May 2013 Answer Guide
Question
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Model Answer
Section A
1 a) As a member of a research group you have been asked to clone the
human gene for a newly discovered enzyme in a bacterial system. Using
the materials below discuss in detail the steps you would perform to
clone this gene.

Multiple copies of the gene amplified by PCR – the ends have been
modified to contain a restriction site for BamH1

The bacterial plasmid, Figure 1 below

The bacterium E.coli
FIGURE 1
Answer
The gene can be cut with restriction enzyme BamHI to create sticky
ends. Cutting the plasmid with BamHI will linearise the plasmid
opening it within the antibiotic resistant gene, tet. The plasmid should
be treated with alkaline phosphatase to reduce the chance of vector
religation.
Ligation – Digested vector and fragment are ligated in the presence of
DNA Ligase. The complementary sticky ends should base pair and
ligase forms the phosphodiester bonds.
The ligation mix would be used to transform bacterial cells, E.coli,.
Probable method would involve calcium chloride and heat shock
(explanation of principles of technique). Control with no DNA
included.
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Question
No.
Model Answer
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After transformation samples from the control (no DNA) are grown
on agar only plate and agar plate containing AMP (all cells should die
due to the antibiotic). Cells containing plasmid can be selected by
plating onto agar containing AMP as they are resistant. To select for
recombinants a copy of the cells is made on nitrocellulose (replica
plating) this is then exposed to tetracycline colonies that are killed
contain the recombinant as insertion of the DNA destroys tet
resistance these colonies are then selected from the original plate
and grown.
To confirm the insert is present a plasmid miniprep is used to isolate
the plasmid. Cut the isolated plasmid with restriction enzymes
appropriate to show the presence of the fragment and its orientation.
Run on agarose gel and visualise.
b) When Eukaryote genes are inserted into E.coli with the aim of producing
a recombinant protein explain why a cDNA template might be used
rather than genomic DNA.
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Answer
Genomic DNA will contain introns which would be spliced out after
transcription in eukaryote cells however this process does not
occur in bacteria and therefore the transcribed mRNA would not
code for the correct protein. cDNA is synthesised using mature
mRNA as the template therefore introns already removed.
2 a) Mutations in proto-oncogenes can cause a cell to divide in an unregulated
manner. Discuss this statement using two examples.
10
Answer
Oncogenes are cancer causing genes ie. expression results in
cancer. These are mutated versions of the normal gene, (protooncogene) genetically dominant, one copy can change the cells
behaviour. They are cellular genes whose function is to promote
normal growth and division of cells. Expression of these genes is
tightly controlled in a normal cell. Changes to the genes may
increase their expression resulting in increased cell division.
Oncogenes may be cell surface receptors, growth factors,
molecules involved in transmembrane signalling, second
messengers or nuclear proteins.
Two examples should be discussed.
b) A DNA library is a useful resource for a scientist. Describe the method(s)
that would be used to create:
i) a genomic library and
ii) a cDNA library
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Question
No.
Model Answer
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Answer
Genomic library is a collection of clones, which between them
contain the DNA of an entire organism. The genomic DNA is
isolated, cut with restriction enzymes, the fragments are inserted
into vectors before transforming host cells.
A cDNA library is synthesized from mRNA by reverse transcriptase.
cDNA fragments are ligated into vectors before transforming host
cells.
c) Briefly outline the difference between the two types of library.
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Answer
cDNA collection is smaller representative only of coding DNA and
then only genes that are expressed in the cell the mRNA is isolated
from – no upstream or downstream sequences or introns. Genes
that are highly expressed will be well represented in the library.
gDNA is the entire genome found in every cell.
3 a) Discuss the differences in technique and applications of standard PCR
and real time PCR.
10
Should describe basic PCR, components in reaction and 3 steps.
End point analysis by gel electrophoresis detects presence or
absence of product but difficult to estimate amount of initial
template. Real time PCR include e.g. SYBR green which binds to
double stranded DNA as it forms emitting fluorescence. The
reaction can therefore be monitored throughout. This can be used
to quantitate the amount of starting template based on the
relationship between Ct value and amount of template. Diagrams
may be used. Could include estimation of transcript using RT
qPCR.
b) Select one genetic disease. Describe the mutation or chromosomal
abnormality, the disease and the diagnostic test.
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Answer
Can select any mutation or chromosomal abnormality. The
mutation should be described and the effect on the protein and/or
disease caused. The test used in labs to detect the disease should
be described e.g. FISH for chromosomal abnormalities, PCR or
sequencing for many mutations.
Section B
4 All three types of RNA are involved in translation (protein synthesis).
Discuss the role of all three in the mechanism of translation.
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Question
No.
Model Answer
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Answer
The answer should describe the function of all mRNA, tRNA and
rRNA in protein synthesis. The mechanism of translation should be
described.
5 a) It is essential that the integrity of the DNA in the cell is maintained.
Discuss the mechanisms that enable this to be achieved.
10
Answer
DNA replication process using complementary base pairing ensures
a low error rate. Proofreading activity of DNA Polymerase during
replication. Repair enzymes should describe recognition and repair
process, DNA Photolyase.
b) In replication both strands of DNA are replicated simultaneously. Describe
how this is achieved. Include a description of the role of the proteins
involved.
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Answer
Description of mechanism used to replicate the leading and lagging
strand in terms of continuous and discontinuous synthesis.
Outlining the role of DNA Polymerase, SSB, DNA Ligase, Helicase,
Topoisomerase and Primase.
6 a) Using examples describe both constitutive and regulatory control of
gene expression in E.coli.
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Answer
Constitutive control being inbuilt e.g. the sequence of the promoter
– strong to weak.
Regulatory may use the lac or tryp operon as examples. Should
describe the operon structure and the control at the level of
transcription.
b) Compare and contrast the organisation of the genome in prokaryotes
and eukaryotes.
Answer
Should include the following minimum:
Bacterial chromosomes circular are found in the nucleoid region of
a bacterial cell. Made more compact by the formation of loop
domains and DNA supercoiling. Generally smaller with large
amount of coding DNA.
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Question
No.
Model Answer
Eukaryote DNA linear strands organised in chromosomes as
chromatin, a DNA protein complex. Eukaryotic DNA wraps around
an octamer of histone proteins to form nucleosomes. The linker
histone, H1, plays a role in nucleosome compaction. Nucleosomes
are further compacted to form a 30-nm fiber. Solenoid and zigzag
models have been proposed. Larger in size with large percentage
of non-coding, repetitive DNA.
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