‘A’ LEVEL BIOLOGY PAPER 3 ESSAY OUTLINES AND NOTES

COMMON BIOLOGY ESSAY OUTLINES AND NOTES

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 ‘A’ LEVEL BIOLOGY PAPER 3 ESSAY OUTLINES AND NOTES

© Gladmore Taruvinga 2018

First edition 2018 – Biology Made Simple: Common Biology Essay Outlines and Notes.
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 ‘A’ LEVEL BIOLOGY PAPER 3 ESSAY OUTLINES AND NOTES

CONTENTS

TOPIC TITLE PAGE

Tips on answering essay type questions 4
Mistakes that you should always avoid 4
Interpreting command words in essay questions 5

1 Cell structure and function ×

2 Biological molecules and water 
3 Cell and nuclear division 
4 Genetic control 
5 Inherited change and evolution 
Gene technology 
Pedigree charts ×
6 Natural and artificial selection 
7 Energetics – ATP structure and synthesis 
Energetics – Photosynthesis 
Energetics – Respiration 
8 Mammalian circulatory system 
Transport systems in plants ×
9 Nervous control ×
10 Reproduction in plants ×
Reproduction in humans 
11 Ecology ×
12 Biodiversity ×
13 Drug and substance abuse ×
Global distribution of Diseases ×
Immunity ×

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BIOLOGY MADE SIMPLE

BIOLOGY PAPER 3 (6030 / 3) QUESTIONS AND NOTES
TIPS ON ANSWERING ESSAY TYPE QUESTIONS

1. Understand the question

• Read the question carefully two or three times. Be sure to distinguish between the relevant information and the
extraneous information.
• Underline or highlight the key points in the question. This is particularly important for essay questions that ask you
to address several points.
2. Plan out your answer before you start writing
• This may seem like a waste of your time. However, it is a greater waste of time to write unnecessary information or
to erase and rewrite.
• Jotting down a quick outline will remind you of the key points that you want to make.
• Making a quick diagram can also help you focus your thoughts.
3. Convey your thoughts in an organized manner
• Write your answers in prose.
• The key points to your answer should be clearly stated and be the focus of your answer.
• The key points should be obvious to the reader / examiner and not buried amongst peripheral material.
• Do not include extra information if it does not directly support your answer.
4. Use relevant / appropriate technical / scientific terminology to answer the question
• Correctly use the relevant biology and science terms that you learn from your courses.
• Technical / scientific terms are highly specific and reduce the total number of words that you will need to write.
• Using technical / scientific terms to communicate will be essential in your professional life.
5. Making a drawing can often assist you in your answer
• However, your written answer must explain what is in the drawing.
6. Support your answer with examples where necessary.
• Some examination questions require thorough descriptions of examples that were learnt during the course.

MISTAKES THAT YOU SHOULD ALWAYS AVOID

1. Do not write too much

 Do not try to write everything that you have ever heard related to the question.
 Answer the question directly, without excess information.
2. Do not write a good answer to the wrong question
 In other words, make sure that you answer the question that is asked and not something else on the related topic.
3. Do not expect the examiner to figure out what you mean
 Do not just make a drawing and expect the examiner to figure out what you were thinking from this. (Unless the
question only asks you to make a drawing).
 Do not expect the examiner to find the relevant information in a sea of irrelevant information.
 Do not expect the examiner to read between the lines and make connections that you should be making.

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INTERPRETING KEYWORDS / COMMAND WORDS IN ESSAY QUESTIONS

Command words are the words and phrases used in exams that tell students how they should answer the question.

Here is a list and meanings / definitions of the command terms used in science examination papers.
The definitions will help you understand what the words / questions are asking you to do.
1. Annotate. Add notation or labeling to a graph, diagram or other drawing.
2. Calculate is used when a numerical answer is required. Working should be shown.
3. Comment. Present an informed opinion.
4. Compare. Identify/comment on similarities and/or differences.
5. Contrast. Identify/comment on differences.
6. Compare and Contrast. The question will always involve two or more related items. "Compare" means that you
should explain the similarities/ between the two items. Use words ―whereas‖, ―while‖, ―however‖ etc. Ordinarily,
examiners do not want you to simply list the similar characteristics, but explain the characteristics and/or how they are
similar. "Contrast" means that you should explain the differences between the two items.
7. Deduce means that the candidate is expected to draw logical and valid conclusion from given information.
8. Define (the term(s)…) is intended literally. Only a formal statement or equivalent paraphrase being required.
9. Describe requires candidates to state in words (using diagrams where appropriate) the main points of the topics. It is
often used with reference either to particular phenomena or to a particular experiment. In the former instance the term
usually implies that the answer should include reference to (visual) observations associated with the phenomena.
10. Design. Set out how something will be done e.g. an experiment or investigation.
11. Determine implies that the quantity concerned cannot be measured directly but is obtained by calculation,
substituting measured or known values of other quantities into a standard formula.
12. Diagram / Draw / Illustrate. Make a drawing. Keep it simple. Labels should be used whenever possible.
13. Discuss. Present key points.
14. Distinguish. List the differences between different items / identify the differences between two or more factors. Use
words such as ―while‖, ―however‖, ―whereas‖ etc.
15. Estimate implies an approximate calculation of the magnitude or quantity concerned.
16. Explain may imply reasoning or some reference to theory, depending on the context.
17. Find means that the candidate is expected to calculate measure or determine.
18. Give. Produce an answer from recall or from given information.
19. Identify. Name or otherwise characterize. Here, a simple list of concepts or terms should be sufficient.
20. Interpret means to put the data or figure into words. In other words, write an explanation of the meaning of the data
or figure.
21. Label. Provide appropriate names on a diagram.
22. List requires a number of points, generally each of one word, with no elaboration. Where a given number of points is
specified, this should not be exceeded.
23. Measure means to establish the quantity concerned using a suitable measuring instrument.
24. Name. Write the correct term or label.
25. Outline means to give the essential points / give a brief description.
26. Predict implies that the candidate is expected to state what is likely to happen by analysing given information.
27. Sketch, when applied to graph work, implies that the shape and/or position of the curve need only be qualitatively
correct.
28. State implies a concise answer, with little or no supporting argument, e.g. a numerical answer that can be obtained
‗by inspection‘.
29. What do you understand by/What is meant by (the term(s)…) normally implies that a definition should be given,
together with some relevant comment on the significance or context of the term(s) concerned, especially where two or
more terms are included in the question. The amount of supplementary comment intended should be interpreted in the
light of the indicated mark value.

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TOPIC 5 INHERITED CHANGE AND EVOLUTION

Key words in Genetics
 Heredity – is the passing of characteristics from one generation to the next.
 Genetics is the study of heredity.
— Inheritance: Process by which characteristics are passed on from parent to progeny.
— Variation: Degree by which the progeny differs from its parent.
 Gene – A unit of heredity. A section of DNA sequence encoding a single protein.
 Genome – The entire set of genes in an organism.
 Allele – one of a number of alternative forms of a gene. Two genes for the same trait that occupy the same position on
homologous chromosomes (like versions of a trait).
 Locus – the specific position of a gene on a chromosome, the two alleles of a gene are found at the same loci on the
chromosome pairs.
 Haploid (n) – haploid cells carry a single set of chromosomes in the nucleus. These include sperm and egg cells.
Produced through meiosis.
 Diploid (2n) – diploid cells carry two sets of chromosomes in the nucleus. Produced through mitosis.
 Phenotype – The physical appearance of an organism (observable characteristics of an organism which are as a result
of genotype and environment )
 Genotype – the genetic makeup of an organism. (The alleles present within cells of an organism, for a particular trait
or characteristic).
 Dominant – The allele of a gene that masks or suppresses the expression of an alternate allele. Indicated by a capital
(uppercase) letter (e.g. T). Only a single allele is required for the characteristic to be expressed, that is, the allele is
always expressed in the phenotype.
 Recessive – An allele that is masked by a dominant allele. Represented by a small (lowercase) letter (e.g. t). The
characteristic is only expressed if there is no dominant allele present.
 Homozygous – Having two identical alleles for a particular characteristic (e.g. TT or tt).
 Heterozygous – Having two different alleles for a particular characteristic (e.g. Tt).
 Homologous chromosomes – a pair of chromosomes, one from the father and one from the mother. Each has the same
genes in the same position, although the alleles of these genes may be different.
 Linkage is the phenomenon where genes for different characteristics, located at different loci on the same chromosome
are linked.
 Monogenic inheritance – when a phenotype or trait is controlled by a single gene. For instance, cystic fibrosis where
the individuals with doubly recessive phenotype are affected.
 Monohybrid cross – a genetic cross involving inheritance of a single pair of genes (one trait).
 Dihybrid cross – a genetic cross involving inheritance of two genes.
 Punnett square – a tool to do genetic crosses and predict the possible genotype and phenotype outcomes and
probabilities.
 Sex linkage – expression of a gene whose locus is on the X chromosome. Expression of the allele depends on the
gender of the individual as the gene is located on a sex chromosome, for instance, males are more likely to inherit an
X-chromosome linked condition because they only have a single copy of the X chromosome. An example of sex
linkage is haemophilia which is a recessive condition (hh).
 Autosomal linkage – genes which are located on the same chromosome (not the sex chromosomes) and tend to be
expressed together in the offspring.
 Codominance – when both alleles are expressed in a heterozygote, that is, both alleles contribute towards the
phenotype. Examples AB blood type. Alleles IA and IB are both dominant (i.e. codominant), so they will both be
expressed to create the phenotype AB.
 Multiple Alleles – More than two alleles for a single gene. Example there are three alleles (multiple alleles) of blood
types namely IA IB and io (or simply, A, B and O).

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MENDELIAN GENETICS / CLASSICAL GENETICS

Briefly describe Gregor Mendel’s key ideas about inheritance?
 Mendel postulated transmissible factors—genes—to explain the inheritance of traits. He discovered that genes exist in
different forms, which we now call alleles.
 Each organism carries two copies of each gene. During reproduction, one of the gene copies is randomly incorporated into
each gamete (Law of segregation). When the male and female gametes unite at fertilization, the gene copy number is
restored to two. Different alleles may coexist in an organism.
 During the production of gametes, they separate from each other without having been altered by coexistence.
 Mendel‟s four laws of inheritance are:
 Principle (law) of unit characters: every genetic character of an organism is controlled by unit factors existing in
pairs.
 Principle (law) of Dominance: some alleles are dominant and others are recessive.
 Mendel referred to traits that showed up in F1 generation as dominant and those that didn‟t show up (were masked)
in the F1 generation as recessive.
 Principle (law) of Segregation: Two alleles for a trait separate during meiosis and each gamete receives only one
allele.
 Principle (law) of Independent Assortment: Genes for two different traits are inherited independently.
MENDEL’S MONOHYBRID CROSS-Mendel's experiments with single trait crosses.
 Monohybrid cross a.k.a. single trait cross– a genetic cross involving inheritance of a single pair of genes (one trait).
 Mendel‟s experiments and observations with pea plants can be analysed using genetic cross diagrams and Punnett
squares.
GENETIC CROSS
 Mendel crossed pure breeding lines (homozygous parents) of peas. In his first experiment Mendel crossed a homozygous
purple flower plant with a homozygous white flower plant to get the F1 generation. He further crossed the F1 offspring
to get the F2 generation. Purple flowers are dominant over white flowers in pea plants. The possible outcomes of F1 and
F2 offspring can be predicted using a genetic cross diagram as illustrated below.

 Conclusion:
1. NB: All / 100% phenotypes express the dominant trait when a homozygous dominant parent and a homozygous
recessive parent are crossed.
2. Principle (law) of Dominance: some alleles are dominant and others are recessive. Purple (P) is dominant over
white (p). White (p) is recessive.
3. Principle (law) of Segregation: Two alleles for a trait separate during meiosis and each gamete receives only one
allele. The alleles are on separate homologous chromosomes.
P and p separated (segregated) during meiosis, and each gamete received only P or p.
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Continued ……
 The F1 generation were self-pollinated. The genetic cross diagram is as follows:


 F2 phenotypic ratio is 3 purple : 1 purple
 [NB. Always 3:1 phenotypic ratio when both parents are heterozygous]
 This 3:1 ratio is called the monohybrid ratio.

Punnett Square
 Named after Reginald C. Punnett, who devised the approach.
 The Punnett square is a square grid used to:
1. predict the genotypes of a particular cross or breeding experiment.
2. determine the probability of an offspring having a particular genotype.
 For the first examples, we will only be testing the complete dominance condition (where one allele completely
dominates over the other).

Rules for predicting monohybrid crosses using a Punnett square:

1. Determine parent genotypes (capital letter for dominant, small letter for recessive).
2. Segregate alleles and place alleles of each parent on top and side of four-squared grid (mom‘s on one side and dad‘s on
the other).
4. Combine parent alleles inside boxes (letters inside boxes show POSSIBLE genotypes of offspring- not ACTUAL).
5. Determine phenotypic ratio and possible genotypes in the following format:
 Fraction, probability statement, phenotype (genotype)
 Ex. ¾ cbetb (Could Be Expected To Be) purple (PP, Pp), ¼ cbetb white (pp).
 MUST BE LIKE THIS EVERY TIME!!!!

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Continued ….
Examples of monohybrid crosses illustrated using PUNNETT SQUARES

1. Mendel crossed pure breeding lines (homozygous parents) of peas. Purple flowers are dominant over white flowers in
pea plants. In his first experiment Mendel crossed a homozygous purple flower plant with a homozygous white flower
plant to get the F1 generation. He further crossed the F1 offspring to get the F2 generation. Use Punnett squares to
predict the possible outcomes of F1 and F2 offspring.

Answer
P = Purple
p= White

P1 phenotypes: Purple flowers X White flowers

P1 genotypes (2n): PP X pp
Meiosis (segregation):
Gametes (n):

F1 genotypes and phenotypes:

 F1 Phenotypes: All (100%) expected to be purple.

 F1 genotypes: All (100%) expected to be Pp (Heterozygotes/Hybrids).
 Conclusion: Principle of Dominance: some alleles are dominant and others are recessive. Purple (P) is dominant
over white (p). White (p) is recessive.

F1 offspring (progeny) were self-pollinated:

Parental (F1) phenotypes: Purple flowers X Purple flowers

Parental (F1) genotypes (2n): Pp X Pp
Meiosis (Segregation):
Gametes (n):

F2 genotypes and phenotypes:

F2 Phenotypes: ¾ expected to be purple. ¼ expected to be white.

(or75% expected to be purple. 25% expected to be white).
 F2 phenotypic ratio: 3 purple: 1 white. [NB. Always 3:1ratio when two heterozygotes are crossed]
 F2 genotypes: ¼ expected to be PP, ½ expected to be Pp and ¼ expected to be pp.
 F2 genotypic ratio 1PP:2Pp:1pp.
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Consistency of Mendel’s results

 Mendel performed monohybrid crosses with seven of pure breeding pea plants and always got consistent results
each time with a ratio of 3 dominant : 1 recessive in the F2 generation (see table below).

Results of Mendel‟s monohybrid crosses for seven characteristics in pea plants

Reasons for Selection of Garden Pea by Mendel:

1. Garden pea is an annual plant and completes the life cycle within three or four months. Due to this short lifespan, he
was able to take three generations in a year.
2. It is a small herbaceous plant that produces many seeds and so he could grow thousands of pea plants in a small plot
behind the church.
3. It is naturally self-pollinating and was available in the form of many varieties with contrasting characters. There
were no intermediate characters.
4. Flowers are large enough for easy emasculation required for artificial cross and produce fertile offspring.

The Reason of Success of Mendel‟s Experiment:

1. Mendel studied the inheritance of one character at a time whereas earlier scientists had considered the organism as a
whole. Initially, Mendel considered the inheritance of one trait only (Monohybrid). Then he studied two traits
together (dihybrid) and then three (Trihybrid).
2. He started with pure line i.e. true breeding. He maintained a complete statistical record by counting an actual
number of offspring.
3. He carried out experiments up to the second and third generations.
4. He conducted ample crosses and reciprocal crosses to eliminate chance.
5. He dealt with a large sample size.

 Mendel‟s findings on pea plants are also applicable to some characteristics in other organisms, including human
beings. However some characteristics deviate from Mendelian / classical genetics to give what we call non-
Mendelian genetics.

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In humans, having dimples is dominant to not having dimples. Use a Punnett square to predict the
genotypic and phenotypic ratios of a cross between a man heterozygous for dimples and a woman
without dimples. [5]

D = dimples
d = no dimples

Parental phenotypes: Dimples X No dimples

Parental genotypes (2n): Dd X dd
Meiosis (segregation):
Gametes (n):

Offspring genotypes and phenotypes:

Offspring Phenotypes: ½ chance of having dimples. ½ chance of having no dimples.

(or 50% chance of having dimples. 50% chance of having no dimples).
Offspring phenotypic ratio: 1 Dimples: 1 No dimples. [NB. Always 1:1ratio when heterozygous x homozygous recessive]
Offspring genotypes: ½ chance of being Dd and ½ chance of being dd.
Offspring genotypic ratio 1 Dd : 1 dd.

In humans the allele for albinism is recessive to the allele for normal skin pigmentation. If two
heterozygotes (carriers) have children,
(a) what is the chance that a child will have normal skin pigment?
(b) what is the chance that a child will be albino?
(c) what is the chance that the child would be a carrier of albinism?
(d) If the child is normal, what is the chance that it is a carrier (heterozygous) for the albino allele? [6]
A = normal skin pigment
a = albino
Parental phenotypes: Normal X Normal
Parental genotypes (2n): Aa X Aa
Meiosis / segregation:

a) Normal skin pigment = ¾ or 75% (for AA and Aa)

b) Albino = ¼ or 25%
c) Carrier = ½ or 50%
d) If child is normal, chance that it is a carrier = or 66.7% (CAREFUL! 3 children are normal and out of these 2 are
carriers).

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WORKING BACKWARDS
 Sometimes we only know about the offspring and we want to learn about the parents. If you have been paying
attention, you should have started to notice a pattern. For example:
 when both parents are heterozygous the phenotypic ratio always comes out 3:1.
 if one parent is heterozygous and the other is homozygous recessive the phenotypic ratio is always 1:1.
 when a homozygous dominant parent and a homozygous recessive parent are crossed, all / 100% phenotypes express
the dominant trait.
 Keeping this in mind see if you can solve the next three problems.
In pea plants, yellow seeds (Y) are dominant and In another cross, a yellow seeded plant was crossed
green seeds (y) are recessive. A pea plant with yellow with another yellow seeded plant and it produced
seeds is crossed with a pea plant with green seeds. offspring of which about 25% were green seeded
The resulting offspring have about equal numbers of plants. What are the genotypes of both parents?
yellow and green seeded plants. What are the Use a Punnett square to prove your answer.
genotypes of the parents? Use a Punnett square to
prove your answer.
Equal numbers means the ratio of the phenotypes is 1:1. Phenotypic ratio of Yellow : Green = 75% : 25% = 3:1
Therefore the parental genotypes are Yy (heterozygous) and Therefore the parental genotypes are both Yy
yy (homozygous recessive). (heterozygous).

In yet another cross, a yellow seeded plant was crossed with a green seeded plant and all / 100% offspring
produced where yellow seeded. What are the genotypes of the parents? Use a Punnett square to prove your
answer.
All / 100% yellow means yellow is dominant over green.
Therefore the genotypes of the parents are YY (homozygous dominant) and yy (homozygous recessive).

Notes:

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The following diagram shows the procedure and results of Mendel’s Monohybrid experiment with flower colour
of the pea plant. Describe stages 1 to 8 and use Punnett squares to show F1 and F2 generation results. Make
reference to the Law of dominance and the Law of segregation in your answer. [12]

Notes:

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TEST CROSS
 DEFINITION: A test cross is the cross of an individual to a homozygous recessive parent; used to
determine if the individual is homozygous dominant or heterozygous
 If the phenotype of a parent in a breeding experiment shows the recessive trait then it is pretty obvious that
their genotype is homozygous recessive as it is the only genotype that will give that phenotype.
 However, when a parent‟s phenotype shows the dominant trait it means their genotype could either be
homozygous dominant or heterozygous (you can‘t tell by looking at it).
 Therefore a test cross is preformed to find out whether the parent is homozygous dominant or
heterozygous.
 How is this done? The parent is crossed with the „tester‟ who is homozygous recessive.
 If all offspring are of one variety then it means the parent is homozygous dominant.
 If we obtain offspring of both varieties (in the ratio 1:1) then it means the parent is heterozygous.
 A test cross involving a single trait is called a monohybrid test cross and gives a phenotypic ratio of 1:1
as explained in the examples below.
 A test cross involving two traits is called a dihybrid test cross and it gives a phenotypic ratio of 1:1:1:1.
We will look at this later in this document under the topic called ‗Dihybrid cross‘.

Examples of monohybrid test crosses

1. A test cross determines whether the dominant character is coming from homozygous dominant genotype or
heterozygous genotype. (e.g., tallness in pea plants coming from TT or Tt).

 When TT (homozygous dominant) is crossed with tt, we obtain all Tt (tall) individuals in the progeny. Whereas when
Tt (heterozygous) is crossed with tt, we obtain Tt (tall) and tt (dwarf) individuals in the progeny in the ratio 1:1.
 Therefore, if tallness is coming from TT (homozygous dominant), then we obtain all tall progenies in a test cross. We
obtain both tall and dwarf varieties in the ratio 1:1 in a test cross, if tallness is coming from Tt (heterozygous).

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Continued ……
2. In Dalmatian dogs, the gene for black spots is dominant to the gene for liver coloured spots. If a breeder has a black
spotted dog, how can she find out whether it is a homozygous dominant (BB) or heterozygous (Bb) spotted dog?
*B = black spots and b = liver spots.

 If the breeder finds a black spotted dog, whose ancestry is not known, she cannot tell by looking at the dog if it is BB
or Bb.
 She should find a liver spotted dog, whose genotype must be bb and mate it with the black spotted dog in question.

 If all puppies / offspring come out as black spotted then it  If both black spotted and liver spotted puppies /
means the parent dog in question is homozygous offspring are obtained (in the ratio 1:1) then the
dominant BB. parent dog in question is heterozygous Bb.
B = black spots, b = liver spots. B = black spots, b = liver spots.
If homozygous dominant: BB x bb If heterozygous: Bb x bb

 All puppies should have black spots.  ½ have black spots. ½ have liver spots.
 Parent dog is homozygous dominant BB / true breed BB /  Parent must be heterozygous Bb.
double dominant BB.  (Not good for breeding if breeder does not want
 (Good for breeding if breeder wants dominant recessive phenotype).
phenotype).

Conclusion: If any of the breed offspring has liver spots, then she can say that she had a heterozygous black spotted
dog. If all the offspring had black spots then she can say that the suspect dog was homozygous dominant.
NB: Test cross always uses recessive to test!

3. You find a wild, black mouse. Explain how you would determine the genotype of this mouse. Hint: in mice white fur is
recessive.
(a) Draw Punnett squares for your possible crosses.
(b) You have 24 offspring, 23 with black fur and 1 with white fur. What was the genotype of the parent mouse? Give a
reason for your answer.
(c) If you only had 3 black offspring, can you tell what the genotype was of the suspect mouse? Explain why or why
not.
Solution
a) Punnett squares for possible crosses.
To determine if black mouse is BB or Bb you would cross it to a bb / white (recessive) mouse.
B = black, b = white. B = black, b = white.
If homozygous dominant: BB x bb If heterozygous: Bb x bb

100% (all) black. Therefore, parent is homozygous dominant, 50% black, 50% white. Therefore parent would be
BB. heterozygous, Bb.
b) Genotype: Bb.
Reason: Both parents had to be heterozygous to have recessive alleles to produce the recessive white (bb).

c) With only 3 offspring, the number is statistically too small to give a valid conclusion about the genotype.
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MENDEL’S DIHYBRID CROSS-Mendel's experiments with double trait crosses.

 A dihybrid cross is a cross between parents differing in two pairs of contrasting characters.
 Mendel studied the inheritance of round and wrinkled characters of seed coat along with the yellow and green colours of
seeds. Round (R) and Yellow (Y) are dominant alleles while wrinkled (r) and green (y) are recessive alleles.
 He found that a cross between round yellow (RRYY) and wrinkled green seeds (rryy) produced only round yellow seeds
in the F1 generation because round (R) and yellow (Y) are dominant alleles.
 When he cross-pollinated (selfed) the F1 offspring to get the F2 generation seeds, four phenotypes were observed. Two of
these phenotypes were similar to the parental combinations (yellow round and green wrinkled), while the other two were
new combinations (yellow wrinkled and green round).
 The following Punnett squares summarize Mendel‘s experiment:

R = Round. r = wrinkled. F1 genotypes and phenotypes:

Y = Yellow. y = green.

Parental phenotypes: Round yellow seeds X Wrinkled green seeds

Parental genotypes (2n): RRYY X rryy
Meiosis (segregation):

F1 genotypes: All RrYy

F1 phenotypes: All heterozygous round yellow
seeds
R = Round. r = wrinkled. Cross-pollination of F1 hybrids to produce F2
Y = Yellow. y = green. Generation:

Parental phenotypes: Round yellow seeds X Round yellow seeds F2 genotypes and phenotypes:
Parental genotypes (2n): RrYy X RrYy
Meiosis (segregation):

F2 genotypes: (As listed in each square).

F2 phenotypes: 9Round yellow:3Round
green:3Wrinkled yellow:1Wrinkled green
Observations of the Dihybrid Cross Experiment:
Expectations: Mendel expected the ratio of yellow and round seeds to green and wrinkled seeds to be 3:1.
Outcome:
 He found seeds of four types, round yellow, wrinkled yellow, round green and wrinkled green in the ratio 9:3:3:1.
 Out of these four types, two were parental combinations. Viz. yellow round and green wrinkled and two were new
combinations like yellow wrinkled and green round.
 In all Mendelian dihybrid crosses the phenotype ratio was always 9:3:3:1. This ratio is called dihybrid ratio.
Conclusion: Law / principle of independent assortment : Genes of two different traits are inherited independently. There
is no connection between them, e.g. seed colour and seed texture were inherited independently.

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SUMMARY OF MENDEL’S DIHYBRID CROSS

Notes:

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What is Mendel’s Law of Independent Assortment?

• Law of independent assortment: Genes for two different traits are inherited independently.
• There is no connection between them (e.g., seed colour and seed texture in pea plants are inherited independently).
Mathematical Explanation of Mendel‟s Law of Independent Assortment:
 The meaning of the word assortment is ‗randomly and freely‘. Thus probability theory is applicable to the dihybrid
cross experiment. By the basic law of probability, ―Probability of two independent events occurring simultaneously is a
product of their individual probabilities‖.
 The probability of the first trait is 3:1 while that of the second trait is also 3:1. Thus the dihybrid ratio should be (3:1) x
(3:1) = 3 x 3 : 3 x 1 : 1 x 3 : 1 x 1 = 9:3:3:1 and Genotypic ratio RRYY: RrYY: RRYy: RrYy: rrYy: rrYy: RRyy: Rryy:
rryy is 1:2:2:4:1:2:1:2:1.
 Mendel performed many dihybrid crosses and reciprocal crosses with different combinations. Every time he got the
same pattern of results.
 The uniform expression was both dominant in F1 generation. In F2 generation always he got both dominant in large
numbers.
In soyabean plants, the green colour allele (G) is dominant over yellow colour allele (g) for seed colour and tall
(T) is the dominant allele in plant height. Parents heterozygous for both traits are cross-pollinated. Determine
the frequency for the four different phenotypes of the offspring. [6]
Parental phenotypes: Green seeds, tall plant X Green seeds, tall plant
Parental genotypes (2n): GgTt X GgTt
Meiosis (segregation):

Genotypes Phenotypes
Green seeds, tall plant, = 9/16.
Yellow seeds, tall plant = 3/16.
Green seeds, short plant = 3/16.
Yellow seeds, short plant = 1/16

Now, let us try a shorter way of solving this same dihybrid cross. Because of Mendel‘s Law of independent Assortment,
we can work with colour gene and height gene separately since these are inherited independently.
So set up two separate monohybrid crosses from those heterozygous parents.

Now use the laws of probability to calculate the frequencies of each trait individually and combined (“Probability of
two independent events occurring simultaneously is a product of their individual probabilities”):

Seed colour Height =P

Green seeds, tall plant ¾ ¾ 9/16
Yellow seeds, tall plant ¼ ¾ 3/16
Green seeds, short plant ¾ ¼ 3/16
Yellow seeds, short plant ¼ ¼ 1/16

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Dihybrid test cross

 DEFINITION: A test cross is the cross of an individual to a homozygous recessive parent; used to determine if the
individual is homozygous dominant or heterozygous.
 If all offspring in a dihybrid cross are of one variety then it means the parent is homozygous dominant.
 If we obtain offspring of four varieties (in the ratio 1:1:1:1) then it means the parent is heterozygous.
 Dihybrid test cross gives a phenotypic ratio of 1:1:1:1.
 Below is an example of a dihybrid test cross involving seed colour and seed shape in peas.

You find a pea plant with yellow, round seeds. Explain how you would determine the genotype of this pea plant in
terms of colour and texture of seeds. Hint: In pea plants green seed colour and wrinkled seeds are recessive.

To determine if yellow, round seed pea plant is YYRR (homozygous) or YyRr (heterozygous) you would cross it to a yyrr
(recessive) plant.
If homozygous dominant, YYRR If heterozygous, YyRr
Y=yellow, y=green, R=round and r=wrinkled. Y=yellow, y=green, R=round and r=wrinkled.

 100% (all) yellow, round seeds means the parent

pea plant is homozygous dominant, YYRR.

 Offspring of four varieties in the ratio 1:1:1:1 means the

parent pea plant is heterozygous, YyRr.

Explain why a test cross is a back cross but a backcross is not necessarily a test cross. [4]
 A backcross is mating between parent and offspring to preserve the parental genotype in a breeding programme.
 A test cross is a way to determine whether an organism that expressed a dominant trait was homozygous or
heterozygous.
 Test cross is a backcross but a backcross is not necessarily a test cross. It is because in a backcross F1 generation can
be crossed with either dominant or recessive parent. But in a test cross, F1 generation is crossed with a recessive parent
only or with a recessive similar to the parent. Thus, test cross is a backcross but backcross is not necessarily a test cross.
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NON-MENEDELIAN GENETICS – DEVIATIONS FROM MENDELIAN GENETICS

 Deviations from Mendelian genetics or violations of Mendel‘s laws of inheritance results in what is known as Non-
Mendelian genetics.
 Examples of non-Mendelian genetics include:
 Incomplete dominance
 Codominance
 Multiple alleles
 Sex linkage / sex-linked traits.

INCOMPLETE DOMINANCE – Blending of parental traits

 In Snapdragon the two alleles for flower colour express themselves equally to give a blend, therefore neither
dominates over the other. We call this condition incomplete dominance and it violates Mendel‘s principle / law of
dominance which states that some alleles are dominant and others are recessive.
 In incomplete dominance, F1 generation has a phenotype that does not resemble either of the two parents, but is a
mixture/blend of the two.
 With incomplete dominance, a cross between organisms with two different phenotypes produces offspring with a
third phenotype that is a blending of the parental traits.

 Flower colour in snapdragon (dog flower), Antirrhinum majus, is an example of Incomplete Dominance and
is like this:

RED Flower x WHITE Flower  PINK Flower

where:
 CRCR − Red flowers
 CWCW − White flowers
 CRCW − Pink flowers (Incomplete dominance – blending of red and white to give pink).

• It's like mixing paints, red + white will make pink. Red doesn't totally block (dominate) white, instead there is
incomplete dominance, and we end up with something in-between (intermediate colour) i.e. pink.
• A capital letter represents the gene and superscripts represent the alleles. Since there is no dominant trait we use two
different superscripts / little letters for the genotype.
• Snapdragon flower/petal colour is controlled by gene C with two alleles, written with superscripts as CR and CW
or sometimes simply as R and W.
 C is the allele for red petals/flowers and C is the allele for white petals.
R W

 When a cross is made between red-flowered snapdragon (C C ) and white-flowered snapdragon (C C ) varieties, F1
R R W W

(CRCW) offspring produced is all pink flowered. When these F1 pink-flowered are self-pollinated or crossed among
themselves to raise F2 generation, they produce red (CRCR), pink (CRCW) and white (CWCW) flowers in the 1:2:1 ratio
respectively. This phenotypic ratio is identical with genotypic ratio because heterozygotes are phenotypically
intermediate between two homozygous types.
— Phenotypic Ratio is 1 Red : 2 Pink : 1 White.
R R R W W W
— Genotypic Ratio is 1 C C : 2 C C : 1C C . (Or simply 1 RR : 2 RW : 1 WW).

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Continued ……
• This is represented in the cross diagrams that follow.
• There are two ways of representing the cross. The diagram on the right uses alleles written in superscript form (use any
one of these in the exam!!):

 Phenotypic Ratio − 1:2:1 that denotes Red: Pink: White

 Genotypic Ratio − 1:2:1 that denotes RR: RW: WW respectively.
OTHER EXAMPLES OF INCOMPLETE DOMINANCE
 Flower/petal colour in Four o’clock plant (Mirabilis jalapa)
In Four o‘clock plant (Mirabilis jalapa) just as in snapdragon or dog flower (Antirrhinum majus), there are three types of
flower colours i.e., red, Pink and white

 When a cross is made between red-flowered Four o‘clock plant (C C ) and white-flowered Four o‘clock plant (C C )
R R W W

varieties, F1 (CRCW) offspring produced is all pink flowered. When these F1 pink-flowered are self-pollinated or
crossed among themselves to raise F2 generation, they produce red (CRCR), pink (CRCW) and white (CWCW) flowers in
the 1:2:1 ratio respectively. This phenotypic ratio is identical with genotypic ratio because heterozygotes are
phenotypically intermediate between two homozygous types.
— Phenotypic Ratio – 1 Red : 2 Pink : 1 White.
— Genotypic Ratio – 1 CRCR : 2 CRCW : 1CWCW. (Or simply 1 RR : 2 RW : 1 WW).
 This is represented in the cross diagrams that follow.

 There are two ways of representing the cross. The diagram on the right uses alleles written in superscript form
(use any one of these in the exam!!):
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Continued….

 Phenotypic Ratio − 1:2:1 that denotes Red: Pink: White

 Genotypic Ratio − 1:2:1 that denotes RR: RW: WW respectively.
 Colour of feathers in Andalusian fowl

The Andalusian fowl is found in three colours: Black, white and blue. Pure forms (homozygotes) are black (BB) and
white (WW). If these two forms are crossed, F1 individuals appear blue (BW) coloured. The blue hybrids on crossing with
each other (BW x BW) give rise to black, blue and white in 1:2:1 ratio respectively.

 TASK: Present this information about colour of feathers in Andalusian chickens using genetic diagrams.

Interpreting incomplete dominance questions

The trick is to recognize when you are dealing with a question involving incomplete dominance. There are two steps to
this:
1) Notice that the offspring is showing a 3rd phenotype. The parents each have one, and the offspring are different from
the parents.
2) Notice that the trait in the offspring is a blend (mixing) of the parental traits.
Now answer the questions that follow.
1. A cross between a blue blahblah bird & a white blahblah bird produces offspring that are silver. The color of blahblah
birds is determined by just two alleles.
a) What are the genotypes of the parent blahblah birds in the original cross?
b) What is/are the genotype(s) of the silver offspring?
c) What would be the phenotypic ratios of offspring produced by two silver blahblah birds?

2. The color of fruit for plant "X" is determined by two alleles. When two plants with orange fruits are crossed the
following phenotypic ratios are present in the offspring: 25% red fruit, 50% orange fruit, 25% yellow fruit. What are
the genotypes of the parent orange-fruited plants?
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Codominance – both of parental traits appear together in offspring (Offspring resembling both
parents)
 In short-horn cattle the two alleles for fur colour are both equally dominant and are expressed together but
independently, therefore neither dominates over the other. We call this condition codominance and it violates
Mendel‘s principle / law of dominance which states that some alleles are dominant and others are recessive.
 With codominance, a cross between organisms with two different phenotypes produces offspring with a third
phenotype in which both of the parental traits appear together.
 Let me point out that the meaning of the prefix "co-" is "together", e.g. Cooperate = work together. Coexist = exist
together. Cohabitate = habitat together.
 That is, with codominance, a cross between two different homozygotes produces heterozygotes in which both
parental traits are expressed together.
 DEFINITION: Codominance is whereby two alleles fail to dominate each other in heterozygosis and their effects
are expressed together in the offspring.
 In codominance, the F1 progeny resembles both the parents.

Roan fur in short-horn cattle is an example of codominance and is like this:

red x white ---> red & white spotted

i.e. short-horn cattle fur can be red (RR = all red hairs), white (WW = all white hairs), or roan (RW = red & white hairs
together).

Red bull crossed with a white cow produces all roan F1 The roan F1 offspring crossed produces red, roan and white
offspring. offspring in the ration 1 red: 2 roan: 1 white.

Conclusion : Probability (chance) of getting:

Red phenotype = = ¼ = 0.25 or 25%

Roan phenotype = = = ½ = 0.5 or 50%

White phenotype = = ¼ = 0.25 or 25%

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Other examples of codominance

Sickle cell haemoglobin
 Another example of codominance is sickle cell haemoglobin in humans. The gene for haemoglobin Hb has two co-
dominant alleles: HbA (the normal gene) and HbS (the mutated gene). There are three genotypes and phenotypes:
 HbAHbA Normal haemoglobin. All haemoglobin is normal, with normal red blood cells.
 HbAHbS Sickle cell trait. 50% of the haemoglobin in every red blood cell is normal, and 50% is abnormal. The
red blood cells are slightly distorted, but can carry oxygen, so this condition is viable. However these red blood
cells cannot support the malaria parasite, so this phenotype confers immunity to malaria.
 HbSHbS Sickle cell anaemia. All haemoglobin is abnormal, and molecules stick together to form chains,
distorting the red blood cells into sickle shapes. These sickle red blood cells are destroyed by the spleen, so this
phenotype is fatal.
 The genetic diagram that follows shows the inheritance of sickle cell haemoglobin – an example of codominance.

Wavy hair in humans

 Hair type is another example of codominance in humans.
 The gene for hair type H has two co-dominant alleles: HS (allele for straight hair) and HC (allele for curly hair).
 There are three genotypes and phenotypes:
 HSHS or simply SS for straight hair.
 HCHC or simply CC for curly hair.
 HSHC or simply SC for wavy hair (codominance).
 The genetic diagram that follows shows the inheritance of wavy hair – an example of codominance.

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Now try the following question:

In humans straight hair and curly hair are codominant traits that result in hybrids who have wavy hair. Cross a curly
haired female with a wavy haired male.
a) Complete a punnett square for this cross.
b) What are the chances of having a curly haired child?
AB blood type
AB blood type is another example of codominance in humans. A and B alleles are codominant. Read the details of blood
types under multiple alleles below.
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MULTIPLE ALLELES
 Multiple alleles occur when more than two alleles control a character, as in human blood groups and coat colour
in rabbits.
 So far we have studied traits or genes that are coded for by just two alleles. Like in pea plants, there was one allele for
yellow seed colour and one for green seed colour. However, some traits are coded for by more than two alleles. One of
these is blood type in humans. This is a violation of / deviation from Mendel‟s Principle of unit characters which
states that every genetic character of an organism is controlled by unit factors existing in pairs.

HUMAN BLOOD GROUPS are controlled by multiple alleles

 In humans, there are four types of blood, type A, type B, type AB, and type O.
 ABO blood groups are controlled by gene I. Gene I has three alleles, I , I and i . A person possesses any two of the
A B O

three alleles.
 The i allele is sometimes simply written as i.
O

 I and I dominate over i . But with each other, I and I are co-dominant.
A B O A B

 The ABO blood types are shown in the following table.

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You are blood type O and you marry a person with blood type AB.
(a) Complete a Punnett square for this cross.
(b) List the possible blood types (phenotypes) of your offspring.
(a) Blood type O x Blood type AB.

(b) Possible blood types / phenotypes of your offspring are A or B.

Notes:

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A young woman called Natasha sued a man named Billy for parental support of her illegitimate child.
Billy’s blood type was already on record as type AB. The mother of the child had type A and her son
had type O blood.
(a). Complete Punnett squares for the possible crosses between Billy and Natasha.
(b). The judge ruled in favor of Natasha and ordered Billy to pay child support costs of the child.
Was the judge correct in his decision based on blood typing evidence? Explain why or why not.
Refer to the Punnett squares to support your answer.
(a) Billy = IAIB
Natasha = IAIA or IAiO
IAIB x IAIA IAIB x IAiO

½ = 50% Blood type A ½ = 50% Blood type A

½ = 50% Blood type AB ¼ = 25% Blood type AB
¼ = 25% Blood type B
(b) No. The judge was not correct.
In order to produce a child of blood type O each parent has to be heterozygous for blood type A or B (i.e. each
parent should be either be IAiO or IBiO) – absolutely Billy was not heterozygous because he has blood type AB.

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COAT COLOUR IN RABBITS is controlled by multiple alleles
 How is coat color in rabbits inherited?
 Coat color in rabbits is inherited as a series of multiple alleles. This means that there can be more than just 2 alleles for a
single gene.
 In the case of coat color in rabbits, there are four alleles, and each one is expressed with a different phenotype (see table
below).
 Analysis:
 Examine the table below. Use this information to answer the questions. Remember, each rabbit can only have 2
alleles for coat color.

1. List all possible genotypes for a

ch h
a. dark gray coated rabbit. CC, Cc , Cc and Cc.
ch ch ch h ch
b. chinchilla rabbit. c c , c c , and c c.
h h h
c. Himilayan rabbit. c c , c c.
d. white rabbit. cc.
ch h
2. Predict the phenotype for a rabbit with a c c genotype. Explain.
Phenotype: Chinchilla.
ch h
Explanation: The Chinchilla allele (c ) is dominant over the Himalayan allele (c ).

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h
3. Predict the phenotype for a rabbit with a Cc genotype. Explain.
Phenotype: Dark gray coat.
h
Explanation: The dark gray coat allele (C) is dominant over the Himalayan allele (c ).

4. Would it be possible to obtain white rabbits if one parent is white and the other is chinchilla? Explain.
Yes it‘s possible, for the cross between homozygous white (cc) and chinchilla (cchc). The white alleles (c) in both
genotypes will segregate and combine to form a homozygous white genotype.

White x chinchilla
cc x cchc

cch c
c cchc cc
c cchc cc
Phenotype: 2 chinchilla, 2 white
Phenotypic ratio: 1 chinchilla : 1 white.

5. Would it be possible to obtain chinchilla rabbits if one parent is Himalayan and the other is white? Explain.
No, it‘s not possible because both Himalayan and white are both recessive to chinchilla.

6. A chinchilla rabbit is mated with a Himalayan. Some offspring are white. What are the parents‘ genotypes?
cchc (chinchilla) and chc (white).

Table summarizing coat colour inheritance in rabbits

Now, try this question:

Suppose you cross a Chinchilla rabbit (cchch) with a dark gray rabbit (Cch). What are the possible
offspring? Use a Punnet square to explain your answer. [6]

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SEX-LINKED TRAITS
 It is well known that boys are different from girls. In humans Some Examples of sex-linked / X-linked traits
sex is determined by the twenty third pair of chromosomes • hemophilia
known as ‗sex chromosomes‘. • white eye colour in Drosophila (fruit fly)
 If you have two X-shaped (XX) chromosomes you are destined • yellow body colour in Drosophila
to be a female. If you have an X-shaped and a Y-shaped (XY) • non-functional sweat glands
chromosome you are destined to be a male. • absence of central incisors
 Since the X and Y carry different information, any genes found • some types of deafness
on the X chromosome are referred to as sex-linked genes. • white forelocks
Therefore women will have two alleles for these genes because • juvenile glaucoma
they have two (XX) chromosomes. On the other hand, men • glucose-6-phosphate dehydrogenase (G6PD)
have only one allele for each of these genes because they have deficiency
only one X chromosome (XY). This is clearly a violation of • juvenile muscular dystrophy
Mendel‘s Principle of unit characteristics, which implies that • retinitis pigmentosa
you receive one set of alleles from each parent. • coat colour in cats.

1. White eye colour in Drosophila (fruit fly).

In fruit flies, the gene for eye colour is carried on the X chromosome which is a sex chromosome (sex-linked). The
allele for red eyes (R) is dominant over the allele for white eyes (r). If a white-eyed female fruit fly is mated with a red-
eyed male, predict the possible offspring.
 For sex-linked traits we need to list the genotypes in a different fashion. We must identify the individual as
being male or female according to their sex chromosomes. Females are XX and males are XY.
 Sex-linked traits are only found on the X chromosome, therefore the letters are placed as superscripts on
(above) the X chromosome:
r r
 Since the female has white eyes, her genotype is X X .
 The male is red-eyed and because he has only one X chromosome, he has only one allele for eye colour, so he
R
his genotype is X Y.
 The genetic diagrams for the cross are as follows:

Full genetic cross: Punnett square:

Parental phenotypes: White-eyed female x Red-eyed male

r r R
Parental genotypes: XX x X Y.

r r R
Meiosis / segregation: X X X Y

Fertilisation:

R r R r r r
Offspring genotypes: X X X X XY XY

R r r R r r
Genotypic ratio: 1X X :1X Y Genotypic ratio: 1X X :1X Y

Offspring phenotypes: 50% of flies are red-eyed.

50% of flies are white-eyed.
100% of females are red-eyed.
100% of males are white-eyed.

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 Thomas Hunt Morgan‟s was the first to clearly recognize sex-linkage in his Drosophila (fruit fly) breeding
experiments:
 When a white-eyed male fruit fly is mated with its red-eyed sister, all the F1 flies will have red eyes. Thus white-
eyes are recessive to red-eyes.
 When the F1 flies are allowed to interbreed, red-eyed and white-eyed flies appear in a 3:1 ratio in the F2
generation but all the white-eyed flies were male.
 The alleles controlling eye colour are situated on the differential (unpaired) segment of the X-chromosome:
 The cross is as follows:

Notes

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In fruit flies, Drosophila, the gene for long wing length and for eye colour are sex-linked. Normal wing and red eye are
dominant to miniature wing and white eye.
(i) Define the term sex-linked.
When gene is on part of the X chromosome and it is passed on to the next generation. [1]
(ii) A cross between a miniature wing, red-eyed male and homozygous normal wing, white-eyed female was carried
out.
Using symbols N for normal wing, R for red eye, r for white eye and n for miniature wing, draw a genetic diagram to
show the genotypes and phenotypes of the F1 generation in the space below. [5]

Ref. #5ScA. 9190/2 J2015

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2. Hemophilia
Hemophilia is a sex-linked trait. A person with hemophilia is lacking certain proteins that are necessary for normal
blood clotting. Hemophilia is caused by a recessive allele so use N for normal and n for hemophilia. Since hemophilia
is sex-linked, remember a woman will have two alleles (NN, Nn or nn) but a man will have only one allele (N or n).

A woman who is heterozygous (a carrier) for hemophilia marries a normal man.

(a) What are the genotypes of the parents?
N n N
Female: X X . Male: X Y.
(b) Make a Punnett square for the above cross. (c) What is the probability that a male offspring will
N = normal, n = hemophilia have hemophilia?
50%
N n (d) What is the probability of having a hemophiliac
X X
female offspring?
N N N N n
X X X X X
0 (zero).
N n
X Y X Y
Y

¼ of offspring has hemophilia = 25% has hemophilia.

½ males are normal = 50% males are normal
½ males have hemophilia = 50% males have hemophilia
100% females are normal.
½ females are carriers = 50% females are carriers.

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3. Red-green colour-blindness.
 Red-green colour blindness is caused by a sex-linked recessive allele.
 The possible genotypes and phenotypes of red-green colour blindness are;
 XN XN - female with normal vision (homozygous dominant).
 XN Xn - female with normal vision (heterozygous carrier of colour-blindness).
 Xn Xn - female with red-green colour-blindness (homozygous recessive).
 XN Y - male with normal vision.
 Xn Y - male with red-green colour-blindness.
Can a color blind female have a son that has normal vision?
N = normal, n = colour blind n
n n N No. All males will receive the X allele from mom only.
X X x X Y

n n
X X
N N n N n
X X X X X

Y n n
X Y X Y

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X-linked inheritance in birds
 In birds (and also in butterflies and moths) the females are the heterogametic sex (XY) and the males are the
homogametic sex (XX). The same principles apply as in the examples of X-linkage above but remember that the pattern
of inheritance will be reversed in the sexes, so that the females will tend to show the linked character most frequently
and the males will be the carriers.
 Examples of X-linked inheritance characters in birds are:
• certain plumage colours in budgerigars (albino, cinnamon, lutino, opaline).
• red and white plumage colours in some breeds of poultry.
• barred plumage in chickens.
 To distinguish the sex chromosomes of birds from the X –Y system the symbols Z and W are often used to identify the
sex chromosomes. ZZ is male and ZW is female.

Y-linked inheritance
It is incorrect to say that the Y-chromosome is ‗genetically empty‘ although it is true to say that the Y-chromosome
contains few alleles. There are very few known examples of Y-linked conditions due to alleles carried on the
holandric/non-homologous section of the Y-chromosome. Examples that are suspected to be Y-linked are:
• porcupine skin
• webbed toes
• hairy external ears
• some forms of male sexual dysfunction
These conditions can only occur in men since women do not possess Y chromosomes.
They could only be passed from father to son.
Notes

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QUESTIONS ON SEX-LINKED INHERITANCE IN BIRDS, MOTHS AND BUTTERFLIES

One variety of domestic chickens has either black feathers or barred feathers. Barred feathers are black with white bars.
The feather colour is controlled by a gene on the X chromosome. The dominant allele B, results in barred feathers while
the recessive allele, b, results in feathers that are black. Females are heterogametic while males are homogametic.
A breeder crossed a black female with a barred male. Fourteen offspring were produced:
3 barred males
4 barred females
4 barred males
3 black females
Using a genetic diagram, show this cross.

½ or 50% offspring have barred feathers

½ or 50% offspring have black feathers
½ or 50% males are black.
½ or 50% males are barred.
½ or 50% females are black.
½ or 50% females are barred.

In pigeons, the colour gene is found on the sex chromosome Z and the allele for blue is dominant to
red. Females are heterogametic while males are homogametic.You have a prize-winning red coloured
female bird. Describe both the phenotype and genotype (using correct notation) of the males required
such that, if you were to breed him with your red female bird, you would get:
a) Offspring that are all blue in color.
b) Offspring where 50% are blue in color and 50% are red in color.
c) Offspring that are all red in color.
In birds, ZW = female, ZZ = male. Let B = blue allele, b = red allele. Therefore, the genotype of the prize-
winning red bird = ZbW
(a) Cross with a homozygous dominant / blue male bird:

Red coloured female x Blue male

ZbW x ZBZB

Zb W
ZB Z Z ZB W
B b

ZB ZB Zb ZB W
All F1 offspring will exhibit the blue phenotype.

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Continued……
(b) Cross with a heterozygous / blue male bird.

Red coloured female x Blue male

ZbW x ZBZb

Zb W
ZB Z Z ZB W
B b

Zb Zb Zb Zb W
50% of the offspring are blue and 50% are red in colour.

(c) Cross with a homozygous recessive / red male

Red coloured female x Red male

ZbW x ZbZb

Zb W
b
Z Zb Zb Zb W
Zb Zb Zb Zb W
All F1 offspring will exhibit red phenotype.

Notes

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THE CHI-SQUARED (χ2) TEST

 By the end of this section you should be able to: use the chi-squared test to test the significance of differences
between observed and expected results.

 One of the most important statistical tests you can carry out in genetics is the chi-squared (χ2) test (also known as
Pearson's chi-squared test because it was developed by Karl Pearson).

 Why would you need to carry out a χ2-test?

 The χ2 test is used to find out whether any differences between expected results and observed results of a genetic
cross are due to chance, or whether the difference is significant (significant, here means having a low probability of
occurring by chance).
 Let's go all the way back to our yellow/green, round/wrinkled peas:
 From our theoretical crosses, we figured out that when you cross two heterozygous parents (RrYy), you would
EXPECT to see a phenotypic ratio of 9 round yellow peas : 3 round, green peas : 3 wrinkled yellow peas : 1
wrinkled green pea in the offspring.
 But what if we actually did this cross for real? Would the numbers we were EXPECTING to see be the same as
the numbers we'd actually OBSERVE in real life? And how would you PROVE that what you did see wasn't
down to random chance? That's where your χ2–test comes in!
 So chi-squared (2) test is a statistical test which allows us to compare our observed (actual) results with what
we would expect if the phenotypic ratio held true (e.g. Mendelian dihybrid 9:3:3:1 ratio or some other
phenotypic ratio such as the 1:1:1:1 testcross ratio etc).
 It allows us to objectively say whether our observed results are as we expected.
 Chi-squared is a measure of the combined error of our results from the expected.
 It will always be conducted from a dihybrid cross at A-level.
 Let's say we did our cross, counted the numbers of peas and we actually got:
Yellow Round Peas Green Round Peas Yellow Wrinkled Peas Green Wrinkled Peas

219 81 69 31

 Is that a 9:3:3:1 ratio? Possibly, but let's prove it.

 Because this is a test, you have to have a hypothesis that can be tested and proved either right or wrong: for the
χ2–test, this is known as the null hypothesis (H0).
 The null hypothesis basically states that there is no difference between the results you observed and the ones you
were expecting, in this case a 9:3:3:1 ratio.

So first, we must write up a Null Hypothesis (H0).

 A null hypothesis always states that
„There is NO significant difference between the observed and expected result. Any variation is totally due to
chance events‟

 But how do we do that mathematically?

Here's how: below is the formula for Chi-squared

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 Little bit scary at first sight, isn't it? Let's break it down into its component parts so it doesn't look so bad:
 First, we need to calculate the number of each phenotype we would expect to see if we did get a perfect
9:3:3:1 ratio.
 You do this by finding the total number of your actual peas and multiplying it by the expected ratio.

Total number of actual peas = 219 + 81+ 69 + 31 = 400

Total ratio = 9 + 3 + 3 + 1 = 16

 9/16 Yellow round = 9/16 x 400 = 225

 3/16 Green round = 3/16 x 400 = 75
 3/16 Yellow wrinkled = 3/16 x 400 =75
 1/16 Green wrinkled = 1/16 x 400 = 25
Now we complete the following table:

 Next, we need to work out the difference between the Observed and Expected (O – E), and then (to get rid of any
inconvenient negative numbers), square that result.

 Now we can calculate the rest of the equation, by dividing the (O – E)2 result by the Expected number (E) for that
phenotype and adding them all together:

 So for our example, the calculated chi-squared (χ2) value = 2.56.

 But it's just a number: how does it prove or disprove our null hypothesis, that there is no difference between the
observed and expected results?

 Now we need to bring in another factor, something known as the degrees of freedom (df). These take into account
the number of different classes represented by the cross, in this case = 4 different phenotypes. The more classes you
have, the greater the variation you are likely to see from any expected numbers.
 Calculating the degrees of freedom (df) is straight forward.
 It is the number of actual different phenotypic groups for the cross (n) minus one. The formula looks like this;
df = n – 1
 Therefore in this case df = 4 – 1 = 3

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 Nearly there! Now we have our calculated χ2 value (2.56) and our degrees of freedom (3), we then go and take a
look at a χ2 probability table (there's an example of one here (see below)).
 We want to know if our result is statistically significant, and for most genetics tests, a significance (or confidence)
level of 0.05 is used (which means that 95 times out of 100, you would see exactly what you were EXPECTING to
see, with variation occurring 5 times out of 100 purely due to random chance).
 What is the critical value? Critical value of χ2, where p=0.05, is the level at which we are 95% certain that the result
is not due to chance.
 If you look at the example χ2 probability / statistical table for a critical value at 0.05 (i.e. 5%) significance level
(along the top) with 3 degrees of freedom (down the left hand side), the χ2 value = 7.81.
 Step by step, how do we do it?
 Select the correct row of critical values to compare your chi-squared value against.
 Each row indicates the level of degrees of freedom. In this case it is row number 3 as we have 3 degrees of
freedom.
 Then we move along the critical values and check that our chi-squared value is higher or lower than the
critical value at 0.05 (5%) significance level.
 If the calculated chi-squared value is higher than the critical value at the 0.05 significance level then we reject the
null hypothesis,
 Stating that
„Observed results are significantly different from expected results‟
 If the calculated chi-squared value is lower than the critical value at the 0.05 significance level then we accept the
null hypothesis,
 Stating that
„Observed results are not significantly different from expected results. Any variation is due to chance‟.
 Back to our example:
 Because our calculated value (of 2.56) is lower (less) than the critical value (of 7.81) we can accept our null
hypothesis and say with 95% confidence that there is no significant difference between the expected and
observed results!
If our χ2 value had been greater than 7.81, then we would have to accept that there was a statistically significant difference
between the numbers we observed and the numbers we were expecting, and that yellow/green and round/wrinkled were
not behaving in the way we had predicted.

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χ2 probability / statistical table

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Summary
Basically there are 5 stages followed when solving chi-squared statistical problems in genetics. The stages are:
1. Formulate the null (H0) hypothesis.
2. Construct the table.
3. Statistical parameters.
4. Interpreting the result.
5. Conclusion.

Study and master the worked examples that follow.

χ2 probability / statistics questions in genetics
1) A true heterozygous pea plant for round yellow seeds was crossed with another true heterozygous pea plant for round
yellow seeds.
R – allele for round seeds
r – allele for wrinkled seeds
Y – allele for yellow seeds
y – allele for green seeds

RrYy x RrYy
Round yellow Round yellow

The observed F1 results of pea plants were as follows.

 9/16 Yellow round = 169
 3/16 Green round = 54
 3/16 Yellow wrinkled = 51
 1/16 Green wrinkled = 14

Use the chi-squared (2) test to test the significance of the difference between observed and expected results.

Solution 1
1. Formulate the null (H0) hypothesis:
There is NO significant difference between the observed result and expected result. Any variation is totally due to
chance.

2. Construct the table:

The total number of pea plants = 169 + 54 + 51 + 14 = 288
The expected number (E) in each case:
9/16 Yellow round = 9/16 x 288 = 162
3/16 Green round = 3/16 x 288 = 54
3/16 Yellow wrinkled = 3/16 x 288 = 54
1/16 Green wrinkled = 1/16 x 288 = 18

Enter the Expected numbers in the table

The difference between the observed (O) and expected (E) values is calculated (O-E) and squared [(O-E)2]. This value
is divided by the expected number (E) and the sum of these values gives the value χ2.
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Therefore the Chi-squared (x2) value is 1.358

3. Statistical parameters:

Number of classes, n = 4
Degrees of freedom, df = n – 1
= 4-1
=3
The level of significance or probability level (p) is 0.05, corresponding with 95% confidence limits.
Using chi-squared tables, for df = 3 and p = 0.05, the critical value for χ2 = 7.81.

4. Interpreting the result:

The calculated value of 1.358 is lower (less) than 7.81. Therefore the null hypothesis is accepted.
The observed results are not significantly different from expected results. Any variation is due to chance.

2) Dianthus (Campion) has flowers of three different colours, red, pink and white. Two pink flowered plants were
crossed and the collected seeds grown to the flowering stage. The observed results of the cross where as follows: 34
red flower plants, 84 pink flower plants and 42 white flower plants. The cross is shown below. Use the chi-squared
(2) test to test the significance of the difference between observed and expected results.

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1. Formulate the null (H0) hypothesis:

There is NO significant difference between the observed result and expected result. Any variation is totally due to
chance.

2. Construct the table:

Total number of observed plants = 34 + 84 + 42 = 160

The expected number in each case:

0.25 x 160 = 40
0.5 x 160 = 80
0.25 x 160 = 40

3. Statistical parameters:
There are 3 classes (red, pink and white)
Degrees of freedom, df = 3 – 1 =2

4. Interpreting the result:

The nearest figures to 1.2 comes after 0.25 (or 25%). A 2 value of 1.2 shows that it is at least 25% probable that the
result is by chance alone.

From the χ2 probability table the critical value is 5.99 at 0.05 (or 5%) significance level.
The calculated value of 1.2 is lower (less) than 7.81. Therefore the null hypothesis is accepted.
The observed results are not significantly different from expected results. Any variation is due to chance.

3) In maize, Zea mays, a single F2 cob is covered in very many kernels, each of which contains a seed which is the result
of a single fertilisation.
The kernels show a number of characteristics, for example texture and colour, as is shown in the picture below.

The phenotypes shown by the cob were identified and the kernels having each phenotype were counted. The results were
entered in the table that follows.

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a) Identify the theoretical Mendelian ratio that is closest to the counts that you have made.
b) Use the χ2 test and probability table to test if the observed ratio is statistically equivalent to the theoretical Mendelian
ratio.
c) Deduce the genotype of the parent plants.

Solution

Null hypothesis:
There is NO significant difference between the observed and expected (theoretical) Mendelian phenotypic ratio which is 9
purple and smooth: 3 purple and wrinkled: 3 yellow and smooth: 1 yellow and wrinkled. Any variation is totally due to
chance.
Table:
Total number of observed plants = 112 +37 + 38 +13 = 200
Total ratio 9+3+3+1 =16
The expected number in each case:
Purple and smooth = 9/16 x 200 = 112.5
Purple and wrinkled = 3/16 x 200 =37.5
Yellow and smooth = 3/16 x 200 =37.5
Yellow and wrinkled = 1/16 x 200 = 12.5

Calculated χ2 value = 0.0356

Statistical parameters:
Since there are 4 classes, n=4
df = n – 1
=4–1
=3
The level of significance or probability level (p) is 0.05, corresponding with 95% confidence limits.
Using chi-squared tables, for df = 3 and p = 0.05, the critical value for χ2 = 7.81.

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Interpreting the result:

The calculated χ2 value (0.0356) is less than the critical value (7.81).
Therefore, the null hypothesis is accepted at the 0.05 level of significance
Therefore the ratio is 9:3:3:1 and any deviation from this theoretical Mendelian ratio is due to chance.
Conclusion:
The phenotype of the sample is determined by two alleles each of two unlinked genes, where ―Purple (P)‖ is dominant
over ―yellow (p)‖ and ―Smooth (S)‖ is dominant over ―wrinkled (s)‖.
The sample is an F2 generation, produced by self-crossing heterozygous F1 maize plants (PpSs X PpSs). Inheritance is
Mendelian.

Notes:

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TOPIC 5 GENE TECHNOLOGY a.k.a. Recombinant DNA technology

Define the phrase gene technology / recombinant DNA technology. [2]
 Gene technology, which is also called genetic modification (GM) or recombinant DNA technology, is the transfer of
genes from one species to another or within the same species in order to produce new varieties of organisms with
useful or desirable characteristics.
 Genes from one organism are inserted into another. This may be done within the same species (e.g. in gene therapy) or
genes may be transferred from one species to another.
 Gene technology covers techniques such as genetic engineering, DNA fingerprinting, the creation of genomic libraries
of DNA and gene therapy.
What is Genetic Engineering?
— changing the genetic make-up of an organism's DNA by adding or removing a gene.
— the new DNA formed is called Recombinant DNA.
- The resultant organism is called a Genetically Modified Organism (GMO) or a transgenic organism.
- A Genetically Modified organism is called a transgenic organism; since genes are transferred from one organism to
another.
- Example transgenic bacteria that produce human insulin.
- DNA sequencing - the determination of the precise sequence of nucleotides in a sample of DNA or even a whole
genome e.g. the Human Genome Project.

GENE SPLICING
- Gene splicing is a genetic engineering technique which involves cutting DNA out of one organism and inserting it
into another organism.
- A gene is transferred from one organism (the donor) to another (the recipient).
- A trait will be transferred from one organism to another.
- For example: the human insulin gene can be removed from a human cell. It can be put into a bacterial cell. The
bacterial cell will now make human insulin.
- This is called transformation: when a gene from one organism is transferred to different organism.
- The organisms that have DNA transferred to them are called transgenic organisms.
 trans: means different,
 genic: refers to genes.

Why do we Genetically Engineer Animals?

 to give them additional characteristics.

 so they can make useful products (proteins).

Examples of genetic engineering in animals?

 additional characteristics
 add gene for disease resistance.
 add gene for growth hormone for growth.
 making useful products
 use to produce anti-thrombin = protein used to make blood clot (people with certain genetic disease may not
produce), use milk producing animal to produce, add gene for anti-thrombin next to milk producing gene in animal,
therefore anti-thrombin protein will be made in the milk (easily extracted).

Why do we Genetically Engineer Plants?

 to give them additional characteristics.
 so they can make useful products (proteins).

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Examples of genetic engineering in plants?

 additional characteristics
 add gene for disease resistance.
 add gene for pest resistance.
 add gene for pesticide resistance.
 add gene to promote growth for high yield.
 produce genetically modified tomatoes = prevented from softening therefore remain hardened (easy for storage and
transport), involves preventing formation of softening enzyme, a gene is added that is complementary to the
softening enzyme gene, so its mRNA will bind to the mRNA of the softening enzyme preventing translation of the
softening enzyme.
 making useful products
 used to make golden rice (rice that contains beta-carotene, a pre-cursor to vitamin A to treat vitamin A deficiency)
 used to make protein raw material for polymers.

Recombinant DNA technology requires 3 key molecular tools to cut DNA (gene) from one organism and paste it
into another. These are:
 restriction enzyme (aka restriction endonuclease).
 DNA ligase.
 Vector, such as a plasmid.

— Functions of the molecular tools:

1. Restriction enzyme (restriction endonucleases):

— Cuts DNA at a specific site – called a restriction site or target sequence leaving either staggered cuts called
“sticky ends” or straight cuts called “blunt ends”.
— The sticky ends can anneal to each other via hydrogen bonding between complementary bases on the single-stranded
overhangs.
— The source of restriction enzymes is bacteria in which they provide defense by cutting invading viruses called
bacteriophages.
— Examples of restriction enzymes are:
 EcoRI isolated from E. coli bacteria
 BamHI is isolated from Bacillus amyloliquefaciens
 Sau3A is isolated from Staphylococcus aureas.

2. DNA ligase

— Ligates/joins DNA fragments with the same compatible ―sticky ends‖.

— How? By creating covalent phosphodiester bonds between any two DNA fragments that have been cut by the same
restriction enzyme, or have the same compatible ―sticky ends‖.
— One of the DNA fragment comes from the donor organism and the other from bacteria plasmid (host organism‘s
plasmid).

3. A “vector”, such as a plasmid,

— A vector is used to transport the donor organism DNA into the host bacterial cell.
— is used to insert a new segment of DNA via restriction enzyme cutting and ligation.
— The plasmid containing the inserted DNA segment will replicate in host cells.

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The diagram that follows explains the roles of restriction enzymes and DNA ligase in cutting and pasting DNA.

Notes

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Why do we Genetically Engineer Bacteria?

— so they can make useful products (proteins)

Genetically engineering bacteria

 to make useful proteins e.g. Insulin
 normally used animal sources (problems = limited supply, infection risk, immunorejection)
 Insulin is a small protein. It is a hormone secreted by β cells in the islets of Langerhans in the pancreas in
response to raised blood glucose concentration.
 In type I diabetes, no or insufficient insulin is secreted, and the person has to inject insulin. This used to be
obtained from animals such as pigs and cows. Today, almost all insulin used in this way is obtained from
genetically modified bacteria.

GENE TECHNOLOGY FOR HUMAN INSULIN PRODUCTION

 involves adding human insulin gene to a plasmid, then inserting this into a bacteria = the bacteria now has the
gene/code to produce the human insulin protein

Involves 6 steps =

1. Isolation, 2. Insertion, 3. Transformation, 4. Identification, 5. Growth/Cloning, 6. Purification

1. Isolation

 either by Reverse Transcriptase (RT) or Restriction Enzyme (RE) or Gene Machine (GM).
 RT = enzyme found in virus, converts RNA into DNA, obtain mRNA for insulin, the RT will convert it into cDNA
(single stranded complementary DNA), DNA Nucleotides and DNA Polymerase added to make it double stranded
 RE = enzyme found in bacteria, cuts DNA at certain base sequences (called recognition sites) by breaking bond
between sugar and phosphate, can cut straight or staggered, staggered used in Genetic Engineering as it leaves
exposed bases called 'sticky ends' [cuts staggered at 6 base pair palindromes, were the 6 bases read forward are
identical to 6 bases read backward on both strands].
 GM = build DNA base sequence from known Amino Acid Sequence of the Protein (uses oligosaccharides) .

End result = Isolated Human Insulin Gene

2. Insertion

 cut plasmid using the same RE from isolation stage

 leaves complementary sticky ends
 join human insulin gene with plasmid via the sticky ends
 use DNA Ligase to join the sugar-phosphate backbone

= Recombinant plasmid (carrying human insulin gene)

3. Transformation

 mix recombinant plasmid with bacteria

 add Ca2+ ions and heat shock / pulses of electricity.
 bacteria will become permeable and take up the recombinant plasmid.
 Genes conferring resistance to an antibiotic are also introduced into the plasmids, next to the insulin gene.
 Not all of the plasmids take up the gene. Some just close back up again without it.
= Genetically Modified Bacteria (carrying recombinant plasmid with human insulin gene).

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4. Identification

Identify which of the bacteria have taken up the recombinant plasmid and of these which ones have accepted the new gene
(human insulin gene).
— How? Antibiotics are then added to the culture of E. coli bacteria. The only bacteria that survive are the ones that had
successfully taken up the plasmids containing the antibiotic resistance gene. Most of these plasmids would also
contain the insulin gene. Most of the surviving E. coli bacteria are therefore ones that now contain the human insulin
gene.
— End result = Genetically Modified Bacteria
Markers
The antibiotic resistance genes added to the plasmids along with the human insulin gene act as markers. They make it
possible to identify the bacteria that have taken up the gene. There is a concern that using antibiotic resistance genes as
markers could increase the likelihood of the development of populations of harmful bacteria that are resistant to
antibiotics. Today, most common markers used are genes that code for the production of fluorescent green protein. The
gene for this protein can be inserted along with the desired gene. Cells that fluoresce green are therefore likely to have
taken up the desired gene.

Promoters
In bacteria, each gene is associated with a region of DNA called a promoter. The enzyme RNA polymerase must bind to
the promoter before it can begin transcribing DNA to produce mRNA.
It is therefore important to ensure that there is a promoter associated with the human insulin gene when it is inserted into
E. coli.

5. Growth/Cloning

 grow genetically modified bacteria (carrying human insulin gene) in fermenters.

 Nutrients and oxygen provided in the fermenters allow the bacteria to reproduce very fast forming large populations.
 The bacteria will produce/synthesise the human insulin.

6. Purification
— The insulin is then purified and packaged into bottles for sale/distribution to patients with diabetes.

The advantages of treating diabetics with human insulin produced by gene technology
Until bacteria such as E. coli were used to produce human insulin, people with insulin-dependent diabetes were injected
with insulin derived from pigs or cattle. Although this type of insulin works in the human body, pig or cow insulin does
not have exactly the same primary structure as human insulin, so its amino acids sequence, while similar to human insulin,
is not identical.
There are a number of advantages of using the human insulin produced by genetically engineered bacteria:

1. it is chemically identical to the insulin that would have been produced had they not been diabetic, so there is little
chance of an immune response where human antibodies fight the cow/pig insulin.
2. because it is an exact fit in the human insulin receptors in human cell surface membranes, it brings about a much
more rapid response than pig or cow insulin,
3. like natural human insulin, the duration of the response is much shorter than pig or cattle insulin,
4. it overcomes problems related to the development of a tolerance to insulin from pigs or cattle,
5. it avoids any ethical issues that might arise from the use pig or cattle insulin, for example, religious objections to
the use of pig insulin or objections from vegetarians to the use of animal products.
6. large quantities of insulin can be made continuously using E. coli, and this can be done under controlled
conditions. Only small quantities of insulin can be obtained from the pancreas of an animal, and it is not easy to
purify the insulin to produce a standard product that is safe for medicinal use.

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Describe the stages in human insulin production by gene technology (recombinant DNA technology). [8]
1) Use either Reverse Transcriptase (RT) or Restriction Enzyme (RE) or Gene Machine (GM) to isolate human
insulin gene from B cells in the islets of Langerhans in the pancreas.
2) How? :
Either;
(a) mRNA is extracted from β cells in the islets of Langerhans in the pancreas.
The mRNA is then incubated with the enzyme reverse transcriptase, which build single-stranded cDNA
(complementary DNA) molecules against it. These are then converted to double-stranded DNA- the insulin
gene.
Some extra single-stranded DNA are then added to each end of the DNA molecules. This gives a human
insulin gene with sticky ends.
Or;
(b) Take the DNA from B cells in the islets of Langerhans in the pancreas. Use the restriction enzyme EcoRI to
cut this DNA for human insulin at GAATTC restriction sites to give sticky ends.
Or;
(c) Use a Genetic Machine to build DNA base sequence of the human insulin gene from known Amino Acid
Sequence of the Protein. Then use the restriction enzyme EcoRI to cut the DNA for human insulin at
GAATTC restriction sites to give sticky ends.
3) A small piece of circular DNA called a plasmid is extracted from bacteria such as E. coli or yeast cell.
4) A small section is then cut out of the circular plasmid by EcoRI restriction enzymes (‗molecular scissors‘) to
produce sticky ends.
5) The gene for human insulin with sticky ends is inserted into the gap in the plasmid. DNA ligase seals the sticky
ends and this plasmid is now genetically modified.
6) The genetically modified plasmid is introduced into a new bacterial cell. How? Mix modified / recombinant
plasmids with bacterial cells. Add Ca2+ ions and heat shock / pulses of electricity. Bacteria cells will become
permeable and take up the modified / recombinant plasmids. But not all the bacteria cells will take up the
modified plasmid.
7) Genes conferring resistance to an antibiotic are also introduced into the plasmids, next to the insulin gene.
This is used to identify bacteria which have taken up the modified plasmid. How?
Antibiotics are then added to the culture of bacterial cells. The only bacteria that survive are the ones that had
successfully taken up the plasmids containing the antibiotic resistance gene plus the human insulin gene. The
surviving bacteria are therefore the ones that now contain the human insulin gene.
Alternatively, fluorescent marker genes can also be introduced into the plasmids, next to the insulin gene.
This can also be used to identify bacteria which have taken up the modified plasmid. How? Cells that fluoresce
green are therefore likely to have taken up the modified plasmid with the human insulin gene.
8) These bacterial cells then divide rapidly and start making insulin.
9) To create large amounts of the cells, the genetically modified bacteria or yeast are grown in large fermentation
vessels that contain all the nutrients they need. The more the cells divide, the more insulin is produced.
10) When fermentation is complete, the mixture is filtered to release the insulin.
11) The insulin is then purified and packaged into bottles and insulin pens for distribution to patients with diabetes.

Explain the advantages of treating diabetics with human insulin produced by genetic engineering [6]
- constant/reliable supply all year round/unlimited supply;
- less risk of contamination/infection;
- identical to insulin produced in the body;
- less/no risk of allergic reaction;
- does not stimulate the immune system;
- fewer side effects;
- can be produced without the killing of animals/ethical reason;
- cheaper/easier to extract and purify;
- more available/large amount;
- more rapid response;
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Use the following diagram to summarise human insulin production by recombinant DNA technology.

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Through advances in biotechnology, human insulin can be mass produced by using genetically
modified bacterium. The principle steps are outlined below:

(a) How is the insulin gene removed from the chromosome (stage 1)?
Through the use of restriction enzymes.
(b) What is the ring of DNA called?
Plasmid
(c) How are stages 2 and 3 accomplished?
Stage 2: Use of DNA ligase to join the sticky ends of plasmid and insulin gene.
Stage 3: Mixing bacteria with recombinant plasmids and Ca2+ ions. Pulses of electricity are passed through bacterial
cells / heat shock; to increase chances of uptake of the plasmids.
(d) Stage 4 is carried out in a fermenter. Suggest 2 precautions that the technicians handling the fermenter should take
note of and explain why you think the precautions are important.
Ensure factors are kept optimal; factors such as
- oxygen (aeration)
- pH (maximum growth of bacteria)
- temperature (to prevent death of bacteria from overheating)
- nutrient concentration (nitrogen, carbon content and mineral salts should be sufficient for growth and multiplication
of bacteria) [Any 2 of the above points]
Technicians should ensure that culture broth is sterile to prevent contamination of bacterial culture and product.
Ensures that impeller is working so that nutrient broth and oxygen are well distributed.
(e) The bacterium multiplies to produce many copies of itself. What is another term for each copy of the original
bacterium?
Clone
(f) How does stage 4 contribute to the mass production of human insulin?
The insulin gene is multiplied as transgenic bacterium multiplies. Each clone of the transgenic bacterium is able to
produce insulin.

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EXAMPLES OF GENETICALLY MODIFIED (GM) CROPS AND LIVESTOCK

Examples of GM livestock / animals meant to improve quality and yield as well as solving the demand of food in the
world
GM Salmon fish Lactating cows injected with Bovine somatotropin
• Salmon are bred for food in many parts of the world. (BST)
• A GM variety of salmon was developed that contains a  Large commercial quantities of BST are produced by
growth hormone (GH) gene from Chinook salmon, and a gene technology using E. coli bacteria.
promoter from a different species of fish called ocean pout.  The BST is extracted, purified and injected into lactating
• The resulting salmon are transgenic / genetically modified. cows.
 NB: The resulting cows are not transgenic / genetically
• Effect modified, but the E. coli used to produce the BST are
1) This makes the GM salmon grow much faster and larger genetically modified.
than normal (higher growth rate).
2) The higher growth rate is due to higher feed conversion  Effect
efficiency. 1) BST increases milk to feed ratio by 5 – 15%.
3) Environmental tolerance: GM salmon expresses GH 2) BST increases milk yield by up to 25%.
gene continuously throughout the year (not just during 3) BST increases cattle weight by 10 – 15%.
warmer seasons as in normal salmon).
 Advantages
• Advantages 1) Increased milk yields in cows
1) More salmon meat is produced faster and is cheaper to 2) Increased profits for farmers over same period of
consumers. time.
2) Increased profits for salmon breeders.
 Disadvantages
• Disadvantages 1) Increased incidence of mastitis (infection of udder).
1) If GM salmon escape into the wild / ocean, they may 2) Constant injections of BST may incur more costs.
outcompete the wild fish and affect ecological balance / 3) Critics say we show no respect for animals.
populations of other marine organisms.
 Solution Pigs injected with Porcine somatotropin (PST)
1) Breeding only sterile, triploid female fish could solve  Large commercial quantities of PST are produced by
the above problem. gene technology using E. coli bacteria.
2) Triploid GM salmon are sterile because they do not  The PST is extracted, purified and injected into pigs.
produce viable gametes.  NB: The resulting pigs are not transgenic / genetically
3) The GM salmon fish should only be grown in land- modified, but the E. coli used to produce the PST are
based water tanks, so that they are much less likely to genetically modified.
escape into the wild / ocean.
 Effect
GM pigs that produce transgenic pork that is heart-healthy 1) PST increases feed efficiency by 15 – 20%.
 Fat-1 gene from roundworm, C. elegans, is transferred into 2) PST reduces fat deposition in pigs, producing leaner
pigs. meat.
 The resulting pigs are transgenic / GMOs.
 Advantages
 Effect 1) Increases growth rate in pigs.
1) GM pork contains 8% omega-3 fatty acids compared to 2) Improves nutritional qualities of pork – less fat,
typical pork that contains only 1%. leaner meat.
3) Increase profits for farmers over same period of time.
 Advantage
1) Higher level of omega-3 fatty acids protects against heart  Disadvantages
disease. 1) Pigs have increased joint and skeletal problems.
2) Constant injections of PST may incur more costs.
3) Critics say we show no respect for animals.

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Examples of GM crops / plants meant to improve quality and yield as well as solving the demand of food in the world
Golden Rice Insect-resistant maize, cotton and tobacco
 Golden Rice is a type of GM rice that has had genes • Yields of maize, cotton and tobacco are reduced by insect
encoding vitamin A added to it. larvae namely the maize borer, the cotton bollworm and the
 This GM rice is crossed with local rice varieties so that it tobacco budworm, respectively.
will grow well in different countries. • Pesticides sprayed onto the crops can kill these insects.
However, the pesticides can also harm other, beneficial
 Effect insects. The insects also evolve resistance to the pesticides.
1) Golden Rice makes beta-carotene in its endosperm, • Genes coding for Bt toxin, a protein from Bacillus
which is needed by humans to make vitamin A, so as thuringiensis, are inserted into maize, cotton and tobacco.
to prevent vitamin A deficiency. • Effect
 This is particularly important in countries where rice 1) The plants therefore produce the protein, which is
is a staple food and forms a major part of the diet. converted into the toxin once inside the gut of insects that
 In such countries children can suffer from vitamin A have eaten the plant leaves.
deficiency if they eat rice lacking beta-carotene.  This means that the toxin breaks down the gut walls of
 This causes night blindness and severe deficiencies insects that feed on the plants and kills them, but not
of the immune system, which can result in death. other insects.
Advantages include the following:
 Advantages 1. Less loss of crop to insect pests, so higher yields are
1) The poor in developing countries who cannot afford obtained.
vitamin supplements in the form of pills can obtain 2. Negative effects of pesticides are avoided e.g. high costs,
vitamin A through eating Golden Rice. laborious application, killing of beneficial insects such as
2) Golden Rice becomes well adapted to local pollinators.
conditions as it is crossed with local rice varieties. 3. Only kills insects that eat the sprayed plants, not other
insects.
Herbicide resistant soyabean (glyphosate resistant 4. Does not harm humans (since they do not have the enzyme
soyabean) that converts the protein to toxin).
• EPSP gene coding for resistance to herbicides ( a 5. It is less likely that insect pests will evolve resistance to the
glyphosate herbicide) from Agrobacterium was introduced Bt toxin than to pesticides.
into soyabeans.  However, there are signs that resistance can develop in
• Effect some pest species. This can be counteracted by using
1) GM soyabean plants express enzymes that degrade the slightly different forms of the Bt toxin, or a
glyphosate herbicide. combination of two different Bt toxins, in GM crops.
2) Weeds do not have the glyphosate degrading enzyme  Disadvantages
and so are affected by the herbicide. Possible detrimental effects include the following:
Advantages 1. Bt maize seed and Bt cotton seed costs more for farmers to
1) Higher yield and quality of soyabean. buy than non-GM seed.
2) Herbicides can be freely applied to kill weeds without 2. Genes from the Bt crops might be transferred (e.g. in
danger of harming crop plants. pollen) to species of wild plants growing nearby.
Tomatoes with improved shelf-life (Flavr Savr tomatoes)
• Polygalacturonase (PG) is an enzyme responsible for the ripening of fruits. It hydrolyses pectins in plant cell walls making
cells flaccid and making fruits softer.
• Antisense gene coding for PG is transferred into tomato plants through Agrobacterium mediated gene transfer.
• Effect
• 1. GM plant produces antisense PG mRNA transcript for PG, hence
• (a) ribosomes cannot gain access to mRNA,
• (b) duplex RNA quickly degraded by ribonucleases in the cells.
• 2. Prevents translation of PG mRNA into PG protein.
• Advantages
1) Delays ripening so that tomatoes ripen on the vine for a long time. This gives a fuller flavour.
2) Tomato fruits remain quite firm after transportation. This reduces loss and increases profits to farmers.
3) Increases shelf-life resulting in increased profits to retailers.
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THE BENEFITS AND HAZARDS OF GENE TECHNOLOGY

Explain the potential benefits and hazards / risks of gene technology. [8]
Benefits of gene technology:

 Creates insulin used to treat diabetes.

 Improves crop yields or crop quality, which is important in reducing hunger around the world.
 Introduce herbicide resistance in crops, which results in less herbicides being used, as weeds are quickly and
selectively killed.
 Insect and pest resistant GM crops will not require expensive pesticides. The plant produces toxins, which would
discourage insects from eating the crop.
 GM crops can be made that will grow in very dry environments.
 Genetically modified (GM) insects, could be created to combat diseases. For example GM mosquitoes could be
used to combat malaria. The GM male mosquitoes released to mate with wild females could produce offspring
that cannot survive to adulthood.
 Genetic modification is a faster and more efficient way of changing organisms than selective breeding.
 GM animals can be made disease resistant.
 Bioremediation. This is the removal of toxic compounds, carcinogens and pollutants, such as industrial solvents,
contaminating ground waters and crude oil spills by genetically engineered bacteria which breakdown these
substances into safer molecules.
 Biomining. This is the use of genetically modified organisms (such as bacteria, fungi and plants) in the extraction
of metals from ores.

Hazards / Risks of gene technology:

 GM crops could escape and breed with weeds making ‗super weeds‘ that are resistant to pesticides.
 GM plants are more resistant against pests and chances are that they will displace local natural plant species in the
long run.
 GM crop seeds are often more expensive and so people in developing countries cannot afford them.
 GM crops could be harmful, for example toxins from the crops have been detected in some people's blood.
 GM crops could cause allergic reactions in people.
 Pollen produced by the GM plants could be toxic and harm pollinating insects such as bees and butterflies.
 Residues / remains of genetically modified plants persist in the soil of fields for many months. This affects the
activity of decomposers which can lead to a loss in fertility of the soil.
 GM bacteria damage / decompose useful materials such as oil or plastics.
 Bioterrorism: Governments are worried that terrorists will use gene technology to create new superbugs,
infectious viruses, or toxins, for which we have no cures.

Notes

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THE SOCIAL AND ETHICAL IMPLICATIONS OF GENE TECHNOLOGY

 Social means relating to society or to the way society is organized.
 The implications of something are the things that are likely to happen as a result, i.e. the outcome.
 The phrase “social implications” of gene technology refers to effects that gene technology will have on the people in
society / community.
 Ethical means relating to beliefs about right and wrong.
 Moral means relating to principles of right and wrong.
 The phrase “ethical implications” refers to the implied moral outcome of any particular action or decision. It is about
the application of moral frameworks concerning the principles of conduct governing individuals and groups, including
what might be thought to be right or wrong, good or bad.
 To “consider the ethical implications” of gene technology means to question whether or not the results / impact of
gene technology will be morally good or bad in the eyes of society.

THE SOCIAL IMPLICATIONS OF GENE TECHNOLOGY

Explain the social implications of gene technology / recombinant DNA technology / genetic
engineering. [8]
The social implications of gene technology / recombinant DNA technology / genetic engineering are the potential,
perceived and actual impact / effects that gene technology will have on human society or individual people i.e. both
positive (beneficial) and negative (harmful) effects of gene technology on human society or individuals:

Positive (beneficial) effects of gene technology on society i.e. advantages of gene technology for society are:
 improved crop yields and food supplies for humans and livestock.
 improved nutritional quality of foods e.g. increasing the level of vitamins in crops to help people overcome vitamin
deficiency. An example is Golden rice, a type of rice produced through genetic engineering to allow the plant make
beta-carotene, needed by humans in order to make vitamin A, so as to prevent Vitamin A deficiency. The vitamin A
is essential for good vision.
 permit crops to grow outside their usual location or season so that people have more food.
 improved crop yields since GM crops are resistant to all kinds of pests and diseases.
 decrease in the use of harmful chemical pesticides as GM crops are pest resistant.
 improved, cheaper medicines.
 improved treatment of genetic diseases.
 GM fruits and vegetables with longer shelf-life and therefore less spoilage.
 creation of medical foods which may replace some common injections.
 success in fighting many diseases improves the life expectancy of people.
 GM fish that clean-up toxic metals from the water by storing them in their proteins.
 a cleaner environment due to bioremediation.
 bio-mining. The use of genetically modified organisms (such as bacteria, fungi and plants) in the extraction of metals
from ores.

Negative (harmful) effects of gene technology on society i.e. disadvantages of gene technology for society are:
 produce ‗super weeds‘ which may cause ecological disturbances, reducing crop yields so that people have less food.
 farmers made dependent on specific varieties, needing fresh seed annually and expensive fertilizers.
 GM crops reduce crop biodiversity by out-competing natural crops so that people are less well fed.
 reduced natural biodiversity resulting in a reduced possibility of new varieties arising.
 antibiotic resistance genes used as markers during gene transfer could spread to bacteria. This may lead to serious
problems since the treatment of diseases with antibiotics will not be effective anymore.
 GM crops engineered to produce plastics or pharmaceuticals could poison animals that feed on the crops or crop
remains after harvesting.
 GM fish that clean-up toxic metals from the water by storing them in their proteins could be harmful if consumed by
other fish or fish-eating birds and mammals.
 bioterrorism: fears that terrorists may use gene technology to create new incurable infectious micro-organisms and
toxins and use them to attack other people.

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THE ETHICAL IMPLICATIONS OF GENE TECHNOLOGY

Explain the ethical implications of gene technology / recombinant DNA technology / genetic
engineering. [8]
The ethical implications of gene technology / recombinant DNA technology / genetic engineering (whether or not the
results / impact of gene technology will be morally good or bad in the eyes of society):

Why gene technology may be regarded as morally good / right:

1. Genetically modified crops are created by modifying the DNA of the plants to improve their growth or use for
human consumption.
 This can improve their disease resistance, make crops endure drought and improve yields.
 This has many important effects, and could help to alleviate poverty by improving food supplies.
2. Inserting animal genes in plant crops makes them to grow faster, improves their quality (e.g. size, taste, nutritional
value), shelf-life and yields.
3. Genetic modification can increase the yield from farm animals, for example cows can be engineered to produce
more milk.
4. Genetically modified farm animals are being used to produce important medicinal products, such as antibodies,
insulin and hormones, in large quantities. These products can be used for the treatment of many different human
conditions.
5. Genetically modified bacteria can be used to produce human insulin which is used to treat diabetes.
6. Improved treatment of genetic diseases by gene therapy such as cystic fibrosis and severe combined
immunodeficiency (SCID).
7. Genetically modified pigs can be used as sources of human spare parts in surgeries and transplants.

Why gene technology may be regarded as morally bad / wrong:

Religion
1. According to some religious views, gene technologists are ‗playing God‘ by editing genomes of organisms.
 Some religions believe it is morally wrong to alter the natural genetic makeup of organisms for economic benefit.
 However, scientists reject statements, such as ‗man should not play at being God‘ / ‗man should not interfere with
God‘s creation‘ because they regard them as vague and non-scientific.
2. Using genes of animals in plants for faster growth pose philosophical and religious problems e.g.:
 insertion of human genes in tomatoes and pepper for faster growth may suggest that one can be a vegetarian
and cannibal at the same time.
 insertion of pig genes in vegetables for faster growth. People who do not eat pork for religious reasons may
shun eating the vegetable with pig genes.
Unknown harms / Unforeseen consequences
 Long-term health impacts of gene technology are unknown.
 It is wrong to continue research on gene technology when the potential impact of the technology is unknown and
many aspects of it remain to be understood.
 Errors in gene technology could even lead to the development of new diseases and deformities.
 Thus, research on gene technology may pose serious negative effects, some of them currently even unknown to
scientists.
Bioterrorism
 Fears that terrorists may use gene technology to create new incurable infectious micro-organisms and toxins and
use them to attack other people.
Biological warfare
 The use highly infectious GM bacteria and viruses in war are regarded by many as morally wrong.
Violating animal rights
People who advocate for animal rights believe gene technology violates animal rights by:
1. experimenting with animals and their genes for human benefit.
2. editing genes of animals which may change the phenotype of the animal resulting in unexpected side effects such
as lameness, reduced fertility etc.

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3. using procedures which may cause pain, distress and suffering in the animals.
Bio piracy and patents
1. Who owns genetically modified organisms (GMOs) and their genes?
 Gene technology also brings with it concerns over intellectual property, and patenting of GMOs and/or the
techniques used to create them.
 Patenting preserves intellectual property by giving the inventor exclusive rights to GMOs but it has the
disadvantage of breeding a culture of confidentiality within the scientific community, which in turn limits data
and animal sharing.
2. Bio piracy is the theft and use of organisms and their genes for profit by rich foreign multinational and
pharmaceutical companies, without permission /authorization from poor indigenous people and often with little or
no compensation.
3. Through patenting and bio piracy foreign companies have monopoly and deprive the indigenous people of the
right to commercially exploit their resources.
 In some countries the indigenous people had to take some foreign companies to court to have the patents and
bio piracy reversed.
Labeling
 While some countries have legislation which enforce the labeling of foods containing GMOs (genetically modified
organisms), others do not have. Some people believe that it is unethical not to inform the public of the GMO status of
foods. Labeling allows consumers to make a choice for themselves whether they wish to be exposed to GMO foods.

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POLYMERASE CHAIN REACTION
What is PCR?
 Polymerase chain reaction.
 PCR is a technique used to replicate DNA artificially.
 PCR is a technique used to amplify DNA/ produce more copies of the DNA.
 PCR amplifies DNA from a limited source of DNA so that there is sufficient amount for analysis.
 One of the applications of PCR is to amplify DNA for use in genetic fingerprinting/ DNA profiling.
 Starting with a small sample, PCR can generate billions of copies of a DNA segment in just a few hours, producing
enough DNA to allow a DNA profile to be constructed.
 Steps in PCR:
 step 1: heat DNA to 95oC, hydrogen bonds break, double strand separates, left with 2 template strands.
 step 2: cool to 55oC, primers bind (short single stranded sections of DNA) to start of each template strand,
prevents the templates from rejoining and allows DNA Polymerase to bind to build the new strand.
 step 3: heat to 72oC, DNA nucleotides attach to complementary bases, DNA Polymerase joins sugar-phosphate
backbone of the new strands.
= 2 copies of DNA (each made of 1 original strand, 1 new strand).

Polymerase Chain Reaction vs Semi-Conservative Replication?

 PCR can only replicate short DNA fragments, SCR can replicate whole DNA
 PCR use 95oC, SCR uses DNA Helicase
 PCR uses primers, SCR does not require primers

In-vitro vs In-vivo method of DNA Replication?

 In-vivo refers to carrying out a biological process inside a living organism.
 So using bacteria to replicate DNA (add DNA fragment to the plasmid, then replicate the bacteria to make many
copies of DNA fragment) = In-vivo.
 In-vitro is performing a biological process outside of a living organism.
 So PCR= In-vitro.
 benefits of in-vitro = more rapid, less complex.
 benefits of in-vivo = more accurate (less mutations), less chance of contamination.
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GENETIC SCREENING AND FINGERPRINTING

GENETIC FINGERPRINTING (aka DNA fingerprinting or DNA profiling)
- Everyone‟s DNA is unique - excluding clones and identical twins.
- This means that we can inspect a person‘s DNA to identify them. An individual‘s DNA profile used to identify
them is called a genetic fingerprint. The process of obtaining such a fingerprint is called genetic fingerprinting.
- DEFINITION: Genetic fingerprinting is a technique by which individuals can be identified and compared via
their respective DNA profiles.
- That is, genetic fingerprinting is the analysis of DNA in order to identify the individual from which the DNA
came from.

Uses of genetic fingerprinting include:

1) Solving paternity cases, i.e. to determine whether a particular person could be the child, mother or father of
another.
2) In forensic to identify criminals from crime suspects, i.e. determine if a sample of semen, blood or other tissue
found at a crime scene could have come from the victim or a suspect.
3) Medical diagnosis of genetic diseases e.g. Genetic fingerprints (DNA profiles) can identify individuals at risk of
developing specific diseases, as some VNTRs are correlated with an increased risk of disease e.g. Huntington‘s
disease
4) In forensic to identify accident or inferno (fire) victims after they are burnt beyond recognition.
5) In forensic to identify victims of war and large scale disasters.
6) Tracking genetically modified crops and livestock. This is important in identifying and protecting the commercial
varieties of crops and livestock.
7) Finding the relatedness of livestock breeds so as to prevent inbreeding by not breeding individuals with similar
genetic fingerprints (DNA profiles).
8) Finding the genetic relatedness of biodiversity, i.e. it is used to find out the evolutionary history of an organism
and trace out the linkages between groups of various organisms. This is important in classifying organisms based
on their common ancestry.

VNTR sequences are the ones used in genetic fingerprinting to identify individuals
- Different people have unique sequences of ‗useless‘ or non-coding DNA called introns. (These introns are said
to be ‗useless‘ or non-coding because they are not translated into a protein during protein synthesis).
- These non-coding sequences in DNA are often repeated many times consisting of 20 to 40 base pairs. These
repeating non-coding lengths of DNA are called variable number tandem repeats (VNTR).
- DEFINITION: VNTR sequences are the non-coding sequences in DNA that are unique to an individual.
- Each person‟s VNTR sequence is unique. Only identical twins share identical VNTR sequences.
- Because of the uniqueness of VNTR sequences it means they can be used to identify individuals.
- Like the rest of DNA, VNTR sequences are passed on from parent to offspring and the number of repeats in the
lengths of the non-coding DNA can be used to identify an individual.
- A genetic fingerprint based on VNTR sequences is produced using a technique called gel electrophoresis
(sometimes simply referred to as electrophoresis).

Notes

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GEL ELECTROPHORESIS
— Gel electrophoresis is a way of separating strands of DNA of different lengths.
— This involves exposing DNA to an electric current in a gel medium through which different fragments of the DNA
travel depending on their size.

— The stages in gel electrophoresis are as follows:

1) DNA samples are extracted from different sources and cut into smaller fragments using an enzyme called
restriction endonuclease.
2) The fragments of DNA from each source are placed in separate wells (holes) at one end of a gel.
3) The current is switched on.
Because agarose gel contains a tangle of cable-like threads, it is porous and acts as a molecular sieve.
Because the DNA fragments carry negative charges on their phosphate groups ( ), they move towards the
positive electrode of the gel with smaller fragments moving the furthest. However, longer DNA fragments are
held back by the sieve of polymer fibers within the gel, so they move more slowly than the shorter fragments.
When the current is switched off, this produces a series of bands which are positioned according to the size of the
fragments.
4) The gel trough is then covered with a nylon membrane onto which the fragments are transferred whilst they
maintain their position. The process by which the fragments are transferred is known as Southern blotting.
(Southern blotting is named after Sir Edwin Mellor Southern a British biologist who invented the technique).
5) The DNA fragments are transparent and invisible, so they are treated to make them visible. Two key ways to do
this are staining and hybridisation:
a) Staining all of the DNA fragments, e.g. using ethidium bromide which makes the DNA bands to fluoresce
under uv radiation. Other stains that can be used include methylene blue and Nile blue A.
b) Hydridisation involves adding to the gel some radioactively labelled single strands of DNA with bases
similar to those in the DNA fragments. These then pair up with fragments with complementary base
sequences. The fragments then emit radiation. The radiation then helps to detect the position of the
bands/fragments by developing an effect on a photographic x-ray film. The pattern (or autoradiograph)
captured from this is a pattern of dark and light bands which are unique to that individual. This is the genetic
fingerprint or DNA profile of that person.

What causes DNA molecules to move toward the positive pole during electrophoresis?
The negatively charged phosphate groups of the DNA are attracted to the positive pole;

Why do large molecules move more slowly than smaller ones during gel electrophoresis?
Longer fragments are more restricted by the tangle of fibers in the gel than are shorter fragments.

…..

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THE USE OF GEL ELECTROPHORESIS IN GENETIC FINGERPRINTING

Involves 6 steps: Extraction, Digestion, Separation, Hybridisation, Development, Comparison
1. Extraction
— Extracting the individual's DNA and amplifying it using PCR (Polymerase Chain Reaction).
2. Digestion
— Cutting the DNA down into fragments using Restriction Enzymes (restriction endonuclease) leaving the VNTRs
intact.
3. Separation
— separate out the DNA fragments by gel electrophoresis.
— add alkali to make the separated fragments single stranded.
— transfer the fragments to a nylon membrane by Southern Blotting.
— add UV light so the DNA fragments set.
4. Hybridisation/staining
— The DNA fragments are invisible since they are transparent, so they are made visible either by hybridisation or
staining.
— Hybridisation: involves adding radioactively labelled DNA Probes complementary to the DNA fragments. These
pair up with fragments with complementary bases allowing them to be identified as they emit radiation which will
be developed on an x-ray photographic film.
— Staining: Staining all of the DNA fragments, e.g. using ethidium bromide which makes the DNA bands to
fluoresce under uv radiation. Other stains that can be used include methylene blue and Nile blue.
5. Development
— Add photographic film and take an x-ray to produce the banding pattern picture or autoradiograph.
6. Comparison/Analysis
— Match the banding pattern of fragments on the gel with other samples of DNA.
— Interpret the results e.g. to solve paternity issues or crime cases.
If the bands match then the suspect is the culprit. If there are mismatches then the suspect is exonerated.

DIAGRAM: Use of gel electrophoresis in Genetic Fingerprinting.

……

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Examples of comparison/analysis of genetic fingerprints / DNA profiles

 DNA profiling is commonly used in criminal investigations (forensics) and to settle paternity disputes.
 The procedure involved is common for both:

— A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR
— DNA (with VNTR sequences) are cut with specific restriction enzymes to generate fragments
— Fragment length will differ between individuals due to the variable length of their VNTRs.
— The fragments are separated using gel electrophoresis and the resulting profiles are compared and analysed.
— Paternity testing and forensic investigations are given as examples below.

1. PATERNITY TESTING:
 Children inherit half their chromosomes from each parent and thus possess a combination of parental fragments.
 In other words, all DNA fragments produced in the child should also be produced by either the mother or father.
 DNA from the white blood cells of the mother, (possible) father and child is taken and used to produce DNA
profiles by the process of gel electrophoresis.
 Removing the mother bands from the child pattern leaves the pattern that should be in the fathers fingerprint.
These are compared to determine if the individual concerned is actually the father.
 Half the DNA profile of the child should match the father‘s. If there are some mismatches then the suspect is not
the father of the child.

 EXAMPLE 1: Compare the genetic fingerprints (DNA profiles) of the following three men with that of a mother
and child to determine the biological father.

— Man 3 is the father of the child

— Reason: Half of the child‘s bands match exactly with man 3‘s bands and the other half match exactly with the
mother‘s.

Notes

……

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 EXAMPLE 2: Analyse the following autoradiographs so as to settle paternity cases.

……
2. FORENSIC INVESTIGATIONS:
Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur.

— Suspect 2 is the criminal.

— Reason: suspect 2‘s bands match exactly with the DNA profile taken from the crime scene.

……

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ADVANTAGES AND DISADVANTAGES OF GENETIC FINGERPRINTING / DNA PROFILING

 Genetic fingerprinting can be controversial. The following are some of the advantages and disadvantages of using
genetic fingerprinting aka DNA profiling.
Advantages Disadvantages
DNA evidence is reliable as it is highly unlikely Stored DNA data might get into the hands of insurance, loan
that two people would share the same profile, companies or employers who could analyse your DNA for
except in the case of identical twins predisposition to disease and refuse your business because of it
DNA profiles can be used to determine paternity Storage of DNA profiles can be seen as an invasion of privacy
DNA profiles can be used to identify genetic Theft of DNA profiles from a database is a real threat
disorders early
DNA profiles can be used to place suspects at a It is possible to plant DNA at a crime scene giving false evidence,
crime scene or an innocent person‘s DNA might be at the scene even though
they had nothing to do with the crime
…..
GENETIC SCREENING
 Genetic screening is analysis of a person‘s DNA to check for the presence of a particular allele / gene.
 It can be done in adults, foetuses and embryos.
 Adult woman with family history of breast cancer (Brca-1 and Brca-2 genes) may choose to be screened; positive
result leads to decision of elective mastectomy to reduce their risk of breast cancer.
 Designer baby made by pre-implantation genetic diagnosis (PGD) and IVF.
1) mix father‘s sperm and female egg in a dish.
2) at eight-cell stage, one cell removed to analyse DNA in it.
3) embryo with faulty allele is discarded.
 Foetus can be screened for genetic diseases by:
6. amniocentesis — obtaining sample of amniotic fluid at 15-16 weeks of pregnancy; hypodermic needle inserted
away from fetus, umbilical cord, placenta, aided by ultrasound scanning.
7. chronic villus sampling — carried out between 10-13 weeks of pregnancy; small part of placenta (chorion)
removed by needle, monitored by ultrasound scanning; small increase in miscarriage risk.
— therapeutic abortion: termination of pregnancy due to medical reasons.
— ethical dilemma of knowing that one carries a disease, or live obliviously.
Genetic counseling
 Genetic counselling is giving advice to people based on results of their genetic screening.
 Genetic counselling helps people understand their choices and make informed decisions,
— e.g. financial costs involved in supporting a child with a genetic disease e.g. cystic fibrosis.
— e.g. whether termination of foetus is appropriate if quality of life is poor.
 A genetic counsellor helps people to interpret the results of genetic screening and help them to make informed
decisions.
 This can be very difficult as it involves social, moral, ethical and medical issues. For example, if a pregnant woman‘s
results show that her child has cystic fibrosis, is this sufficient grounds to have her pregnancy terminated, or does the
child have a righy to life.
 Genetic screening can be used:
(a). to identify carriers of a harmful recessive allele for genetic disorders such as sickle cell anaemia, haemophilia
or cystic fibrosis.
— Therefore if a couple in a marriage are both heterozygous they can be advised about the chances of their
children inheriting the disorder.
— i.e. advise a couple who have hereditary disorders about the chances of their children inheriting the disorders.
(b). in prenatal testing ( i.e. checking the genes of an embryo / foetus in the uterus) to inform a pregnant mother
whether the embryo / foetus has genetic disorders or not and hence prepare for its birth or termination.
(c). advise a person in choosing a marriage partner.
(d). to check the genes of an embryo produced in vitro (i.e. by fertilisation outside the body) before it is placed in
the mother‘s uterus for implantation; this ensures that only embryos that are free of genes for a genetic disease are
implanted.
(e). to identify people who will develop a genetic condition later in life; for example, Huntington‘s disease which is

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caused by a dominant allele that manifests itself later in middle age; a person with such a disease could check if
they have the gene before they decide to have children themselves.
(f). to identify people with alleles that put them at risk of developing other diseases; for example, a woman with
family history of breast cancer (Brca-1 and Brca-2 genes) may choose to be screened; positive result leads to
decision of elective mastectomy to reduce their risk of breast cancer.
Ethics of genetic screening
 There are many social and ethical considerations for genetic screening, which include:
 International law prohibiting addition of allele to human egg, sperm, or zygote.
 Some people believe that law is too relaxed, others believe otherwise.
 There is controversy of sex preselection and termination in pregnant mothers.
 Being able to take preventative measures (e.g. elective mastectomy when cancer genes BRCA1 and BRCA2 are
detected) - giving individuals control to prevent illness.
 Using pre-implantation genetic diagnosis to select embryos that do not carry faulty disease-causing alleles. This could
lead to the fear of ‗‗designer babies‘ being created (this includes creating/choosing embryos with tissue matches to
older siblings).
 Pre-implantation genetic diagnosis can be carried out during in-vitro fertilisation (IVF); cells are extracted from the
embryo in an embryo biopsy and genetically screened in order to preselect the embryos without faulty alleles / genes.
 Using genetic counselors to help people understand their choices and make informed decisions (e.g. financial costs,
whether termination of foetus is appropriate if quality of life is poor).
 Risk of miscarriage (which has emotional consequences) due to the procedures used to collect DNA which are not
100% risk-free (i.e. amniocentesis and chronic villus sampling).
 Choosing to terminate a pregnancy (therapeutic abortion) because the embryo has a genetic disorder (e.g. cystic
fibrosis) or even terminating the embryo due to a minor ‗defect‘ that could have seen the child lead an almost normal
life.
 Being able to make informed reproductive decisions (e.g. cystic fibrosis).
 Determining whether it is best to know the risk of having a disease, especially when there is no cure (e.g.
Huntington‘s).
 Deciding at what age screening should begin e.g. whether parents should be able to choose for their children to be
screened.
 The possibility of stigmatization and discrimination. The person may feel stigmatized if they have the disease or
discriminated against by health insurers or employers.
 Confidentiality of the data collected – who will have the right to view the results obtained.
 Genetic screening that inform someone that they have increased risk of developing a fatal disease, e.g. cancer, could
impact upon someone‘s mental health and wellbeing and negatively impact the way they live their life. Conversely,
having this information can empower someone to make better lifestyle choices and be more aware of their health.
……
What are the advantages and disadvantages of genetic screening? [8]
Advantages of genetic screening
1) A sense of relief from uncertainty.
2) A greater understanding of your health and your cancer risk.
3) Information to help make informed medical and lifestyle decisions.
4) Opportunity to help educate other family members about the potential risk.

Disadvantages of genetic screening

1) Results from genetic tests can trigger emotional reactions.
2) Testing may increase your stress and anxiety.
3) The tests can be costly for most patients.
4) Results in some cases may return inconclusive or uncertain. You may not receive a clear result from the test.
(In some cases the results are an estimation of the chances that one will develop a specific condition related to
his/her genetic profile. So the answer will not be a clear yes or no).
5) Negative impact on family and personal relationships.
6) Test results could get put into databases outside of your personal control.
7) You might not be eligible if you do not fit certain criteria required for testing.
……

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EXAM STYLE QUESTIONS

Explain how gel electrophoresis is used to analyse DNA. [7]
Mark scheme
A. Gel electrophoresis separates DNA fragments based on size / number of nucleotides by passing them through a gel in
an electric field / direct current;
B. The porous structure of agarose gel allows it to act as a molecular sieve;
C. Rate at which a DNA fragment travels is inversely proportional to its size / Longer DNA fragment moves at a slower
rate / Shorter DNA fragment moves at a faster rate;
D. Samples are loaded into wells and gel is orientated such that wells are near to the negative electrode;
E. Since DNA is negatively-charged due to its sugar-phosphate backbone, it will move towards positive electrode;
F. DNA fragments of the same length appear at the same band position;
G. The DNA can be visualised by staining the gel with ethidium bromide and when viewed under UV radiation, DNA
bands fluoresce.
Describe the application of DNA profiling (genetic fingerprinting) to determine paternity and also in forensic
investigations. [8]
Mark Scheme
A. sample of DNA / blood / saliva / semen is obtained;
B. satellite DNA / repetitive sequences used for profiling;
C. reference samples of DNA are obtained;
D. PCR used to amplify / produce more copies of the DNA;
E. DNA broken into fragments by restriction enzymes;
F. DNA fragments are separated by gel electrophoresis;
G. separation according to the length of the fragments;
H. pattern of bands obtained / different pattern of bands with DNA from different individuals;
I. bands compared between different DNA samples;
J. if pattern of bands is the same then DNA is (almost certainly) from same source;
K. if some bands are similar then individuals are (almost certainly) related;
L. used for criminal investigations / example of use in criminal investigation;
M. used to check paternity / who is the father / mother / parent;
N. used to check whether two organisms are clones;
……
GENE THERAPY
- Gene therapy is the treatment of a genetic disease by replacing or repairing a faulty gene / allele in a person‘s cells.
- Gene therapy involves the repair or replacement of faulty genes / alleles with healthy versions.
- It is carried out by introducing DNA containing the functional gene into a patient, to correct a disease-causing mutation.
- Gene therapy is only suitable for:
 diseases caused by a single gene,
 diseases caused by a recessive allele of a gene,
 serious diseases for which treatment is limited and no other cure is possible.
— There are two types of gene therapy: somatic gene therapy where the gene / allele is introduced in the target cells of the
body only and germline gene therapy where the allele / gene is introduced in embryonic cells or gametes, thus meaning
every cell contains the normal gene.
- The success in gene therapy has been limited to somatic cells. These cells are the cells that make up our body but are
not inherited, as opposed to the gametes (egg or sperm) that make future generations. This means that any changes
made to the genetic material in a patient are not passed on to the children.
- Therefore, somatic gene therapy is a short-term solution and needs to be repeated, whereas germline therapy is a
permanent solution which will be passed down to the offspring.
How are the therapies delivered?
The DNA for gene therapy is delivered into cells affected by the faulty gene using either liposomes or viruses
(retrovirus/lentivirus, adenovirus and adeno-associated virus). The viruses and liposomes are said to be vectors because
they transfer the gene into a person‘s cells.

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 Using viruses as vectors to transfer the DNA into a person‟s cells.

- One of the most promising methods of DNA delivery is through the use of viruses.
- Viruses are small infectious agents that have evolved mechanisms to be highly efficient at infecting our cells. They can
deliver their own genetic material into host cells, and use the host cellular machinery to synthesise proteins that help
them to produce more viral particles. Their ability to deliver DNA into cells makes them a highly useful tool for
delivering genetic material to patients. Researchers can remove the disease-causing DNA, and add the therapeutic gene.
Now, the virus can infect the required cells (without causing disease) and deliver the healthy gene into the cell‘s
genome. The DNA is expressed by the cell‘s normal transcription and translation machinery, so the cell can produce the
correct protein required to prevent the disease.

DIAGRAM: An overview of gene therapy.

……

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GENE DELIVERY SYSTEMS USED IN GENE THERAPY

The vectors or gene delivery systems used in gene therapy are liposomes or viruses (retrovirus/lentivirus, adenovirus
and adeno-associated virus).
Describe the advantages and disadvantages of gene delivery systems used in gene therapy.
GENE DELIVERY ADVANTAGES DISADVANTAGES
SYSTEM
Liposomes  Non-pathogenic.  Low infection efficiency.
 Does not trigger an immune response.  Low rate of stable integration.
 No limit to size of insert.  Low levels of expression of normal function
 Can be targeted for specific cells or tissues by allele.
modification of liposome membrane by the
addition of specific glycoproteins.
Retrovirus • High infection efficiency. • Random integration may cause insertional
• Integrates into host cell genome providing mutagenesis.
stable gene expression (as it contains • Can trigger an immune response.
integrase). • Limit to size of insert.
• Safety concerns as virus is dangerous.
• Infects dividing cells only e.g. lentivirus.
Adenovirus  High infection efficiency.  No integration into the genome, transient
 Can inject DNA through the nuclear pore and gene expression so DNA is not passed onto
deliver it into the nucleus. daughter cells.
 No integration into the genome so no  Can trigger an immune response.
insertional mutagenesis.  Limit to size of insert.
 Infects dividing and non-dividing cells.
Adeno-associated  High infection efficiency.  Limit to size of insert (smaller than other
virus  Non-pathogenic. viruses).
 Does not trigger immune response.
 Insert into specific site in chromosome 19
 No random integration into genome.
 No insertional mutagenesis.
 Infects dividing and non-dividing cells.

…….
How can the faulty gene be repaired by Gene Therapy?

1. Gene augmentation therapy is used to treat diseases where a mutation in the DNA causes a loss of the function of a
gene, stopping it producing a working protein. Gene augmentation therapy adds DNA containing a functional version
of this gene back into the cell. This type of therapy only works if the lack of the functional protein hasn‘t caused lasting
damage to the body, or wasn‘t required during development.
2. Gene inhibition therapy is used in the treatment of diseases caused by spurious gene activity, either by the gene being
over-active (perhaps the gene is on when it should be off) or by production of a new protein that interferes with the
function of another gene. Gene inhibition therapy aims to reduce or stop the activity of the problematic gene. This is
particularly useful in cancers, which are often caused by the over-expression of genes that promote cell growth and
division.
3. Targeted killing of specific cells is also used in cancer gene therapies. The aim is to deliver DNA into a diseased cell
as before, but this time the DNA is designed as such that it will kill the cell. This can be done in various ways, and one
of the most effective ways is inserting a gene that programmes the cells to die, a ‗suicide‘ gene. This method carries an
extra risk: it is essential that the inserted DNA is targeted appropriately to avoid killing healthy cells.

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An example of successful gene therapy: SCID

- Adenosine deaminase deficiency is a form of Severe Combined Immunodeficiency Disease (SCID).

- It is caused by a defective enzyme, adenosine deaminase called (ADA) and causes problems with the immune system.
- Gene therapy can be used to treat this disease.

The ADA gene can be delivered to affected babies using a modified virus. Firstly, some bone marrow is extracted so that
the correct DNA can be added directly to the affected cells, and the cells are put back into the body. Once the corrected
cells are back in the body, the corrected gene allows the cells to produce the enzyme, generating healthy blood cells that
can largely outcompete the faulty cells in the blood. This improves the health of the baby.
……
CHALLENGES OF GENE THERAPY
Although in principal gene therapy seems simple, there are many technical, biological and ethical challenges. Delivery of
the gene to the correct place and making sure the cell can utilize it appropriately is crucial for success and also to avoid
adverse effects. Similarly, care has to be taken to ensure that the gene does not insert into a place where it can disrupt the
function of other genes.
ETHICAL IMPLICATIONS OF GENE THERAPY
1. Human gene editing in the form of gene therapy is being used to treat genetic diseases such as cystic fibrosis and
severe combined immunodeficiency (SCID).
2. The editing of genes in somatic (body) cells to treat genetic diseases is not in gametes, so there is no risk of them being
inherited and causing some unknown effects on offspring or future generations.
3. Experts generally believe that human gene editing for the purposes of reproduction should not be carried out at
this time, but that studies and research on the matter should continue.
4. Germline gene therapy (editing genes in gametes i.e. sperm and ova) could allow families to prevent their offspring
from developing a genetic disease, but it might have as yet unknown side effects. Additionally, people who would be
affected by germline gene therapy cannot give informed consent, as they are not yet born.
5. Safety is also a big concern. This is due to the possibility of off-target effects of gene editing; meaning edits in the
wrong place could cause harmful effects such as cancers.
6. Some researchers are also concerned that any genome editing may be used for non-therapeutic and ‗enhancement‘
purposes. An example is enhancing sporting performances unfairly through gene doping. This is where the genes are
altered to give an unfair advantage e.g. to provide a source of erythropoietin (the hormone that promotes the formation
of red blood cells).
7. How do we decide which human traits are ―normal‖ and which constitute a disorder that can be corrected with gene
therapy?
8. The expense of treatments as multiple injections of the genes may be required if the somatic cells are short-lived (eg.
Severe combined immunodeficiency). This may make the cost of gene therapy accessible to a limited number of
people.
……
Describe factors that keep gene therapy from becoming an effective treatment for genetic diseases. [8]
1. Short-lived nature of gene therapy and hence the need for multiple rounds of gene therapy to have long term benefits.
— due to death of cells containing normal functional allele.
— due to inability to stably integrate normal, functional allele into genome.
2. Multiple disorders e.g. diabetes, heart disease.
— are difficult to treat as they are caused by the combined effects of several genes.
— disorders that arise from mutations in a single gene are the best candidates for gene therapy.
3. Precise level of gene regulation required for some diseases (e.g. thalassemia).
— Overexpression of a transferred gene can be problematic while low levels of expression will be ineffective.
4. Viral vectors
(a). Can trigger an immune response.
— Use of viral vectors can elicit an immune response which can destroy the vector before it can deliver the normal
functional allele to target cells. Subsequent treatment with the same virus will elicit a faster and stronger immune
response making repeated treatment difficult.
— Immune responses can also include allergic, inflammatory or toxicity responses to the viral vectors resulting in tissue

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damage and discomfort.

— Can be minimised with use of non-viral gene delivery systems.
(b). Can target only specific cells.
— thus different delivery systems need to be developed for targeting different cells.
(c). Can possibly recover its ability to cause disease once in the host.
(d). Can induce tumour formation by insertional mutagenesis.
— When retroviruses are used for delivery, random integration of the viral genome into the target cell genome (i.e.
insertional mutagenesis) may
— Inactivate tumour suppressor genes resulting in increased chance of getting cancer (by disruption of tumour
suppressor genes).
— Active proto-oncogenes resulting in increased chance of getting cancer (as strong regulatory sequence in the vector
can upregulate proto-oncogenes).
…..
CYSTIC FIBROSIS (CF)
 Cystic fibrosis (CF) is a genetic disease where abnormally thick mucus is produced in lungs and other parts of body.
 Person with CF is prone to bacterial infections in lungs because mucus is difficult to remove and allows bacteria to
breeding.
 Thick mucus affects other parts of body; the pancreatic duct is blocked; men are often sterile.
 CF is caused by a recessive allele of the gene coding for transporter protein in alveoli cell membrane, CFTR (cystic
fibrosis transmembrane conductance regulator); this protein should allow Cl– ions to pass out of cells, but doesn‘t in CF.
 Cells lining airways pump out Cl– through channel protein formed by CFTR.
 Results in high conc. of Cl– outside cells, reducing water potential.
 Under normal conditions water should mix with mucus to make it thin enough for easy removal by cilia.
 However, water moves out of cell via osmosis, down water potential gradient.
 There is therefore less water on the outer surface of these cells than there should be. The mucus that is produced on these
cells therefore does not mix with water in the usual way. Thus the mucus becomes thick and sticky. As a result:
1) Abnormally thick mucus collects in the lungs interfering with gaseous exchange and increasing the chance of bacterial
infections.
2) The pancreatic duct also gets blocked with sticky mucus, interfering with digestion in the small intestines.
3) The sperm duct (vas deferens), which is the passage of sperms, may become blocked making a person sterile.
CFTR gene
– CFTR gene is found on chromosome 7. It consists of 250 000 bases.
– Mutations in this gene produce defective alleles; most common is deletion of 3 bases, resulting in missing one amino
acid.
– Faulty CFTR allele is recessive. Heterozygous people are symptom-free but are carriers.
– CF is caused by a single gene, so it is a good candidate for gene therapy.

…….
Problems encountered in using gene therapy to treat cystic fibrosis
a) Trials included insertion of normal alleles into liposomes and spraying them as aerosol into noses. Only a few cells
took up the normal gene, so only these few cells produced normal mucus.
b) Only lasts a week as cells in the respiratory passage have a short lifespan. So treatment would need to be repeated
every week.
c) Cells in the respiratory passage took up the normal gene but not cells in the pancreas and sperm duct.
d) Harmless viruses can also be used as vectors to carry alleles into passages of gas exchange system. This caused side-
effects due to viral infection.
e) Allele needs to enter many cells throughout respiratory system but this has not been achieved.
– In some cases of CF, mutation simply replaced one base with another, creating a stop codon. Translation on
ribosomes stops when this codon is reached, so only a short length of CFTR protein is made. Drug PTC124 can be
used because it allows translation to keep going even after stop codon has been reached.
– In some occasions, DNA is inserted without vectors into tissue. This method removes problems associated with using
vectors.

…….

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This document is intentionally incomplete. It is a SAMPLE COPY from the textbook

„Biology Made Simple: Common Biology Essay Outlines and Notes‟ by G. Taruvinga.

© Gladmore Taruvinga 2018

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