Paper 3 Answers

Restriction enzymes cut DNA molecules only at specific target sites with particular
base sequences. The target sites for enzymes EcoRI, BamHI and EcoRV are
shown in Table 1.1. The lines in each sequence show where the enzyme cuts the
DNA molecule.
Restriction enzyme
Specific target base sequence of DNA
EcoRI

BamHI

EcoRV

G A A T T C
C T T A A G
G G A T C C
C C T A G G
G A T A T C
C T A T A G
Table 1.1

(a)

Some restriction enzymes generate sticky ends, while others generate blunt
ends.
Explain why it is more efficient to clone DNA fragments with sticky ends than DNA
fragments with blunt ends. [1]
a) Sticky ends between two DNA fragments will be
complementary and will anneal to each other via hydrogen
bonds
b) This holds the 2 fragments in place whilst DNA ligase seals
the nicks.
c) Does not need additional steps such as attaching linkers

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Nh e I
Pm a I
Ap a I
Xb o I
Xh t I
No XI
Bst RV
Eco RI
Eco I
X
Bst H I
m
Ba n I
Kp d III
Hin

The pcDNA plasmid shown in Fig.1.1 is a very useful cloning vector used in
recombinant DNA technology. In the commercial production of insulin, the cDNA
of human insulin is inserted into pcDNA.
The recombinant pcDNA is then transformed into Escherichia coli to produce
large numbers of recombinant plasmids. The recombinant plasmids are then
purified and transfected into mammalian cells which will express high levels of the
functional insulin.

Fig.1.1
(b)

A genetic engineer cuts the plasmid shown in Fig.1.1 with EcoRV in order to
insert a human insulin gene.
Describe the steps that must be carried out after digestion with EcoRV to produce
a recombinant plasmid. [4]
a) Digestion of the plasmid with EcoRV produces blunt ends.
b) Linkers containing a restriction site must thus be added to
the blunt ends and the restriction enzyme used to digest the
attached linkers to produce sticky ends
OR
Terminal transferase must add dGTPs to the blunt ends to
the plasmid.
c) The flanking regions of the human insulin gene should be
digested with the same restriction enzyme to generate
complementary sticky ends
OR
Terminal transferase must add dCTPs to the blunt ends of
the human insulin cDNA.
d) The plasmid and the human insulin cDNA are mixed together
with DNA ligase, the sticky ends will anneal by
complementary base pairing,
e) DNA ligase sealing the nicks by catalyzing the formation of
phosphodiester bonds between adjacent nucleotides on the
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sugar phosphate backbone.

(c)

The pcDNA contains two selectable markers, the ampicillin resistance gene
(AmpR) and the neomycin resistance gene (NeoR) which code for resistance to the
antibiotics ampicillin and neomycin respectively. Ampicillin specifically targets cell
wall synthesis whilst neomycin binds irreversibly to ribosomes preventing protein
synthesis. The pcDNA used has been modified to express the NeoR only in the
mammalian cells.
(i)

Explain why the AmpR is not an effective selectable marker in mammalian cells.
[2]
a) Mammalian cells do not have cell walls. Ampicillin will not
be able to inhibit cell wall synthesis in mammalian cells.
b) Mammalian cells will survive in the presence of ampicillin
with or without the AmpR gene present

(ii)

Suggest why the NeoR is only expressed in mammalian cells. [1]

NeoR gene is coupled to a eukaryotic promoter which can only be
recognized by eukaryotic transcription factors and RNA polymerase

(d)

Explain why mammalian cells instead of E.coli were used to produce the
functional human insulin. [2]
a)
Mammalian cells able to carry out post-translational modification
b)
where the C chain is cleaved and disulfide bonds are formed
between A and B
[Total: 10]
Osteogenesis imperfecta (brittle bone disorder), is a genetic disorder
characterised by bones that break easily.
Adult bone stem cells transplanted into children with osteogenesis imperfecta
have stimulated growth of bone in these children. During the 6 months
immediately following the transplant, the childrens growth reached 60% to 94%
of expected normal values for children their age.

(a)

(i)

In terms of differentiation potential, explain how an adult bone stem cell is

different from a fertilized egg of a human. [2]
a) The zygote/fertilized egg is totipotent i.e. it can give rise
to all cells types in the body including extra embryonic
tissue;
b) but the adult bone stem cell is multipotent i.e. it can only
give rise to a limited range of cell types

(ii)

Explain how the adult bone stem cells may increase bone growth in children with
osteogenesis imperfecta. [2]
a) Stem cells can divide by mitosis
b) and in the presence of the appropriate signals can also
differentiate into new bone cells
This thus increases the number and type of bone cells
present which contribute towards bone growth.
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(b)

The polymerase chain reaction (PCR) can be used to make many copies of a
gene. Fig.2.1 shows the main stages in the process.

Stage 1:
Stage 3: DNA is heated
2: DNA
cooled
to 64oC
DNA is heated to 95oC for 5 minutes in the presence of primers, DNA Stage
nucleotides
& is
DNA
polymerase

the cycle is repeated

Fig.2.1
(i)

Describe the unique property of the DNA polymerase used in stage 1. [1]
It is thermostable.

(ii)

Identify stage 3 and state the temperature required at this stage. [1]
Extension , 72oC

(iii)

Explain the role of the primers in Fig.2.1. [3]

Primers are added as their
a) sequences are complementary to the regions flanking the gene of
interest thus determines the segment to be amplified
b) Hence they will anneal to them via hydrogen bonding
c) and provide the free 3-OH group for chain extension by DNA
polymerase so that the gene of interest can be amplified

(iv)

How many cycles of the PCR would be needed to produce 128 molecules of DNA
from a single DNA molecule? [1]
7
(27 =128)

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(c)

The sequence of nucleotides in a sample of DNA can be determined using a

method involving the PCR followed by electrophoresis. In this method, the
primers used in the PCR are radioactively labelled and some of the normal
deoxyribonucleotides are chemically altered.
Fig.2.2 shows a single strand of DNA and the copies produced by the PCR using
a mixture of all 4 DNA nucleotides (dATP, dTTP, dGTP and dCTP) and some
chemically altered dTTP also known as ddTTP. This ddTTP is represented as T*
in Fig.2.2.

T*

T*
T*
T*

Fig.2.2
(i)

In order for this method to work, both electrophoresis and the use of radioactive
primers are necessary. Explain why this is so. [2]
a) Electrophoresis will separate the strands according to their size in the
gel
b) The radioactive primer will allow the visualization and the location of the
various strands (which will appear as bands) via autoradiography
Fig.2.3 shows the chemical structure of deoxyribonucleotide (left) and a
chemically altered deoxyribonucleotide (right).
.

Fig.2.3

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(ii)

With reference to the Figs.2.2 and 2.3, explain why three different lengths of new
strand were produced. [2]
a) Since 3 adenine bases are present on template strand, there are 3
places where dTTP/chemically altered dTTP (ddTTP)/T* can be
incorporated (in copying so three lengths of new strand)
b) Where chemically altered dTTP used, copying stopped/block further
extension of strand due to the absence of the 3OH group
c) Where normal thymine used, copying continued
(Max 2 marks)

(d)

Scientists have found a new method of copying DNA that is faster than PCR. The
new method is called Helicase Dependent Amplification (HDA). HDA uses an
enzyme to separate the two strands of DNA.
This means that DNA can be copied at a constant temperature of 37C. In all
other ways, HDA works in exactly the same way as PCR.
HDA is faster than PCR. Explain why. [1]
No need for heat and cooling cycles thus saves time OR
Primer annealing and extension steps can occur simultaneously

[Total: 15]
Plant physiologists attempted to produce papaya plants using tissue culture.

(a)

They investigated the effects of different concentrations of two plant growth

factors on small pieces of the stem tip from a papaya plant. Their results are
shown in the Table 3.1.

Table 3.1
(i)

With reference to the Table 3.1, give evidence that the cells from the stem tip are
totipotent. [2]
a) At 10 mol dm-3 auxin and 50 mol dm-3cytokinin, whole
plantlets (with leaves and roots) are produced from the stem
tips
b) The stem tip cells are totipotent and hence able to
differentiate into all specialized cell types for form a whole

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plant.
(ii)

Stem tips are used in tissue culture to produce disease-free plants.

Explain why cells are taken from the stem tip instead of flowers or leaves for
tissue culture. [2]
a) This is because there are inhibitors of viral replication
present at apical meristems not present in other meristems
b) The vascular bundles which usually transmit diseases are not
yet established at stem tips but are present in leaves and
flowers.

(iii)

State the recommended cytokinin to auxin ratio that should be used to produce
papaya plants using stem tip culture. [1]

5 : 1(with 50 mol dm-3cytokinin and 10 mol dm-3 auxin)

(iv)

Explain the advantage of growing papaya plants from tissue culture rather than
from seeds. [3]
a) Growing papaya from tissue culture produces clones which are
genetically identical to the parent
b) Allow mass propagation of clones
c) Hence all the favourable traits/desired characteristics of the parent are
found in these clones
d) Whereas in seeds these favourable traits of the parent may not be found
in the progeny due to genetic variation from independent assortment/
crossing over at meiosis/ random fusion of gametes at fertilization
AVP) Tissue culture allows genetic modification/engineering to form
favourable trait/phenotype.
(3 max)

(b)

In 1989, a transgenic salmon line was created by injecting eggs of the Atlantic
salmon with a gene construct containing the gene of the Chinook salmon growth
hormone. Transgenic salmon were grown as sterile, all-female populations in
tanks on land located away from the sea.
Salmon reaches a marketable value at 4kg. However, this line of salmon was not
allowed to be commercialized. The growth of the transgenic salmon and the
standard Atlantic salmon is shown in Figure 3.1.

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(i)

Fig.3.1
With reference to Fig.3.1, explain the benefits of farming transgenic
salmon as compared to the standard salmon. [3]
a) The transgenic salmon reaches marketable value at 4kg much earlier at
600 days from first feeding compared to the standard salmon which
reached marketable value at 830 days from first feeding / Ref to higher
rate of growth (quote data) / Ref to higher mass within a certain time
period (quote data)
b) The faster growth allows farmers to save on cost of feeding the fish/
higher feed conversion efficiency
c) The also allows a higher turnover rate of fish farmed thus increasing the
supply/yield of salmon meat in a certain period

(ii)

Give reasons why transgenic salmon are grown as sterile, all-female

populations in tanks on land away from the sea. [2]
a) Prevent transgenic salmon from escaping into the sea/river in a land
based facility.
b) Prevent reproduction of transgenic salmon
Or
Prevent transgenic salmon crossing with standard salmon pass on the
transgenes to wild salmon
c) Prevent it from outcompeting the standard salmon for resource and
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The gene construct used is shown in Fig.3.2. It contains a promoter and
termination region from the ocean pout antifreeze gene and a growth hormone
from a Chinook salmon.

Fig.3.2
(iii)

Explain why the promoter from the ocean pout antifreeze gene is used in this
construct as opposed to the original growth promoter of the Atlantic salmon. [2]
The ocean pout antifreeze promoter allows the growth hormone gene to be
constitutively expressed/continually expressing/always on/even during
the colder/winter months/all year round. (Accept reference to active
promoter)
The growth hormone gene under the control of the original promoter is not
expressed during the colder/winter months (Accept reference to inactive
promoter)
[Total:15]

Planning question
You are required to plan, but not carry out, an investigation into the effect of substrate
(hydrogen peroxide, H2O2) concentration on the rate of enzymatic activity of yeast cells.
Yeast cells contains enzyme catalase which catalyses the decomposition of H2O2.
H2O2 H2O + O2
When droplets of a yeast-alginate mixture are dropped into calcium chloride solution,
yeast-alginate beads are produced and the yeast is immobilized into alginate beads.
When the one of the yeast-alginate beads is then transferred to a test-tube containing
H2O2, it will naturally settle at the bottom of the tube. Over time oxygen bubbles will form
around the bead. The bubbles provide buoyancy and will cause the bead at the bottom of
the test-tube to float to the top.
The rate of catalase activity can be determined by measuring the time taken for the yeastalginate bead to float.
Your planning must be based on the assumption that you have been provided with the
following equipment and materials, which you must use:
1M hydrogen peroxide
Yeast-alginate mixture
Calcium chloride solution
Droppers
Forceps
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Test-tube
A variety of different sized beakers, measuring cylinders or syringes for measuring
volumes.
Stop watch
Hot water (80oC)
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, if necessary.
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,
include relevant risks and precautions taken,
the correct use of technical and scientific terms.
[Total: 12]
1. Aim : To investigation into the effect of substrate (hydrogen peroxide, H2O2)
concentration on the rate of enzymatic activity of yeast cells.
2. Theory :
At low substrate concentration, an increase in H2O2 (substrate)
concentration will increase the frequency of effectively collision
between enzyme catalase and substrate, H2O2, to form enzyme-substrate
complex. [1m] - a

Hence increase the rate of enzyme reaction.

There will be an increase rate of O2 (product) production, as the O2
bubbles formed around the beads provide buoyancy to rise the beads,
hence faster rate / shorter time taken for the beads to rise (expected
trend). [1m] - b

However at high substrate concentration, a further increase in H 2O2

(substrate) concentration, rate of reaction will plateau/ remain constant.
As enzyme concentration is limiting, as all active sites are already
saturated with substrates.
3. Procedure
(a) Pilot test 1M - PT

Conduct a pilot experiment to determine the suitability of apparatus,

range of H2O2 concentration (independent variables), suitable
catalase conc optimum conditions and amount of materials used.
(b) Well-annotated diagram

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(c) Procedure
(1) Sucrose concentration is the independent
concentrations of H2O2 solution by dilution:

Concentration of H2O2 (M)

0.2
Volume of 1M H2O2 solution 20
added (cm3)
Volume of water added (cm3) 80

[1m] I
Must show
dilution table

variable.

Prepare

0.4
40

0.6
60

0.8
80

1.0
100

60

40

20

Preparation of the yeast-alginate beads:

(2) Using a measuring cylinder, add 20cm 3 of calcium chloride to a small
beaker.
(3) Using a dropper, by holding it level with the rim of the beaker as shown in
the diagram above, drop by drop release the yeast-alginate mixture into
the calcium chloride solution.
(4) Repeat this step for to produce 20 yeast-beads of similar sizes.
(5) Using a forcep, pick up the yeast-beads.

[1m] P

(6) Using syringe / measuring cylinder, fill a test-tube with 5cm 3 1M H2O2
solution.
(7) Using the 800C hot water provided, prepared water bath of 30oC , and
place the test-tube in thermostatically controlled water bath of 30oC.
(8) EQUILIBRATION TIME : Allow 1 min for the contents in test tube to reach
temperature of water bath. 1M -E
(9) Drop 1 of the yeast-bead into the test-tube, and as soon as the yeast-bead
touches the bottom of the test-tube, immediately start timing with a
[1m] D
Points 9&10
stopwatch.*
(10)
Stop the timing as soon as the bead rises from the bottom
or 12 alone
of the test-tube.
(11)
Conduct 2 more replicates to check for anomalies present.
(12)
DEPENDENT VARIABLE : time taken for the yeast-bead to
rise from bottom of test-tube (in seconds). 1M
(13)
INDEPENDENT VARIABLE : repeat the steps 6 to 9, each
time with a new yeast-beads with 0.2, 0.4, 0.6 & 0.8M H2O2 solution.
*NB : there will be some beads that will not sink to the bottom of the H2O2 solution, and
these should be discarded.

Constant Variables (any 2) [1M] V1/V2

1 bead per
Volume of H2O2
Temperature e.g.
3
test-tube
e.g 3 - 10cm
(between 20 400C)

Beads of
constant
size

pH

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(d) Control [1M] - C
1. Prepare 1 control with 0M H2O2 solution / water in place of H 2O2 solution /
use alginate beads with boiled and cooled yeast
2. This is to confirm any bubbles produced around the yeast-bead is due to
reaction with H2O2 solution and not due to other factors.

[1m] C

`
(e) Repeats and Replicate 1M
1. Repeat the experiment to ensure reproducibility
2. Conduct 2 replicates to check for anomalies present.

[1m] R2
4.

Data recording and processing. [Table 1M, Graph 1M all with correct
units and heading]

Table showing effect of different H2O2 concentration/M on the rate rising of yeast-bead/s-1
Concentration of
H2O2 / M

Time taken for rising of yeast-bead / s

Replicate 1

Replicate 2

Replicate 3

Average

Rate of
rising of
yeastbead / s1

0.2
0.4
0.6
0.8
1.0
Graph showing effect of H2O2 concentration /M on the rate of rising of yeastbead /s-1

Rate of rising of
yeast bead / s-1

NB : Graph and table must have the

H2O2 concentration/ M

same units.

5.

Risk and precautions [1M]

a) H2O2 is corrosive, ensure gloves are worn when handling H 2O2.

Free-response question
Write your answer to this question on the separate paper provided.
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Your answer :
should be illustrated by large, clearly labelled diagrams, where appropriate
must be in continuous prose, where appropriate.
must be set out in section (a), (b)., as indicated in the question.
5

(a)

Describe the genetic basis of cystic fibrosis and, compare liposomal and viral
delivery systems in the treatment of cystic fibrosis using gene therapy.
[8]
A. Most common mutation is a deletion mutation (3 nucleotides on
chromosome 7), resulting in the loss of phenylalanine in the polypeptide.
B. Follows an autosomal recessive inheritance pattern
C. Affects membrane protein, cystic fibrosis transmembrane conductance
regulator (CFTR) such that it is either defective
Scientist use either viral (e.g. adenovirus) or non-viral delivery (liposomes)
methods to introduce a normal functional CFTR allele into the human.

Point
of Adenovirus-mediated
comparison
therapy

gene Liposome-mediated gene therapy

Differences
D1

Immune
response

Adenovirus more likely

evoke immune response

to Less likely
response

D2

Effectivenes
s on evoking
immune
response

Less likely to be effective after Remains effective as body does

the body has developed not develop immunity to the vector
immunity to the viral vector

D3

Disease
Adenoviral vector (which does
causing
/ not integrate into the genome
risk
can cause throat infections but
assessment
not
human
malignancies
(cancer)

Do not cause disease at all / safer

(Liposomes have a small degree of
toxicity so many rounds of
treatment can be harmful to the
cells or tissues being treated)

D4

Specificity

More specific targeting of cells

Less specific targeting of cells

D5

Transfection
efficiency

High transfection efficiency.

Low transfection efficiency.

D6

Delivery
of
normal
functional
CFTR allele
into nucleus

Greater chances of delivering No ability to transfer normal

normal functional allele into functional allele into nucleus.
nucleus via nuclear pore as
adenovirus directly injects
DNA at nuclear pore.

D10

Entry
cell

into Virus
enters
cell
via
endocytosis, delivering normal
functional CFTR allele into
target cell.

to

evoke

immune

Fusion of lipid bilayer of liposome

with cell membrane leads to
delivery of normal functional CFTR
allele into cell.

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Similarities
S1

Integration

Liposomes and adenoviruses not able to integrate normal

functional CFTR allele into genome (extrachromosomal) / do not
cause insertional mutagenesis;
And hence the treatment is short-lived;

S2

Duration of Treatment is transient as epithelial cells are constantly being shed,

therapeutic
so repeated therapy is needed;
effects

S3

Target
organs

Cystic fibrosis affects other organs that are less accessible e.g.,
pancreas and small intestines, and the epithelial cells of the
pancreas is a challenge to target by both liposomal or viral delivery;

S4

Target cells

Difficult to target the stem cells of the epithelium of every organ

S5

In vivo / ex
vivo
approach

Both systems are carried out using an in vivo approach because it

is difficult to extract epithelial cells from respiratory tract.

S6

Level
of Fine-tuning the expression of normal functional CFTR allele within
expression
target cells is difficult; under-expression of normal functional CFTR
allele in target cells would render gene therapy ineffective.

Point
of Retroviral-mediated
comparison
therapy

gene Liposome-mediated gene therapy

D7

Insertional
mutagnesis

Retroviral vector may induce a No insertional mutagenesis.

tumour
via
insertional
mutagenesis of e.g. tumour
suppressor
gene
by
integrating normal functional
allele into the genome

D8

Integration
of
normal
functional
CFTR allele
into
host
genome

Retrovirus
possesses Absence of integration of normal
integrase which integrates functional CFTR allele into host
normal functional CFTR allele genome.
into host genome.

D9

Stability
of Stable gene expression.
gene
expression

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(b)

Discuss how the human genome project can help parents "customise" their babies
and the possible social and ethical implications of doing so.
[6]
Info from HGP [1] must have both to gain 2 marks.
One can use the information from Human Genome Project to identify a wide
range
/genome-wide range of disease causing alleles, (important to state that it is a
genome-wide range, since prior to HGP, other genetic traits would have been
known).
alleles that give desirable traits such as height, intelligence, strength
endurance etc
How info use to create Designer Babies [1] either one is a necessary point.
Based on info from HGP, Designer babies could 1) be selected for using Preimplantation Genetic Diagnosis (PGD)/(anyword to that effect) that are
disease-free or have desirable traits
Or based on the HGP, designer babies could 2) go through genetic
modification for enhancements in desirable traits/removal of undesirable traits
discovered through info from HGP
Possible Benefits (at least 2 points to gain full marks)
Populations with designer babies can benefit from more individuals with
desirable traits e.g. less physical defects, good health, better physical
strenght, HIV resistance, longevity and a higher IQ etc; (at least one example)
Babies that are born would not have genetic defects as those that
have disease-causing alleles are not implanted or have their genes modified to
correct the disease
Designer siblings of children who have genetic diseases may be created to
provide matching and healthy stem cells for therapy
Determine the sex of the child as this might prevent babies who have sexlinked diseases like haemophilia and Duchenne Muscular Dystrophy, Fragile X
syndrome, only show themselves in male babies
Social and Ethical concerns (at least 2 points to gain full marks)
For the selection of embryos to implant in PGD, those that are not selected are
discarded or unused which will raise ethical concerns since these embryos
have been fertilized.
Designer siblings may be feel that their existence was for a cure rather than
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parents wanting a child/feel inferior to the child who is sick

May lead to a social divide between between those who can afford designer
babies/designer genome and those that cannot.
AVP - e.g., discrimination against non-designer individuals
(c)

Describe how RFLP can be used in detecting sickle-cell anaemia in a human

population.
[6]
a) in sickle-cell anaemia, the disease-causing mutation occurs at a
restriction site for MstII within the -globin gene
b) In the disease allele, this MstII restriction site is eliminated
c) Genomic DNA from individuals in a population with sickle-cell anaemia
and a normal individual are digested with MstII resulting in different-sized
restriction fragments are produced
d) Agarose gel electrophoresis, and then Southern blot using a probe
complementary to part of the -globin gene region, is carried out;
e) presence of a disease allele in an individual can be seen from
characteristic banding pattern in the autoradiography of Southern blots;
f) Description of banding patterns at least the disease genotype: A normal
person (homozygous dominant) would have 2 bands, a carrier
(heterozygous) would have 3 bands and a person with homozygous
recessive genotype for the gene and hence the disease will have 1 band
(can draw banding pattern)

[Total:20]

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