2011 A Level Paper 3 Suggested Answers

PAPER 3

Question 1
(a)
Isolate insulin mRNA from -cells of islet of Langerhans in the pancreas;
Using reverse transcriptase, synthesise a DNA strand using mRNA as the template;;
Degrade the mRNA using RNAse;
Using DNA polymerase, synthesise a second DNA strand using the first DNA strand as the
template;;
Forming a double-stranded cDNA;

(b)
Examiners Comments: Many candidates tried to use a cDNA library to obtain the required
DNA. Since cDNA is only formed using mRNA, this approach is incorrect. Candidates who
used the information in the question that somatostatin is a chain of 14 amino acids were able
to give valid suggestions.

Since somatostatin is a short polypeptide chain of 14 amino acids, its DNA can be
chemically synthesised;;
Nucleotides can be derived using the codon dictionary of the genetic code; and added one
by one before ligation;;

(c)(i)
Cuts and destroy foreign DNA (e.g that of invading viruses) that enters the bacteria;;
thus protecting the bacteria;;

(ii)
Sticky end 1: AATT
Sticky end 2: CTAG

(iii)
To ensure the correction orientation of the gene (promoter upstream of stop triplet) when
inserted into a vector;;

(d)
Incubate the plasmid with EcoRI and BamHI to allow restriction enzymes to cut at specific
restriction sites, creating sticky ends complementary to that on the somatostatin gene;;
Incubate somatostatin (with sticky ends added) with the cut plasmid to allow complementary
ends to anneal via H bonding;;
Add DNA ligase to catalyse the formation of phosphoester bonds between adjacent
nucleotides, thus forming the recombinant plasmid;;

(e)
Small size facilitates the vectors entry into host cells and the biochemical manipulation of
DNA;;
Presence of origin of replication allows for the vector to replicate itself and the inserted gene
of interest within the host cell;;








2011 A Level Paper 3 Suggested Answers
Question 2
(a)(i)
Normal allele of Rpe65 gene can be targeted;; by the viral vector to pigment cells beneath
the retina;;
Normal allele of Rpe65 gene can be integrated into the host cell chromosome;; and be
passed on to daughter cells during replication, offering an opportunity for long term stablilty
of the allele;;
Normal allele of Rpe65 gene can be introduced into the nucleus;; offering an opportunity of
long-term stable expression of the allele;;

(ii)
Liposomes have no viral genes that may cause diseases in host cell;;
Liposomes would not trigger attack by host immune system, thus normal allele of Rpe65
gene is not destroyed;;

(b)
Alleles delivered by adenoviruses are not integrated into chromosomes, thus can only
function sporadically;;
Alleles delivered by retroviral vectors integrate randomly into host cell chromosomes, so
might disrupt useful genes in the host cell and cause disease;;
This also means that therapeutic alleles would not be passed on the new cells, and would be
lost as the cells die;;
Reject: genes die or do not live for very long

(c)(i)
Examiners Comments: The question did not ask how a genomic DNA library was
constructed or what it was used for.

A random sample of all the DNA sequences in an organism;;
Entire genome is cleaved with a specific restriction endonuclease;;
Resulting fragments are inserted into plasmids before introducing into bacteria;;

(ii)
Examiners Comments: The question did not ask how a cDNA library was constructed or
what it was used for.

cDNA library contains only regions of the genome that have been transcribed into mRNA;;
Total RNA from a specific tissue is converted, using reverse transcriptase, to a double-
stranded cDNA;;
These molecules are inserted into plasmid and cloned;;

Question 3
(a) (i)
Cells were grown in culture vessels containing nutrients and plant growth regulators;;
Auxin/cytokinin is added to the nutrient agar to stimulate the cells to divide by mitosis
forming a callus;;
The number of calli increased by subculturing;;
The cells of the callus are induced by auxin and cytokinin/gibberellin to differentiate into a
plantlets;; producing large number of genetically identical plants






2011 A Level Paper 3 Suggested Answers
(a) (ii)
Examiners Comments: When beads of tungsten are fired at very high speed at corn cells,
the cell wall, cell surface membrane or nuclear envelope will not be too strong to prevent
entry.

as the tungsten were fired at very high speed at corn cells, most of the cells will be damaged
and only some cells will take up the plasmids;;
The probability of the plasmid reaching the nuclei and be incorporated into the chromosome
is low;;

(b) (i)
Growing a Bt crop could affect food chain.
All of the Bt-susceptible caterpillars that fed on the Bt leaves died before the wasps could
complete their development and emerged;
as compared to 63% of Bt-susceptible caterpillars that fed on non-Bt leaves survived long
enough for the adult wasps to emerge;

(b) (ii)
There is evidence that the Bt toxin kill wasp larvae;
For Bt-resistant caterpillar that fed on Bt leaves, there are 54% of the parasitized caterpillars
from which adult wasps emerged; which is 2% lower than for those fed on non-Bt leaves;

Question 4 Planning Question

An explanation of the theory to support your practical procedure [1]
1. The plasmid contains 2 selectable marker: ampicillin (Amp
R
) and tetracycline resistance
(Tet
R
) genes / Name the different types of cells present after transformation
2. There are multiple cloning sites in Tet
R
gene. Restriction enzyme is used to cut at Tet
R

gene to insert the gene of interest.
3. Insertion of gene of interest will inactivate the gene conferring resistance to tetracycline.
4. Transformed cells with recombinant plasmid will be Amp
R
but Tet
S

5. Transformed cells with non-recombinant plasmid will be Amp
R
but Tet
R
.
6. Non-transformed cells will be Amp
S
.
7. Nucleic acid hybridisation to identify the cells with anti-thrombin gene inserted into the
plasmid.

Description of the method used including the scientific reasoning behind the method
and the type of data generated by the experiment [9]

(I) Identification of E. coli transformant cells that carry recombinant plasmids
8. Plating: Plate E. coli cells on agar plate containing ampicillin (master plate).
a. to identify the transformed cells which have taken up plasmid from the non-
transformed cells. Transformed cells will be Amp
R


9. Replica-plating: use sterile velvet to replica-plate the cells obtained from above onto
tetracycline-containing nutrient agar.
a. to identify the transformant cells that have picked up the non-recombinant plasmids
instead of the recombinant plasmids.
b. the restriction fragments are inserted into the tet
R
gene in the plasmid. This will
inactivate the gene conferring resistance to tetracycline/ insertional inactivation of
an antibiotic resistance gene. Recombinant cells are Amp
R
but Tet
S
.
c. cells that carry non-recombinant DNA are resistant to both ampicillin and
tetracycline.

2011 A Level Paper 3 Suggested Answers
10. Identification: Colony hybridisation to identify the transformed cells that carry the anti-
thrombin gene.

(II) Identification of E. coli transformant cells that carry the gene of interest by colony
hybridisation.
11. Blotting: All the amp
R
tet
S
colonies collected from the ampicillin master plate are
transferred onto the nitrocellulose filter and lysed to release DNA (using alkaline solution).
a. the lysis treatment/alkaline solution results in the denaturation of DNA.
b. makes it single-stranded for subsequent hybridization with probes

12. Probing: nitrocellulose membrane is exposed to radioactively-labelled, single-stranded
DNA probes.
a. radioactively-labelled, single-stranded DNA probes are designed to be
complementary to the gene of interest, can hybridise via complementary base
pairing.

13. Autoradiography: a sheet of photographic film is laid over the nitrocellulose membrane
and developed after a suitable period of exposure.
a. radioactive probes that are bound to the gene of interest form an image on the
photographic film
[total: 14, max 9]

How results will be analysed including how the bacteria with recombinant plasmid
can be determined [1]
14. Compare the master plate and replica plate, identify bacteria that are resistant to
ampicillin but susceptible to tetracycline. These are the bacteria that have taken up
recombinant plasmid. Colonies of recombinant bacteria can be isolated by going back to
the original cultures on the ampicillin-containing agar. []
15. Compare the photographic film with the master plate. The colonies contain the gene of
interest can be identified. []

Scientific terms [1]

Question 5
(a)
1. loading of DNA fragments at one end of the agarose gel;

Ref to addition of loading dye to DNA fragments:
2. They increase the density of the sample, ensuring that the DNA sinks into the well;
3. They add colour to the sample to make it easy to load into the wells;
4. They contain tracking dyes that, in an electric field, move toward the anode just like the
DNA fragments so it serve as visual markers for migration during electrophoresis and
indicates when to turn off the power;

5. The gel is bathed in an buffer solution which contains ions that conduct electricity;
6. electric field or voltage applied across the gel;
7. DNA being negatively charged;
8. will move towards the positive electrode/anode;
9. rate of movement/speed/distance travelled in a given time depends on the size of DNA
fragments;
10. larger DNA fragments travel slower and smaller fragments traveling faster;
11. more resistance for the larger fragment to move through the pores of the gel;
12. DNA ladder is added to calibrate;
13. use of ethidium bromide / fluorescent dyes and UV light to see the DNA bands;

2011 A Level Paper 3 Suggested Answers
(b)
Definition of RFLP
1. Variations in gDNA sequences among individuals of a species;
2. Caused by mutations found within coding or non-coding regions;
3. Variety of genotype / inherited genetic differences among individuals in a population/the
varied alleles of the genes in the gene pool;
4. Giving rise to variations in length of DNA fragments when cut by the same restriction
enzyme;;

Detection of RFLP
5. Separation of restriction fragments of different sizes by agarose gel electrophoresis;

Southern blot:
6. DNA fragments transferred onto nitrocellulose membrane;
7. Alkaline condition used to denature DNA, making it single-stranded;

Probing:
8. Nitrocellulose membrane exposed to radioactively-labelled, single-stranded DNA probes;
9. Probes designed to be complementary to VNTRs;
10. Can hybridise / attach to restriction fragments of interest via complementary base-pairing;

Autoradiography
11. Photographic film laid over nitrocellulose membrane and developed after suitable period
of exposure;
12. Radioactive probes bound to restriction fragments form an image on photographic film;


(c)
Examiners Comments: The majority of answers were in terms of the disease mentioned in
the syllabus - sickle cell anaemia and these were often extremely detailed. Candidates who
wish to describe other diseases should ensure that the complete details of restriction
enzymes, fragment lengths etc. are known to the same degree as they are known for the
well documented example of sickle cell anaemia.

-globin gene
1. Sickle-cell anaemia is caused by a base substitution within a restriction site in the human
-globin gene;;
2. A thymine nucleotide was substituted by an adenine nucleotide; / triplet base code
change from CTT to CAT;;

RFLP analysis
3. A restriction enzyme recognises and cuts DNA molecules at a specific base sequence
(i.e. restriction site;;
4. The point mutation destroys one of the DdeI restriction sites at the 5 end of the -globin
gene;;
5. When the normal and sickle-cell alleles of the -globin gene are cut with the same
restriction enzyme DdeI;;
6. different mixture of fragments from each allele gives its own band pattern in gel
electrophoresis;;
7. normal allele contains 2 restriction sites within the coding region, producing 3 bands;;
8. sickle-cell allele contains 1 restriction site within the coding region, producing 2 bands;;
9. At the end of nucleic acid hybridization, the band patterns of the person homozygous for
normal alleles, homozygous for sickle-cell allele and heterozygote will be different;;
2011 A Level Paper 3 Suggested Answers
10. i.e. person homozygous for normal allele, produces 3 bands; person homozygous for
sickle-cell allele, produces 2 bands; heterozygote, produces 4 bands;





Alternative restriction enzyme, e.g. MstII (For details pls ref to N08/P3/Q1)