1.

Plasmids are small circular DNA, found in many bacteria, which can be used for
cloning foreign genes in suitable bacteria. Bacteria can be genetically modified
to produce hormones eg insulin for human use. To achieve this, human genes
for specific hormones are transferred into bacteria.
The fragments of human DNA and the cut plasmids were mixed together with
DNA ligase. Several types of plasmid were formed. Some contained human
DNA in the centre of the gene coding for resistance to tetracycline. The different
types of plasmid are shown in Fig.1.1.

Fig 1.1
(a)

Explain what causes several types of plasmid to be formed

[2]

1. Same restriction enzymes

2. all cut DNA have same/complementary sticky ends;;
3. random process by which sticky ends join;;

(b)

Describe two properties of plasmids that allow them to be used as

DNA cloning vectors.
[2]
1. circular, double stranded DNA, so they can form sticky ends
2. separate from main bacterial DNA, easy to isolate
3. contains only a few genes, so small and will not introduce too
many unwanted effects in target cell.
4. Small, so easy to manipulate
5. replicate on its own/independently of the chromosomal DNA, so
will only express the genes of concern and not entire bacterial
genome
6. has an origin of replication, to indicate the start of DNA
replication.

7. contain selectable genetic markers, to help in the screening of

transformed bacteria
8. high copy number so many copies of recombinant DNA can be
obtained
9. MCS

any 2 of the above

(c)

The plasmids are mixed with the bacteria. Some bacteria take up
the plasmids.
(i)

Explain how it is possible to distinguish between bacteria

which have taken up a plasmid with human DNA and those
which have taken up a plasmid without any extra DNA
[4]
1.
2.
3.
4.
5.
6.
7.
8.
9.

(ii)

Insertional inactivation of genes

replica plating;
use of pad/velvet surface to transfer bacteria;
use of agar plate containing ampicillin/no tetracycline and
agar plate containing tetracycline;
in bacteria with human DNA tetracycline gene no longer
functional/not
resistant to tetracycline;
bacteria with human DNA grow on plate with ampicillin/no
tetracycline
but are killed by tetracycline;
bacteria without DNA inserted into tetracycline resistance
gene in plasmid not killed;

Insulin obtained from cattle is described as being strongly antigenic.

Explain why insulin obtained from cattle is more strongly antigenic
than insulin produced by genetic engineering.
1. Genetic engineering produces human insulin/insulin very
similar to human insulin;
2. Difference in amino acids which make up cattle insulin;
3. Therefore will not be accepted as self/treat it as
foreign/rejection;

[2]

[Total:10]
2.

The pedigree below shows the inheritance of an autosomal recessive disorder, sickle
cell anaemia, in human beings.
Fig. 2.1 shows the pedigree obtained using the Southern blot data for two restriction
fragment length polymorphism (RFLP) loci.
RFLP 1 has alleles of either 7 kb or 5 kb and is revealed by digestion with the
restriction enzyme BamHI; RFLP 2 gives either a 4 kb or 3 kb EcoRI fragment.

Fig. 2.1
(a)

Using information from Fig. 2.1, state and explain which RFLP(s) is/are
linked to the diseased gene.
[3]
The BamHI polymorphism/ 5kb RFLP is likely linked to the disease gene;;
where D are the wildtype alleles ;
and d are the recessive disease gene alleles;
The children with the disorder are d 5/d 5 (double homozygotes,
affected);;

(b)

For the RFLP loci identified in (a), suggest and explain which individuals
are likely to be recombinants?
[2]
individuals II-3 and II-10;;
3 is affected but he does not have the RFLP, 10 is not affected but he has
the genotype for the disease.

(c)

Explain briefly the basis behind RFLP.

Different individuals have different sequence of nucleotide bases in their
genome;
Different number/location of restrictions sites;
When digested by restriction enzymes;
Restriction fragments of different sizes or diff no of RF are generated;;
That can be differentiated/separated when;
analysed through an agarose gel;
according to different sizes with the larger fragments migrating at a slower
rate
smaller fragments migrating at a higher rate.

(d)

Suggest two additional applications of RFLP analysis besides genetic

[3]

fingerprinting.

[2]

Paternity test;
identification of human remains;
genetic tests to establish evolutionary relationships
(f)

Explain why genes especially housekeeping genes are seldom used as

markers for genetic fingerprinting.
[2]
House keeping genes are structural genes that encode for important
proteins;
Thus they are highly conserved;
With very few mutations;
Hard to tell related individuals apart.

A 6Kb PCR amplified fragment includes three RFLPs in a family that is affected by a
disease. The restriction sites in the RFLPs are labelled A, B, and C. A and C can be
cut in the mutant allele, but not in the normal allele. B can be cut in the normal allele,
but not in the mutant allele. The restriction cut sites are mapped on the PCR
fragment as shown in Fig 2. 2:

Fig. 2. 2
Mutant = 2 kb, 3kb, 1 kb

Normal = 4 kb, 2kb

A restriction digest was carried out on the PCR products of the family members. The
results of restriction digest were run on an agarose gel.
Based on the agarose gel (Fig. 2.3) and the restriction map Fig. 2.2. What are the
genotypes of each family member?
Indicate on Fig 2.3, homozygous mutant (-/-), heterozygous carrier (+/-), or
homozygous normal (+/+) in the last row (genotype).

+/-

+/-

+/+

-/-

-/-

+/+

Fig. 2. 3
[Total 15]

3.

(a)

As shown in Fig. 3.1 tissue culture is a technique used for artificial

propagation of plants. A callus can be created artificially by placing
meristematic tissue in a suitable growth medium. The callus cells will
develop into plantlets when grown in conditions coordinated by plant growth
substances.

Fig. 3. 1
(a)

(i)

State one advantage of using meristematic tissue in plant tissue [1]

culture.
Stem cells; have high rates of cell division;
Or able to differentiate into different tissues with the appropriate
stimulus/stimuli.

(ii)

With reference to Fig 3.1, explain how a large number of [2]

plantlets can be produced from one explant.
One explant is grown on a culture with suitable nutrients/growth
substances;
To simulate callus development;
Calli are separated and subcultured by;
Put into different cultures for growth into plantlets
Plantlets were then subjected to different rooting and growth
conditions with plant hormones and growth factors;;
high auxin/cytokinin ratio induces root regeneration;
whereas a low ratio promotes shoot induction;
To encourage the growth of plantlets

(iii)

Explain why these plants are described as clones.

Asexual reproduced; from one parent;

[2]

genetically identical to one another and parents;;

(b) Explain the advantages of tissue culture over more traditional methods
of cloning plants, such as taking cuttings or grafting.
[3]
The plants can be genetically manipulated/sexually incompatible
plants can be hybridised;
If protoplast PTC is used;
Plantlets can be grown from a single plant cell;;
Mass production;
Is possible with the faster rate of growth in PTC;
The space required for PTC is lesser compared to traditional
methods;;
(c)

The following experiments were carried out to investigate the effects of plant
growth substances on callus growth:
-

The callus was subcultured into 8 equal batches.

Each batch was placed in separate culture vessels containing
culture medium (with plant growth substances). Each culture
vessel was given a different treatment.

Table 3.1 shows the results of this investigation. Each tick represents one of
the eight batches of callus.
Table 3.1

The batches of callus were left at 25 oC in identical conditions and the

percentage of callus growth was calculated.
Fig 3.2 shows the results of this investigation.

Fig 3.2
(i)

Suggest how abscisic acid affects callus growth.

[1]

It probably results in the development of the callus tissues into

specialised tissues, resulting in no callus growth;;
(ii)

With reference to Fig 3.2, describe the effects of plant growth

substances on callus induction
[3]
The presence of gibberellin will stimulate/increase the callus to
grow;
as seen by the increase in plant growth from 10% at 0 moldm-3
to 90% at 5 moldm-3;;
Abscisic acid works antagonistically with gibberellin; to inhibit
callus growth; as seen by the growth remaining constant at 0%
when abscisic acid is added;;

(d) Plant cells with cell walls removed are termed protoplasts. Cultured
protoplasts can be used in protoplast plant tissue culture for taking up
foreign DNA. Generally, protoplast plant tissues can be used for the transfer
of useful genes such as disease resistance, abiotic stress resistance or
genes of industrial use.
(i)

Describe and explain the benefits of genetic engineering of [2]

plants.
Introduction of novel genes; that are not possible via sexual
reproduction;
Or hybridisation;
Traits that can increase the yield/flavour/nutritional;

(ii)

Discuss on the ethical implications of producing genetically [2]

engineered plants.

Breaking the species integrity;

Scientists playing the role of gods;
the rights of consumers;
to be informed about the food one is eating;
there might be unknown risks for consumers;
such as allergens;
[Total 10]
Planning question
You are required to plan, but not carry out an investigation into the role of carbon
dioxide in living organisms and to determine the compensation point of a plant.
Carbon dioxide is a gas found in air at 0.04%. Carbon dioxide when dissolved in
water forms carbonic acid thus reducing the pH. When bicarbonate indicator
solution is equilibrated with air, it turns red/ orange. Bicarbonate indicator
changes colour with different levels of pH.
Your planning must be based on the assumption that you have been provided
with the following equipment and material, which you must use:

green seedlings
Drosophila fly maggots
bicarbonate indicator solution
distilled water
boiling tubes
test tubes
perforated gauze
rubber bung
syringes of varying sizes
lamp
ruler

Your plan should:

have a clear and helpful structure such that the method you use is able to be
repeated by anyone reading it,
be illustrated by relevant diagram(s) to show, for example, the arrangement of
the apparatus used,
include layout of results tables and graphs with clear headings and labels,
include full details and explanations of the procedures that you would adopt to
ensure that the results obtained were as quantitative, precise and reliable as
possible.
[Total: 12]

4.

Background Max 2 marks

Carbon dioxide is a gas found in air at 0.04%

Carbon dioxide dissolves in water to to form carbonic acid thus reducing the pH.

When bicarbonate indicator solution is equilibrated with air, it turns orange/red

Bicarbonate indicator changes colour in different levels of pH

In respiration, glucose + oxygen to produce carbon dioxide + water + energy

Photosynthesis, carbon dioxide + water produces glucose + oxygen

The point where carbon dioxide released by plant from respiration, = carbon
dioxide absorbed by plants for photosynthesis is the plants compensation point

Rate of photosynthesis is also affected by light intensity. The compensation point

is that light intensity where PS= respiration, at atmospheric CO2

Hypothesis:
Living organisms produce carbon dioxide during respiration and uses CO2 during
photosynthesis. Plants photosynthesize and the compensation point of a plant is
the light intensity at which production of CO2 = consumption of CO2
Variables (1 mark)
Independent variable: amount of CO2 present
Dependent variable: colour change in bicarbonate indicator
Controlled variable: rate of respiration and photosynthesis, temperature, age of
and no of living organism
Precautions: (1 mark)
Bicarbonate solution is corrosive, Wear gloves when handling and handle with care.
Method (max 6 marks)
1.

2.

3.

4.

5.

6.

Rinse 3 boiling tubes with distilled water, and then with bicarbonate indicator
solution
Using a syringe place 3-5cm3 of bicarbonate indicator solution into each boiling
tube.
Carefully place a piece of perforated gauze in each boiling tube so that it is just
above the indicator solution.
Place a rubber bung into the first boiling tube.
Carefully place 3 green bean seedlings onto the gauze in the second boiling
tube and seal with a rubber bung.
Carefully place 3 maggots onto the gauze in the third boiling tube and seal with

a rubber bung.
7.

8.

9.

10.

11.

Place the three boiling tubes near a light source and note the distance (A) from
the light source, using the ruler.
Check that the colour of bicarbonate indicator solution in each boiling tube is
red/orange at the start of the experiment.
Leave the tube for at least 30mintues, comparing the colour of each indicator
solution every ten minutes.
When the colours look different in all three tubes, note the final colour of the
indicator in each of the 3 boiling tubes.
Record the results in a table as shown below:
Time/ mins

Colour of indicator after 30mins

Green seedlings

Drosophila fly
maggots

Control
(0.04% CO2)

0
10
20
30

Distance from light source: _______X_______ cm

12.

The tube without living material serves as the control to show that CO2 is
produced by living organisms as well as to serve as the indication of point of
compensation in the respiring green seedlings, where there is no net exchange
of CO2

13.

The time at which the colour of all 3 tubes is different indicates

14.

Use the control tube as the reference for determining compensation point.

15.

Set up 3 boiling tubes as in step 5.

16.

Place the boiling tubes at the same distance from the light source as in step 7.

17.

18.

19.

Leave the tube for 30minutes, checking the colour of the solution every ten
minutes.
After each colour check, move the light source 10cm further from the tube.
Record your results in a table below;
Time/ min

Distance from light

Colour of indicator

source/ cm

Tube 1

Tube 2

Tube 3

0
10
20
30

20.

Repeat steps 15-19 two times.

Expected results: (1 mark)

The compensation point of the green seedlings is that distance when the indicator is
orange / red, indicating that there is no net change in CO2. At this distance, the light
intensity is at the level where the rate of photosynthesis is equals the rate of
respiration.
Suggestions (1 mark)
Limitations:
Observation of colour change is subjective.

Varying light intensity by increasing distance is not a very accurate method to

determine the compensation point

Improvements:
Use of colorimeter to track the colour change that would match atmospheric CO2
concentration

(a)

Use a light meter to measure the light intensity at each interval to determine the
exact light intensity of the compensation of the seedling.

Explain the role of stem cells in the treatment of a named disease.

Description of the disease (max 2 marks)

Sickle cell anemia

Mutation of coding sequence from CAA to CAT;

resulting in substitution of valine for glutamic acid;

Haemoglobin sickle shaped under low oxygen condition;

Homozygote suffers from anemia;

[6]

Or Cystic Fibrosis;

Autosomal recessive disorder;

Deletion of triplet bases in chromosome 7;

Loss of amino acid phenylalanine;

Results in a loss of function of transmembrane regulator CFTR

protein;

Inability to pump chloride ions out of the cell;

Loss of water in the extracellular environment;

Production of sticky mucus

Accumulation of bacteria;

Clogged up respiratory tract;

Or Severe Combined Immuno Deficiency

Sex linked recessive disorder;

Diseased allele coding for non functional common gamme chain;
Immune cells are not able to differentiate and respond to pathogenic
invasion;
Child susceptible to opportunistic infections;
Autosomal recessive disorder
Diseased allele coding for non functional enzyme adenosine
deaminase
Not able to degrade deoxynucleoside adenosine, resulting in a build
up of these substances;
Toxicity to the immune cells;

Treatment (max 2 marks)

Isolation of correct gene

Hematopoietic stem cells extracted from the bone marrow or a valid

source of stem cells;

Inserted with the corrected functional gene through recombination;

Restriction digest

Choice of vector;

Select for stem cells that have successfully taken up the functional
gene;

Inject the stem cells back to the patients bone marrow/ target
organ;

Ex-vivo therapy;

Nature of stem cells (max 2 marks)

(b)

It serves as a host cell;

High replicative potential;

The ability to make multiple clones within a short time;

Replicating the therapeutic gene several times and passing them

down to the other stem cells;

Production of normal haemoglobin to mask the effects of the

recessive allele;

Multipotent;

Ability to differentiate into a variety of cell types;

Discuss the process of polymerase chain reaction.

Denaturation: Heating of DNA at 94C (accept: 90 - 100C);;

causes denaturation of DNA double helix hydrogen bonds

between complementary bases on DNA molecule break to form
single stranded DNA;

Annealing: This is done at 54C (accept: 30 - 65C);

Primers OR oligonucleotides anneal to the single-stranded DNA at

the 3 ends of the target sequence by complementary base
pairing;;

Elongation This is done at 74C (accept: 60 - 75C);;

Optimal functioning temperature of Taq DNA polymerase attaches to

[6]

one end of each primer and adds nucleotides to the 3 end of each
primer;

New strands of DNA are synthesized, complementary to the

template DNA molecules (target DNA);
Note that the bold and underlined phrases are compulsory to
be awarded the mark.

(c)

Discuss how molecular techniques of DNA analysis can be used to [8]

provide evidence for molecular homology.

quantify the degree of nucleotide/ amino acid similarity;

analyzing their VNTR profiles;

between the different organisms with the use of southern blotting

techniques;;

Spontaneous mutation

Molecular homology: Molecular evidence for evolution includes

similarities at the gene, proteins and chromosomal levels;;

Gene sequence differences can be placed in a timeframe derived

from mutation rates;;

Closely related organisms exhibit high percentage of similarity

between their nucleotide sequences or VNTR profiles;;

A large proportion of their sequences are identical as the closely

related species have a short time scale to accumulate independent
mutations as divergence event took place recently;;

Use different restriction enzymes

Restriction fragment length polymorphism: refers to a molecular

technique that exploits variations in homologous DNA sequences;;

Differences between samples of homologous DNA molecules of

different species, specifically differing locations of restriction enzyme
sites;;

would generate different sized molecular fragments when subjected

to restriction digest of the specific restriction endonuclease;;

On the contrary, closely related species who exhibit high percentage

of similarity between their nucleotide sequences, due to low
mutation rate would have similar RFLP profile;;

The different fragments would be separated via the process of gel

electrophoresis;

Whereby the migration distance of the fragment is inversely

proportional to the fragment size;

Nucleic acid hybridisation: The process of establishing hydrogen

bonding between specific two complementary strands of nucleic
acids; (technique of Southern blotting)

In this case, a complementary oligonucleotides/ gene probe

sequence tagged with a molecular marker of either radioactive or
fluorescent molecules. Detection of the presence of target DNA
sequences is possible as the probe sequence is complementary to
the target DNA;

Autoradiogarphy

Subject nitrocellulose paper to X ray, DNA fragments appears as

black bands

Use of NaOH to denature DNA, use of capillary action of

nitrocellulose paper
[Total: 20]