BABS2213 Principles of Genetics

BABS2213 Principles of Genetics_2019v0
Department of Bioscience, FOAS, TARUC
FACULTY OF APPLIED SCIENCES

KUALA LUMPUR CAMPUS
BABS2213 PRINCIPLES OF GENETICS
LABORATORY MANUAL
BACHELOR OF SCIENCE (HONOURS) IN

BIOSCIENCE WITH CHEMISTRY
LABORATORY SAFETY RULES
The following laboratory safety rules apply specifically to practical and other
activities in the undergraduate teaching laboratories.
For all practical work to be undertaken in this course:

• Health, safety and environmental aspects of the practical have been considered
and risk assessments have been carried out on the chemicals and procedures
involved.
• Safety equipment has been provided where necessary. Appropriate information
and supervision necessary for the safe execution of each practical is provided.
Specific information about particular hazards and how to avoid, eliminate or
minimize your exposure to them is given in the relevant places in the laboratory
manual.
Students are required to:

• Avoid, eliminate or minimize hazards of which they are aware.
• Comply with all occupational health and safety instructions.
• Make proper use of all safety devices and personal protective equipment.
• Not willfully place at risk the health and safety of themselves or any other
person.
• Seek information or advice where necessary or when in doubt before carrying
out new or unfamiliar work.
• Wear protective clothing and footwear.
• Be familiar with emergency and evacuation procedures. Report and record all
accidents and near miss incidents.
Emergency Evacuation and Safety Equipment

• In an emergency and during practice evacuations, move quickly and carefully
from the laboratory to the external stairwell or nearest emergency exit. Proceed
to the designated assembly area (tutor will advise) and wait there until
permission is given to re-enter the building. Never run in the laboratory or
along corridors. Be aware of the position of emergency and other exits.
• Ensure you are aware of the safety facilities of the laboratory including the
location and use of safety showers and eyewash stations.
Laboratory dress code

• Enclosed footwear must be worn at all times in the laboratory. ‘Enclosed’
requires that the heel and upper foot be covered. You will be denied
participation in that session unless wearing suitable footwear. If you are
changing shoes before or after the practical this must be done outside the
laboratory.
• A clean laboratory coat must be worn when conducting practical work and
must be fastened (all buttons done up). Sleeves must be rolled down. It must be
removed when leaving the laboratory for any reason. You must store your
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laboratory coat in a plastic bag between practical to reduce the risk of
transferring contaminants to your books and other articles. Laboratory coats on
which chemicals or biological materials have been spilled must not be taken out
of the laboratory and must be left with the Preparation Laboratory staff for
decontamination. It is not necessary to wear a laboratory coat in the case of
tutorials, exams (except practical exams) or poster sessions.
• Safety glasses must be worn in practical classes where you are instructed that
eye protection is required. Prescription glasses alone are not sufficient – safety
overglasses must be worn in addition to these. You are required to provide
your own safety glasses or overglasses. Contact lenses do not provide any eye
protection for hazardous operations and must be worn in conjunction with
approved safety eyewear. If you wear contact lenses during practical involving
potential exposure to chemical fumes, vapours or splashes, you should remove
them at the first sign of any eye redness or irritation.
• Where gloves are required, they must be worn. They must be removed if you
leave the laboratory for any reason.
• Long hair must be tied back. If a headscarf is worn, the ends must be tucked
into the front of the lab coat. Peaked caps are not permitted in practical classes
where gas burners are being used (unless worn backwards) as they constitute a
fire risk.
• Cuts and other skin wounds must be covered.
Food and Drink

• Eating and drinking is prohibited in the laboratory. This includes sweets, gum
and drinking from water bottles. Any food or drink must remain in your bag on
the bag racks – you may not remove it inside the laboratory. This includes
drinking from water bottles while standing at the bag racks. The staff and tutors
have authority to dispose of any items of food or drink as necessary. Take off
your laboratory coat and gloves, wash your hands and exit the laboratory with
your bag if you need to access items of food and drink during the practical.
General Rules
• Leave bags, jackets etc on the bag racks near the entrance to the laboratory. Do
not block passageways or fire exits. Keep valuables with you.
• Do not enter the Preparation Room without permission from laboratory staff or
tutors.
• Keep hands, pens and other material that may become contaminated away from
your face. Ensure that workbooks, lab manuals etc do not become contaminated.
• Mobile phones should be turned off and should not be used during the practical.
They should be kept in your bag or pocket and not placed on the bench where
they could become contaminated.
• The use of ear-plug/headphone audio devices (IPods, MP3 players etc) is
prohibited in the teaching laboratories.
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• Experiments may only be performed under the direct supervision of a tutor,
during the scheduled hours of opening of the laboratory. Follow the instructions
of the tutor at all times.
• Unauthorised experimentation is strictly forbidden. Inappropriate conduct and
disruptive behaviour may result in denial of further laboratory access.
• No reagent, specimen or equipment is to be removed from the laboratory
without permission of the tutor or staff member in charge.
• Be aware of the conditions required for the safe handling of substances.
Information is provided in the laboratory notes. If in doubt, ask your tutor.
• Sitting on laboratory benches is prohibited.
• Pipetting by mouth is prohibited. Automatic pipettes or other pipetting aids are
provided. Take care not to contaminate these.
• Report any defective equipment or broken glassware to the tutor.
• Spills must be reported to the tutor immediately. A staff member or tutor will
clean up if the spill is hazardous. If microbiological cultures are spilled, remain
seated and ask someone else to report to the tutor so that the contamination is
not spread. Contaminated lab coats must be left with the Preparation
Laboratory staff for decontamination.
• UV transilluminators and other radiation sources must only be used under the
direction of the tutor or member of staff.
• Correct use of gas burners will be demonstrated. Be aware of the potential
danger of unattended burners as the pilot light is often difficult to see. Before
leaving the laboratory, turn the gas burner off at the tap.
• Handle dissecting equipment with care, store scalpel blades covered and
secured inside the dissecting kit and always remove blade from handle.
• Regard all microbiological cultures as potential pathogens. Work practices
should aim to minimize the production of aerosols when working on the open
bench.
• In the case of practical using microorganisms, swab the bench with 70%
ethanol when you have finished your work.
Waste Disposal
• Wastes must be disposed in accordance with the charts displayed in the
laboratory, unless you are given special instructions in your laboratory notes or
by the tutor or member of staff. Do not dispose of waste via the sink unless you
are authorized to do so.
• Hazardous chemical waste is to be disposed of into the correct labeled
containers which are provided.
• Sharps (including pasteured pipettes and plastic pipette tips) are to be disposed
of into the yellow plastic sharps bins provided on the benches.
Leaving the Laboratory
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• At the end of the practical session and if you leave the laboratory for any reason
during the practical, you must REMOVE your laboratory coat and gloves and
wash your hands. Hands-free hand washing sinks and skin disinfectant are
located at both ends of the laboratory.
• If you are changing shoes at the end of the practical, this must be done
OUTSIDE the laboratory.
• The wearing of gloves and/or lab coat when using the water cooler in the
corridor is prohibited.
• Exercise care when opening and closing doors on entering and leaving the
laboratory.
First Aid
• Report all accidents, injuries and illnesses to the tutor immediately. If required,
trained personnel will administer first aid. All accidents, injuries and illnesses
must be recorded. Non injury-causing incidents such as spills, electrical shorts
etc must also be reported.
• Eye injuries (chemical/biological splash or mechanical injury) are serious.
Treatment requires immediate and prolonged flushing with water (20 minutes
minimum) at the eyewash station. Notify tutor immediately. If contact lenses are
worn, remove these as soon as practical but do not delay irrigation while waiting
for contact lens removal. Medical advice should always be obtained for an eye
injury – MSDS should accompany student.
• Chemical or biological spills on skin - thoroughly wash affected area with
copious quantities of water. Notify tutor immediately. Laboratory staff will
consult MSDS to determine appropriate first aid. MSDS should accompany
student if necessary to seek medical treatment.
• Burns including chemical burns – cool burnt area under running water (10
minutes for thermal burns, 20 minutes for chemical burns). Do not apply ice or
any lotions or creams. Seek medical advice (take MSDS if chemical burn).
• Sharps injuries – notify tutor immediately. Wash the wound and encourage
bleeding
• If you are feeling unwell or dizzy when participating in a practical class, stop
immediately, sit down and notify tutor.
Pregnancy and Allergies

Students who are pregnant or trying to fall pregnant may be at higher risk from
exposure to certain chemicals and hazards. In addition, some students may develop
allergies or may be sensitive to particular chemicals. It is important that you contact
the lecturer or person running the practical class if this applies to you.
Student Safety Declaration Form for Laboratory Work
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This form must be completed by the Student during the first practical class. No
experimental work should start until this form has been completed.
Name: _______________
Student ID Number: _____________
Course Code: _________
I have read and agreed on the following:
 I have read and I understand the LABORATORY SAFETY RULES.

 I am aware of my Health and Safety responsibilities.
 The lecturer/lab instructor has explained what personal protective
equipment (PPE such as lab coat and goggles) is required. I agree to wear it
when required.
 I understand that if I am not wearing appropriate PPE, I can be excluded
from the laboratory for that class.
 I agree to follow all safety procedures explained to me by the lecturer/lab
instructor.
 I understand that I must not eat food or drink in the laboratory.
 I understand that inappropriate conduct can result in the denial of further
laboratory access.
 I understand that all faulty or broken equipment needs to be brought to the
attention of my lecturer/lab instructor / lab staff immediately.
 I am familiar with the emergency procedures for the laboratory and are
familiar with the location of the eye wash and safety shower.
Student signature:
Date:
6
BABS2213 PRINCIPLES OF GENETICS
CONTENTS
NO. EXPERIMENT PAGE NO.
1 Probability of Inheritance 8
2 Testing the Genetic Ratios -- Chi-Square Analysis 15
3 Hardy Weinberg Equilibrium 19
4 The Mendelian Principles, Gene Interaction and Polygenic 26

Inheritance
5 Mendelian Genetics: Drosophila 33
6 The Sex Check 43
7 The Effect of Colchicine on Chromosome 47
8 Chromosomes: Polytene Chromosomes from Drosophila Salivary 51

Glands and Human Chromosomes
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Experiment 1: Probability of Inheritance
Background:
In 1866 Gregor Mendel, an Austrian monk, published the results of his study of inheritance
on garden peas. Although Mendel did not understand the mechanics of inheritance, his work
became the basis for the modern study of genetics. From his studies on the inheritance of
certain traits in pea plants, Mendel formulated three laws of inheritance:
1. the law of dominance,
2. the law of segregation and
3. the law of independent assortment.
Mendel thought that every trait was controlled by a pair of factors, which we now called
genes. The law of dominance states that one gene, the dominant gene, prevents the
appearance of the trait controlled by the other gene, the recessive gene. The law of
segregation states that during gamete (egg and sperm) formation, the pair of genes for a trait
separate, so that each gamete has only one of the genes for the trait. The law of independent
assortment states that as gametes are being formed, the genes for various traits separate
independently of one another. In this activity you will learn some principles of probability.
You will use these principles and Mendel’s law to predict the inheritance of traits.
Objectives:
In this activity you should be able to
1. Predict the probability of the occurrence of a single event.
2. Predict the probability of two independent events occurring at the same time.
3. Apply Mendel’s law to predict the occurrence of certain traits on the offspring of
parents exhibiting particular traits.
I. The Idea of Chance
First of all, consider a simple demonstration of the operation of chance (i.e., probability) in
the tossing of coins. It is usually impossible to measure or control the many factors involved
when a coin is tossed and allowed to return to the resting position. The end result is said to
occur by chance. The coin returns to a resting position with one or the other of its faces
upturned, that is, heads or tails. When a single coin is tossed many times, it is expected that
about half of the tosses will result in heads facing up and the other half in tails.
Table 1: Results of Tossing a Coin 30 times
Results Observed Expected Deviation (O - E)2 / E

Number (O) Number (E) (O - E)
Heads (H)
Tails (T)
Total 30 0 2 =
1. Toss a coin 30 times. Record the results in Table 1. Calculate the expected number of
heads and tails and determine the deviations (O-E) between observed and expected.
Be sure to indicate whether each deviation is a positive or a negative number. What is
the sum of the deviations?
2. If the deviations (O-E) obtained are small, you can attribute them to chance. If they
are large, you must attribute them to some cause other than chance. After you study
the chi-square test in Investigation 4, you may wish to return to the data in Table 3.1
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to decide whether or not the deviations are too large to be attributed reasonably to
chance alone.
3. What biological and genetic situations are analogous to, and result in, the 50:50
expectation one observes when flipping a coin? Consideration of the following
questions will indicate a few biological parallels to flipping a coin:
a. What is the probability that the child expected in the Smith family will be a
boy? A girl?
b. If you randomly select 100 families having only one child, in how many of
these families might you expect the child to be a boy? A girl?
c. What is the probability that if the mating Aa x aa is made, the offspring will
have the genotype Aa? Genotype aa?
II. Independent Events Occurring Simultaneously
1. Now toss two coins together 40 times. Record the results in Table .2. When
calculating the expected number in each category, keep in mind that when two or
more coins are tossed together, each is independent and has an equal chance of falling
heads or tails. The results obtained from a series of tosses vary with the number of
coins tossed together. The expected results can be postulated on the basis of the
following generalization: The probability of two or more independent events
occurring simultaneously is the product of their individual probabilities.
2. When two coins are tossed together, the chance for each falling with heads up is ½ .
Likewise the chance for tails is ½ . Therefore, the chance that both will be heads is ½
x ½ (= ¼ ).
Table 2: Results of Tossing Two Coins 40 times
Results Combinations Observed Expected Deviation

Number (O) Number (E) (O - E)
Heads on both HH
coins
Heads on one, HT, TH
tails on the
other coin
Tails on both TT
coins
Total 4 40
3. Note that this situation is similar to what one sees in a simple monohybrid cross.
When Aa produces gametes, the probability is that ½ of the gametes will contain the
gene A and ½ will contain a. When an Aa female is crossed with an Aa male and
progeny are produced, the probability is ¼ that an A egg and an A sperm will come
together to produce an AA offspring. Similarly, the probability is ½ for Aa and ¼ for
aa progeny. In studying this monohybrid cross you are considering a situation in
which independent events (the union of different kinds of gametes) occur
simultaneously. Thus, a basic probability principle underlies Mendel's first law.
4. The same law of probability applied to the experiment with two coins may be used to
calculate the expected ratios when 3, 4, or more coins are tossed together. For
example, calculate the expected results from tossing three coins together. Let H
represent the individual coins that fall heads and let T indicate tails for the same coins.
Complete Table 3, showing the various possible combinations and the chances of
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their occurring. The first combination of three heads, which can occur only one way,
that is, when all three coins show heads, is worked out as an example. Finally, toss
three coins 40 times and record the frequency of occurrence of each of the classes.
Calculate the expected numbers and deviations.
Table 3: Expected Results from Tossing Three Coins Together
Classes Combinations Probability of Observed Expected Deviation

Each Class Number Number (O - E)
Occurring (O) (E)
3 heads HHH ½ x ½ x ½ =
1/8
2 heads: HHT, HTH
1 tail THH
1 head:
2 tails
3 tails
Total
5. A parallel situation would be observed if you were to study families consisting of

three children. If you were to select randomly 160 families in each of which there
were three children, in how many would the three children be expected to be
a. boys?
b. Two boys and a girl?
c. One boy and two girls?
d. Three girls?
6. Calculate the expected results for tossing four coins together. Complete Table 4,
showing the probability of the different combinations.
Table 4: Expected Results from Tossing Four Coins Together
Classes Combinations Probability of Each Class

Occurring
4 heads HHHH
3 heads : 1 tail HHHT, HHTH, HTHH, THHH
2 heads : 2 tails
1 head : 3 tails
4 tails
7. Assuming that the probability that a boy will be born is 1/2 and that the chance for a
girl is also 16, answer the following questions:
a. If four babies are born in a given hospital on the same day, what is the probability
that all four will be boys?
b. What is the probability that three will be boys and one a girl?
c. What is the probability that two will be boys and two girls?
d. What combinations of boys and girls among the four babies is most likely to
occur? Why?
e. What is the probability that if a fifth child is born it will be a boy? A girl?
III. Binomial Expansion
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In Sections I and II you determined combinations empirically by actually tossing coins
(first a single coin and then two coins) and observing the results. Section II discussed a
generalization that could be carried over to larger numbers of coins. In this section, you
obtained additional empirical data to compare with expected numbers calculated on the
basis of the principle first developed. Finally, you saw that if a 1:1 ratio, similar to that for
heads and tails on coins, is assumed for boys and girls in human populations, the same
combinations would be expected for babies as for coins.
Expectations for various combinations in groups of given size (n) can be obtained math-
ematically. Mendel and others recognized that combinations can be calculated by
expanding .the binomial (a + b)n, in which n is the size of the group, a is the probability
of the first event, b is the probability of the alternative event, and a + b = 1. In the
example involving babies, a = probability of girls = 1/2 and b = probability of boys = 1/2.
Now if you wish to consider the case in which four babies are born in the hospital in one
day, expand (a + b)4 = a4 + 4a3b + 6a2 b2 + 4ab3 + b4.
Expectation of 4 girls = a4 = (½ )4 = 1/16.

Expectation of 3 girls: 1 boy = 4a3b = 4(½ )3(½ ) = 4/16 = ¼ .
Expectation of 2 girls: 2 boys = 6a2b2 = 6(½)2(½)2 = 6/16 = 3/8.
Expectation of 1 girl: 3 boys = 4a b3 = 4(½)(½)3 = 4/16 = ¼ .
Expectation of 4 boys: = b4 = (½)4 = 1/16.
1. If five babies are born in a given hospital on the same day, what is the chance of
a. 5 boys;
b. 4 boys, 1 girl;
c. 3 boys, 2 girls;
d. 2 boys, 3 girls;
e. 1 boy, 4 girls;
f. 5 girls?
Note: To answer this question you expand the binomial (a + b)5
When the probability of only a certain combination in a given size group is required,
factorials may be employed as indicated in the following formula:
P
n!
 p x q n x
x!(n  x)!
where P is the probability to be calculated; n! is the product of the number of integers in

the group of items [n! = n x (n - 1) x (n - 2) x (n - 3) x ... X 1 (i.e., if a group consists of
five items (such as the five babies in part 111-1), n! = 5! = 5 x 4 x 3 x. 2 x 1 = 120]; x ! is
the product of the integers for one class (p) and (n - x)! is the product of the integers in the
other class (q); p is the probability for one occurrence (i.e., boys) and q is the probability
for the other (i.e., girls)).
Note: Factorial 0(0!) = 1 and any number raised to the 0th power = 1.
2. If six babies are born in a given hospital on the same day, what is the probability that
two will be boys and four will be girls? Substitute in the following formula:
P
n!
 p x q n x
x!(n  x)!

2!(4!) 2
  
6! 1 2 1 4
2
=15 x ¼ x 1/16 = 15/64
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Now, using the same procedure, calculate the probability that among six children
born in a givens hospital on a particular day
a. one will be a boy and five will be girls.
b. three will be boys and three will be girls.
c. all six will be girls.
3. Albinism in humans is controlled by a recessive gene (c). From marriages between

two normally pigmented carriers (Cc), what is the probability of having a normal
child?___________An albino?______ Assume that from such a marriage (Cc x Cc)
four children will be produced.
a. What is the probability that all four will be normal?
b. Three will be normal and one albino?
c. Two normal and two albino?
d. One normal and three albino?
e. All four albino?
[Note that the theoretical expectation from the cross Cc x Cc is a 3:1 ratio. Yet when
only four progeny are produced, the probability is actually less than one half (27/64)
that the expected 3:1 ratio will be obtained! Thus, geneticists generally favor
experimental organisms that produce many progeny so that theoretically expected
ratios can be more readily approximated.]
IV Either–Or Situations (Mutually Exclusive Events)
An additional probability principle is useful in solving certain problems: The probability

of either one or the other of two mutually exclusive events occurring is the sum of their
individual probabilities.
Three examples will illustrate how this principle has implications for genetic studies.
1. What is the probability that an individual with the genotype Cc will produce either C
or c gametes?
Obviously the answer must be I (i.e., 100%); that is, Cc can produce only two kinds
of gametes-those containing C and those containing c. On the basis of the principle
just presented, ½ = probability that the gamete will contain C and ½ = probability that
the gamete will contain c. Therefore, the probability that the gamete will contain
either C or c is ½ + ½ = 1.
2. If Aa is mated to Aa, what is the probability that the offspring will have either the
genotype AA or the genotype Aa?
The probability for AA = ¼ ; that for Aa = ½ . Therefore, ¼ + ½ = ¾ = probability of
either AA or Aa.
3. Probability can also be a useful tool in predicting the results of dihybrid and trihybrid
matings. For example, in mating AaBb x AaBb, one would expect ¼ of the progeny to
be AA, ¼ Aa, and ¼ aa, or ¾ A- to ¼ aa. Likewise, ¼ should be BB, 2/4 Bb, and ¼ bb,
or ¾ B- to ¼ bb. Now if the A(a) and B(b) genes are independently assorting, one
would expect ¾ x ¾ = 9/16 of the progeny to be A-B-, ¼ x ¼ to be AAbb, and so forth.
Now use this approach in answering the following series of questions. If AaBb is
mated to AaBb, what is the probability that the offspring will have
a. either the genotype AABb or the genotype AaBB?
b. either the genotype AaBb or the genotype aaBb?
c. either the genotype Aabb or the genotype aaBB?
d. either the phenotype A-B- or the phenotype aaB-?
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V. Probability and Genetic Counseling
In many genetic counseling situations the counselor will prepare a pedigree for the family
or families seeking advice. Insofar as possible, the counselor will, determine the
phenotype and the genotype for each person in the pedigree. The counselor can then apply
probability principles to determine the probability that a defective child will be produced
among the offspring of a certain marriage.
To illustrate the application of these concepts, consider the pedigree shown in Figure 1.
For the purposes of this problem your instructor will indicate which members of the
pedigree express the genetic abnormality. Unless there is evidence to the contrary, assume
that individuals who have married into this family do not carry the recessive gene for the
trait.
For a specific example, you might assume that four members of this pedigree are albinos:
the woman in the first generation, her third daughter (the mother of individuals 9, 10, and
11), and individuals 6 and 12 in the third generation. Now assume that you are a genetic
counselor and that individuals 1 and 10 in the third generation of this pedigree come to
you and ask, "What is the probability that if we marry and have a family, an albino child
will be born to us?"
First, the counselor must determine the probability that individuals 1 and 10 are
heterozygous carriers of the recessive gene for albinism. The counselor must also
consider the probability of two heterozygous carriers producing a homozygous recessive
child. First of all, the mother of individual 1 must be heterozygous (Hh) because her
mother is an albino (hh). One can further assume, because there is no evidence to the
contrary, that the father of individual 1 is homozygous normal (HH). Thus, the probability
is ½ that individual 1 is heterozygous. The probability that individual 10 is heterozygous
is 1 (i.e., 100%), because the mother of individual 10 is an albino (hh). Furthermore, if
two heterozygous carriers marry (Hh x Hh), the probability of their having a recessive
albino child (hh) is ¼ .
Finally, the genetic counselor may advise individuals 1 and 10 that if they have children,
the probability of their having an albino child will be ½ x 1 X ¼ = 1/8. This is the
probability that the three independent events will occur simultaneously.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
16 17
Figure 1: Human pedigree showing four generations. Circles represent females and
squares represent males.
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Continue to assume that the same three individuals in the pedigree are albinos, and
calculate the probability of the albino trait appearing in the offspring if the following
cousins and second cousins should marry and reproduce (show your calculations):
a. 3 x 6 ________________________________________________
b. 2 x 11 _______________________________________________
c. 6 x 13 _______________________________________________
d. 5 x 14 _______________________________________________
e. 2 x 7 ________________________________________________
f. 16 x 17 ______________________________________________
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Experiment 2: Testing the Genetic Ratios -- Chi-Square Analysis
Objectives:
The students will be able to:

i. Calculate 2 to determine whether a data set approximates a theoretically
expected ratio.
ii. Interpret a calculated 2 value at an appropriate number of degrees of freedom.
Introduction:
The Chi Square Test

The extent an observed data set fits or differs from the predicted or expected occurrences can
be evaluated by testing the goodness of fit of the data. When the data do not fit exactly, we
will want to find out how much deviation can be allowed before we reject a null hypothesis.
One of the simplest statistical tests to assess the goodness of fit of the null hypothesis is chi-
square analysis (2), which tests the difference between observed (O) and expected (E) values.
This 2 value is then used to estimate how frequently the observed deviation can be expected
to occur strictly as a result of chance, allowing us to determine whether progeny phenotype
ratios fit our assumptions about their genotypes. The formula for calculating the chi-square
value is:
n
(Oi  Ei ) 2
 = 
2
i 1 Ei
where O = the observed number of individuals in a particular phenotype,

E= the expected number in that phenotype, and
Σ= the summation of all possible values of (O-E)2/E for the various phenotypic
categories
The degree of freedom (df), which is equal to n – 1, where n is the number of categories (the
number of phenotypes considered), will then be determined. Degrees of freedom must be
taken into account because the greater the number of categories, the more deviation is
expected by chance alone. The calculated 2 value can now be interpreted at a corresponding
probability value (p), which can be obtained from 2 table (Appendix) for (n–1) degree of
freedom.
The p value can be thought as a percentage. For example, a p value of 0.26 indicates that were
the same experiment repeated many times, 26% of the trials would be expected to exhibit
chance deviation as great as or greater than that seen in the initial trial. Conversely, 74% of
the repeats would show less deviation than initially observed as a result of chance. The
interpretation of the p value reveals that a hypothesis is never proved or disproved absolutely.
Instead, a relative standard must be set to serve as the basis for either rejecting or failing to
reject the hypothesis. This standard is often a probability value of 0.05. If the calculated 2
value is smaller than the critical value given in the table at (n–1) df, then we accept the null
hypothesis and conclude that the difference between observed and expected results is not
significant, and vice versa.
The following is an example of how one might apply the 2 test to genetics. In a cross of tall
tomato plants to dwarf ones, the F1 consisted entirely of tall plants, and the F2 consisted of
102 tall and 44 dwarf plants. Does this data fit a 3:1 ratio? To answer this question, a 2 value
can be calculated (see table 1).
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Table 1: Summary of Calculation of Chi-Square Value in Hypothetical Plants

Phenotype Genotype O E (O-E) (O-E)2 (O-E)2/E
Tall T- 102 109.5 -7.5 56.25 0.5137
Dwarf tt 44 36.5 7.5 56.25 1.5411
Totals 146 146 2.0548
The calculated 2 value is 2.0548. What does this mean?
If the observed had equaled the expected, the value would have been zero. Thus, a small 2
value indicates that the observed and expected ratios are in close agreement. However, are the
observed deviations within the limits expected by chance?
In order to determine this, one must look up the 2 value on a chi square table (see table 2).
Statisticians have generally agreed on the arbitrary limits of odd of 1 chance in 20 (probability
= .05) for drawing the line between acceptance and rejection of the hypothesis as a
satisfactory explanation of the data tested. A 2 value of 3.841 for a two-term ratio
corresponds to a probability of .05. That means that one would obtain a 2 value of 3.841 due
to chance alone on only about 5% of similar trials. When 2 exceeds 3.841 for a two term
ratio, the probability that the deviations can be attributed to chance alone is less than 1 in 20.
Thus, the hypothesis of compatibility between the observed and expected ratios must be
rejected. In the example given the 2 value is much less than 3.841. Therefore, one can
attribute the deviations
to chance alone.
Cautions When Using Chi-Square

Statisticians suggest that calculating 2 is inappropriate when the sample size in any class is
less than five. With certain exceptions, 2 calculations must be based on numerical
frequencies and not on percentages or ratios.
The P-value is the probability of observing a test statistic at least as extreme in a Chi-square
distribution. Accordingly, since the cumulative distribution function (CDF) for the
appropriate degrees of freedom (df) gives the probability of having obtained a value less
extreme than this point, subtracting the CDF value from 1 gives the P-value. The table below
gives a number of P-values matching to χ² for the first 10 degrees of freedom. A P-value of
0.05 or less is usually regarded as statistically significant.
Table 2: 2 values at various degrees of freedom

Degrees of freedom (df) 2 value
1 0.004 0.02 0.06 0.15 0.46 1.07 1.64 2.71 3.84 6.64 10.83
2 0.10 0.21 0.45 0.71 1.39 2.41 3.22 4.60 5.99 9.21 13.82
3 0.35 0.58 1.01 1.42 2.37 3.66 4.64 6.25 7.82 11.34 16.27
4 0.71 1.06 1.65 2.20 3.36 4.88 5.99 7.78 9.49 13.28 18.47
5 1.14 1.61 2.34 3.00 4.35 6.06 7.29 9.24 11.07 15.09 20.52
P value (Probability) 0.95 0.90 0.80 0.70 0.50 0.30 0.20 0.10 0.05 0.01 0.001
Nonsignificant Significant
Materials:
Non-biological materials: container of equal quantities of colored and white beads, petri
dishes, two equal-value coins and a calculator.
16
For Activity 1 to 4, you are to work in a group of 2-3 students.
Activity 1
You will be given a beaker containing 150 colored and 150 white beads that have been
thoroughly mixed. Proceed with the following:
1. Remove one petri dish-full random sample of beads from the beaker.
2. Segregate and count the beads of the different colors.
3. Record your data in Table 1 and then calculate the expected numbers based on the
size of the sample and the known ratio of colored to white beans in the entire
population. Complete Table 1 and calculate 2.
Table 1
Classes Observed Expected Deviations (OE)2 (OE)2/E
(Phenotypes) (O) (E) (OE)
Colored
White
Total: 2 =
Degrees of freedom: ____________

P value: _________________; Critical value: _________________
I cannot reject /reject the hypothesis that the data approximate the expected ratio.
Activity 2
1. Toss two equal-value coins together 30 times.
2. Record your data in Table 2 and then calculate the expected numbers. Note that the
probability of two or more independent events occurring simultaneously is the
product of their individual probabilities.
3. Complete Table 2 and calculate 2.
Table 2
Heads on both coins
Heads on one, tails on
the other coin
Tails on both coins
Total: 2 =

I cannot reject /reject the hypothesis that the data approximate the expected ratio.
Activity 3
You will be given a beaker containing 100 beads of two different colors in the ratio of 3:1.
The beads have been thoroughly mixed. Proceed with the following:
17
1. Remove one petri dish-full random sample of beads from the beaker.
2. Segregate and count the beads of the different colors.
3. Record your data in Table 3 and then calculate the expected numbers based on the size of
the sample and the known ratio of colored to white beans in the entire population.
Complete Table 3 and calculate 2.
Table 3
Colored
White
Total: 2 =

I cannot reject reject the hypothesis that the data approximate the expected ratio.
Activity 4
The following are the approximate frequencies of the various ABO blood groups in a
hypothetical population: 41% A, 9% B, 3% AB, and 47% O. Determine if the data given in
Table 4 represent a satisfactory sample of the hypothetical population. Calculate E and 2
values to two decimal places, complete the table, and answer the following question.
Table 4
Phenotype Observed Expected Deviations (OE)2 (OE)2/E
(O) (E) (OE)
A 326
B 82
AB 27
O 404
Total: 2 =

I cannot reject/reject the hypothesis that the data approximate the expected ratio.
18
Experiment 3: Hardy Weinberg Equilibrium
Background:
In 1908 Hardy in England and Weinberg in Germany independently demonstrated a principle
on which population genetics is founded. This principle is concerned with the frequencies of
genes or alleles present in a gene pool. Hardy and Weinberg 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. That is if the gene
frequencies of the parental population is known, the expected zygotic frequencies could be
predicted.
Hardy and Weinberg went on to develop a simple equation that can be used to discover the
probable genotype frequencies in a population and to track their changes from one generation
to another. This has become known as the Hardy-Weinberg equilibrium equation. In this
equation (p² + 2pq + q² = 1), p is defined as the frequency of the dominant allele and q as the
frequency of the recessive allele for a trait controlled by a pair of alleles (A and a). In other
words, p equals all of the alleles in individuals who are homozygous dominant (AA) and half
of the alleles in people who are heterozygous (Aa) for this trait in a population. In
mathematical terms, this is
p = AA + ½Aa
Likewise, q equals all of the alleles in individuals who are homozygous recessive (aa) and the
other half of the alleles in people who are heterozygous (Aa).
q = aa + ½Aa
Because there are only two alleles in this case, the frequency of one plus the frequency of the
other must equal 100%, which is to say
p+q=1
Since this is logically true, then the following must also be correct:
p=1-q
There were only a few short steps from this knowledge for Hardy and Weinberg to realize that
the chances of all possible combinations of alleles occurring randomly is
(p + q)² = 1
or more simply
p² + 2pq + q² = 1
In this equation, p² is the predicted frequency of homozygous dominant (AA) people in a

population, 2pq is the predicted frequency of heterozygous (Aa) people, and q² is the
predicted frequency of homozygous recessive (aa) ones.
From observations of phenotypes, it is usually only possible to know the frequency of
homozygous recessive people, or q² in the equation, since they will not have the dominant
trait. Those who express the trait in their phenotype could be either homozygous dominant
(p²) or heterozygous (2pq). The Hardy-Weinberg equation allows us to predict which ones
they are. Since p = 1 - q and q is known, it is possible to calculate p as well. Knowing p and
q, it is a simple matter to plug these values into the Hardy-Weinberg equation (p² + 2pq + q²
19
= 1). This then provides the predicted frequencies of all three genotypes for the selected trait
within the population.
Objectives:
Upon completion of the lab, students should be able to
1. Derive the Hardy-Weinberg Equation.
2. Calculate genotypic and allelic frequencies.
3. Discuss the conditions under which a population is in Hardy-Weinberg Equilibrium.
4. Determine if a population is in Hardy-Weinberg Equilibrium.
5. Design and perform a test of a disruption to Hardy-Weinberg Equilibrium.
6. Identify why the Hardy-Weinberg Equilibrium is difficult or impossible to demonstrate
with living organisms.
Materials:
Two different colors of beans that are approximately the same size (e.g., black and white) will
be provided. For each group you will need 400 beans of each color.
I. Testing the Hardy-Weinberg equilibrium
Procedure:
1. These beads represent the gametic contribution of the parents 'to form zygotes. The black
beads from one parent and the white from the other parent. The colors represent the
dominant and recessive alleles in a gene pool. Let the black beads represent the allele for
the dominant character and the white represent the allele for the recessive character.
2. Mix the beads thoroughly and pick out one pair of beads without looking. This is to
ensure that no selection is made on the basis of colour and there is random selection. Note
the color of the beads. Replace the beads back into the container, mix well and then make
your next pick randomly, note the color of the beads.
3. If you get 2 black beads then the zygote is homozygous dominant (AA).
4. If you get 2 white beads then the zygote is a homozygous recessive (aa) and if you have
picked I black and 1 white then a heterozygote is obtained (Aa).
5. Keep repeating the process for a large number of times (ie. 100 times) so that the
frequencies of the 3 genotypes in the next generation cab be obtained and compared with
the expected frequency calculated from the gene frequency in the parental population.
6. Answer the following questions:

a. What is the proportion of black homozygous and heterozygous if the frequency of the
white allele is 0.001?
b. If the black homo-zygote is 310, white homozygous is 50 and heterozygous is 240.

What is the expected frequency, of black and white alleles.
c. Assuming that 410 blacks homozygous, 320 heterozygous and 70 white homozygous
were picked. Is the population at equilibrium?
20
II. Genetic Drift
Introduction:
Genetic drift or allelic drift is the change in the frequency of a gene variant (allele) in a
population due to random sampling. The alleles in the offspring are a sample of those in the
parents, and chance has a role in determining whether a given individual survives and
reproduces. A population's allele frequency is the fraction of the copies of one gene that share
a particular form.
Genetic drift is an important evolutionary process, which leads to changes in allele

frequencies over time. It may cause gene variants to disappear completely, and thereby reduce
genetic variation. In contrast to natural selection, which makes gene variants more common or
less common depending on their reproductive success, the changes due to genetic drift are not
driven by environmental or adaptive pressures, and may be beneficial, neutral, or detrimental
to reproductive success.
The effect of genetic drift is larger in small populations, and smaller in large populations.
Procedure:
1. Using the same 400 black and 400 white beads of same size, weight and texture the
genetic drift by selection can be demonstrated. Let us assume that the white (aa) which
represents the recessive is lethal. So we shall eliminate the white beads for a number of
“generations" so that it is completely removed.
2. In the first generation there are 400 black and 400 white. Mix both the beads in a
container. Pick a pair of beads. If both the beads are black (BB - homozygous dominant)
put them back into the container, mix and pick another pair. If the pair is one black and
one white (Bb - heterozygous dominant) put them hack into the container, mix and pick
another pair. If the pair consists of 2 white beads, remove them and leave them separately
in another container. These white beads represent the homozygous recessive (lethal). Do
this for 20 picks for 5 times. Discuss the trend observed, with regards, to homozygous
dominant, heterozygous and homozygous recessive.
III. Detection of Gene Frequencies in Human Populations
Introduction:
Hardy-Weinberg Law Derivation

The Hardy-Weinberg Law:
p2 + 2pq + q2=1
Recall that alleles are alternative forms the same gene, which is located at a specific position
on a specific chromosome. The genetic composition of each individual is that individual’s
genotype. Each individual will carry two alleles of every gene, one from their mother and one
from their father. This composition of alleles for all individuals in a population is called the
gene pool.
The Hardy-Weinberg Law (HWL) states that when a population is in equilibrium, the
genotypic frequencies will be in the proportion p2, 2pq and q2. In a hypothetical population
where the frequency of allele A is p and the frequency of allele a is q, each genotype passes
on both alleles that it possesses with equal frequency. Therefore, in a population with just 2
alleles of a gene, the possible combinations are
21
This table shows the relationship between the allelic frequencies (p and q) and the genotypic
frequencies (p2, 2pq, and q2), which form the basis of the HWL. For example, the frequency
of the genotype AA is p2; the frequency of the genotype Aa is 2pq.
The HWL states that the genotypic frequencies remain constant generation after generation if
the population is large, mates randomly, and is free from evolutionary forces. For the above
example, that would mean that after many generations the frequency of AA is still p2 and the
frequency of Aa is still 2pq.
We can demonstrate this by considering a hypothetical randomly mating population from

table above. To do this, first consider all the possible matings from every genotypic outcome
above. These matings are listed in Column A below.
Next, since we are assuming that this population is subject to HWL, we can assume that all
matings are random and therefore will occur with equal frequency. For instance, AA x AA
matings don’t occur more often than aa x aa matings. This means that the frequency of
mating between any two genotypes is the product of how frequently these genotypes are
present in the population. Therefore, the mating frequency of AA x AA, for instance, is p2 x p2
or p4, which is already filled in for Column B.
Now that we know how frequent each of these matings are, we can determine the alleles that
the offspring of that mating will contribute to the gene pool. Columns C-E are the genotypic
frequencies resulting in the next generation. In our example of AA x AA, 100% of the
offspring will have the genotype AA, so the frequency of that genotype in the next generation
is p4.
Tally down the totals in each column. If Hardy-Weinberg is correct, the total of Column B
should equal the totals of D, C, and E combined, which should come out the same as the
frequencies of the original generation. Put another way, the combined totals of C, D, and E,
which makes up the entire population of the next generation, should still result in the same
Hardy-Weinberg equation: p2+2pq+q2 =1.
22
Materials:
sterile lancet
filter paper strip
PTC paper strip
paper towels
sterile cotton
70% ethyl alcohol
anti-A and anti-B antisera
Procedure:
1. PTC-tasting – one of the most thoroughly documented genetic traits is the ability or
inability to taste the chemical, phenylthiocarbamide (PTC, C7H8N2S), also known as
phenylthiourea. The PTC taster gene is designated as T. Tasters are homozygous (TT) or
heterozygous dominant (Tt). Non-tasters carry the tt genotype.
2. Tongue-rolling – the inheritance of several tongue movement traits has been documented,
researched and disputed throughout the last century. The ability to roll the tongue
upwards from the sides has received the most emphasis. In 1940, A.H. Sturtevant reported
two classes within the human population, roller and non-roller. The roller phenotype is
dominant (R_), while individuals unable to perform the maneuver are homozygous
recessive (rr) for the trait.
3. Facial dimples – the occurrence of natural indentations at the corners of the mouth
(dimples) is controlled by a dominant allele (D_). Persons without facial dimples
generally possess the homozygous recessive phenotype, dd. Facial dimples are sometimes
inherited as an irregular dominant.
4. Record your data here for the inherited human traits in the Table below:
Phenotype Your Class Data (N = )

Possible Homozygous Dominant Homozygous Recessive
Genotype or Heterozygous
1. PTC taster
(T_)
Non-PTC taster
(tt)
2. Tongue roller
(R_)
Non-tongue
roller (rr)
3. Facial dimples
(D_)
No facial
dimples (dd)
5. Calculate the frequencies of the dominant and recessive alleles for

a. the ability of tasting PTC
b. for the ability of rolling tongue.
c. the occurrence of facial dimples.
23
IV. Determining Gene Frequencies Where Three Alleles are Involved
Introduction:
The Hardy-Weinberg Law provides a basic algebraic formula for describing the expected
frequencies of various genotypes in a population. As discussed in the ealier section, the HWL
states that gene frequencies in a population remain constant from generation to generation if
no evolutionary processes like migration, mutation, selection and drift are operating. Thus if
matings are random, and no other factors disturb the reproductive abilities of any genotype,
the equilibrium genotypic frequencies are given by the square of the allelic frequencies.
If there are only two alleles A and a with frequencies p and q respectively, the frequencies of
the three possible genotypes are
(p + q)2 = p2 + 2pq + q2
If there are 3 alleles say A1, A2 and A3 with frequencies p, q and r, the genotypic frequencies
would be
(p + q + r)2 = p2 + q2 + r2 + 2pq + 2pr + 2qr
This square expansion can be used to obtain the equilibrium genotypic frequencies for any
number of alleles.
The blood group genes are inherited in the Mendelian fashion in the case of A, B and O
groups. The AB group however, results from codominance of A and B alleles. Thus blood
groups of offspring can be determined from their parent’s genotypes in the following way.
Therefore, the frequencies of the different phenotypes are as follows:
Phenotype Genotypes Frequency

A IA IA and IA IO p2 +2pr
B IB IB and IBIO q2 +2qr
AB IA IB 2pq
O I OI O r2
24
Based on this, the gene frequencies may now be estimated.
1. Calculation of the frequency of the IO allele (r).
r2 = frequency of the O phenotype

r= r2
2. Calculation of the frequency of the IA allele (p).
(p + r)2 = p2 + 2pr + r2 = frequency of the A phenotype plus the frequency of the O

phenotype.
Therefore,
p+r= p 2  2 pr  r 2
p = p 2  2 pr  r 2 - r
Procedure:
1. On a clean slide, put a drop of serum A and a drop of serum B.
2. Clean your index finger with cotton wool wet with ethyl alcohol.
3. Using the sterile lancet make a prick so that a drop of blood can be added to each of the
test sera.
4. After adding the blood, rock the slide gently for a few minutes so that the serum mixes
with the blood.
5. If neither of the blood samples clump the blood is type O. If both samples clump, the
blood is AB. If only the anti-B serum mixture clumps, the blood is group B. If only' the
anti-A serum mixture clumps, the blood is group A.
6. Using the class data, calculate the allelic frequencies (p, q and r).
25
Experiment 4: The Mendelian Principles, Gene Interaction and Polygenic Inheritance
Introduction:
Although the Mendelian principles are robust in explaining the inheritance of genetic
variability for a large number of discrete traits, single-gene and two-gene characteristics, the
classical patterns of Mendelian inheritance (e.g., the 3:1 and 9:3:3:1 F2 ratios) are often
modified because of gene interaction (epistasis) or the action of multiple genes on a single
trait. Indeed, the inheritance of quantitative traits (also known as continuous traits), such as
skin color, height, intelligence in human, and the predisposition of various human diseases
(obesity, heart diseases, movement disorders), is best explained by a polygenic model of
transmission. Despite the fact that polygenic traits are at the heart of several disciplines in
genetics, including human genetics, plant breeding, livestock breeding, wildlife management,
and quantitative trait locus (QTL) mapping, they tend to be neglected in the classroom and
laboratory. As such, we will study the Mendelian principles in maize, gene interactions, and
polygenic inheritance in this investigation.
I. Mendelian Inheritance: Maize

Seed Color in corn is variable. One gene that affects color has two alleles. For the color gene
one allele is for yellow and one allele for purple for this color gene. Purple color is dominant
to yellow which is recessive. A second trait is for seed shape. One allele for seed shape is
smooth which is dominant to a wrinkled kernel which is recessive.
The inheritance of variability in both maize seedlings and aleurone (endosperm) obeys the
Mendelian principles. An expected 3:1 ratio has been observed in a recessive mutant allele for
albinism (absence of chlorophyll) in the maize seedlings and a recessive allele for colorless
aleurone segregating according the Mendel’s first law.
Mendel also postulated that independent assortment of genes occurs when two traits are
considered simultaneously, yielding an expected ratio of 9:3:3:1 among the dihybrid F2
26
progenies and dihybrid testcross ratio 1:1:1:1. Indeed, the dihybrid F2 kernels of corns yield a
ratio of 9:3:3:1 for the phenotypes starchy (smooth) endosperm, sugary (wrinkled) endosperm,
colorless starchy endosperm, and colorless sugary endosperm, respectively.
In this investigation, students will be given commercially prepared F2 kernels of corns

because maize has a relatively long life cycle (three months or more).
Objectives:
The students will learn to recognize and interpret maize F2 data that illustrate Mendel’s law of
segregation and law of independent assortment.
Materials:
Commercially prepared monohybrid and dihybrid F2 kernels of corns.
For Activity 1 to 4, you are to work in a group of 2-3 students.
Activity 1: Mendel’s Law of Segregation (suggested time: 20 mins)

Without removing the kernels from the corn ears, count the number of kernels in the different
phenotypes. Record your data in Table 1 and then formulate hypotheses to explain the data
collected. Two possible illustrations of endosperm traits are given in Table 2.
Table 1 – Record of F2 data for maize endosperm trait.

Phenotypes* Number of Kernels Number of Kernels Genotypes of
Observed Expected Kernels
Total:
*Phenotypes will vary depending on the trait studied.
Using Chi-square analysis, determine if the observed and expected values differ at P = 0.05.
Table 2 – Two possible endosperm traits.

Parents F1 F2
WW  ww Ww 3 W__ : 1 ww
starchy sweet starchy starchy sweet
(smooth) (wrinkled) (smooth) (smooth) (wrinkled)
CC  cc Cc 3 C_ : 1 cc
pigmented non-pigmented pigmented pigmented non-pigmented
(purple) (white) (purple) (purple) (white)
Activity 2: Mendel’s law of independent assortment (suggested time: 20 mins)

Group the kernels into four different phenotypes. Without removing the kernels from the corn
ears, count and record the number of kernels in each different phenotype in Table 3.
Table 3 – Data of independent assortment in maize.

Phenotypes Observed Expected Deviation Genotypes**
Number (O) Number (E) (O–E)
Total:
**Select the appropriate gene symbols to represent the two alleles of each gene.
Using Chi-square analysis, determine if the observed and expected values differ at P = 0.05.
27
Activity 3: Gene Interaction (suggested time: 20 mins)

The color and characteristics of maize endosperm are controlled by numerous gene loci
scattered over the 10 pairs of chromosomes of maize. If two independently assorting genes
both affect the same characteristic such as the maize endosperm color, the classical 9:3:3:1
may be modified to produce such variations as 9:3:4, 9:7, 9:6:1, 12:3:1, 13:3, and 15:1. Such
interaction is called epistasis, and it is a common phenomenon in both plants and animals. An
example is the coat color in mice with a modified ratio of 9 agouti (wild-type) : 3 black : 4
albino.
In this exercise you will be given an ear of corn containing F2 kernels of two or more colors.
Table 4 shows some possible kinds of ears of corn that you may be getting.
Table 4 – Ears of corn that may be used in this exercise.

Phenotypes of the true-breeding Phenotype of the Phenotypes of the F2 kernels
parents F1 kernels
Purple  white Purple Purple, red, white
Yellow  yellow Yellow Yellow, purple
White  white Purple Purple, white
Purple  yellow Purple Purple, yellow, white
Without removing the kernels from the corn ears, count the number of kernels of each color.
Based on the format of Table 1 and Table 3, design a table of your own to record the
observed and expected data. Determine, using Chi-square analysis, if the observed and
expected values differ at P = 0.05.
II. Polygenic Inheritance

In contrast to single-gene traits and chromosome abnormalities, polygenic traits are controlled
by many independently assorting gene loci exhibiting a wide and continuous range of
measurable expression. Phenotype is determined by the sum of all the active alleles affecting
the trait. The inheritance of polygenic inheritance can neither be analyzed by the pedigree
method used for single-gene traits nor by chromosome studies as done in the study of
suspected chromosomal abnormalities. Analysis of polygenic traits involves quantitative
measurements and the outcome is expressed as a frequency diagram that usually yields a
normal (bell-shaped) distribution. A large sample size is essential in the analysis of polygenic
trait because variation in small sample size can be due strictly to chance. Polygenic traits are
also referred to multi-factorial traits because their expression is often markedly affected by
the environment.
Objectives:
The students will construct a histogram using the class data of total ridge counts (TRC) of
fingerprints and discuss the characteristics of this polygenic model of inheritance.
Materials:
2B pencils, plain white paper, graph paper, cellophane tape, magnifying glass or dissecting
microscope, and graphite squares or inkpad.
28
Activity 4: Fingerprint Ridge Count (suggested time: 60 mins)

There are three major groups of fingerprint patterns of dermal ridges: arches, loops, and
whorls (Figure 1).
Figure 1 – Three principal types of fingerprint patterns: (a) arch with no triradius and a
ridge count of 0; (b) loop with one triradius and a ridge count of 12, and (c) whorl with two
triradii and ridge count of 15 (the higher of the two possible counts).
The arch is the least frequent pattern, and it may be sub-classified as “plain” when the ridges
rise slightly over the middle of the finger or “tented” when the ridges rise to a point. The loop
pattern consists of a triradius and a core. A triradius is a point at which three groups of ridges
from three directions meeting at angles of about 120 degrees. The core is essentially a ridge
that is surrounded by fields of ridges, which turn back on themselves at 180 degrees. Loops
can be either radial or ulnar. A finger possesses a radial loop if its triradius is on the side of
the little finger for the hand in question, and the loop opens toward the thumb. A finger has an
ulnar loop if its triradius is on the side of the thumb for that hand and the loop opens toward
the little finger. The whorl pattern has two triradii, with the ridges forming various patterns
inside. The frequencies of these finger-print pattern types in the general population are as
follows:
5.0% arch, 5.4% radial loop, 63.5% ulnar loop, and 26.1% whorl.
For an arch the ridge count is 0. The ridge count on a finger with a loop is determined by
counting the number of ridges between the triradius and the center or core of the pattern. For a
whorl, a ridge count is made from each triradius to the center of the fingerprint, but only the
higher of the two possible counts is used (Figure 1).
Each student will prepare his/her own fingerprints and determine his/her own TRCs and
individual fingerprint patterns as follows:
1. Rub one of your fingers in a circular motion on the graphite square, making certain you
have covered all of the triradii on the fingerprint. It is important that the sides of your
finger are covered with graphite. Now carefully place a piece of cellophane tape onto
your blackened finger so that the tape comes in contact with the entire print. Make certain
that you include any triradii on the outer edges of the finger by rolling the finger over the
tape in one continuous motion. Peel away the tape and affix it to the appropriate place on
your data sheet (Table 5).
2. Repeat this process, preparing a print of each of your 10 fingers, and fill up Table 5.
3. Examine each print carefully; if a print is incomplete, prepare a new one. Use a
magnifying glass or dissecting microscope to classify the pattern (arch, loop, or whorl),
and to determine the ridge count for each print.
29
4. Record your fingerprint pattern data, total ridge count and sex in Table 6. Make the
calculations indicated in the table.
5. Using the class data, construct a histogram in which frequencies (no. of students) are
plotted against total ridge count.
Briefly describe the histogram, and answer the following questions:

Question 1: Is there a difference between male and female average TRCs?
Question 2: If so, what might account for this difference?
Each group of students will have to submit Table 5 (individual fingerprints), Table 6 (class
data), histogram (class data) and answers to the questions to your instructor for grading.
30
Table 5 – Data sheet for fingerprints.
Right hand
Thumb Second Third Fourth Fifth
Pattern
Ridge Count
Total:
Place left
fingerprints in
this space
Left hand
Thumb Second Third Fourth Fifth
Pattern
Ridge Count
Total:
Place right
fingerprints in
this space
31
Table 6 – Class data for fingerprint patterns, total ridge count, and sex of students.
Student Number of Fingers with TRC Sex

Loop Whorl Arch
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Totals
% of Totals
Mean TRC
Mean Female
TRC
Mean Male
TRC
32
Experiment 5: Mendelian Genetics: Drosophila
Introduction:
The fruit fly, Drosophila melanogaster, has been widely used for demonstrating the classical
Mendelian laws. This small fly undergoes a complete metamorphosis from egg, larva, pupa,
through imago in 10 to 14 days at 25oC (Figure 1). The fertilized eggs normally hatch into
white larvae in 22–24 hours after they have been laid. The larvae burrow in the culture
medium and feed on it for about four days. They then pupate on a dry spot, usually at the
sides of the container. The pupation lasts for another four to seven days before the adults
emerge from the pupa case. The newly emerged adults are pale in color with unexpanded
wings, but mature in about 8–10 hours with a characteristic darkened body. The females
usually do not mate before 8 hours after emergence and do not lay eggs until they are two
days old. In addition to a short life cycle, Drosophila is highly prolific, and has a lot of
genetic variability with many morphological variants.
Figure 1. Life cycle of Drosophila melanogaster
Physical appearance
Wildtype fruit flies have brick red eyes, are yellow-brown in color, and have transverse black
rings across their abdomen. They exhibit sexual dimorphism: females are about 2.5
millimeters (0.098 in) long; males are slightly smaller and the back of their bodies is darker.
Males are easily distinguished from females based on color differences, with a distinct black
patch at the abdomen, less noticeable in recently emerged flies (see Figure 2), and the
sexcombs (a row of dark bristles on the tarsus of the first leg). Furthermore, males have a
cluster of spiky hairs (claspers) surrounding the reproducing parts used to attach to the female
during mating.
33
Figure 2: Male (left) and female D. melanogaster
Culture Media for Drosophila

Numerous media have been developed for the culture of Drosophila. Perhaps the easiest is to
use commercially prepared instant medium whereby one need only add water to the
concentrate to produce a medium which can be used immediately. However, this medium
may prove too expensive for a large class. In this investigation you will be provided with the
culture media of banana with the following ingredients which are sufficient for 10 cultures:
water 100 ml
banana 100 g
Modex (methyl-para-hydroxyl-benzoate), or Tegosep, or Dowicil-200, 0.2 g
dissolved in 20 ml 95% ethanol as mold inhibitor
agar 3g
yeast 0.9 g
The mixture will then be poured into the culture bottles, autoclaved, and allowed to cool. Do
not contaminate the sterilized culture media.
Objectives:
In this investigation, students will be able to
1. Distinguish between male and female D. melanogaster
2. Categorize mutant flies based on aberrant phenotypes
3. Prepare controlled genetic crosses of D. melanogasterto and make crosses to
demonstrate the Mendelian laws. .
Materials:
Drosophila flies
Vials
Culture Medium Yeast
Ether and etherising bottle Fine brush
White Tile/Index card
Microscope
Light
Scalpel
Cotton plug (absorbent)
Pasteur pipettes
34
Week 1
Activity 1: Etherizing Flies and Distinguishing Their Sexes (Sexing)
1. Place a few drops of ether on the absorbent material (cotton or sponge) of the
etherizer.
2. Tap the culture bottle lightly to allow the flies drop to the bottom.
3. Remove the culture bottle plug, quickly replace it with the mouth of the etherizer,
invert the bottle over the etherizer, and shake flies into the etherizer.
4. Etherize the flies for about 30 seconds after they stop moving. Over-etherizing will
kill the flies. Do not kill the flies if they are to be used for further mating. Over-
etherized flies will hold their wings vertically over their body in contrast to the
normal at-rest position.
5. Transfer the etherized flies to a clean white card or a petri dish.
6. Examine the etherized flies with a dissecting microscope at 10 or 20 magnification.
Use a soft brush or teasing needle to move the flies about on the stage of the
microscope.
7. If the flies revive before you finish examining them, add a few drops of ether to the
absorbent pad on the re-etherizer and cover flies on the microscope stage for a few
seconds.
8. If the flies are not needed for observation, they may be immediately discarded.
Etherized flies to be used for further mating should be permitted to recover in a dry
vial or on a dry surface in the culture bottle before they come into contact with the
moist medium.
Activity 2: Fly Characteristics & Observation and Handling of D. melanogaster mutants
You will compare the morphological traits of wild and mutant flies. Complete Table 1,
recording the difference you see between mutant and wild-type flies with appropriate symbols,
sketches, or descriptions. First, examine wild-type flies that are considered normal for all
traits listed below. Place a plus sign in the appropriate space to signify the normal condition.
Carefully examine flies from four mutant stocks prepared as unknowns in the laboratory and
compare them with the wild type. If the unknown flies are wild-type for a given trait, place a
plus sign in the appropriate space. If they are different from wild-type, diagram the mutant
trait or use one or two key words to describe it briefly.
Observe and sketch each of the following:

Phenotype Genotype Location Chromosome
Wildtype +/+
vestigial vg/vg wing 2
apterous ap/ap wing 2
dumpy dp/dp wing 2
black b/b body 2
yellow y/y body 1 (sex-linked)
ebony eb/eb body 3
brown bw/bw eye 2
white w/w eye 1 (sex-linked)
sepia se/se eye 3
eyeless ey/ey eye 4
vermillion vn/vn eye 1 (sex-linked)
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Table 1 – Comparison of wild-type and mutant Drosophila.
Trait Wild-type Unknown Unknown Unknown Unknown
1 2 3 4
Body color
Eye color
Eye shape
Wing shape and size
Antenna shape and size
Bristle shape and size
Activity 3: Monohybrid, Sex-linked, Dihybrid and Reciprocal Crosses

Since female flies are capable of storing viable sperm for several days after they have been
inseminated, using virgin females for mating will ensure a controlled mating between
different genetic stocks. The most common method of obtaining virgin females is to select
those that have recently emerged from their pupa cases. These flies do not mate for at least
eight hours after emerging from the pupa case. Thus, 8-hour-old females will still be virgin
even if male flies are present. In this investigation, you will be provided with virgin female
flies of various variants for the first mating. To cross between different mutants, two to three
virgin females from one strain should be placed in a bottle containing culture medium with a
corresponding number of males from the other strain.
For each type of crosses (monohybrid, reciprocal, sex-linked and dihybrid), follow the steps a
to c shown below during Week 1 and steps d to f during Week 2 to Week 3:
a. Etherize the flies, examine and confirm the strains.

b. Gently place two or three flies of each strain (different sex for each strain) into the culture
bottle.
c. Label the bottle with your name, the date, and the type of crossing, e.g.,
P: e vg ♂  ++ ♀
i) A number of monohybrid crosses might be used to demonstrate Mendel’s first

law, the law of segregation. Some examples include:
P1 (parental) female  P1 male

Sepia eye color  wild type (red)
White eye color  wild type (red)
Vermilion eye color  wild type (red)
Apricot (yellow) eye color  wild type (red)
Bar eyes  wild type (oval)
Vestigial wings  wild type (long)
Curly wings  wild type (long)
Apterous (wingless)  wild type (long)
Ebony body color  wild type (gray)
Black body color  wild type (gray)
ii) Meanwhile, make a reciprocal cross for the strain that you have selected for the
monohybrid cross. For example, if you choose to cross vestigial female with
wild-type male as your monohybrid cross, then your reciprocal cross will consist
of breeding vestigial male and wild-type female.
36
iii) For sex-linked inheritance, mutant females are crossed with wild-type males, and
vice versa for the reciprocal cross. Possible mutants are: white-eye and
vermillion-eye.
iv) The gene for vestigial wing (vg) is located on the second chromosome, and the
gene for ebony body color (e) is located on the third chromosome. For dihybrid
crosses, mating may be made from these two stocks. Be certain to use virgin
female flies. Make reciprocal crosses as well, i.e., crosses between vestigial
female (vg vg; e+e+)  ebony male (vg+ vg+ ee) and between ebony female 
vestigial male.
Week 2 to Week 3
d. Remove the parents within 5 to 7 days (after the emergence of larvae). Examine and
reconfirm the correct strain and numbers of the parental flies as labeled, then discard the
parental flies in bottle containing spirit.
e. The phenotype of the offspring can be determined within 10 – 14 days.
f. Record in Table 2 the phenotypes of the first filial generation (F1) flies of each sex.
Activity 4: Generating F2 Offspring

g. Place three males and three females of these F1 flies in a fresh bottle of medium (Be sure
to label the bottle). This mating will allow for the production of a second filial generation
(F2). It is not necessary that the F1 female flies be virgins for this mating.
Week 3 to Week 5
h. Remove the F1 parental flies in step g within 5 to 7 days (after the emergence of larvae).
Examine and reconfirm the correct strain and numbers of the F1 parental flies as labeled,
then discard the F1 parental flies in bottle containing spirit.
i. The phenotype of the offspring can be determined within 10 – 14 days
j. Record in Table 2 the phenotypes of the second filial generation (F2) flies of each sex.
Activity 5: Chi-Square Analysis

For each cross, use Chi-square analysis to determine if the observed phenotype differs
significantly from the expected ratio at P = 0.05.
37
Name(s): ______________________________
Table 2a – Drosophila record for monohybrid crosses.

___________________________________________________________________
1. Cross __________________ female  _____________ male
2. Date P1s mated: _______________________
3. Date P1s removed: _____________________
4. Date F1s first appeared: _________________
5. Phenotype of F1 males: __________________
6. Phenotype of F1 females: _________________
7. Date F1 male and female placed in fresh bottle: __________
8. Date F1 flies removed: _____________________
9. Date F2 progeny appeared:
10. Record F2 data in the following table

__________________________________________________
Males Females
___________________ ___________________
Phenotype Number Phenotype Number
__________________________________________________
a. _________ _______ _________ _______

b. _________ _______ _________ _______
c. _________ _______ _________ _______
d. _________ _______ _________ _______
e. _________ _______ _________ _______
f. _________ _______ _________ _______
g. _________ _______ _________ _______
h. _________ _______ _________ _______
___________________________________________________________________
38
Name(s): ______________________________
Table 2b – Drosophila record for reciprocal crosses of monohybrid inheritance.

___________________________________________________________________
Cross __________________ female  _____________ male
1. Date P1s mated: _______________________
2. Date P1s removed: _____________________

__________________________________________________
Males Females
___________________ ___________________
__________________________________________________
a. _________ _______ _________ _______

b. _________ _______ _________ _______
c. _________ _______ _________ _______
d. _________ _______ _________ _______
e. _________ _______ _________ _______
f. _________ _______ _________ _______
g. _________ _______ _________ _______
h. _________ _______ _________ _______
___________________________________________________________________
39
Name(s): ______________________________
Table 2c – Drosophila record for sex-linked crosses.

___________________________________________________________________
2. Date P1s mated: _______________________
3. Date P1s removed: _____________________

__________________________________________________
Males Females
___________________ ___________________
__________________________________________________
a. _________ _______ _________ _______

b. _________ _______ _________ _______
c. _________ _______ _________ _______
d. _________ _______ _________ _______
e. _________ _______ _________ _______
f. _________ _______ _________ _______
g. _________ _______ _________ _______
h. _________ _______ _________ _______
___________________________________________________________________
40
Name(s): ______________________________
Table 2d – Drosophila record for dihybrid crosses.

___________________________________________________________________
2. Date P1s mated: _______________________
3. Date P1s removed: _____________________

__________________________________________________
Males Females
___________________ ___________________
__________________________________________________
a. _________ _______ _________ _______

b. _________ _______ _________ _______
c. _________ _______ _________ _______
d. _________ _______ _________ _______
e. _________ _______ _________ _______
f. _________ _______ _________ _______
g. _________ _______ _________ _______
h. _________ _______ _________ _______
___________________________________________________________________
41
Name(s): ______________________________
Table 2e – Drosophila record for reciprocal crosses of dihybrid inheritance.

___________________________________________________________________
2. Date P1s mated: _______________________
3. Date P1s removed: _____________________

__________________________________________________
Males Females
___________________ ___________________
__________________________________________________
a. _________ _______ _________ _______

b. _________ _______ _________ _______
c. _________ _______ _________ _______
d. _________ _______ _________ _______
e. _________ _______ _________ _______
f. _________ _______ _________ _______
g. _________ _______ _________ _______
h. _________ _______ _________ _______
___________________________________________________________________
42
Experiment 6: The Sex Check
Introduction:
Barr body
A Barr body is the inactive X chromosome in a female somatic cell, or the inactive Z in a
male, rendered inactive in a process called lyonization, in those species (including humans) in
which sex is determined by the presence of the Y or W chromosome rather than the diploidy
of the X or Z. The Lyon hypothesis states that in cells with multiple X chromosomes, all but
one are inactivated during mammalian embryogenesis. This happens early in embryonic
development at random in mammals, except in marsupials and in some extra-embryonic
tissues of some placental mammals, in which the father's X chromosome is always
deactivated. Barr bodies are named after their discoverer, Murray Barr. In men and women
with more than one X chromosome, the number of Barr bodies visible at interphase is always
one less than the total number of X chromosomes. For example, men with a 47,XXY
karyotype have a single Barr body, whereas women with a 47,XXX karyotype have two Barr
bodies. Barr bodies can be seen on the nucleus of neutrophils.
X-Inactivation
Human females inherit two copies of every gene on the X chromosome, whereas males inherit
only one (with some exceptions: the 9 pseudoautosomal genes and the small number of
"housekeeping" genes found on the Y). But for the hundreds of other genes on the X, are
males at a disadvantage in the amount of gene product their cells produce? The answer is no,
because females have only a single active X chromosome in each cell.
During interphase, chromosomes are too tenuous to be stained and seen by light microscopy.
However, a dense, stainable structure, called a Barr body (after its discoverer) is seen in the
interphase nuclei of female mammals. The Barr body is one of the X chromosomes. Its
compact appearance reflects its inactivity. So, the cells of females have only one functioning
copy of each X-linked gene — the same as males.
X-inactivation occurs early in embryonic development. In a given cell, which of a female's X

chromosomes becomes inactivated and converted into a Barr body is a matter of chance
(except in marsupials like the kangaroo, where it is always the father's X chromosome that is
inactivated). After inactivation has occurred, all the descendants of that cell will have the
same chromosome inactivated. Thus X-inactivation creates clones with differing effective
gene content. An organism, whose cells vary in effective gene content and hence in the
expression of a trait, is called a genetic mosaic.
Mechanism of X-inactivation
Inactivation of an X chromosome requires a gene on that chromosome called XIST. XIST
encodes a large molecule of RNA (of a type different from those, e.g., mRNA, used in protein
synthesis). XIST RNA accumulates along the X chromosome containing the active XIST
gene and proceeds to inactivate all (or almost all) of the other hundreds of genes on that
chromosome. XIST RNA does not travel over to any other X chromosome in the nucleus.
Barr bodies are inactive X chromosomes "painted" with XIST RNA.
The Sequence of Events

During the first cell divisions of the female mouse zygote, the XIST locus on the father's X
chromosome is expressed so most of his X-linked genes are silent. By the time the blastocyst
has formed, the silencing of the paternal X chromosome still continues in the trophoblast but
in the inner cell mass (ICM) transcription of XIST ceases on the paternal X chromosome
allowing its hundreds of other genes to be expressed. The shut-down of the XIST locus is
43
done by methylating XIST regulatory sequences. So the pluripotent stem cells of the ICM
express both X chromosomes. However, as embryonic development proceeds, X-inactivation
begins again. But this time it is entirely random. There is no predicting whether it will be the
maternal X or the paternal X that is inactivated in a given cell.
X-Chromosome Abnormalities
People are sometimes found with abnormal numbers of X chromosomes. Unlike most cases
of aneuploidy, which are lethal, the phenotypic effects of aneuploidy of the X chromosome
are usually not severe.
Examples:
Females with but a single X chromosome: the most common cause of Turner's syndrome. The
phenotypic effects are mild because each cell has a single functioning X chromosome like
those of XX females. Number of Barr bodies = zero. XXX, XXXX, XXXXX karyotypes: all
females with mild phenotypic effects because in each cell all the extra X chromosomes are
inactivated. Number of Barr bodies = number of X chromosomes minus one. Klinefelter's
syndrome: people with XXY or XXXY karyotypes are males (because of their Y
chromosome). But again, the phenotypic effects of the extra X chromosomes are mild because,
just as in females, the extra Xs are inactivated and converted into Barr bodies.
Figure 1: Barr body as indicated by arrow in the female cells (XX).
Objectives:
The students should be able to:
1. perform buccal smear test for investigation of the existence of Barr's body
Materials:
Microscope equipped with oil immersion objective
Clean slide
smear from oral mucosa
95% ethyl alcohol, 70% ethyl alcohol, 50% ethyl alcohol
absolute ethyl alcohol
5 or 6 N hydrochloric acid
xylene
distilled water
Procedure: [a modification of one developed by Ludwig and Klinger (1958)]
A. Obtaining the Smear

1. Two difficulties may be encountered in preparing a suitable smear from oral mucosa:
If the slide is not scrupulously clean, the epithelial cells will be lost in the course of
the staining procedure. Consequently, it is suggested that the slides be washed in a
detergent, rinsed thoroughly in distilled water and given a final rinse in 70% ethyl
alcohol. The slide may then be flamed dry.
44
2. A second problem, which is more difficult to overcome, is that of contamination of

the epithelial cells with oral bacteria. These are especially troublesome because they
stain readily with thionin and may be confused with sex chromatin if they are, by
chance, super-imposed on the nucleus of an epithelial cell. To remedy this problem it
is suggested that the cell donor (student) vigorously rinse his mouth with tap water to
remove loose epithelial cells and bacteria. This rinsing should be repeated two or
three times for best results.
3. The smear is prepared by gently scraping the lining of the cheek with the end of a
clean slide and smearing the material thus obtained on a second clean slide. Both
slides should be air dried for thirty seconds and then processed according to the
schedule described below.
B. Staining and Mounting

1. Place each slide in 95% ethyl alcohol for two minutes then in 70% ethyl alcohol for
two minutes, 50% ethyl alcohol for two minutes, and distilled water for two minutes.
2. Upon removing the slide from the distilled water, place it in 5 or 6 N hydrochloric
acid for approximately 10 seconds. This is a very critical part of the procedure
preparatory to staining. The acid hydrolyzes RNA in the nucleus of the cell and thus
eliminates RNA-rich bodies which also stain with thionin. Excessive hydrolysis may
disrupt the DNA as well as the RNA, while insufficient hydrolysis may leave such
RNA-rich bodies as the nucleolus intact. Thionin is regarded as a differential stain for
the nucleic acids staining DNA a dark blue-black and RNA violet. When dealing with
objects as small as the nucleolus and sex chromatin bodies in these cell, however, it is
desirable to avoid having to distinguish between the two on the basis of color
differences. Proper hydrolysis will eliminate RNA-rich bodies and prevent their being
confused with the DNA-rich chromatin.
3. The slide is transferred from the hydrochloric acid to distilled water for 10 - 15
seconds to remove the acid. It is then stained in 1% aqeous thionin for 10 to 15
minutes after which it is again rinsed in distilled water and then dehydrated according
to the following schedule:
50% ethyl alcohol - 30 seconds,

70% ethyl alcohol - 30 seconds
95% ethyl alcohol - 30 seconds,
absolute ethyl alcohol 30 seconds.
4. The slide is then transferred to xylene for clearing for one minute. A drop of balsam
mounting medium should then be placed over the cells followed by the application of
a coverslip. Care should be taken in placing the cover slip so as to avoid trapping
bubbles in the mounting medium.
5. Examination of smears prepared in this way should reveal cells with virtually
colorless cytoplasm and a pale blue nucleus. The sex chromatin bodies most
frequently located adjacent to the nuclear membrane, should be stained a dark blue-
black. Locate cells to be examined with the low power objective, then check for
chromatin using the oil immersion objective. Care must be taken not to get balsam on
the microscope objective.
45
Conclusion Notes:
1. A few sex chromatin bodies are occasionally observed in tissues taken from males.
The statistical incidence of sex chromatin in male cells normally ranges from about
2% in oral smears to as high as 20% in liver preparations. In fact, for an accurate
determination of nuclear sex, a statistically significant number of cells would have to
be examined and the percentage of incidence of sex chromatin computed. For oral
smears, made from females, most investigators find between 50 and 65% of the
healthy cells will demonstrate sex chromatin. Only normal-looking nuclei that stain
evenly can be validly considered. Nuclei that are folded, have granulated nucleoplasm,
or demonstrate any sign of degeneration cannot be included in a critical study.
2. Investigate the following:

a. Examine 50 squamous epithelial cells from a human female. How many
exhibit sex chromatin?
b. What percentage of these 50 cells reveals sex chromatin?
c. What is the incidence of sex chromatin among 50 oral mucosal cells obtained
from a human male?
d. Did you find any cells of the human female which have more than one sex
chromatin body?
3. In most mammals studied, and in humans in particular, the evidence has accumulated
to show that the Y chromosome is male-determining and that whenever it is present
the phenotype of the individual will be male. Female-determining genes are
apparently X-linked. The normal human female has a somatic chromosome
complement of 46, consisting of 44 autosomes and two X chromosomes; while the
normal male has 44 autosomes plus an X and a Y chromosome. On the basis of this
information, complete the following table, indicating the probable sex chromosome
complement and the total number of chromosomes anticipated in the somatic cells of
the individual (assuming the presence of the normal 44 autosomes).
Number of sex Phenotypic sex of Probable sex Total number

chromatin bodies individual from chromosome of
observed in somatic cell whom cell was complement chromosomes
obtained in somatic cells
0 Male (normal)
0 Female (defective)
1 Male (defective)
1 Female (normal)
2 Male (defective)
3 Male (defective)
a. Which of the individuals has classical Turner’s Syndrome?

b. Which has classical (the most common form of) Klinefelter’s Syndrome?
c. Which individuals might be regarded as metafemales?
46
Experiment 7: The Effect of Colchicine on Chromosome
Introduction:
A typical cell has a cell-wall which encloses the cytoplasm and a nucleus. The nucleus is
regarded as the site of hereditary. It is in the nucleus where the hereditary, material is stated in
the form of chromatin materials or chromosomes. Each species has its own definite number of
chromosomes. Example the Drosophila has 4 chromosomes.
These chromosomes carry the genes, the hereditary determiners, that are transmitted from one
generation to another. If any change were to occur in the chromosomes whether in number or
structure, this change or aberration can be passed on through the gametes to the progeny. The
change in the chromosome number usually leads to polyploidy where more than 2 sets or
chromosomes or genomes - e.g. triploid (3n) tetraploid (4n), pentaploid (5n) - are seen.
Polyploidy
Polyploidy can be caused EITHER by somatic doubling whereby cells undergo irregularities
at mitosis and give rise to meristematic cells that transmit these irregularities to the new
generations OR reproductive cells may have an irregular division in which the sets of
chromosomes fail to separate completely to the poles at anaphase.
The former somatic doubling at mitosis is well demonstrated in most plant cells. Mitosis
consists of four stages: the prophase, the metaphase, the anaphase and the telophase. The
prophase and telophase are longer than the metaphase and the anaphase. When mitosis starts
there is duplication of chromosomes in the prophase and the arrangement on the equatorial
plate. If mitosis is arrested at late prophase, the cell with the double sets of chromosomes
(tetraploid) at the time goes into the interphase. Later the cell may undergo normal cell
division and produce daughter cells which have sets of the normal chromosomes. If the
process of mitosis stops at the metaphase stage in the growing tip of a plant, than a shoot,
which will be having cells that are exclusively tetraploid, may be produced. This branch may
be propagated by cutting or through grafting to continue the tetraploid nature. These
tetraploids usually are larger, and seen to produce better fruits and other products of
commercial value. As a result, nowadays, polyploids are artificially induced so as to increase
vegetative and floral parts.
Colchicine
A number of chemicals and heat stock treatments have been employed to induce polyploidy in
plants; but now a poisonous chemical known as colchicine has replaced practically all the
techniques used to double the number of chromosomes in plants. This method of induction
was developed by Blakeslee, A.G. Avery and B.K. Nebel. These investigations found that an
alkaloid, colchicines, extracted from a autumn crocus plant, Colchicum Autumnale, could
produce a disturbance in spindle formation during cell division. This colchicine is water
soluble and if used in proper concentration, it is capable of changing the nuclear processes at
metaphase.
Colchicine generates the cell wall (plants) very rapidly. Effects may be noticed within
seconds after the chemical comes in contact with the nucleus. But the rate of penetration
depends on the concentration exposure, mitotic stage, kind of cell and general growth
condition of the mitotic cell.
Every mitotic cycle gives rise to new spindle fibers. Cytoplasmic separation, a function of
cytokinesis is closely related with the functions of the spindle fibers. Colchicine prevents the
formation of a spindle and delays chromosomal separation; inhibits daughter nuclei and
blocks cleavage processes. Due to this arrest at the metaphase stage, more metaphases are
47
seen in tissues treated with colchicine. This change caused by colchicine can be classified as
mutation but NOT as a gene mutation.
The application of colchicine has permitted the production of large number of polyploids
from diploids. It is used in horticulture since the products of tetraploids or polyloids are of
superior quality with the usual changes associated with multiplication of chromosomes,
gigantic characters in leaf, flower, fruit and seeds. i.e., the tetraploid (4n) Zea maize produces
about 20% more vitamin A than the normal (2n). Usually colchicine is added in the form of
liquid at the seedling stage and the plant grows through the addition of new cells at the tips of
their stem.
In animals, induction is difficult since animals grow in many different parts of the body at the
same time. Polyploidy has brought about new species and many of the commercially valuable
plants like wheat and cotton tobacco, grapes, oats and many others. One of the greatest
achievements in the process of induction is the work of Kihara and others to produce seedless
watermelons. Tetraploid parents were produced by colchicine. These watermelons have 4N
chromosomes and are easily distinguished from the diploid by the increase in seed size, pollen
size and other characteristics. The tetraploids produced become the seed parent were crossed
with the diploids as pollinations to make the triploids. These triploids gave fruits without
seeds.
2X treated with colchicines
♀ 4x x 2X ♂
3X - seedless watermelons
Objective:
The aim of this practical is to illustrate the effect of colchicine on onion root tip.
Materials:
forceps
watchglass
glass slides and cover slips
microscopes
a pair of scissors
glass rod
spirit lamp
white paper
Pasteur pipettes
Blotting paper
young onion root tips
young onion root tips treated with colchicines for 1-3 hours
3 parts alcohol and 1 part glacial acetic acid
10% HCl
45% acetic acid
aceto-orcein stain (2.2% solution of stock solution in glacial acetic acid, dilute to 1% before
use)
1 part of N HCl to 9 parts of 1% aoeto-orcein
48
Procedure:
I. Squash Preparation of untreated onion root tip
1. The onion roots are fixed in a solution of 3 parts alcohol and 1 part glacial acetic acid
and hydrolysed in 10% HC1 at 60°C for 6 minutes, so that the cell wall is softened
and will easily be squashed. These onion roots can be kept in 45% acetic acid.
2. The stain to be used is the aceto-orcein. Orcein stains root tips chromosomes well but
since it deteriorates very rapidly in dilute acid it is always advisable to use freshly
prepared stain. Prepare a 2.2% solution of stock solution in glacial acetic acid and
dilute to 1% before use. Stock solution can be stored in the refrigerator.
3. Select a few root tips (3 - 4) and place them in a clean watch-glass. Add about 9 drops
of freshly prepared aceto-orcein and 1 drop of HCl to the roots (1 part of N HCl to 9
parts of 1% aoeto-orcein).
4. Warm the watch-glass over a spirit lamp for about one minute till white vapors are
seen. Stop heating. DO NOT BOIL for it will cause clumping of chromosomes and
fragmentation.
5. Close the watch-glass with another watch-glass and allow it to cool for about 5 - 10
minutes. Remove the roots from the stain onto a white paper. Cut about 1 - 2 mm of
the root tip and discard the rest. Clean a slide well before transferring the root tips
(use forceps) and add 1% aceto--orcein stain (preferable without acid). A drop or two
will do. Do not flood the slide.
6. Using a glass rod, tap lightly on the root tips so that the cells will get separated from
one another. Now cover the squash with a coverslip. There should be no air-bubbles.
Carefully blot the excess stain without moving the overslip. Heat the slide gently over
a spirit lamp. The warming will help the chromosomes to spread. DO NOT BOIL.
Too much heat will cause shrinkage and clumping of the cells and also excess stain in
the cytoplasm. The heat should be gradual and should not exceed 60°C. A good
estimate is to heat the slide till it is too warm to hold.
7. Invert the slide very carefully onto a filter paper placed at the edge of the table. Press
very gently with your thumb on top without shifting the slide. This will help to flatten
the cells so that the chromosomes will be clearly seen.
8. Now invert it back to the normal position with the coverslip on top. If there are any
air bubbles introduce a drop of stain near the edge of the covorslip. The slide is now
ready to be observed under the microscope. Examine the slide and try to identify the
different stages in mitosis. Draw and label the stages. Make sure you see a
metaphase stage. How many chromosomes are there?
II. Squash Preparation of Colchicine treated onion root tip

1. The onion root tips are placed in a solution of colchicine (about 0.3% ) for about 1 - 3
hours. The mitotic stage at which colchicine is most effective in lowest concentration
is late prophase. There is no doubt that the drug interferes with the karyolymph since
the regular linear arrangements of fibers do not develop. These spindle are normally
formed 20 minutes after disappearance of nuclear membrane (in a normally dividing
cell); but in the presence of colchicine the fibers do not appear. Among plants the
inhibition begins at the polar cap stage.
49
2. Make a squash preparation with the treated root tips following the same method as for
untreated root tips. Compare the 2 metaphase stages of the treated and untreated
onion root tips. Draw and label clearly, showing the difference.
[Source: by J. G. O'Mara (1939). Observations on the immediate effects of colchicines. J

Hered 30(2): 34-37]
50
Experiment 8: Chromosomes: Polytene Chromosomes from Drosophila Salivary Glands

and Human Chromosomes
I. Polytene Chromosomes from Drosophila Salivary Glands

Polytene chromosomes from the salivary glands of Drosophila larvae are unusually large and
multi-banded. These chromosomes are formed by the process of endomitosis, in which the
chromosomal strands (chromonemata) repeatedly replicate but without dividing into daughter
nuclei. Correlation between crossover data and the positions of the bands suggest that certain
genes are located in certain bands.
Objectives:
Students will learn to prepare and identify Drosophila polytene chromosomes.
Materials:
1. Biological materials: Drosophila larvae at third instar.
2. Chemicals: aceto-orcein or acetocarmine stain with dropper, saline solution.
3. Equipment: stereo-microscope, compound microscope, microscope slides, cover-slips,
teasing needles, scalpel blades and spirit lamps.
For Activity 1 and 2, you are to work in a group of 2-3 students.
Activity 1 (suggested time: 60 mins)

1. Transfer a Drosophila larva (third instar) from a culture bottle into a drop of saline
solution on a microscope slide.
2. Dissect the larva under a stereo-microscope. Place one teasing needle in the middle of
the larva and the other needle at the anterior end, near the black mouth parts. Be sure
you can tell the anterior from the posterior end.
3. While holding the larva with the first needle, pull outward with the needle at the
anterior end of the larva. This will cause the internal organs of the larva to be pulled out
of the body wall.
4. Identify the two granular, transparent salivary glands, which are located anteriorly
(Figure 1). Detach these elongated glands and remove as much fat as possible.
5. With a teasing needle, transfer the isolated, fat-free salivary glands to a drop of
acetocarmine or aceto-orcein stain on a clean microscope slide. Place a cover glass over
the drop of stain.
6. Gently heat the slide over the flame of a spirit lamp. Do not overheat. Boiling will
destroy the salivary glands.
7. Cover the slide with a paper towel and push down on the cover glass with the handle of
a needle or the eraser end of the pencil. Be careful not to let the cover glass slide while
pressing down on it. The pressure you apply will squash the glands, rupture the nuclear
membranes, and free the chromosomes.
8. Observe the slide under the compound microscope using the low- and then the high-
power objectives.
In a well-prepared slide, the chromosomes will have been expelled from the nucleus so you
can see them readily. Draw a labeled diagram of what you see on the slide that you have
prepared. Tape your prepared slide to one side of the diagram and submit it to your instructor
for grading before leaving the lab.
51
Figure 1 – Dissection of anterior end of Drosophila larva showing the two salivary glands
(large cells) with attached fat bodies.
II. Human Chromosomes

Studies of human chromosomes were made feasible after scientists managed to culture human
cells outside the body. Dividing cells in culture could be treated with the alkaloid colchicine,
which frees the chromosomes from the spindle apparatus of the dividing cell. Cells in culture
can then be exposed to a hypotonic solution that causes the cell to swell, thereby separating
the chromosomes so that they can be observed individually and counted. Investigations of
human chromosomes routinely employ blood leukocytes, which are convenient to obtain,
culture, and induce to undergo mitosis. Human chromosomes vary in length; the largest being
about 10 μm long and the smallest about 2 μm long. During karyotyping, chromosomes are
normally arranged in order by length and centromere position into seven groups (A to G) of
autosomes and one pair of sex chromosomes. Banding patterns are used to identify the two
chromosomes that are the members of the each pair. Three different staining techniques (G, R,
and Q) have been used to reveal the cross bands on chromosomes. However, usually only one
type of banding, the G banding which can be revealed by Giemsa blood stain, is required for
identification of all metaphase chromosomes. The human chromosome number was
established at 2n = 46. Abnormal chromosomal numbers have long been known to be the
causes for several disorders, including Down’s syndrome (trisomy 21), Klinefelter’s
syndrome (XXY, XXXY, XXXXY), and Turner’s syndrome (2n = 45).
Objective:
Students will learn to describe the morphology of human chromosomes with reference to size,
centromere position, and presence or absence of satellites.
Materials:
Three karyotype pictures per group of students (2-3 persons), scissors, karyotype forms
(entitled “Report of Chromosome Study”, Figure 3), cellophane tape or glue.
Activity 2 (suggested time: 60 mins)
52
1. Examine the picture of normal human karyotype (Figure 2). With the help of a genetics
textbook, familiarize yourself with the following terms: centromere, satellites,
metacentric, submetacentric and acrocentric chromosomes.
2. You will be given three pictures of karyotype illustrating a set of human metaphase
chromosomes from a leukocyte culture of patients with chromosomal abnormalities.
Chromosomes in one of the pictures have been labeled for you.
3. Cut out every chromosome in the picture given (Figure 3). Arrange them in order by
centromere position, length and banding patterns. Then paste them on the karyotype
forms (Figure 4) with cellophane tape or glue.
4. Interpret the data by identifying the chromosomal abnormality, and submit the
completed forms to your instructor for grading.
Figure 2 – Karyotype of human metaphase chromosomes from a leukocyte culture.

Chromosomes are arranged in order by length and centromere position into seven groups of
autosomes and one pair of sex chromosomes. Banding patterns are used to identify the two
chromosomes that are members of each pair. This karyotype is of a normal 46, XX female.
53
Figure 3(i)
54
Figure 3 (ii)
55
Figure 3 (iii)
56
Name: ______________________________
REPORT PF CHROMOSOME STUDY

CYTOGENETIC LABORATORY
1 2 3 4 5
A Group B Group
6 7 8 9 10 11 12
C Group
13 14 15 16 17 18
D Group E Group
19 20 21 22 X Y
F Group G Group SEX
Chromosome counts:
Karyotype(s):
Interpretation:
Figure 4 – Karyotype form.

Exercise:
57
The following shows the karyotypes of different individuals (A to E) from different families:
Chromosome counts:
Karyotype:
Interpretation:
Chromosome counts:
Karyotype:
Interpretation:
58
Chromosome counts:
Karyotype:
Interpretation:
Chromosome counts:
Karyotype:
Interpretation:
59
Chromosome counts:
Karyotype:
Interpretation:
Case study:
A 2-week-old baby is hospitalized for inadequate feeding and poor growth. The parents are
concerned by the child’s weak cry which sounded like a cat’s meow. The baby doesn’t look
much like either parent. The baby was later noted to have a small head size (microcephaly)
and subtle abnormalities of the face. A karyotype was then ordered by the physician. The
following was obtained:
60
What can you tell about the syndrome the baby has? ______________________________
61

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BABS2213 Principles of Genetics_2019v0

Department of Bioscience, FOAS, TARUC

FACULTY OF APPLIED SCIENCES

BACHELOR OF SCIENCE (HONOURS) IN

For all practical work to be undertaken in this course:

Students are required to:

Emergency Evacuation and Safety Equipment

Laboratory dress code

Food and Drink

Leaving the Laboratory

Pregnancy and Allergies

Student Safety Declaration Form for Laboratory Work

Student ID Number: _____________

Course Code: _________

I have read and agreed on the following:

 I have read and I understand the LABORATORY SAFETY RULES.

BABS2213 PRINCIPLES OF GENETICS

2 Testing the Genetic Ratios -- Chi-Square Analysis 15

3 Hardy Weinberg Equilibrium 19

4 The Mendelian Principles, Gene Interaction and Polygenic 26

5 Mendelian Genetics: Drosophila 33

6 The Sex Check 43

7 The Effect of Colchicine on Chromosome 47

8 Chromosomes: Polytene Chromosomes from Drosophila Salivary 51

Experiment 1: Probability of Inheritance

I. The Idea of Chance

Table 1: Results of Tossing a Coin 30 times

Results Observed Expected Deviation (O - E)2 / E

II. Independent Events Occurring Simultaneously

Table 2: Results of Tossing Two Coins 40 times

Results Combinations Observed Expected Deviation

Table 3: Expected Results from Tossing Three Coins Together

Classes Combinations Probability of Observed Expected Deviation

5. A parallel situation would be observed if you were to study families consisting of

Table 4: Expected Results from Tossing Four Coins Together

Classes Combinations Probability of Each Class

III. Binomial Expansion

Expectation of 4 girls = a4 = (½ )4 = 1/16.

where P is the probability to be calculated; n! is the product of the number of integers in

3. Albinism in humans is controlled by a recessive gene (c). From marriages between

IV Either–Or Situations (Mutually Exclusive Events)

An additional probability principle is useful in solving certain problems: The probability

The students will be able to:

The Chi Square Test

where O = the observed number of individuals in a particular phenotype,

Table 1: Summary of Calculation of Chi-Square Value in Hypothetical Plants

The calculated 2 value is 2.0548. What does this mean?

Cautions When Using Chi-Square

Table 2: 2 values at various degrees of freedom

For Activity 1 to 4, you are to work in a group of 2-3 students.

Degrees of freedom: ____________

Degrees of freedom: ____________

Degrees of freedom: ____________

Degrees of freedom: ____________

Experiment 3: Hardy Weinberg Equilibrium

In this equation, p² is the predicted frequency of homozygous dominant (AA) people in a

I. Testing the Hardy-Weinberg equilibrium

1. Cross ______ female  _ male

a. _____ _ _ _

Cross ______ female  _ male

a. _____ _ _ _

1. Cross ______ female  _ male