygous genotype, and q
2
represents the homozygous recessive
genotype. This equation, used to calculate genotype frequency, equals 1 or 100% of the
population.





















I. Testing the Hardy-Weinberg Equilibrium

Materials:
1. 400 of white colour beads. (Two different colours of beans that are approximately the
same size)
2. 400 of black colour beads. (Two different colours of beans that are approximately the
same size)

Methodology:
400 of black colour beads and 400 of white colour beads were mixed together into a beaker.



One pair of bead was picked up without looking.



The colour of the pair of bead was observed and recorded down.



The beads were placed back into the beaker and mixed well.



The steps 2, 3 and 4 were repeated 100 times and all the colour of beads were observed and
recorded down.

Results:
Assumption:
Black bead = Dominant allele, A
White bead = Recessive allele, a

Genotype
Observed number, O Average observed
number, O
Genotype Frequency
Expected
number, E G
1
G
2
G
3
G
4

AA 17 30 23 18 22 p
2
= 0.5
2
= 0.25 25
Aa 57 50 49 53 52 2pq= 2(0.5)(0.5)= 0.5 50
aa 26 20 28 29 26 q
2
= 0.5
2
= 0.25 25
Total, n 100 100 100 100 100 1 100

Calculation:
- p = 400/800 = 0.5
- q = 400/800 = 0.5
- AA + 2 Aa + aa = 1
- p2 + 2pq + q2 = 1









Applying chi square formula

( )

( )

( )









Interpretation:
Failed to reject the null hypothesis, as the P value is more than the critical value of 0.05 at degree
of freedom to 2, the hypothesis that there was no migration, no mutation and no selection in a
large population, the frequencies of any pair of gene allele will tend to remain constant from
generation to generation. There is 70% ~ 80% of the time that the deviation of the observed
number from the expected is due to the chances.

Discussion:
The Hardy-Weinberg theorem provides a mathematical formula for calculating the
frequencies of alleles and genotypes in populations. We begin with a population with two alleles
at a single gene locus - a dominant allele, A, and a recessive allele, a - then the frequency of the
dominant allele is p, and the frequency of the recessive allele is q. Therefore, p + q = 1. The
frequency of one allele, p, is known for a population, the frequency of the other allele, q, can be
determined by using the formula q = 1 - p. During sexual reproduction, the f frequency of each
type of gamete produced is equal to the frequency of the alleles in the population. If the gametes
combine at random, the probability of AA in the next generation is p
2
, and the probability of aa is
q
2
. The heterozygote can be obtained two ways, with either parent providing a dominant allele,
so the probability would be 2pq. These genotypic frequencies can be obtained by multiplying p +
q by p + q. The general equation then becomes
(p + q)
2
= p
2
+ 2pq + q
2
= 1
Male
Female
pA qa
pA ppAA pqAa
qa pqAa qqaa
To summarize:
p
2
= frequency of AA
2pq = frequency of Aa
q
2
= frequency of aa

When a population meets all of the of the Hardy-Weinberg conditions, it is said to be in
Hardy-Weinberg equilibrium. How far a population deviates from Hardy-Weinberg equilibrium
can be measured using the goodness of fit or chi-squared test (
2
).

Mathematically the chi-squared test is represented:

2
= [(observed value expected value)
2
/ expected value]

Since we have three genotypes, therefore we have 3 minus 1, or 2 degrees of
freedom. Degrees of freedom is a complex issue, but we could look at this in simple terms: if we
have frequencies for three genotypes that are truly representative of the population then, no
matter what we calculate for two of them, the frequency of the third must not be significantly
different for what is required to fit the population.



Looking across the distribution table for 2 degrees of freedom, we find our chi-squared
value of 0.48 is more than that required to satisfy the hypothesis that the differences in the O and
E data did not arise by chance. Since the chi-squared value falls above the 0.05 (5%) significance
cut-off, we can conclude that the population does differ significantly from what we would expect
for Hardy-Weinberg equilibrium.

Conclusion:
The hypothesis is accepted. At df = 2, there is 70% ~ 80% of the time that the deviation of the
observed number from the expected is due to the chances. There was no migration, no mutation
and no selection in a large population, the frequencies of any pair of gene allele will tend to
remain constant from generation to generation.


II. Genetic Drift

Materials:
1. 400 of white colour beads. (Two different colours of beans that are approximately the
same size)
2. 400 of black colour beads. (Two different colours of beans that are approximately the
same size)

Methodology:
400 of black colour beads and 400 of white colour beads were mixed together into a beaker.



One pair of bead was picked up without looking.



The colour of the pair of bead was observed and recorded down.



One pair of beads with different colours (white and black) or black in colour were placed back
into the beaker and mixed well. One pair with white colour beads represented lethal allele were
excluded and taken out from the beaker.



Steps 2, 3 and 4 were repeated 100 times and all the colour of beads were observed and recorded
down.



The whole experiment was repeated again without replace the excluded white beads of the first
batch.

Results:

Assumption:
The homozygous recessive (aa) gene is lethal.
n
1
= First batch of 100 picks;
n
2
= Second batch of 100 picks





Genotype
Observed
number, O
Expected
number, E
Expected genotype
frequency during n
1

Expected genotype
frequency during n
2

n
1
n
2
n
1
n
2

AA 30 36 25 44 p
2
= 0.5
2
= 0.25 0.442
Aa 49 47 50 45 2pq= 2(0.5)(0.5)= 0.5 0.446
aa 21 17 25 11 q
2
= 0.5
2
= 0.25 0.112
Total, n 100 100 100 100 1 1

Random
mating
Genotype frequency
Progeny frequency
AA Aa aa
AA x AA 0.33 x 0.33= 0.109 0.109 - -
AA x Aa 2(0.33 x 0.67) = 0.442 0.221 0.221 -
Aa x Aa 0.67 x 0.67= 0.449 0.112 0.225 0.112
Total 1 0.442 0.446 0.112

Calculation:
- p = 400/800 = 0.5
- q = 400/800 = 0.5
- AA + 2 Aa + aa = 1
- p
2
+ 2pq + q
2
= 1
- AA = p
2
= (0.5)
2
= 0.25
- Aa = 2pq = 2 (0.5) (0.5) = 0.5
- aa = q
2
= (0.5)
2
= 0.25

Expected genotype frequency during n
2
:
- aa (Lethal allele) = failed to reproduce
- AA = 0.25/0.75 = 0.33
- Aa = 0.5/0.75 = 0.6
Discussion:
Genetic drift describes random fluctuations in the numbers of gene variants in a
population. Genetic drift takes place when the occurrence of variant forms of a gene, called
alleles, increases and decreases by chance over time. These variations in the presence of alleles
are measured as changes in allele frequencies.

Normally, genetic drift occurs in small populations. If genetic drift continues, involved
allele is either lost by a population or left the only allele present in a population at a particular
locus. Both possibilities decrease the genetic diversity of a population. Genetic fixation, the loss
of all but one possible allele at a gene locus in a population, is a common result of genetic drift in
small natural populations. Genetic drift is a significant evolutionary force in situations known as
the bottleneck effect and the founder effect.

Besides, in this experiment, the homozygous recessive (aa) gene is lethal. A lethal allele's
phenotype, when expressed, causes the death of an organism. Lethal alleles arise when a
mutation to a normal allele disrupts the function of an essential gene. Without this essential gene,
the organism dies. Lethal alleles can be embryonic or postnatal. Embryonic lethals cause the
death of the fetus, and fertility studies are often required in order to positively determine that an
embryonic lethal exists. An example of an embryonic lethal is the AY allele in mice. Meanwhile
for postnatal lethal alleles cause abnormalities in the progeny that cause them to die early on in
development. An example is parrot jaw. Lethal alleles can be dominant or recessive and can be
sex linked or autosomal. If the allele is dominant, then both homozygous dominant and
heterozygous individuals will die. If it is a recessive allele, then only the homozygous recessive
individuals will die.

In this experiment, 400 black beads represent dominant trait and 400 white beads as
recessive trait, and the homozygous recessive 2 white beads is the lethal allele. This allele will
cause an organism to die thus failed to reproduce to next generation and failed to contribute to
the gene pool.

In a narrower sense, genetic drift refers to the expected population dynamics of neutral
alleles (those defined as having no positive or negative impact on reproductive fitness), which
are predicted to eventually become fixed at zero or 100% frequency in the absence of other
mechanisms affecting allele distributions. So, in this part of experiment, if we continue after 100
times, genetic drift will occur. From the beginning, n
1
and n
2
are close with the expected number,
n
1
and n
2
. This is because there are large population that support the Hardy-Weinberg
Equilibrium and the deviation and error are low by first generation, n
1
with the ratio of 1:2:1. But
once the offspring ends with a lethal allele, it is no longer survived in the population.

If the experiment further continues, the homozygous recessive 2 white beads (lethal allele)
will eventually no survived in this population. As the population is getting smaller, genetic drift
will having effect. And genetic drift causes populations to lose genetic variation. This is because
the number of black beads has increased and the number of white beads has reduced as it is not
replacing back. The homozygous trait and heterozygous have an increase of genetic ratio and at
the same time the homozygous recessive decrease. Hence, the black phenotype will increase if
continue the experiment with the homozygous dominant becomes the major allele in that
population with the heterozygous and homozygous recessive will reduce and become extinction.

Conclusion:
The hypothesis is accepted. As stated in Hardy-Weinberg Equilibrium, large population can
avoid the affect the genetic drift. However, if the experiment continues without replacing back
the white beads, the hypothesis is rejected. This is due to the large population is getting smaller
and genetic drift occurred. Genetic drift reduces the amount of genetic variation in a population.
With less genetic variation, there is less for natural selection to work with. And, alleles face a
greater chance of being lost. As Hardy Weinberg Equilibrium states that it is only valid when
there is no genetic drift.






III. Detection of Gene Frequencies in Human Population

Materials:
1. PTC paper strip
2. 33 members of course mates

Methodology:
(A) PTC-tasting
The whole course members tasted phenylthiocarbamide (PTC) by using PTC paper.



The results were recorded down the tasters as homozygous dominant or heterozygous and non-
tasters were homozygous recessive.

(B) Tongue-rolling
Course members who were able to roll their tongue were recorded as homozygous dominant or
heterozygous.



The course members who were not able to roll their tongue were recorded as homozygous
recessive.

(C) Facial dimples
The course members with facial dimples were recorded down as homozygous dominant or
heterozygous.



The course members without facial dimples were recorded down as homozygous recessive.















Results:
Phenotype Genotype
Observed
number, O
Estimated genotypic
frequencies
Estimated
number
PTC- Taster
TT
18
0.1 3
Tt 0.44 15
Non- PTC
Taster
tt 15 0.45 15
Tongue roller
RR
28
0.37 12
Rr 0.5 16
Non-tongue
roller
rr 5 0.15 5
Facial dimples
DD
9
0.02 1
Dd 0.25 8
No Facial
dimples
dd 24 0.73 24

Calculation:
(A) PTC taster
- q
2
= 15/33
q = 15/33
q = 0.67

- p = 1 q
p = 1 0.67
p = 0.33

Estimated genotypic ratio of
- TT = p
2
= (0.33)
2
= 0.1
- Tt = 2pq = 2 (0.33) (0.67) = 0.44
- tt = q
2
= (0.67)
2
= 0.45

Estimated number of
- TT = 0.1 33 = 3
- Tt = 0.44 33 = 15
- tt = 0.45 x 33 = 15

(B) Tongue Roller
- q
2
= 5/33
q = 5/33
q = 0.389

- p = 1 q
p = 1 0.389
p = 0.611


Estimated genotypic ratio of
- RR = p
2
= (0.611)
2
= 0.373
- Rr = 2pq = 2 (0.611) (0.389) = 0.475
- rr = q
2
= (0.389)
2
= 0.151

Estimated number of
- RR = 0.373 33 = 12
- Rr = 0.475 33 = 16
- rr = 0.151 x 33 = 5

(C) Facial Dimples
- q
2
= 9/33
q = 9/33
q = 0.853

- p = 1 q
p = 1 0.853
p = 0.147

Estimated genotypic ratio of
- DD = p
2
= (0.147)
2
= 0.02
- Dd = 2pq = 2 (0.147) (0.853) = 0.25
- dd = q
2
= (0.853)
2
= 0.73

Estimated number of
- DD = 0.02 33 = 1
- Dd = 0.25 33 = 8
- dd = 0.73 x 33 = 24

Discussion:
In this experiment, the class population has higher dominant trait in PTC taster and
tongue roller, and higher recessive trait in facial dimples.

PTC is known as Phenylthiocarbaminde Taste Paper. Besides, it is also known as
Phenylthiocarbamide, or phenylthiourea, is an organic compound that either tastes very bitter, or
is virtually tasteless, depending on the genetic makeup of the tasterThe genetic taste phenomenon
of PTC was discovered in 1931 when a DuPont chemist named Arthur Fox accidentally released
a cloud of a fine crystalline PTC. . The ability to taste PTC is a dominant genetic trait. The test to
determine PTC sensitivity is one of the most common genetic tests on humans. The genetic
correlation was so strong that it was used in paternity tests before the advent of DNA matching.
There are three SNP's (single nucleotide polymorphisms) along the gene that may render its
proteins unresponsive. There is conflicting evidence as to whether this trait is a result of either
dominance or incomplete dominance. Any person with a single functional copy of this gene can
make the protein and is sensitive to PTC. Hence, PTC tasting is largely determined by a single
gene, TAS2R8, with two common alleles, and the allele for tasting is mostly dominant over the
allele for non-tasting. However, both classical family and twin studies, and modern molecular
genotyping, show that there are other genes or environmental factors that influence PTC tasting.
As a result, there is a continuous range of PTC tasting, not absolute separation into tasters and
non-tasters.

Next, the ability to roll one's tongue is a genetic trait that historically with the concepts of
dominant and recessive traits. However, the dominant nature of the "tongue-rolling gene" has
been disproved. From some researches, family studies clearly demonstrate that tongue rolling is
not a simple genetic character, and twin studies demonstrate that it is influenced by both genetics
and the environment. The percentage of the human population that has this skill ranges from 65
to 81 percent. A slightly higher percentage of females can do this than males.

When some people smile, they have dimples in one or both cheeks. Other people don't
have dimples. This is occasionally said to be a simple genetic trait, which dimples are controlled
by one gene with two alleles, and the allele for dimples is dominant. Dimples are caused by a
fault in the subcutaneous connective tissue that develops in course of the embryonic
development. A variation in the structure of the facial muscle may also cause dimples. Transfer
of dimples from parents to children occurs due to just one gene. The dimple creating genes are
present in the sex cells prior to the process of reproduction. Each parent provides one of these
genes to the child. So, if both the parents have dimples, the children have 50-100% chances of
inheriting dimple genes. If, however, only one parent has dimple genes, the chances of the
children inheriting the genes are 50%. If neither of the parents has the dimple genes, their
children will not have dimples. But this is not always be, because their inheritance isn't
completely predictable, dimples are considered an irregular dominant trait. Having dimples is
probably controlled mainly by one gene but also influenced by other genes. However, the
presence of dimples may change during an individual's lifetime, and there is no published
evidence for a genetic basis for dimples. Therefore, dimples cannot be said as basic genetics.

Conclusion:
The hypothesis is rejected. This is because the Hardy-Weinberg Equation is difficult or
impossible to demonstrate with living organisms as living organisms may be mutated and they
are natural selection. For example, tongue rolling and dimples. The genes can be mutated by
other genes or influenced by environment.














IV. Determining Gene Frequencies Where Three Alleles are Involved

Materials:
1. Sterile lancet
2. Sterile cotton
3. 70% ethyl cotton
4. Anti-A and anti-B antiserum
5. Microscopic slide

Methodology:
A drop of each anti-A serum and anti-B serum were put on a clean slide.



Finger index was cleaned by using a piece of 70% ethyl alcohol paper.



The finger index was pricked by using sterile lancet and drop of blood was added to each test
sera.



The blood was mixed with the serum after few minute and the result was recorded down.

Results:
Phenotype
of Blood
group
Genotypes Observed
number
Observed genotypic
frequencies
Estimated
number
A I
A
I
A
7 0.017 1
I
A
I
O
0.194 6
B I
B
I
B
8 0.022 1
I
B
I
O
0.220 7
AB I
A
I
B
0 0 0
O I
O
I
O
18 0.546 18
Total: 33 1 33

Calculation:

Estimated number
- Blood type A: p
2
+ 2pq = 7/33
- Blood type B: q
2
+ 2qr = 8/33
- Blood type AB: 2pq = 0/33
- Blood type O: r
2
= 18/33

r
2
= frequency of the O phenotype,
r = r
2

r = 18/33
r = 0.739

p = ( p
2
+ 2pr + r
2
) r
p = ( 7/33 + 18/33) 0.739
p= 0.131

q = ( q
2
+ 2qr + r
2
) r
q = ( 8/33 + 18/33) 0.739
q= 0.149

Estimated genotype frequency:
- AA: p
2
= (0.131)
2
= 0.017
- AO: 2pr = 2 0.131 0.739 = 0.194
- BB: q
2
= (0.149)
2
= 0.022
- BO: 2qr = 2 0.149 0.739 = 0.22
- OO: r
2
= (0.739)
2
= 0.546

Estimated genotype number:
- AA: 0.017 33 = 1
- BB: 0.022 33 = 1
- AO: 0.194 33 = 6
- BO: 0.22 33 = 7
- OO: 0.546 33 = 18




















Discussion:
The blood groups refer to the presence on human red blood cells of certain antigens, the
blood group factors. One very important group of factors present on the red blood cells is the
ABO system. The ABO group of a person depends on whether his/her red blood cells contain
one, both, or neither of the 2 blood group antigens A and B. Hence, there are 4 main ABO
groups: A, B, AB and O.

Antibodies (agglutinins) for the antigens A and B exist in the plasma and these are
termed anti-A and anti-B. The corresponding antigen and antibody are never found in the same
individual since, when mixed, they form antigen-antibody complexes, effectively agglutinating
the blood.

In this experiment, we used anti-A serum and anti-B serum. Anti-A serum and anti-B
serum are used in the forward typing of ABO blood grouping. Anti-A sera contain antibodies to
the A blood group, while Anti-B sera contain antibodies to the B blood group. So, when doing a
blood type, if anti-B serum reacts with the patient's blood, this means that the person is group B
or their blood cells at least contain the B antigen. When the patient's blood containing the B
antigen is mixed with anti-B serum this causes clumping. They could be type B. If the blood
causes clumping with anti-A serum and anti-B serum, this means the person is group AB blood
type. However, there is no agglutination in anti-A serum and anti-B serum in type O blood. As
blood type O contains anti-A antibodies and anti-B antibodies, which is no containing antibodies,
it wont causes clumping with anti-A serum and anti-B serum.

By referring diagram below:
1. Blood group type B
2. Blood group type A
3. Blood group type AB
4. Blood group type O



ABO blood group is an example of three allele involved. In the class population, blood
groups of O have the highest probability compared with other blood types. Meanwhile, there is
no population that have blood group of AB. When two copies of gene (type A with type O or
type B with type O) are fused to form the next generation, the one with blood group type A is
IAIA or IAIO while blood group B forms genotype of IBIB or IBIO. Both of this is dominant.
However when genotype IAIB gene form, this is a codominant trait which indicates in AB blood
group. If an individual consist of genotype IOIO, the individual are said to be homozygous
recessive and show as blood group O.
Conclusion:
The hypothesis is rejected. Although p + q + r = 1 as Ab group is the codominance, so 2pq is not
included, but, in reality, no population satisfies the Hardy-Weinberg Equilibrium completely
because human always choose their mates (no random mating) and always migration. It can be
seen that phenotype of Blood AB group is absence in this experiment.

References:
1. Crow, J. F. Hardy, Weinberg and language impediments. Genetics 152, 821-825 (1999).
2. Edwards, A. W. F. & Hardy, G. H. 1908 and Hardy-Weinberg
Equilibrium. Genetics 179, 1143-1150 (2008).