CATHOLIC JUNIOR COLLEGE

JC2 PRELIMINARY EXAMINATIONS

Higher 2


STUDENT
NAME


CLASS 2T
INDEX
NUMBER



BIOLOGY 9648/03
Applications Paper and Planning Question MONDAY 02 SEPTEMBER 2013
Paper 3 2 hours

Additional Materials: Answer Paper

READ THESE INSTRUCTIONS FIRST
Write your index number and name on all the work you hand in.
Write in dark blue or black pen on both sides of the paper. [PILOT FRIXION ERASABLE PENS ARE NOT ALLOWED]
You may use a soft pencil for any diagrams, graphs or rough working.
Do not use staples, paper clips, highlighters, glue or correction fluid.

Answer all questions.

At the end of the examination, fasten all work securely together.
The number of marks is given in brackets [ ] at the end of each question or part of the question.






























This document consists of 24 printed pages.
[Turn over
For Examiners Use
1 [15]
2 [10]
3 [15]
Planning
4 [12]
Free-response
5a [7]
5b [6]
5c [7]
TOTAL
72
2
Answer all questions.

1 (a) Explain what is meant by a restriction enzyme. [2]

[L1]
Any two points:
1. A restriction enzyme is an endonuclease which detects palindromic restriction site which is
4-6 base pairs in length.
2. Restriction enzymes bind to the restriction site, i.e. a specific sequence of bases of DNA and
hydrolyse the phosphodiester bonds within polynucleotide strands of the DNA molecule.
3. They hydrolyse covalent phosphodiester bond of both DNA strands, often in a staggered way
creating single-stranded ends called sticky ends. Some restriction enzymes make a simple
cut across both strands at a single point, forming blunt or flush ends.

(b) Fig. 1.1 illustrates part of a DNA sequence that is recognised by a restriction enzyme.
Complete the sequence by filling up the blanks in Fig. 1.1. [1]








Fig. 1.1

[L2]








(c) What do the 5 and 3 ends in Fig. 1.1 represent? [2]

[L2]
Any two points
1. The 5 end represents a phosphate group linked to the sugar-phosphate backbone via carbon
5 of deoxyribose sugar.
2. The 3 end represents the hydroxyl group linked to the sugar-phosphate backbone via carbon
3 of the deoxyribose sugar.
3. Both strands of a DNA molecule are antiparallel. As seen in Fig. 1.1, the top strand of a
DNA molecule runs from 5 to 3direction and the complementary bottom strand runs
from 3 to 5 direction.

5 A G C _ _ _ 3
3 _ _ _ _ _ _ 5

5 A G C G C T 3
3 T C G C G A 5

3
Fig. 1.2 illustrates a typical plasmid used in genetic engineering.











Fig. 1.2

(d) The plasmid shown in Fig. 1.2 lacks two features that are essential for genetic engineering.
Add these two features to the plasmid in Fig. 1.2 and label them. [2]

[L1]
1. Label ori (origin of replication)

[L2]
2. Label Multiple Cloning Site / any restriction site with a relevant example within either of the
antibiotic resistance genes.

(e) Explain the significance of one of the features added in part (d). [1]

[L2]
1. Presence of origin of replication within the plasmids allow the recombinant plasmids to
multiply independently from the host DNA and make many copies of the inserted
gene/gene of interest.
2. Multiple cloning sites / Restriction site with a relevant example. This serves as a convenient
site into which foreign DNA may be inserted.

Ampicillin resistance
gene
Tetracycline resistance
gene
4
A dot blot is a simplified version of Southern Blotting. It can be employed to test for the presence of gene
mutations. In this molecular technique, undigested DNA is applied directly onto a membrane through a
vacuum. These dots containing DNA will be hybridised to a sequence-specific oligonucleotide probe and
be subjected to autoradiography. Dots which successfully hybridise to a probe will be seen as a dark dot
on the autoradiogram.

(f) Describe two key differences between the process of obtaining a dot blot and a Southern blot. [2]

[L3]
Any two points
1. For the dot plot, the entire undigested DNA is being used, whereas for Southern Blot, DNA
digested with restriction enzymes are being used.
2. For the dot plot, DNA can be transferred directly onto a membrane, whereas for Southern Blot,
DNA fragments must go through gel electrophoresis first before transference.
3. For the dot plot, transfer of DNA is through a vacuum, whereas for Southern Blot, transfer
of DNA is through capillary action.
Also accept: if student only stated the difference in dot plot without a point-by-point
comparison.

Cystic fibrosis (CF) has been linked to several mutations. Two probes have been synthesized to
hybridise with a mutated sequence associated with CF as well as the corresponding normal sequence.
The dot blots of nine patients clinically diagnosed with CF are shown in Fig. 1.3.


Probe for normal sequence
Probe for mutated sequence
CF patient

Adapted from: http://www.glowm.com/resources/glowm/cd/pages/v5/ch076/framesets/006f.html
Fig. 1.3 Dot plot of 9 CF patients.

(g) State one mutation linked to cystic fibrosis. [1]

[L1]
1. delta F508
OR
1. Deletion of codon 508 of the CFTR gene.

(i) Account for the presence of two dots in individuals 1, 7 and 9. [2]

[L3]
1. Human cells are diploid / have homologous chromosomes.
2. These individuals are heterozygous for the sequence, i.e. they have a copy of the normal
sequence as well as a copy of the mutated sequence.

(j) With reference to Fig. 1.3, explain why the mutation associated with CF is most likely to be
recessive. [2]

[L2]
1. Most CF patients / 5 out of 9 patients/ CF patients 2, 3, 4, 5 and 8 have only one dot shown,
2. indicating that 2 copies of the CFTR mutated allele is needed for the manifestation of cystic
fibrosis.
[Total: 15]
5
2 A patient was found to have a defective gene that results in the failure to produce an important chemical
substance, chemical substance X.

Three experiments were carried out to compare three different techniques for introducing chemical
substance X into such patients:

Experiment 1: The chemical substance X was injected directly into the patient.

Experiment 2: The gene coding for chemical substance X was packaged into a viral vector that was
modified to render it harmless. The viral vector containing the gene was injected into the
patient.

Experiment 3: The gene coding for chemical substance X was placed into a protein capsule, which was
then inserted into the tissues of the patient.

Fig. 2.1 shows the results from these experiments.

Experiment 2
Experiment 3
Experiment 1
Direct injection of chemical
substance X
Experiment 2
Injection of viruses carrying
gene coding for substance X
Experiment 3
Injection of protein capsule
containing gene coding for
substance X

Fig. 2.1

(a) (i) Describe the results of the three experiments. [3]

[L2]
1. For Experiment 1, direct injection of substances results in a rapid / sudden / sharp
increase / rise in the concentration of the substance in the tissues, followed by a rapid
decrease / short time in the harmful concentration range / effective concentrate range.

2. For Experiment 2, injection of viruses carrying the gene for the substance / viral gene vector
shows a slower increase / rise of the concentration of the substance in the tissues; the
concentration decreases gradually / remains in the effective/harmful range for a longer
period of time

3. For Experiment 3, using protein capsule containing the gene, results in the slowest
increase / rise in the concentration of the substance; the substance remains at the
effective concentration range over the longest period of time / (accept: idea of)

Additional Note: Both experiments 1 and 2 results in having the substance at concentrations
that can cause harmful side effects but not for experiment 3.
6
(ii) Using the information in the graph, suggest two advantages and one disadvantage of the
protein capsule method, as compared to the other methods.

[L3]
Advantage: [2]
1. The chemical substance never reaches / does not reach harmful levels / concentration
that may cause possible harmful side effects to occur, hence less likely to cause / little
or no damage / harm to the organism.

2. The chemical substance remains longer in the effective concentration range, hence can
provide longer relief from the symptoms / needs less frequent treatment.

Disadvantage: [1]
3. The concentration of substance in tissues increases / appears and reaches effective range
very slowly, hence treatment takes a longer time to take effect / to be become effective.

Duchenne muscular dystrophy (DMD) is a common lethal X-linked human genetic disease caused by
the absence of the protein dystrophin in muscle fibres.

Two possible treatments have been proposed to correct the deficiency of dystrophin:
(1) Transplant normal cells from genetically compatible donors into the patient;
(2) Gene therapy.

Gene therapy involves inserting the gene coding for dystrophin into an adenovirus and injecting the
recombinant adenovirus into the patients muscle.

(b) Gene therapy using the recombinant adenoviruses did not provide a permanent cure for DMD. The
trials showed that the transgene expression declined after about 2 weeks and was negligible after
only 4 weeks. Explain this phenomenon. [2]

[L2]
1. Transfected / modified muscle cells differentiated and non-dividing cells, are replaced
when worn-out / die. The therapeutic gene that the transfected muscle cells carry is hence lost
when these muscle cells die.

2. Transgene / gene coding for dystrophin fails / does not integrate into the genome / remains
extrachromosomal, affecting the extent and effectiveness of the gene expression.

(c) Suggest a suitable vector for delivery of genes designed to kill cancer cells. Explain your choice. [2]

[L3]
1. using retroviruses / modified retroviruses (accept: modified HIV)
Reject: adenovirus since it can infect all kind of cells)

Any one of the following:
2. Can only infect dividing cells, thus can target actively dividing cancer cells
3. Cannot target non-dividing cells / cells that have stopped dividing, hence will not damage or
harm to surrounding normal cells

Reject: retrovirus ability to integrate gene into genome, leading to long-term stable gene
expression this is out of context

[Total: 10]
7
3 (a) Genetic engineering has been used to improve the quality and yield of many crop plants.
One method of genetic engineering involves modifying Ti plasmid from a bacterium, Agrobacterium
tumefaciens, and transferring the modified Ti plasmid containing the transgene into crop plants. This
method has been used for the production of Bt corn.

(i) State one other method that can be used for the genetic engineering of crop plants. [1]
[L1]
1. Gene gun.

(ii) The Ti plasmid is usually modified to contain an antibiotic resistance gene (e.g. ampicillin
resistance gene).

Explain the purpose of this modification. [2]

[L2]
1. The antibiotic resistance gene acts as a selection marker.
2. To identify the Agrobacterium tumefaciens colonies that has been successfully
transformed with recombinant Ti plasmid containing the transgene/foreign gene.

The successfully genetically engineered crop plant cells may be stimulated to grow into plantlets
using tissue culture.

(iii) Describe how a plantlet may be produced from cells in tissue culture. [2]

[L1]
1. Plant cells may be induced to grow into a callus by adding specific ratio of plant growth
regulatory factors such as auxin and cytokinin and addition of nutrients;
2. The callus is stimulated to develop shoots and roots by altering the concentration of
auxin and cytokinin such that it is at the specific ratio for shoot and root growth;

(iv) Describe two advantages of using tissue culture to clone genetically modified plants. [2]

[L3]
Any two of the following:
1. Once a new gene / transgene is successfully introduced / transformed into a plant cell,
the transformed / modified plant cell can be grown into a whole plant and then cloned
to produce many genetically identical plants, all containing that transgene (GM plants)
2. Allows for rapid production of large numbers / mass production of GM plants from just
one or a few stock plants
3. Production of virus-free / disease-free plants by selecting only meristematic tissue of
the GM plant / explants containing meristematic tissue for propagation;
4. Allows all year round production of GM plants / can be produced at any time of the year /
not affected by seasons higher productivity / annual yield
5. Reliability since quality control can be achieved by standardising growing
conditions such that batch after batch of standard GM plants can be produced.
6. Land area needed for initial growth of these GM plants through tissue culture is much
less, hence saving valuable arable space
7. Prevents the loss of transgene due to sexual reproduction / through gamete formation;
preservation of transgene within the entire crop
8. Prevents GM plants from being completely wiped out by changing environmental factors;
as the plants are always available as stored in the cold storage;
9. AVP

8
(b) A number of different crop plants have been genetically engineered to express a gene for an
insecticidal toxin (Bt toxin) from a bacterium, Bacillus thuringiensis.

The Bt toxin binds to cell membranes of the cells in the insect gut. The receptor for the toxin has
been identified as a membrane-anchored protein encoded by the cadherin gene. Bt toxin forms ion
channels that cause an efflux of potassium ions from the insect gut cells, leading to cell lysis.

The cotton bollworm, Helicoverpa armigera, is one of the most serious insect pests of cotton in Asia,
Australia, and Africa. A major deletion of the cadherin gene was associated with a high level of Bt
toxin resistance in H. armigera. Three alleles (r1, r2, and r3) of the cadherin gene in the resistant
strain have been identified:
r1 allele lacks 24-bp
r2 allele has a 202-bp deletion
r3 allele has a 126-bp deletion

(i) Explain how deletions of the cadherin gene could confer resistance to Bt toxin in H. armigera.
[4]
[L2]
1. Deletion of the cadherin gene results in the removal of corresponding mRNA codons;
2. Leading to missing / removal of certain amino acids in the polypeptide chain / a
truncated / shorter polypeptide chain
3. Affecting the overall folding of polypeptide chain / changes in protein / receptor protein
conformation
4. Bt toxin unable to bind to cell membrane, unable to exert its effect, hence prevent / no
cell lysis

A survival test was conducted on larvae of different genotypes to assess their resistance to Bt toxin.
F
r
e
q
u
e
n
c
y

o
f

s
u
r
v
i
v
a
l
Cadherin genotype of larvae
Legend:
s - s allele coding for normal
cadherin gene
r1 - r1 allele lacks 24-bp
r2 - r2 allele has a 202-bp deletion
r3 - r3 allele has a 126-bp deletion

Fig. 3.1

(ii) Suggest why larvae of genotype ss was included in this test. [1]

[L3]
1. It acts as a negative control to ensure that any survival of the larvae in Bt cotton is due
to the effect of r1, r2 or r3 alleles and not due to any other factors.

(iii) Describe the effects of the genotypes r1r1, r1r3 and r3r3 on the resistance of larvae to Bt toxin.
[3]

[L2]
1. r1r1 increased the resistance of larvae by a frequency of 0.38 as compared to the ss
genotype;
2. r1r3 conferred the greatest increase in the resistance of larvae, by conferring an increase
in resistance by a frequency of 0.56 as compared to the ss genotype;
3. r3r3 was the least effective and increased the resistance of larvae only by a frequency
of 0.8 as compared to the ss genotype.
[Total: 15]
9
4 Planning question

You are required to investigate the rate of photosynthesis on a certain species of algae,
Scenedesmus Chlorella.

Algae are tiny and difficult to work with directly in water. Hence, sodium alginate is usually used to
trap large numbers of algal cells into agar beads so that the cells are all contained in one piece.


Source: Copyright Science & Plants for Schools: www.saps.org.uk

Fig. 4.1

Hydrogen carbonate indicator can be used to measure the rate of photosynthesis as it is very
sensitive to changes in carbon dioxide level. The indicator is red when the amount of CO
2
in
the solution is in equilibrium with atmospheric air, becomes orange to yellow with
increased CO
2
in the solution and changes from red through magenta to deep purple as
CO
2
is removed from the solution. This is represented in the diagram below.

PURPLE MAGENTA RED ORANGE YELLOW
Carbon dioxide
concentration
0.04% CO
2
Atmospheric air
Low
High

Source: Copyright Science & Plants for Schools: www.saps.org.uk
Fig. 4.2

10

The colour changes in hydrogen carbonate indicator can be measured objectively using a
colorimeter. A colorimeter measures the absorbance of the solution in the cuvette. The
cuvette holds the working solution.

0.20A Absorbance
reading
Cuvette
holder
Cuvette


Source: Copyright Science & Plants for Schools: www.saps.org.uk
Fig. 4.2

Different colours absorb different wavelengths of light to a different extent. The darker the
colour, the greater the absorbance value (A) will be. The known absorbance values (A) of
the following colours of the hydrogen carbonate indicator are shown in the table below.

Table 4: Table of known absorbance values

Color of Hydrogen Carbonate Indicator Absorbance of Colorimeter/ A
Yellow 0.10
Orange 0.20
Red 0.30
Magenta 0.50
Purple 0.70
A = Absorbance units

11

Using all the information above, you are required to plan, but not carry out, an investigation into:

The effect of light intensity on the rate of photosynthesis in the algae,
Scenedesmus Chlorella

Your planning must be based on the assumption that you have been provided with the following
equipment and materials, which you must use:

150 Scenedesmus Chlorella algal beads
100 cm
3
of hydrogen carbonate indicator solution containing 387 parts per million (ppm) CO
2
, which is
at equilibrium with the 0.04% atmospheric CO
2
.
7 cuvettes
Colorimeter with a digital display to 2 decimal places and a cuvette holder.
7 glass tubes with screw-on-lids. The walls of these tubes are transparent.
A 150 W bench lamp which emits very little heat.
5 cm
3
syringe
Stop watch.
Ruler.

Your plan should:

have a clear and helpful structure such that the method proposed can 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]
12
ANSWERS

Planning question

MARKS TIME
PLAN 6
AIM
0 0.5
HYPO
0 1
INTRO
2 3min
VARIABLES
2 3
APPARATUS
0 0.5
PROCEDURE
5-7 10.5
SAFETY
1 1.5
RESULTS
1 2.5
INTERPRETATION
1 1.5
Correct Use/
Scientific Reasoning
1
MAX 12 30


13

AIM

AIM
MARKS TIME
0 0.5 min

APPROACH

AIM can be derived directly from the Question Paper:
you are required to plan, but not carry out, an investigation into the effect of light intensity on
the rate of photosynthesis in a certain species of algae, Scenedesmus Chlorella.

This experiment aims to investigate the effect of light intensity on the rate of
photosynthesis in a certain species of algae, Scenedesmus Chlorella.

HYPOTHESIS
HYPO
MARKS TIME
0 1 min

APPROACH

State your STAND with reference to the AIMS.
State the General Hypothesis.
State the Hypothesis specific for experiment.

[General Hypothesis]

Increase in light intensity increases the rate of photosynthesis in Scenedesmus
Chlorella.
Note:
The rate of photosynthesis will continue to increase until the light saturation point is reached.
After the light saturation point, light is either no longer the limiting factor.

[Hypothesis specific for experiment]

The closer the distance of the Scenedesmus Chlorella algal beads to the
bench lamp, the higher the absorbance value being shown on the colorimeter,
14

INTRODUCTION [1 - 4 MAX 2m]
INTRO
MARKS TIME
2 3min

APPROACH

Give THEORETICAL EXPLANATIONS to SUPPORT HYPOTHESES made.

LIGHT
=> CO
2
intake
=> Hydrogen Carbonate turns RED MAGENTA PURPLE
=> Absorbance Value on Colorimeter
=> RATE

NO NEED to give additional information that is UNRELATED to the hypothesis.
e.g. The types of chlorophyll pigments present in the plant or the theory of chemiosmosis.

LIGHT
=> CO
2
intake
=> Hydrogen Carbonate turns RED MAGENTA PURPLE
=> Absorbance Value on Colorimeter
=> RATE
1] As light intensity is increased, more light is absorbed by the photosynthetic pigments
located at the thylakoid membranes of the chloroplasts.
2] This would increase the rate at which the electrons are being excited from the primary
reaction centres/ primary pigment molecule of the photosystems, resulting in a faster rate of
production of ATP and NADPH, which are needed in the Calvin Cycle.
3] This makes a greater amount of ATP and NADPH available for CO
2
fixation by RuBP in the
Calvin Cycle.
4] This leads to a higher rate of absorption of CO
2
from the hydrogen carbonate indicator,
leading to a higher absorbance value on the colorimeter.

15

VARIABLES [5 - 9 MAX 2m]

VARIABLES
MARKS TIME
2 3 min

APPROACH

Independent variable: A variable that can be changed by the investigator [i.e. light intensity].
*Clearly state HOW you would vary it and at least 5 independent variables
at fixed intervals must be given.

Dependent variable: The variable being investigated in this experiment [i.e. rate of
photosynthesis].
*Clearly state HOW it is being measured.

Controlled variables: Ensure that all OTHER variables are controlled and
*Clearly suggest HOW you would control these variables.

Negative Control: Ensures that any change in the dependent variable is due to the independent
variable
and NOT any other variable.

Positive Control: Ensures that the reagents and apparatus used in the experiment are in working
condition.

PRIORITY goes to Independent Variable, Dependent Variable and Controlled Variable.


5] Independent Variable:
Light intensity,
Varied by the distance of the bench lamp from the algal beads 5cm, 10cm, 15cm, 20cm,
25cm.

6] Dependent Variable:
Rate of photosynthesis,
Measured by the absorbance value shown on the colorimeter.

7] Controlled Variables:
a] Number of agar beads used,
b] Duration of experiment,
c] Concentration of CO
2
in hydrogen carbonate solution, controlled by ensuring that the
hydrogen carbonate indicator is red at the start of each experiment.
d] Temperature of the surroundings kept constant by holding experiment in a thermostatically
regulated room and through use of bench light that emits very little heat.

8] Negative control:
Set up the glass tube placed in a dark box/ wrapped in black paper.
This is to ensure that the change in absorbance values in the colorimeter is due to light
and not other environmental factors.
OR
This is to ensure that the change in colour of the hydrogen carbonate indicator is due to
light and not other environmental factors.
16


9] Positive control:
Set up glass tube that has 0cm distance between the bench lamp and the glass tube
containing the algal beads.
This simulates 100% light reaching the algal beads.
This is to ensure that all reagents and apparatus used in the experiment is in working
condition.

APPARATUS

APPARATUS
MARKS TIME
0 0.5 min

APPROACH

DO NOT REWRITE all apparatus given in question.

You cannot request for a larger quantity of a given apparatus.


As given in question, with addition of a dark box/ black opaque paper with 0% light
transmission.

17
PROCEDURE [11 - 18 MAX 6m]

PROCEDURE
MARKS TIME
5-7 10.5 min

APPROACH

ALL apparatus should be mentioned/ used.

QUANTITY used should be stated.

PURPOSE of the step should be stated [when applicable].

REPLICATES VS REPEATS

REPLICATES - additional measurements of the dependent variable made to CALCULATE
AVERAGES.

REPEATS - additional runs of the experiment made to ensure results are REPRODUCIBLE.


Basic Experimental Procedure [11 - 21 MAX 5m]

10] Label 7 glass tubes and 7 cuvettes A, B, C, D, E, positive control and negative control.

11] Transfer 10 agar beads of Scenedesmus Chlorella to all 7 glass tubes.

12] Using a 5 cm
3
syringe, transfer 3 cm
3
of hydrogen carbonate indicator to all 7 glass
tubes.

13] Darken the laboratory to ensure that there is no external light source influencing the rate
of photosynthesis.

14] Allow 2 minutes for the algal beads to equilibrate to the experimental conditions [e.g.
hydrogen carbonate solution].

15] Using a ruler, position the glass tubes at their respective distances from the bench lamp, in
the table shown below.

Glass
Tube
A B C D E Positive
control
Negative
control
Distance
of Tube
from
bench
lamp/
cm
5 10 15 20 25 0 0
Glass
Tube
Transparent Transparent Transparent Transparent Transparent Transparent Placed in
dark box/
Fitted with
black
opaque
paper
Cuvette
A B C D E Positive
control
Negative
control
18

16] Turn on the bench lamp and start the stop watch.

17] Turn off the bench lamp when the time reaches 5 minutes.

18] Fill the cuvettes with the indicator from their respective glass tubes, and record their
respective absorbance values from the colorimeter.

Labelled Diagram [19 MAX 1m]

19]

1 10 5 15 20 25 30
Positive
Control
Negative
Control
A C
B D
E
150W
bench
lamp
Glass
Tubes
hydrogen
carbonate
solution
Scenedesmus
Chlorella
algal beads
Ruler/cm

Modified from Source: Copyright Science & Plants for Schools: www.saps.org.uk

Ensure Reliability of Experiment [20 - 21 MAX 1m]
20] Obtain 3 replicates, replenishing the set-up with 3 cm
3
of fresh hydrogen carbonate indicator
solution between each replicate.

Calculate the average absorbance reading for each glass
tube.

21] Repeat steps 10- 19 [the entire experiment] using another 10 agar beads to ensure
reproducible results.

22] Plot a graph showing the relationship between light intensity and rate of photosynthesis.

19
SAFETY [23 MAX 1m]

SAFETY
MARKS TIME
1 1.5 min

APPROACH

Risk and Precaution must be stated.
DO NOT write blindly Read question carefully.


23]
*Risk: Glass bottles are fragile and glass bits can cause injury when bottles are broken.
*Precaution: Handle glassware carefully by placing them away from the edge of the
workbench
Rationale: So as not to break them and accidentally injure oneself.

Reject: Care not to touch hot lamp near the bulb so as not to get scalded.

RESULTS [24 25 MAX 2m]
RESULTS
MARKS TIME
1 2.5 min

APPROACH

Table should contain INDEPENDENT VARIABLE [Light] and DEPENDENT VARIABLE
[Rate of P/S]

VALUES/TRENDS MUST BE SHOWN to SUPPORT HYPOTHESIS.




24] Table showing relationship between distance from lamp/cm and absorbance reading/ A

Tube
Distance from
bench lamp/
cm
Absorbance reading from colorimeter/ A
Average
reading /
A
Reading 1 Reading 2 Reading 3
Positive
Control
0 0.70 0.71 0.69 0.70
A 5 0.60 0.61 0.59 0.60
B 10 0.50 0.51 0.49 0.50
C 15 0.40 0.41 0.39 0.40
D 20 0.30 0.31 0.29 0.30
E 25 0.20 0.21 0.19 0.20
Negative
Control
NIL 0.10 0.11 0.09 0.10
Reject: No values in the table.
Reject: If average was not calculated.


20
25] Graph showing relationship between distance from lamp/cm and absorbance reading/ A
Distance from
bench lamp/ cm
Absorbance
reading/ A

*PRIORITY: TREND must be shown.
*May include values from table but do not spend too much time scaling the points.
Also accept: Straight line

INTERPRETATION [26 MAX 1m]

INTERPRETATION
MARKS TIME
1 1.5 min


APPROACH

Reference MUST be made to RESULTS.
i.e. As shown by the graph, ..

SUPPORT Hypothesis.



26] As shown by the graph, the smaller the distance between the bench lamp and
the algal beads, the higher the absorbance reading.

This supports the hypothesis that an increase in light intensity increases the
rate of photosynthesis.

27] Correct use of scientific terms/ correct scientific reasoning.

21
Free-response question

Write your answer to this question on the separate answer paper provided.

Your answer:

should be illustrated by large, clearly labelled diagrams, where appropriate;
must be in continuous prose, where appropriate;
must be set out in sections (a), (b) etc., as indicated in the question.


5 (a) Describe the unique features of stem cells and explain the normal functions of stem cells in a living
organism. [7]

[L1]
Planning outline:
Qn Key
Wd
Level Example
Characteristic Stem
Cells
Function Living Org
Ans Key
Wd
1] Growth of
entire
organism
1]
Embryonic
stem cells
2] Unspecialised Are pluripotent /
totipotent;
To become
3] Can divide and
grow indefinitely
create organs and
specialised layers
4] able to
Differentiate
Give rise to 3 germs
layers: endoderm,
mesoderm and
ectoderm;
5]
Replenishment
due to wear and
tear
Adult SC
5]
Hematopo
etic stem
cells/
blood
stem cells
6] Unspecialised Are pluripotent;

7] Can divide and
grow indefinitely
to replenish numbers
Neural /
Epidermal
/
Epithelial.
8] able to
Differentiate
Can differentiate into all
blood cell types;
To form granulocytes /
monocytes /
erythrocytes /
megakaryocytes

22
(b) Explain what is meant by restriction fragment length polymorphism (RFLP) and how it can be
detected. [6]

[L2]
What is meant by RFLP [max 2m]

1. Restriction fragment length polymorphism refers to inherited genetic differences in a single
locus found within a population.
2. These differences may be due to mutations such as substitution, addition or deletions; or
due to differences in the number of variable number of tandem repeats (VNTR) or short
tandem repeats (STR).
3. These genetic differences in nucleotide sequences lead to a difference in the number or
location of restriction sites between individuals, which will leads to a variation in the length
and/or number of restriction fragments formed when the same specific restriction enzyme
is used on different individuals.

How it can be detected [max 4m]
RFLP between individuals can be detected by RFLP Analysis, which consists of restriction digest,
gel electrophoresis and Southern Blotting (DNA transfer, nucleic acid hybridisation and
autoradiography).

4. The same specific restriction enzyme is first used to hydrolyse the genomes of different
individuals within a population, resulting in the production of different restriction fragments
lengths and/or numbers;
5. The digested DNA samples are then separated by their restriction fragment lengths by
agarose gel electrophoresis;
6. Restriction fragments are then transferred from the agarose gel to the nitrocellulose
membrane by capillary action;
7. The single stranded DNA fragments will then undergo nucleic acid hybridisation, where the
DNA fragments on the nitrocellulose membrane are hybridized with specific radioactive
labelled probes that are complementary to the polymorphic locus. The excess probes are
washed away.
8. The membrane is laid over a photographic film and autoradiography is then used to
visualize the relative positions of the DNA fragments bound to their probes.



23
(c) Using a named example, explain how RFLP analysis can be used for disease detection. [7]

[L3]

Detecting Sickle Cell Anaemia using DdeI Restriction Site

Note: Candidates may choose to illustrate
points using labeled drawings.
1 For Sickle cell anemia, base pair substitution of the normal beta-globin
gene (Hb
A
) from CTT to CAT results in a harmful recessive allele, Hb
S
. The
mutation results in the loss of a DdeI restriction site.
2 Restriction digest of Hb
A

using DdeI would result in 2
restriction fragments, 175
kb and 201 kb. Restriction
digest of Hb
S
would result
in only 1 restriction
fragment of 376kb.
376kb
A]
Hb
A
Hb
S
DdeI
175kb 201kb
CHR. 11
CHR. 11

3 Gel electrophoresis is used to separate the restriction fragments based on their
lengths. Negatively charged DNA will move towards the anode, with shorter
fragments moving further.
4 The separated restriction fragments are being denatured by an alkaline
solution and the single stranded DNA molecules are transferred to a
nitrocellulose membrane via capillary action. Single stranded DNA
molecules cross-link to nitrocellulose membrane.
5 Nitrocellulose membrane is
soaked in radioactive
probes that are
complementary to 175kb
fragment of Hb
A
and
376kb fragment at
chromosome 11.
Note: Also accept any
probe positions that allow
for differentiation between
HbA and HbS.
376kb
A]
Hb
A
Hb
S
DdeI
175kb 201kb
CHR. 11
CHR. 11
Radioactive
probe
Radioactive
probe

6 Autoradiography exposes the radioactive bands.
7 Homozygous normal
(Hb
A
Hb
A
) will have 1 thick
band at 175kb.

Carriers (Hb
A
Hb
S
) will have
1 band at 175kb and 1
band at 376kb.;

Homozygous recessive
(Hb
S
Hb
S
) will have 1 thick
band at 376kb.

C
a
r
r
i
e
r
H
o
m
o
.
r
e
c
e
s
s
i
v
e
H
o
m
o
.
N
o
r
m
a
l
175kb
376kb

8 By comparing the symptomless carrier with the bands of the 3 known
genotypes, one can identify his genotype.
24

OR
Detecting Sickle Cell Anaemia using MstII Restriction Site

Note: Candidates may choose to illustrate
points using labeled drawings.
1 For Sickle cell anemia, base pair substitution of the normal beta-globin
gene (Hb
A
) from CTT to CAT results in a harmful recessive allele, Hb
S
. The
mutation results in the loss of a MstII restriction site.
2 Restriction digest of Hb
A

using MstII would result in 2
restriction fragments,
1150 kb and 200 kb.
Restriction digest of Hb
S

would result in only 1
restriction fragment of
1350kb.
B]
Hb
A
Hb
S
MstII
1150
200
1350
CHR. 11
CHR. 11

3 Gel electrophoresis is used to separate the restriction fragments based on their
lengths. Negatively charged DNA will move towards the anode, with shorter
fragments moving further.
4 The separated restriction fragments are being denatured by an alkaline
solution and the single stranded DNA molecules are transferred to a
nitrocellulose membrane via capillary action. Single stranded DNA
molecules cross-link to nitrocellulose membrane.
5 Nitrocellulose membrane is
soaked in radioactive
probes that are
complementary to 200kb
fragment of Hb
A
and
1350kb fragment at
chromosome 11.
Note: Also accept any
probe positions that allow
for differentiation between
HbA and HbS.
B]
Hb
A
Hb
S
MstII
1150
200
1350
CHR. 11
CHR. 11
Radioactive
probe
Radioactive
probe

6 Autoradiography exposes the radioactive bands.
7 Homozygous normal
(Hb
A
Hb
A
) will have 1 thick
band at 200kb.

Carriers (Hb
A
Hb
S
) will have
1 band at 200kb and 1
band at 1350kb.;

Homozygous recessive
(Hb
S
Hb
S
) will have 1 thick
band at 1350kb.
C
a
r
r
i
e
r
H
o
m
o
.
r
e
c
e
s
s
i
v
e
H
o
m
o
.
N
o
r
m
a
l
175kb
1350kb

8 By comparing the symptomless carrier with the bands of the 3 known
genotypes, one can identify his genotype.
[Total: 20]
END OF PAPER