2012 A level H2 Biology

Paper 3

Question 1
(a)
1. A collection/ set of cloned DNA fragments;
2. of the entire genome of an organism;
3. genome is cleaved by a specific restriction enzyme;
4. restriction fragments are inserted into plasmids before introducing into
bacteria;

(b)
1. Reverse transcriptase; uses mRNA as a template to synthesise cDNA;
2. RNA-ase; to degrade the RNA template;
3. DNA polymerase; synthesizes a complementary strand using the cDNA as
template to produce double stranded DNA;

(c)
1. cDNA library is produced using mRNA which are transcribed at a particular
time in a cell;
2. gene expression might be different at different time;
3. different mRNA might be present;
4. depending on the function and need of the cell;
5. genomic library is produced from the entire set of genome in the cell;
6. which remains the same all the time;

(d)
(i)
1. Annealing of primers;
2. to DNA template;
3. by forming hydrogen bonds;
4. between complementary base pairs;

(ii)
Free deoxyribonucleoside triphosphate (dNTPs);
Unused primers;
Taq polymerase;
Double stranded DNA molecule;
Double stranded DNA molecule with regions annealed to DNA primers;
Double stranded DNA molecule with Taq polymerase bound to it;


(e)
1. A - Denaturation of double stranded DNA;
2. breaking of hydrogen bonds between complementary base pairs;
3. B - Binding of single stranded radioactive probes;
4. to complementary region on a single stranded DNA;









2012 A level H2 Biology

Question 2
(a)
Multipotent

(b)
1. cells from donor trachea might be incompatible to the recipient;;
2. recognized by recipient immune system as foreign;
3. Elicit immune response;
4. resulting in tissue rejection/ destroying the donor trachea;;

(c)
1. stem cells are undifferentiated;
2. when treated with chemicals, trigger the cells to divide indefinitely and
differentiate;;
3. into tracheal cells;
4. causing certain genes to be turned on/ turned off;

(d)
1. Presence of telomerase in stem cells;
2. Lengthen the telomeres at the ends of chromosomes;
3. No telomerase in other cells;
4. DNA daughter strand shortened after every round of mitotic division;
5. when telomeres shortened to a particular length triggers cell death;
6. unable to continue to divide mitotically;

Question 3
(a)
(i)
1. Plasmids have multiple restriction sites;
2. that can be recognized and cleaved by different restriction enzymes;
3. DNA fragment isolated from any species by restriction digestion;
4. can be inserted into the plasmid if cut with the same restriction enzyme;

(ii)
1. Allows plasmid to replicate independently;
2. forming multiple copies within a bacterium;;

(b)
(i) Complementary base pairing;;

(ii)
1. Plasmid reanneals;
2. forming back the original plasmid;
3. Gene of interest anneals at both ends;
4. forming a circular DNA;
5. due to complementary base pairing;
6. between sticky ends;
7. CTTC and GAAG;


(c)
1. Treat bacteria with CaCl
2
;
2. To neutralize the negative charge on the DNA and phospholipid bilayer;
3. Place the mixture of bacteria and recombinant plasmids on ice;
4. Perform heat shock treatment at 42
o
C for 30s;
2012 A level H2 Biology

5. creates temperature imbalance which sweeps the plasmid into the bacteria;

(d)
Grow bacteria in medium containing antibiotic;
Non-transformed bacteria would die;
Transformed bacteria with reannealed plasmid or recombinant plasmid would
survive;
Conduct replica plating;
Grow transformed bacteria in another antibiotic;
Transformed bacteria with recombinant plasmid would die;
due the antibiotic resistance gene being disrupted by insertion of gene of interest;

(e)
Plasmid Q;;
Plasmid contain both PstI and FokI recognition sites for insertion of genes S and T
respectively;
Plasmid Q has medium copy number more efficient than plasmid P;

Question 4

Aim
Effect of concentration of sucrose solution and the rate of respiration in yeast cell.

Reaction
Sucrose Glucose + Fructose
C
6
H
12
O
6
+ 6O
2
6CO
2
+ 6H
2
O
CO
2
+ Ba(OH)
2
BaCO
3
(s) + H
2
O

Background information
1. Sucrose can by hydrolysed into glucose and fructose.
2. Yeast cell undergoes aerobic respiration using glucose.
3. Carbon dioxide is released as by-product of oxidative decarboxylation during
link reaction and Krebs cycle.
4. Reactions are catalyzed by respiratory enzymes

Relate method of measurement to dependent variable.

5. Barium hydroxide can react with the CO
2
to produce barium carbonate
6. The remaining barium hydroxide can be neutralized with hydrochloric acid
7. when all the remaining barium hydroxide is completely neutralised,
8. end point of the reaction can be determined using indicator phenolphthalein
9. as it turns from pink to colourless
10. amount of CO
2
released is inversely proportional to the amount of HCl
required to neutralize the remaining barium hydroxide
11. In the absence of CO
2
, 5 cm
3
of HCl is required to completely neutralize the
10cm
3
of barium hydroxide
12. 1 cm
3
of HCl is equivalent to 2.2mg of CO
2
13. mass of CO
2

can be calculated using the formula below:
14. (5 - Volume of HCl used to neutralise Barium hydroxide) x 2.2mg
15. rate of respiration= mass of CO
2
produced/ fixed duration of experiment

Expected trend:
16. As the concentration of sucrose increases, the mass of CO
2
produced in a
fixed duration increases, thus higher rate of reaction.

2012 A level H2 Biology

17. This is because as concentration of sucrose increase
18. the frequency of successful collision between the active site of respiratory
enzymes and substrates increases,
19. more enzyme-substrate complexes formed per unit time and
20. more CO
2
formed per unit time
21. rate of respiration increases until saturation point.
22. Rate of respiration will remain at maximum when all the enzyme active sites
are occupied by substrates

Experiment variables
23. Independent variable concentration of sucrose
24. Range: 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mol dm
-3
of sucrose

Dependent variable
25. mass of CO
2
produced, based on the volume of HCl required to neutralize the
remaining barium hydroxide

Controlled variables
Controlled variables Quantity
26. Time taken for reaction 1 min
27. Volume of yeast suspension

5.0 cm
3

28. Concentration of yeast
suspension

1%
29. Volume of sucrose solution

10.0 cm
3

30. Volume of barium hydroxide 10 cm
3
31. Concentration of barium
hydroxide
0.025 moldm
-3
32. Concentration of HCl 0.1 moldm
-3
33. Volume of phenolpthalein 0.5 cm
3
34. Temperature 37C


Experimental Procedures
1. Conduct pilot experiment to determine suitability of apparatus, optimum
conditions and amount of material used.
2. You are given 1.0moldm
-3
sucrose stock solution.
3. Label plastic vials 0.2, 0.4, 0.6, 0.8 and 1.0. Dilute the sucrose stock solution,
using labeled plastic vials, to give 30 cm
3
of each concentrations: 0.2 moldm
-
3
, 0.4 moldm
-3
, 0.6 moldm
-3
, 0.8 moldm
-3
, 1.0 moldm
-3


Dilution table of sucrose solution

Concentration
of diluted
sucrose/
moldm
-3

Volume of
stock sucrose/
cm
3

Volume of
water/ cm
3

0.2 2.0 8.0
0.4 4.0 6.0
0.6 6.0 4.0
0.8 8.0 2.0
1.0 10.0 0.0

2012 A level H2 Biology

4. Label test tubes 0.2, 0.4, 0.6, 0.8 and 1.0. Aliquot 10cm
3
of each
concentration of the diluted sucrose from the plastic vials into respective test
tubes.
5. Place test tubes containing diluted sucrose solution and yeast suspension in
a water bath maintained at 37C for 1 minute.
6. Add 10cm
3
of barium hydroxide into a test tube using syringe
7. Add 5 cm
3
of active yeast suspension to the test tube containing 0.2 moldm
-3

sucrose solution and mix well.
8. Cover the test tube with rubber bung connected to delivery tube, place
delivery tube into the test tube containing barium hydroxide immediately and
start the stopwatch.
9. After 1 minute, remove the text tube containing barium hydroxide, add 0.5
cm
3
of phenolphthalein
10. Add HCl to the test tube using syringe, 0.5 cm
3
at a time.
11. Record the volume of HCl required for phenolphthalein to turn from pink to
colourless.
12. Repeat steps 6 to 11 for each concentration of diluted sucrose.
13. Repeat procedures two more times to obtain 2 more readings for each
concentration.
14. Repeat the entire experiment two more times.
15. Carry out statistical test to determine whether there is any significant
difference between the means.

Negative Control
35. A negative control is subjected to the same factors as that for the experiment,
except that the 10 cm
3
of sucrose is replaced with 10 cm
3
of distilled water.
36. It is expected that 5 cm
3
of HCl will be required to neutralize the solution as
no CO
2
is produced.
37. This proves that it is indeed the presence of sucrose and not any other
factors, causes the production of CO
2
.

Data manipulation
38. Calculate the mass of CO
2
produced using the formula below:
(5 volume of HCl required) x 2.2mg
39. The mass of CO
2
produced by each mixture is converted to rate (R) by the
following equation: R = mass of CO
2
produced/ 1min
40.
Table showing the effect of concentration of sucrose solution on the rate of
respiration
Concentration
of sucrose
solution,c
/moldm
-3

Volume of HCl
required, V/cm
3
Mass of CO
2
produced/mg
Rate
respiration
in yeast
cell, R/mg
min
-1

V
1


V
2

V
3

_
V
0.2
0.4
0.6
0.8
1.0




2012 A level H2 Biology


Graph showing the effect of concentration of sucrose solution on the rate of
respiration















Safety precaution

41. Phenolphthalein indicator is toxic. Wear gloves and goggles to avoid contact
with skin and eyes.
42. Barium hydroxide and hydrochloric acid are corrosive. Wear gloves and
goggles to avoid contact with skin and eyes.
43. AVP i.e. Use a piece of rag to hold the beaker containing hot water (when
preparing water bath) to prevent burns / scalds.

Question 5
5(a)

Steps for plant tissue culture Scientific reason
S1. Explants (i.e. shoot tips and root tips)
are excised from the plant to be cloned;
S2 Explant must contain meristematic
cells;
R1. Meristematic cells are totipotent;
R2. Can regenerate into a whole new
plant;
S3. Cells are sterilized using dilute
sodium hypochlorite;
R3. to kill bacteria and fungus/ prevent
contamination;
S4. Cells are grown in culture vessels;
S5. on nutrient agar containing nutrients
and plant growth regulators;
S6. to form callus;
R4. To stimulate cells to divide by
mitosis;
S7. Cut callus into several pieces;
S8. Place the pieces into separate
culture vessel;
R5. To increase the number of callus;
R6. by subculturing;
S9. Different levels of auxin and cytokinin
are added;
S10. Differentiated tissues will grow into
a plantlet;

R7. To induce cell differentiation;
R8. Low level of cytokinin and high level
of auxin triggers formation of roots
growth;
R9. High level of cytokinin and low level
auxin triggers shoot growth;
S11. Transfer of plantlets to soil;

R10. Acclimatisation before transferring
to the field;
Concentration of
sucrose/moldm
-3

Rate of
respiration,
R/ mgmin
-1

2012 A level H2 Biology

5(b)
Positive aspects [max 2]
Inclusion of pest-resistance genes, reduced use of pesticides
- chemical-free plant crops for consumers;;
- lower production cost for farmers since there will not be a need to
purchase pesticides;;

Inclusion of genes to increase the nutritional value of crop plants
- nutritionally enhanced food can overcome the problem of malnutrition in
developing countries;;

Inclusion of gene to increase crop yield
- reduce the problem of food shortage;;

Inclusion of delayed ripening gene
- greater commercial value for crop plant to be freight over longer distances
to consumers;;

Negative aspects [max 4]

Threat to human safety as there is a potential of transfer of allergens;;
Antibiotic resistance gene if not properly broken down by the digestive system
may be passed to E. coli found in the gut, making them resistant to
antibiotics, potential impact to human health;;
GMO may be carried by wind to other places and establish themselves as
weeds and pose difficulties to be eradicated;;
Cross-pollination between GMO and wild relatives, spreading herbicide-
resistance to weeds, resulting in superweeds, unable to eradicate by
herbicides, outcompete with the crop plants and took over agricultural field;;
GMOs outcompete the local native species and thereby upset the balance in
the natural ecosystem and species/ loss of biodiversity;;
Violation of the natural organism's intrinsic values with the mixing genes
among species may evoke strong responses from naturalists;;
Objections from specific religious groups with strict dietary restrictions;;
Patenting of GMOs;;
Labelling of GM food is not mandatory in some countries;;



5(c)
Considered as new species
Biological concept defines species as a group of organisms capable of
interbreeding and producing fertile offspring;
Creation of GMO may result in organism being sterile due to polyploidy;
Unable to interbreed with original population of 2n species to produce fertile
offspring;

Ecological niche concept defines species as a group of organisms sharing the
same ecological niche;
No two species can share the same ecological niche;
GMO are modified to be able to withstand the unfavourable condition in the
environment;
2012 A level H2 Biology

Likely to outcompete the original species when they occupy the same
ecological niche, establish themselves as new species;

Based on morphological concept, a group of organisms should share a
unique set of structural species;
Genes from another organism are introduced into GMO;
giving desired traits, different phenotype from original species;

According to the phylogenetic concept a group of organisms bound by a
unique ancestry and share one or more derived character;
Mixing of genes of more than one species, no evolutionary relationship of
GMO with other species;

Not considered new species
GMO may still exist as a diploid organism, capable of producing fertile
offspring with the original species;
Little/no changes to structural morphology, still resemble original species;