Department of Life Sciences

The University of the West Indies

Mona
Molecular Biology
&
Genetics
BIOL1018

Laboratory Manual
2016/17

THE UNIVERSITY OF THE WEST INDIES

MONA
DEPARTMENT OF LIFE SCIENCES

TABLE OF CONTENT
Page

COURSE OUTLINE

GENERAL INSTRUCTIONS

PRACTICAL EXERCISES

1. The Chemical Organization of Cells

2. Plasmid DNA Isolation and Restriction Endonuclease Analysis

11

3. Polymerase Chain Reaction and Agarose Gel Electrophoresis

20

4. Probability, Monohybrid and Dihybrid Inheritance & Quantitative

25

Traits
5. Binomial Expression and Human Genetics

30

Appendix 1 Chi Squared Table

36

MOLECULAR BIOLOGY TUTORIAL QUESTIONS

37

GENETICS TUTORIAL QUESTIONS

39

MOLECULAR BIOLOGY AND GENETICS (BIOL1018)

COURSE OUTLINE
WEEK
7
(Oct 17 - 22)

LECTURES

PRACTICALS

1. The Structure of DNA

1.The Chemical Organization of Cells
(Fri & Sat lab streams only)

2. The Function of DNA

3. The Structure of Chromosomes

8
(Oct 24 - 29)

4. DNA Replication
5. DNA Repair and

1.The Chemical Organization of Cells

(Mon, Tues, & Wed lab streams only)

Recombination
6. Transcription
9
(Oct 31 Nov 5)

7. Translation
8. Control of Gene Expression
9. Recombinant DNA Technology

10
(Nov 7 - 12)

2. Plasmid DNA Isolation & Restriction

Endonuclease Analysis
(All lab streams)

10. Genetics - Monohybrid

inheritance
3. Polymerase Chain Reaction and
Agarose Gel Electrophoresis
(All lab streams)

11. Modifications in Gene

expression & Dihybrid
inheritance
12. Probability and Statistical tools
11
(Nov 14 - 19)

13. Genetic Interactions

14. Linkage and Crossing over
15. Crossing over and Gene

4. Probability, Monohybrid & Dihybrid

Inheritance & Quantitative Traits
(All lab streams)

mapping
12
(Nov 21 - 26)

16. Interference and coincidence

17. Sex Determination and Sex
Linkage

5.The Binomial Expansion & Human

Genetics
(All lab streams)

18. Sex Related Inheritance

13
(Nov 28)

INCOURSE TEST

Lecturers:

Prof. Paula Tennant (weeks. 7 - 9)

Dr. Dwight Robinson (weeks 10 - 12)

Venue:

IFLT (Morning lectures),

C5 (Afternoon lectures)

Teaching
Assistants:

Ms. Christine Lawson

Mr. Denneko Luke
2

GENERAL INSTRUCTIONS

The objective of the Laboratory Manual is to help you learn to make scientific observations
and to formulate explanations and conclusions. In order to meet these objectives, you
need to do some homework before coming to the laboratory:
Read each exercise before the given practical session. Study the introductory
material and the experimental procedures. In order to obtain a better understanding
of the material you may find it helpful to read through your lecture notes and the
corresponding chapter in the prescribed text before each laboratory session.
Many of the chemicals (reagents) and some of the equipment in the Biology Laboratory
are potentially dangerous.
The following rules of laboratory safety should be studied and should become a
habit.
1. Assume that all reagents are poisonous and act accordingly. If chemicals come into
contact with skin, wash immediately with water.
2. DO NOT:
a. Ingest any reagents.
b. Eat, drink or smoke in the laboratory. Toxic material may be present, and some
chemicals are flammable.
c. Put chemicals in the sink or trash unless instructed to do so.
d. Operate any equipment until you are instructed in its use.
3. DO:
a. Keep your work area neat, clean and organised. Ask your instructor or
demonstrator for assistance in cleaning up broken glassware and spills. Wash your
hands at the end of each exercise and before leaving the laboratory.
b. Stopper all reagent bottles when not in use. Immediately wash reagents off yourself
and your clothing if they spill on you, and immediately inform the instructor or
demonstrator. If you accidentally get any reagent in your mouth, rinse your mouth
thoroughly and immediately inform the instructor or demonstrator.

c. Use caution when using heat, especially when heating chemicals.

d. Use extra care when working with scalpels, knives and glass tubing.
e. Wear clothing which, if damaged, would not be a serious loss, or use a laboratory
coat since some chemicals may damage fabrics.
f.

Be familiar with the experiments you will be doing before coming to the laboratory.
Confusion is dangerous. Completely follow the procedure set out by the
instructor.

g. Note the location of emergency equipment such as a first aid kit, eyewash bottle,
and fire extinguisher. Report all accidents immediately.
h. Report any condition that appears unsafe or hazardous to the instructor or
demonstrator.

PRACTICAL WORK
A. GENERAL
Each student should bring his/her dissecting set and laboratory notebook to each practical
class.
At the end of each practical you must have your work checked and signed by your
demonstrator.
It is your responsibility to see that your demonstrator has marked your attendance.
At the end of the lab session, you must leave the bench clear; return all equipment to the
designated areas, turn off the electric lamps, and throw discarded material into the
appropriate container.
DO NOT throw solids (e.g., animal tissue, cotton wool, etc.) into the sink as they clog the
drains.
B. EXPERIMENTS
When writing up an experiment, try to be orderly and concise (i.e., as brief as possible) in
your presentation. Suggested headings for your notes are as follows:

 Title: says what you did. It should describe the main point of the experiment or
investigation.
Aim (of Experiment): here, you would state what you are hoping to discover.
Introduction: give a brief statement of the theory behind the experiment.
Method: the details of the apparatus and techniques employed will come here.
Results: these can sometimes be presented in the form of a table.
Discussion: here, you would comment on the results you have obtained, the
observations you have made and state how they have helped you fulfil your aims.
C. ASSISTANCE
The lecturer, teaching assistants and demonstrators are there to help you. Do not be
shy, or feel that you will be looked down on if you ask for help. Report rude or unhelpful
demonstrators to the lecturer.

LABORATORY 1: THE CHEMICAL ORGANIZATION OF CELLS

U

EXERCISE ON THE EXTRACTION OF GLYCOGEN, NUCLEIC ACIDS AND

PROTEINS FROM YEAST (SACCHAROMYCES CEREVISIAE) CELLS
This exercise is a basic introduction to the methods used to extract various
macromolecules from whole cells.

Before commencing the exercise it is necessary to draw your attention to the proper
method of using a centrifuge and to give some general instructions.

Centrifugation
Observe the following instructions.
1. Use only plastic centrifuge tubes.
2. Each tube must be balanced with another on the opposite side of the rotor.
This can be gauged by your eye in these experiments, since we are not
centrifuging at high speeds.
3. Close the lid before starting, and do not open it until the rotor is at a complete
stop. Do not brake it by hand to stop.
4. If the centrifuge vibrates strongly, turn it off, and re-balance all the pairs of
tubes.
5. Others are sharing this machine, so use it efficiently. Remove your materials
as soon as it has stopped.
6. If you have any doubts about how to use the centrifuge, please ask the
lecturer, teaching assistant or demonstrator for assistance.

General Instructions

When you need a reagent go to the supply bench and obtain it.

Exercise caution when handling the chemicals in this and subsequent

laboratories. Many are extremely caustic. Do not spill them on your skin or
clothes. If you do, wash them off immediately with lots of water.

Never return solutions, either used or unused, to the stock bottles. Discard them
into the sink.

Try not to waste solutions.

To mix solutions in a test tube, roll the tube in a rotary motion between your
hands, or use a glass stirring rod. Do not use your thumb as a stopper.

Heat solutions only in a hot water bath.

Wash and rinse well all test tubes between experiments and after completing the
final experiment.

Exercise A: Extraction of Glycogen, Proteins and Nucleic Acids with TCA

Stir the yeast culture briskly to remove trapped carbon dioxide.

Next, with a teaspoon, weigh out a 10 g sample into an aluminium foil weighing
boat. (This mass should approximate to a heaping teaspoon.)

Use a small teflon spatula to scrape this mass of yeast into a mortar.
Add a heaping teaspoon of white sand and grind with a pestle for 5 min.
Now add 15 ml of 5 % trichloroacetic acid (TCA). CAUTION: THIS IS VERY
CAUSTIC.
Grind for another 3 min.
Allow the sand to settle for 1 min, and carefully decant the liquid into a plastic
centrifuge tube. Label with your initials and centrifuge for 5 min at 3000 r.p.m.
The liquid phase (supernatant) of your extract will contain dissolved glycogen,
and the precipitate will contain nucleic acids and proteins.
Exercise B: Precipitation and Isolation of Glycogen with Ethanol
After centrifugation, decant the upper supernatant phase into a glass test tube. (Save
the precipitate for Part C.) Add two volumes of 95 % ethanol to the tube. Cool in an ice
bath. Stir with a glass stirring rod for 10 min. The precipitate that forms is glycogen.
Divide the contents of the glass tube evenly into two centrifuge tubes, and
centrifuge both tubes again for 5 min at 3000 r.p.m. Discard the ethanol
supernatant from both tubes. Resuspend the pellets (i.e. put back into solution)
from the two tubes in 1 ml of distilled water (resuspend the contents of one tube
in 1 ml distilled water and then pour this resuspension into the second tube.
Resuspend the contents of the second tube with the resuspension from the first
tube).
Transfer to a glass test tube. Label this tube GLYCOGEN, and add your name or
initials.

Now remove a sample from the tube to a spot plate, and test for glycogen with iodine
solution (see part E). Also test a drop of distilled water as a control. Additionally, test a
sample of 1 % glycogen and compare the results obtained with those obtained with your
own extracted glycogen. Record your results.
Exercise C: Isolation of Nucleic Acids with Hot NaCl Followed by Ethanol
Precipitation
- Take the centrifuge tube from part A that contains only the precipitated nucleic acids
and proteins.
Add 7 ml of 10 % NaCl, resuspend the pellet, and transfer it to a glass test tube.
Now boil the contents for 10 min in a boiling water bath. This will solubilise the
nucleic acids and leave the proteins as a precipitate.
After boiling, allow the tube to cool and then transfer the contents into a
centrifuge tube.
Centrifuge for 5 min at 3000 rpm.
Decant the supernatant of the centrifuge tube, which contains the nucleic acids,
into a separate glass test tube.
DO NOT DISCARD THE PELLETS IN THE CENTRIFUGE TUBE AS IT IS THE
PROTEIN TO BE USED IN PART D.
Next, add two volumes of ethanol and cool for 10 min in an ice bath while stirring
occasionally with a glass rod. This will precipitate the nucleic acids. After about
10 min of cooling and stirring, the nucleic acids will precipitate out from the
ethanol.
Now transfer the contents of the glass tube evenly into two plastic centrifuge
tubes.
Centrifuge at 3000 r.p.m. for 5 min, and pour off the supernatant from both tubes.
Resuspend the contents of one tube in 2 ml distilled water and then pour this
resuspension into the second tube.
Resuspend the contents of the second tube with the resuspension from the first
tube.
- Pour the final mixture into a glass test tube, and label this tube with the words
NUCLEIC ACIDS and your name.
- Apply the Dische test to this fraction to confirm the presence of nucleic acids (see
part E below).

Exercise D: Isolation of Proteins

With the reserved centrifuge tubes from part C do the following:
- Add 4 ml water and mix contents well.
- Transfer your mixture to a glass test tube. Label the tube with the word PROTEIN
and your name.
- Apply the Biuret test (see part E below) to this fraction to confirm the presence of
proteins.
Exercise E: Tests for the Macromolecules Extracted from Yeast Cells

(i) Dische Test for Nucleic Acids

The Diche test is based on the colour change that may be observed when Dische
reagent (diphenylamine) forms a complex with DNA. A blue colour indicates the
presence of DNA.

- Add 2 ml Dische reagent to the NUCLEIC ACID sample.

- Heat in the water bath for 20 min, then cool and note the colour change. Record your
results.
(ii) Iodine Test for Glycogen
A positive test for glycogen gives a dark red-brown colour with iodine.

- With a Pasteur pipette remove 2 drops of the GLYCOGEN sample to a spot plate and
test for the presence of glycogen with 2 drops of iodine.
- Also test a sample of 1 % glycogen and a sample of water (control) on a spot plate to
compare with your unhydrolysed extract. Record your results.

(iii) Biuret Test for Proteins

When mixed with certain specific reagents, proteins may be identified by different colour
reactions characteristic of their active groups. Large protein molecules produce a violet
colour, whereas lower molecular weight peptides produce a pink colour.

- Add 2 ml of 10 % NaOH to the PROTEIN sample. Mix well, then add 2 ml of 0.5 %
copper sulphate and mix again.
- Allow 5 min for the colour to develop. What can you conclude from the colour of your
sample? Record your results. Wash all test tubes and return them to the test tube rack.

A table summarizing the extraction procedures, each test, observations and conclusions
will be collected at the end of the lab session.

10

LABORATORY 2: PLASMID DNA ISOLATION AND RESTRICTION

ENDONUCLEASE ANALYSIS

Molecular biology methods have tremendous value not only in the investigation of basic
scientific questions, but also in disease prevention and treatment, generation of new
protein products, and manipulation of plants and animals for desired phenotypic traits. The
following exercises have been chosen to demonstrate the basic principles in recombinant
DNA: extraction of DNA, digestion of DNA with a restriction endonuclease, and agarose
gel electrophoresis of DNA samples.

MINIPREP ISOLATION OF PLASMID DNA

A plasmid is an independent, self-replicating DNA molecule usually found in bacteria. It is
typically circular and double-stranded and vary in size; from 1 to over 400 kilobase pairs
(kbp). Plasmids often contain genes that confer a selective advantage to the bacterium
harboring them such as the ability to make the bacterium antibiotic resistant Plasmids are
autonomous molecules and exist in cells as extra-chromosomal genomes.

Plasmids used in genetic engineering are called vectors. They are used to transfer genes
from one organism to another. Most also contain a multiple cloning site which is a short
region containing several commonly used restriction sites allowing the easy insertion of
DNA fragments at this location. Plasmids are commonly used to multiply (make many
copies of) or express genes of interest.

There are several methods to isolate plasmid DNA from bacteria. The miniprep can be
used to quickly isolate plasmid DNA for analysis by restriction digestion and for some
cloning techniques.

In this exercise, you will isolate the plasmid from cultures of bacterial cells.
Materials
Sterile microfuge tubes
Micropipettors and tips (200 L & 1000 L)
Bacterial culture grown overnight in LB broth
Luria- Bertani (LB) broth
10 g tryptone
5 g yeast extract

11

10 g NaCl
make to 1 litre after the pH has been adjusted to 7.5)

SOLUTION I
25 mM Tris base [2-amino-2-hydroxymethyl 1, 3-propanediol, pH 8
10 mM EDTA
50 mM Glucose
Store at 4oC and keep on ice when in use
P

SOLUTION II
0.2 M NaOH
1% (w/v) SDS

SOLUTION III
3 M Potassium acetate
5 M Glacial acetic acid

25:24:1 PCI (phenol: chloroform: isoamyl alcohol)

70% ethanol
100% ethanol
RNase (purified from bovine pancreas, 20 g/uL dissolved in TE buffer

TE Buffer
10 mM Tris base pH 8
1 mM EDTA
General instructions
Molecular biologists frequently use very small volumes of liquids in their work, even getting
down to 0.1 L (thats one ten thousandth of a millilitre, or one ten millionth of a litre!). For
such small volumes, they need to use a micropipettor. Some micropipettors deliver fixed
volumes, however the majority are adjustable. We will be using two micropipettors; one
that measures between 20 to 200 uL and the another that measures between 100 and
1000 uL. In using the micropipettors, set the desired volume by holding the micropipettor
body in one hand and turning the volume adjustment knob until the correct volume is
shown on the indicator. Attach a disposable tip to the shaft of the pipettor. Depress the
plunger to the first stop, holding the micropipettor vertically, immerse the tip a few mms

12

into the sample liquid, allow the push bottom to return slowly to the up position. Never
allow it to snap up. Withdraw the tip from the sample liquid. To dispense the sample,
place the tip against the wall of the receiving vessel, and depress the plunger slowly to the
first stop, and then after a few second (2-3) to the second stop, to expel any residual liquid
in the tip. With the plunger still depressed, carefully withdraw the micropipettor.
Then, allow the plunger to return to the top position. Discard the tip by depressing the tip
ejector button. Collect some water in a microtube and practice using the micropipettors
before starting the experiment.

Remember the four important DO NOTS when using a micropipettor:

1. DO NOT attempt to use the micropipettor without a tip in place. This can ruin the
precision piston that determines the volume of the fluid.
2. DO NOT lay down a micropipettor that has a filled tip. Fluid in the tip could run back
into the micropipettor and ruin the precision piston.
3. DO NOT let the plunger snap back after withdrawing or delivering the fluid.
4. DO NOT dial past the limits of the micropipettor.

Method
1. Shake culture to resuspend bacterial cells.
2. Label one 1.5 mL microfuge tubes with the group number. Use a micropipettor
transfer 1000 L of the overnight culture suspension into each tube.
3. Close the cap and centrifuge at 5,000 rpm for 5 minutes.
4. Pour off supernatant into the waste beaker. Be careful not to disturb cell pellets.
Invert tubes and gently tap on the surface of a clean paper towel to drain
thoroughly.
5. Add 100 L of ice cold SOLUTION I to the tube. Re-suspend pellets by pipetting
solution in and out several times. The suspension should be homogenous with no
visible clumps. Hold the tube to the light and check that there are no clumps.
6. Add 200 L of SOLUTION II. Close the cap and mix by inverting rapidly 5 times.
Stand tube on ice for 5 minutes. The suspension will become relatively clear.
7. Add 150 L of SOLUTION III. Close cap and mix by inverting rapidly 5 times. A
white precipitate will appear immediately. Stand tube on ice for 5 minutes.
8. Centrifuge 14, 000 rpm in a balanced microfuge for 15 minutes.
9. Transfer 400 L of supernatant into a clean 1.5 mL microfuge tube. Avoid the
precipitate. Discard old tube containing the precipitate in the waste beaker.

13

10. Add one volume (400 L) of PCI. Close caps and mix carefully by inverting slowly
5 times.
11. Centrifuge for 5 minutes. You will note the formation of three layers: an upper
phase (aqueous phase), a thin interphase, and a lower phase. The upper phase
contains your DNA, the interphase contains remnants of the cells and the lower
phase will be mostly phenol, chloroform, proteins, and a composite of
biochemicals.
12. Transfer 300 L of the top aqueous phase into a clean 1.5 mL microfuge tube
13. Add two volumes 100% ethanol. Close the cap and mix by inverting slowly 5 times.
14. Centrifuge for 5 minutes at 14, 000 rpm. Align tubes so that cap hinges point
outward. Nucleic acid (visible or not) will collect on the tube side under the hinge
during centrifugation.
15. Pour off supernatant. Be careful not to disturb nucleic acid pellet. Invert the tube,
and tap gently on the surface of a clean paper towel to drain thoroughly.
16. Add 500 L of 70% ethanol. Close cap and flick the tube several times.
17. Centrifuge at 14, 000 rpm for 5 minutes. Pour off supernatant. Align tubes so that
cap hinges point outward. Invert tubes, and tap gently on the surface of a clean
paper towel to drain thoroughly. Allow the pellets to air dry at room temperature for
about 10 minutes. All ethanol must be evaporated before proceeding to the next
step. Hold tube up to the light to check that no ethanol droplets remain.
18. Add 10 L TE. Close caps and flick tubes several times to re-suspend DNA pellet.
Check that all DNA is dissolved and that no particles remain on the side of the tube.
Remember to label the tube with your group number and DNA.
19. Take time for responsible cleanup; ensure that all micropipettor tips and microfuge
tubes have been placed in the waste beaker, wipe down lab bench with 70%
ethanol.
EXERCISE ON THE EXTRACTION PROCEDURE
1. If a P-20 micropipettor is set so that the display reads (from top to bottom) 1-3-7, how
many microliters will the micropipettor pick up when used properly?
2. If a P-1000 micropipettor is set so that the display reads (from top to bottom) 0-8-2,
how many microliters will the micropipettor pick up when used properly?
3. If a P-200 micropipettor is set so that the display reads (from top to bottom) 1-0-2, how
many microliters will the micropipettor pick up when used properly?
4. What are the four major types of biologically important molecules found in cells?

14

5. The solutions/buffers used in the miniprep procedure each target specific biologically
important molecules found in the bacterial cell resulting in cell lysis and the recovery
of plasmid DNA. For each step of the procedure, identify the components in the
solution/buffer and the biological molecule that is targeted. Include a brief description
of the resulting effect.

RESTRICTION ENDONUCLEASE ANALYSIS

Restriction endonucleases are valuable tools for characterizing and manipulating DNA
molecules. Restriction endonucleases are enzymes that recognize specific nucleotide
sequences, often 4 to 8 bp long and cut DNA at these sequences only. The specific
sequence of nucleotides that is recognized is referred to as the "restriction site". There are
many different restriction enzymes that recognize and cut many different nucleotide
sequences. Restriction enzymes are produced by bacteria as a defense against invasion
of foreign DNA, particularly DNA of bacteriophages. Foreign DNA that finds its way into
the bacterial cell is recognized and digested or "restricted" by the cell's restriction
enzymes. A bacterium modifies its own DNA to protect it from endonuclease activity. This
protective mechanism for bacteria is a source of enzyme reagents in molecular biology
experiments.
By cutting a given piece of DNA with specific restriction enzymes, one can determine the
locations of the restriction sites for those enzymes on that DNA, and generate a "restriction
map" of the given DNA. One can identify the different-sized fragments resulting from the
digestion of a given piece of DNA with a certain restriction enzyme by "electrophoresing"
the restriction-digested DNA on an agarose gel, as shown in Figure 2.1. Briefly, the
digested DNA is loaded into one of the sample wells at one end of an agarose
electrophoretic gel. A voltage is set up across the gel such that the sample wells are
closest to the negative electrode, and the far end of the gel is closest to the positive
electrode. DNA has a net negative charge, and thus moves through the gel toward the
positive electrode (this process is called electrophoresis). The gel is composed of a porous
matrix that acts like a molecular sieve. Smaller DNA fragments move through the gel faster
than do larger DNA fragments.
After the electrophoresis of the digested DNA has been carried out for a certain period of
time, it is stopped, and the DNA in the gel is stained to make it visible. The stain ethidium
bromide interchelates between double-stranded DNA and fluoresces brightly when
illuminated with UV light. The DNA is often visible as discrete bands. Each band represents

15

a collection of many DNA fragments that are all of the same size and have thus migrated
the same distance in the gel.

Figure 2.1: Gel Electrophoresis

http://www.stanford.edu/group/hopes/diagnsis/gentest/f_s02gelelect.

If digestion is complete, the sum of the molecular sizes of the fragments should be
equivalent to the size of the undigested plasmid. A partial or incomplete digestion will
result in many bands that are not equimolar and add up to more than the size of the
original plasmid. A smearing pattern occurs when there are many bands of similar size
close together.

In this exercise, you will digest a plasmid DNA with a restriction enzyme. In the next lab,
you will then separate the restriction fragments by agarose gel electrophoresis and
determine the sizes of the restriction fragments by comparing them with molecular
standards.
16

Materials
Gloves
Sterile microfuge tubes
Micropipettors and tips (200 L)
Plasmid DNA
BglI 2U/L
10X digestion buffer
Sterile distilled water
Water baths at 37oC
P

0.8% agarose in 0.5X TBE (90mM Tris-borate, 1mM EDTA)

0.5X TBE
Loading dye (50% v/v glycerol, 0.5% w/v bromophenol blue)
Molecular marker 1 kb DNA ladder (100 g/mL)
Ethidium bromide (1 g/mL)
Transluminator
Method
1. Label a microfuge tube. Set up a digestion of your plasmid DNA in a 1 mL microfuge
tube as shown in the Table below. Add the components in the order shown in the
Table: water first, buffer second, DNA third, and enzyme last. Remember to label the
tube with your group number.

Tube # Distilled water 10X BglI Buffer Plasmid DNA RNase Bgl
1g/mL (2U/L)
1
2L
1L
5L
1L
1L (2U)

2. After all the components have been added to each tube, flick the tubes with your finger
to mix and centrifuge them briefly in the centrifuge to bring the solutions to the bottoms.
3. Incubate the tube in a 37oC water bath. Remove the plasmid DNA digest after 60
P

minutes. All tubes will be kept at -20oC until the next lab.
P

17

EXERCISE ON RESTRICTION DIGESTION

1. The recognition sequence of the restriction enzyme BamHI is given below. The enzyme
cleaves after the first G.
5 GGATCC 3
3 CCTAGG 5

What is the term used to describe the cut and the termini of the fragments generated with
this enzyme? If the DNA sequence given below was cut with BamHI, how many DNA
fragments would you expect? Indicate the cut on the sequences and state the number of
fragments that will be generated.
5 ATTGAGGATCCGTAATGTGTCCTGATCACGCTCCACG 3
3 TAACTCCTAGGCATTACACAGGACTAGTGCGAGGTGC 5
2. The recognition sequence of the restriction enzyme SmaI is given below. The enzyme
cleaves after the third C.
5 CCCGGG 3
3 GGGCCC 5

What is the term used to describe the cut and the termini of the fragments generated with
this enzyme? If the DNA sequence given below was cut with SmaI, how many DNA
fragments would you expect? Indicate the cut on the sequences and state the number of
fragments that will be generated.
5 GATTGAGGATCCTACCCGGGTCGTA 3
3 CTAACTCCTAGGATGGGCCCAGCAT 5

3. Refer to the diagram provided in the lab. It represents the results of a restriction
digestion and agarose gel electrophoresis of the products. The molecular standard was
loaded in lane 1.

The sizes of the fragments are indicated.

Answer the following

questions:
1. What is a likely explanation for the banding pattern of the undigested plasmid in lane
2?

18

2. How many times did the enzyme used in lane 3 digest the plasmid? Give an
explanation.
3. Estimate the sizes of the products in lane 3.
4. Estimate the size of the original plasmid.
5. What is a likely explanation for the smearing in lane 4?
6. Explain the results in lane 5. The same plasmid was analysed in lane 3 and lane 5.
However, they were isolated by 2 different technicians.
7. What is a likely explanation for the absence of fragments in the digestion represented
in lane 6?
The responses to the exercises will be collected at the end of the lab session. The writeup for the experiments will not be collected until all data have been collected (plasmid DNA
extraction, restriction digestion and electrophoresis of the digested samples).

19

LABORATORY 3: POLYMERASE CHAIN REACTION AND AGAROSE

GEL ELECTROPHORESIS

Polymerase chain reaction (PCR) is an extremely useful technique that has revolutionized
the amplification, manipulation and cloning of DNA fragments. The technique is commonly
used to detect and study genes of pathogenic organisms, and in other sciences, PCR is
used for DNA fingerprinting, detection of hereditary genes, cloning of genes, paternity
testing and detection of introduced or foreign DNA.
PCR uses a basic strategy for DNA replication that is similar to the mechanism of
replication in the cell. One major difference between the cellular and PCR reactions is that
the in vitro method is carried out at elevated temperatures (as high as 95C). At this
temperature, DNA polymerase isolated from most cells is inactivated. Thus, in PCR a
thermostable DNA polymerase purified from the thermophilic bacterium, Thermus
aquaticus, is used. This enzyme is referred to as Taq DNA polymerase. Other cellular
components required for DNA replication provided in the in vitro reaction mix include the
four deoxyribonucleotide triphosphates (dATP, dCTP, dTTP, dGTP) and primers. Primers
are very short pieces of DNA that are complimentary to the 3' end of each of the two DNA
strands from the region of interest. They are sufficiently small to enable the quick location
and binding to their complimentary DNA once the large DNA molecule has been denatured
(the two strands of the helix separate into single strands).
The basic steps in a typical PCR are outlined in Figure 3.1.
DNA isolated from tissues is first heated to 94-95C in order to separate its complementary
strands. The reaction mixture is then cooled to 45-60 C and the separated strands anneal
with two synthetic primers that are present in the mixture. These primers, which are
generally between 15 and 20 nucleotides in length, serve to specify the positions that DNA
synthesis can be initiated by Taq polymerase. The annealed mixture is then incubated at
68-72C, the optimal temperature for Taq polymerase catalysis.

20

Figure 3.1: Amplification of a designated sequence by PCR

http://faculty.washington.edu/dovichi/CoursePages/Chem428site/labs/Lab%202%20PCR%20Lab.pdf

21

These three steps (denaturation, primer annealing, and synthesis or extension) represent
a single PCR cycle and takes about 5 minutes. Special machines, called thermocyclers,
have been developed with an automated system to rapidly heat or cool reaction
components. The devise contains a thermal block into which the tubes holding the PCR
reaction mixtures are inserted. The cycler then raises and lowers the temperature of the
block according to the pre-programmed steps.

Notably, each PCR cycle doubles the amount of DNA in the previous cycle. Thus, the
amount of DNA after 1, 2, 3, and 4 cycles is 2, 4, 8, and 16 times the amount of original
template. This exponential amplification of template DNA can result in amplification levels
6

as high as 10 during the course of a typical PCR reaction of 20 to 30 cycles. After PCR
cycling is complete, the amplification products can be subjected to cloning, sequencing, or
analysis via gel electrophoresis.

Two exercises will be conducted in the lab session. First collect your restriction digestion
tubes from last week and run your digested samples on an agarose gel.

1. Prepare the 0.8% agarose gel and running buffer. Melt 0.35 g of agarose in 35 mL of
running buffer in the microwave. When gel is hardened, carefully remove the comb and
put the gel into the electrophoresis apparatus. Add running buffer to the buffer chambers
until the surface of the gel is submerged.
2. Collect practice petri dishes and practice loading 15 L of dye to the wells.
Remember to carefully position the micropipettor tip under the buffer above the
appropriate well (do not insert the tip in the well), slowly release the contents by
pressing the plunger to the last position in the micropippettor. Do not release the
plunger until the micropipettor is lifted out of the buffer.
3. Add 5 L of loading buffer to each digestion and thoroughly mix by pipetting up and
down. Label 2 other tubes and transfer 5 L undigested plasmid DNA and 5 L undigested
plant DNA to the tubes. Add 5 L of loading buffer to each tube, flick the tubes with your
finger to mix. The molecular ladder will be provided. Use 5 L.
4. Carefully load the samples into wells in the agarose gel using micropipettor in the order
indicated below:

22

Lane 1 1 Kb ladder
Lane 2 Undigested plasmid DNA
Lane 3 Plasmid DNA digest

Carefully position the micropipettor tip under the buffer above the appropriate well, slowly
release the contents by pressing the plunger to the last position in the micropippettor.
Do not release the plunger until the micropipettor is lifted out of the buffer.

5. Electrophorese at 100 V until the dye front has traveled approximately 3/4 of the
distance between wells (~ 30 minutes). Turn off the power supply, disconnect the leads
and remove your gel.
6. Stain gel in ethidium bromide (0.5 ug/mL) for 10 minutes. Decant the ethidium bromide
into the appropriate container, add about 100 mL distilled water to destain for 5 minutes.
7. Visualize DNA patterns by removing the gel slab from the mold and placing it on a UV
transluminator. Record the pattern by photography or computer image capture.

Record the number of fragments and the distances (cm) the fragments migrated in your
gel. Estimate the molecular sizes of each band observed by comparison with molecular
marker (range of fragments 500, 1000, 1500, 2000, 3000 [has increased intensity to serve
as a reference band], 4000, 5000, 6000, 8000, 10,000). Estimate the size of the original
plasmid. How many places does the restriction enzyme cut the plasmid?
While the gel is running complete the virtual laboratory which will demonstrate how PCR
is carried out. Answer the following questions on PCR and DNA replication.

EXERCISE ON PCR
1. What components are needed for DNA to be amplified via PCR?
2. What temperatures will the PCR thermocycler cycle through? and what is
accomplished at each temperature?
3. What is the size of the PCR product obtained in the lab exercise?
4. How many DNA strands would you expect to find after 20 cycles of PCR?
5. thalassemia is a disease that is most often caused by a deletion of all or part of the
globin gene. Briefly describe a PCR procedure that you would use to determine
whether a patient is suffering from thalassemia.

23

6. The following DNA is part of a gene that codes for a polypeptide of seven amino acids.
Complete the table to illustrate the salient features of DNA replication, transcription
and translation.

Original DNA DNA after one mRNA tRNA Protein

DNA

cycle of PCR

The responses to the exercises will be collected at the end of the lab session. The writeup for the experiments is due in a week. Results of the gel electrophoresis will be posted.

24

LABORATORY 4: PROBABILITY, MONOHYBRID AND DIHYBRID INHERITANCE &

QUANTITATIVE TRAITS

PART A: PROBABILITY, MONOHYBRID AND DIHYBRID INHERITANCE

To illustrate the relevance of statistical tests to genetics and at the same time to
demonstrate that the standard genetic F2 ratios of offspring do result from random
B

selections, the following exercises are to be done. Students should work in pairs. In these
exercises, we will attempt to assess differences between observed and expected
outcomes in simulated monohybrid and dihybrid inheritance. Consult the section dealing
with the Chi squared test in your genetics notes (available on OurVLE). Bring those notes
with you to the laboratory.

EXERCISES ON PROBABILITY MONOHYBRID INHERITANCE

(A)

Count out into 2 containers equal numbers of coloured beads of two types about
20 of each say yellow (Y) and red (R) or blue (B) and green (G).
Shake the container and without looking select two beads at a time. Put back
the beads and repeat the process 100 times.
Record by totalling on a neatly set out table, the number of times you pick two
yellow beads, a yellow and a red or two red (or two blue beads, a blue and a
green or two green).
Calculate and record the ratio of the three types of results. Show on the basis of
probabilities, what ratios might be expected.
You are now faced with a value judgement: How can you decide whether the ratio
that you obtained differs more than just by chance from the expected ratio? In
some instances it might be obvious that two such values are very similar. In other
cases they might clearly be different. But in many cases it will be hard to decide.
Statistical tests such as the Chi squared (2) test can help you to make that
P

decision.
On the basis of the total numbers of draws made and the expected ratios calculated
above, determine the expected numbers of the three types of result (YY, YR,
RR)/(BB, BG, GG). Tabulate together the observed and expected numbers (Use
the table outline on page 27 as a guide).
25

Calculate the Chi squared value for the difference between the observed and
expected results, and use the table provided in Appendix I to determine whether
the difference is significant at the 0.05 level of probability.
Write out clearly the relationship between the above exercise and the results of
the monohybrid cross, explaining why in both cases the same ratios are
expected.

EXERCISES ON PROBABILITY DIHYBRID INHERITANCE

Get together with another group of students for this exercise. One group should have a
bag with yellow (Y) and red (y) beads and the other group should have a bag with blue
(B) and green (b) beads.
Both groups will now pick two beads from each container at the same time.
Record, by tallying in a clearly set out table, the frequency with which you pick
1. At least one yellow and one blue bead, Y/ (Y or y) + B/ (B or b) - [ Y_ B_ ]
2. At least one yellow and no blue bead, Y/(Y or y) + bb

- [ Y_ bb ]

3. No yellow and at least one blue bead, yy + B/ (B or b)

- [ yy B_ ]

4. No yellow and no blue bead, yy + bb

- [ yy bb ]

Return the beads to their respective containers and shake before the next draw. Make at
least 160 draws in all.
Calculate the ratio of the four different categories of events.

Show on the basis of

probability, what ratio might be expected.

On the basis of the total number of draws and the expected ratio, determine the expected
number of each type of event. Tabulate the observed and expected results together (Use
the table outline on page 27 as a guide).
Calculate the Chi squared value for the difference between the observed and expected
results. Determine from the Table provided (Appendix I), whether the difference is
significant, explaining clearly how you decide this.

26

Explain as clearly and as concisely as you can, the relationship between this exercise
and the results of the standard dihybrid cross.

Class

Observed
Results (o)

Expected
Results (e)

Deviation
(o-e)

Squared Deviation
(o-e)2

(o-e)2
(e)

Chi squared = (o-e)2

(e) .

27

PART B: QUANTITATIVE TRAITS

A quantitative trait in a large population usually displays a symmetrical, characteristically
bell-shaped distribution which is called a normal distribution. It is approximated by the
binomial distribution (p + q)n
Analysis of the inheritance of such a trait therefore requires the use of statistical
methods which are applicable to populations.
In most cases a description of a large population is obtained from samples, which
to be useful, have to be chosen at random.
The following exercise is intended therefore to illustrate the method of obtaining:
(a)

A concise description of the quantitative characteristics of a sample obtained from

a population.

(b)

An estimate of (1) the quantitative characteristics of the population from which the
sample was taken, (2) how well the sample represents the population.

(c)

An expression of the probability that two samples differ significantly in terms of a

particular theory concerning the reason for the observed differences.

EXERCISE ON THE NORMAL DISTRIBUTION:

Collect and record to the nearest centimetre the heights of as many adults males and
females as you can in 3/4 hour. Keep the data for the different sexes separate. You must
ask to make sure that persons you measure have not previously been measured.
Measuring rods will be provided.

At the end of this lab you should understand the use of terms such as:
RANDOM, FREQUENCY DISTRIBUTION, CATEGORY OR CLASS MEAN, VARIANCE,
STANDARD DEVIATION, NORMAL, STANDARD ERROR OF THE SAMPLE MEAN,
STANDARD ERROR OF THE DIFFERENCE IN MEANS, SIGNIFICANCE.

On returning to the lab, you will be required to collate the two sets of data (for males and
females) on the board. You will be required to express the sets of data as frequency
distribution histograms. You will then find the mean and standard deviations for the
heights of the men and women, and try to decide whether differences between the means
are significant or real, or whether they are due simply to inconsequential, random

28

differences between the persons sampled. For the last-mentioned comparison, you will
be expected to determine the standard error to the difference in means. The methods
for calculating the standard deviations, standard errors, standard error to the difference in
means and whether the difference between the means are significant, are presented
below. Results should be tabulated as outlined below.

Height

Midpoint

(x)

X X

X X

X X

The total number of individuals (n) =

The standard deviation (SD) =

(x x )
n

The mean height (x ) =

2

fx

The standard error (SE) =

SD
n

In genetics, it is often necessary to determine whether the difference in the means of two
samples is statistically significant. This usually arises when one has to decide whether
two sample means represent a single population or genetically different populations.
The decision can usually be made by calculating a statistic known as the Standard error
of the difference in means (SEd).

SEd

S S
2

x1

x2

S x1 and S x 2 represent the standard error of the sample mean for the two samples. If
the different in means x 1 x 2 is greater than twice the standard error of the difference of
the sample means (2 SEd), the difference in sample means is considered significant. Here
significance means two different populations. This is because when the probability that
two samples have come from the same population falls below 0.05, the difference in
means is considered significant.

29

LABORATORY 5: THE BINOMIAL EXPANSION & HUMAN GENETICS

PART A: THE BINOMIAL EXPANSION

In genetics, problems frequently arise in which there are several independent events
occurring together, each of which has two possible outcomes - the problem being to
determine the probability (i.e. the expected frequency) for the occurrence of a given
combination of the events. For example, each child born can be a boy or a girl, but if a
couple plans to have three children what is the probability that two will be boys and the
other a girl?

Similarly, a gene often exists in two alternative forms e.g. normal or vestigial wing gene,
or contributing and non-contributing alleles of polygenes, and one might wish to know the
expected frequency of occurrence of particular combinations of various alternatives.

Problems of this sort are analogous to the problem of tossing several coins together and
trying to predict the frequency with which you will get a given combination of heads and
tails.

Predictions of this nature can be made using the binomial expansion.

(a + b)2 = a2 + 2ab + b2
(a + b)3 = a3 + 3a2b + 3ab2 + b3
EXERCISES ON THE USE OF THE BINOMIAL EXPANSION
(A)

1. Take two coins and decide which is heads (H) and which 'tails' (T).
2. Shake them together and record by tallying, in a neatly set out table, the
number of times you get two heads (HH), a head and a tail (HT) two tails (TT).
3. Repeat this about 30 times. Pool your results with the class results on the board.
4. Using the binomial expansion, predict what the probabilities of the different
events should be
5. From these and the total number of 'tosses' calculate the expected number in
each category
6. Carry out a Chi squared test to see if the observed and expected distributions
differ significantly.

(B)

Repeat exercise (A) using three coins.

30

(C)

1. For those students with families with three or more children, we shall try to
find out how many consist of 3 Boys, 2 Boys/1 Girl, 2 Girls/1 Boy, and 3 Girls,
among the first three children.
2. Tabulate these results neatly.
3. Predict using the binomial expansion, what the expected numbers should be.
4. Carry out a Chi squared test to see whether the two distributions differ
significantly (Refer to table on page 27).

PART B: HUMAN GENETICS

SOME INHERITED HUMAN CHARACTERISTICS
Attached ear lobes
In most people the ear lobes hang free but where a person is homozygous for the
recessive gene (a) the ear lobes are attached directly on the side of the head.
Widow's PeakIn some people the hair line drops downward and forms a distinct point in the centre of
the forehead. This is known as widow's peak. It results from the action of a recessive
gene (w).
Tongue rollingA dominant gene (R) gives some people the ability to roll the tongue into a distinct U-shape
when the tongue is extended from the mouth.
Bent little finger
A dominant gene (B) causes the last joint of the little finger to bend inward toward the
fourth finger. Lay both hands flat on the table, relax the muscle and note whether you
have a bent or a straight little finger.
Mid-digital hair
Some people have hair on the second middle joint of the fingers while others do not. The
complete absence of hair from all fingers is due to a recessive gene (m) and its presence
due to a dominant allele (M). There seems to be a number of these alleles which determine
whether the hair shall grow on one, two, three or four fingers. This hair may be very fine,
and you should use a hand lens and look very carefully on all fingers before deciding
whether this hair is absent from any one of your fingers.

31

Interlocking fingers
When the fingers are interlocked, some people will almost invariably place the left thumb
on top of the right and others place the right over the left. Studies of family pedigrees
indicate that the placing of the left over the right is due to dominant gene (F) while the right
thumb on top is due to a recessive (f).
Hitch-hikers thumb
This refers to the ability to fold the distal joint of the thumb back about 90. Evidence
indicates that this characteristic is due to a recessive gene (h). There is some variation in
expressivity, for occasionally this ability will be found in one thumb only. There seems to
be a five percent reduction in penetrance i.e. about one person in twenty who carries the
gene will not express the characteristic.
P.T.C. tasting
Some people detect a distinct bitter taste from the chemical known as P.T.C.
(phenylthiocarbamide) while others do not taste it at all. Nontasters are homozygous for
the recessive gene (t). The ability to detect a bitter taste results from the presence of a
dominant gene (T), but there appears to be two alleles of it - one for early tasting and one
for late tasting.
Tongue folding
This refers to the ability to fold the tongue backward without pressing against the upper
teeth. This characteristic appears to be due to a recessive gene (f).
Short second finger
A dominant gene (S) causes the second finger to be shorter than the fourth.
Darwin's ear point
This is a small point of cartilage on the outer rim of the ear and is due to the effect of a
dominant gene (D)
Long Palmar muscle
Clench your fist and flex the hand at the same time. The presence of triple tendons on the
underside of the wrist is due to the effect of a recessive gene (1). The dominant gene (L)
results in the presence of two tendons.
Heavy eyebrows
Heavy eyebrows as opposed to thin light eyebrows are due to the effects of a dominant
gene (H).

32

SURVEY OF INHERITED HUMAN CHARACTERISTICS

Make a table with four columns. In one, you list the traits referred to previously. In the
second list presence or absence, in the third column list your possible genotype for each
of the traits listed. Remember that with a dominant trait the genotype could be either
homozygous or heterozygous. If you are not certain of the phenotype, use a dash to
indicate that the allele is unknown. In the fourth column list the frequency of the trait in the
class.

33

EXERCISES ON THE ANALYSIS OF INHERITED HUMAN CHARACTERISTICS

(A)

Choose any two characteristics and determine the chance of each showing in
any person picked at random.
Characteristic No. 1:
Total No. of persons in class who show it:
Total No. of persons in the class:
Percentage showing it:
Characteristic No. 2:
No. of persons in the class who show it:
Total No. of persons in the class:
Percentage showing it:

Now consider the percentage showing a characteristic as the probability of any

one person showing it.
Calculate the chance of any one person showing:

Both characteristics
Only one of either of these two characteristics
Neither of these two characteristics

According to the figures obtained above, how many in the class would be expected to
show

Both characteristics

Only one of either of these two characteristics

Neither of these two characteristics

(Give results to 1 decimal place).

How many in the class actually show:

These two characteristics?

Only one of either of these two characteristics?

Neither of these two characteristics?

Is the difference between the calculated figures and the observed distribution of the two
characteristics statistically significant?

34

Does the results indicate that there must be free assortment among the genes responsible
for the characteristics chosen? Explain your answer.
(B)

Choose a characteristic whose expression as a homozygous recessive we can

easily identify e.g. attached ear lobes.
How many people in the class show this trait?
How many express the dominant allele?
What are the frequencies of the alleles (a and b) in the class?
Now if we allow a to represent the dominant allele and b the recessive then
the distribution of these alleles in the class will be given by the binomial
expression:
(a+b)2 = a2 + 2ab + b2 = 1
P

(% homo. dom.)
a2
P

(% hetero. dom.)

(% homo. rec.)
b2

2ab

Determine the frequencies of alleles a and b as outlined below:

Since b2 = frequency of 2 recessive genes.
P

Then b = b2 = frequency of allele b.

P

Therefore a = frequency of allele a = 1 - b.

How many persons in the class have the homozygous dominant gene?

How many persons in the class have the heterozygous form of the gene?
The number of people in the class who are homozygous for a =
a2
P

Convert a2 + b2 to numbers.
P

Then the number in the class left after subtracting a2 + b2 from the total = 2ab = persons
P

heterozygous for the genes.

35

APPENDIX I
CHI SQUARED TABLE

Degrees
of
Freedom
1
2
3
4
5
6
7
8
9
10

P r o b a b i l i t y

0.95

0.90

0.80

0.70

0.50

0.30

0.20

0.10

0.05

0.01

0.001

0.004
0.10
0.35
0.71
1.14
1.63
2.17
2.73
3.32
3.94

0.02
0.21
0.58
1.06
1.61
2.20
2.83
3.49
4.17
4.86

0.06
0.45
1.01
1.65
2.34
3.07
3.82
4.59
5.38
6.18

0.15
0.71
1.42
2.20
3.00
3.83
4.67
5.53
6.39
7.27

0.46
1.39
2.37
3.36
4.35
5.35
6.35
7.34
8.34
9.34

1.07
2.41
3.66
4.88
6.06
7.23
8.38
9.52
10.66
11.78

1.64
3.22
4.64
5.99
7.29
8.56
9.80
11.03
12.24
13.44

2.71
4.60
6.25
7.78
9.24
10.64
12.02
13.36
14.68
15.99

3.84
5.99
7.82
9.49
11.07
12.59
14.07
15.51
16.92
18.31

6.64
9.21
11.34
13.28
15.09
16.81
18.48
20.09
21.67
23.21

10.83
13.82
16.27
18.47
20.52
22.46
24.32
26.12
27.88
29.59

Nonsignificant

Significant

36

MOLECULAR BIOLOGY TUTORIAL QUESTIONS

1. As regards the nitrogen-containing bases in nucleotides, what is the difference
between DNA and RNA?
2. What is the rule for pairing nitrogen-containing bases in the DNA molecule?
3. As regards biological function, what is the difference between DNA and RNA?
4. Briefly outline the experiments by Griffith and Avery and show how the results led to
the conclusion that DNA was the genetic material.
5. Describe the different levels of chromosome folding in eukaryotic cells.
6. Draw a schematic representation of a replication fork indicating the overall direction
of replication and the locations of the following:
(i) leading strand
(ii) lagging strand
(iii) DNA polymerase
(iv) single strand binding proteins
(v) okazaki fragments
(vi) toisomerase
(vii) RNA primer
(viii) DNA ligase
(xi) Helicase
(x) RNA primase
7. Compare the processes of transcription and replication.
8. What are the post-transcriptional modifications required to produce mature mRNA?
9. There are inaccuracies in the DNA molecule shown below.
5' AGTCCGAUGC 3
5' TCAGGCTATG 3'
Correct the inaccuracies in the top sequence and generate the complementary
strand. Assuming mRNA is transcribed from left to right, which strand is the template
strand? Explain your choice. What would be the sequence of the transcript and the
polypeptide? Show the polarity, that, is the 5 and 3 ends of the transcript and the
NH2 and COOH ends of the polypeptide.
10. A partial sequence of a DNA strand is given below. Given that the mRNA is
transcribed from left to right and that the intron splice sites are CUU and AAG, give
the sequence of nucleotides of the transcript from this region of the gene. Show the
polarity (that is the 5 and 3 ends)
5 ACCGAAGATCTTGCTGTTAAGCCAAAGCAGATTGCCCAGGAG 3
3 TGGCTTCTAGAACGACAATTCGGTTTCGTCTAACGGGTCCTC 5
Using the table of the codons specifying amino acids, give the sequence of the
polypeptide encoded by this region of the gene, denoting the NH 2 and COOH ends.
37

11. A nematode gene encoding a polypeptide of 117 amino acids was cloned by a
laboratory studying the contribution of collagen to the body structure of eukaryotic
organisms. What is the minimum number of nucleotides coding for this region of the
gene? Explain your answer.
A partial sequence of one DNA strand in an exon in this gene is given below. What
is the sequence of nucleotides of the mRNA in this region of the gene? Show the
polarity.
3 TGGCTTCTAGGTTTCGTCTAACGGGTCCTC 5
Using the table of the codons specifying amino acids, give the sequence of the
polypeptide encoded by this region of the gene, denoting the NH 3 and COOH ends.

12. Briefly outline the steps involved in splicing a gene into a plasmid and inserting the
plasmid into a bacterial cell.

38

GENETICS TUTORIAL QUESTIONS

1. If a black female guinea pig is test crossed and produces at least one white offspring,
determine (a) the genotype and phenotype of the sire (male parent) which produces
this white offspring, (b) the genotype of this female.
2. Tall tomato plants are produced by the action of a dominant allele D and dwarf plants
by its recessive allele d. Hairy stems are produced by a dominant gene H and hairless
stems by its recessive allele h. A dihybrid tall, hairy plant which is heterozygous for
both genes is testcrossed. What are the expected genotypes and phenotypes in the
F1 generation and the expected genotypic and phenotypic ratios?
3. The colour of the snap dragon flower may be red (RR), pink (Rr) or white (rr), while the
leaves of the plant may be hairy (HH) or smooth (hh). If plants heterozygous for both
genes are crossed, determine the expected phenotypic ratio.
4. The shape of radishes may be long (LL), round (ll) or oval (Ll). The colour may be red
(RR), white (rr) or purple (Rr). If a long white strain is crossed with a round red strain,
what percent of plants will produce long purple radishes in the F 2 generation?
5. Squash plants may be white, yellow or green. The cross of yellow X white produced
an F1 of all white-fruited plants that when crossed gave an F 2 of 12 white: 3 yellow: 1
green. (a) Suggest genotypes for the white, yellow and green phenotypes. (b) Give
genotypes of the P, F1 and F2 of this cross. Use genotype symbols starting with the
first letter of the alphabet.
6. Using probabilities, determine what proportion of the offspring are expected to have
the identical genotype as the parents, if both parents are heterozygous for the two
genes that are being tracked.
7. Red eyes, an ebony body and a striped wing are produced by recessive genes r, e
and s on the third chromosome of Drosophila. When homozygous red eyed, ebony
body females were mated with homozygous striped wing males and the resulting
trihybrid F1 females were test crossed to produce the F2 generation, it was noticed that
2,765 were red eyed with ebony bodies, 2,920 had only striped wings, 456 were red
eyed with striped wings, 378 had the ebony body only, 227 were red eyed only, 215
had striped wings and an ebony body, 21 had all three characteristics and 18 were the
wild type. Using the data presented above, determine how the genes are arranged on
the parent chromosome, the distance between the genes and the level of interference.
8. Baldness and a short index finger are sex-influenced traits that are dominant in men
and recessive in women. If a heterozygous bald man with long index finger fathers
children with a heterozygous long-fingered bald woman, what are the possible
phenotypes of the children?

39