Pure and Applied Genetics
January 2010
SECTION A
Answer at least ONE Question from this section
Q1
a)
Answer
Should outline the main features such as being capable of replication
in the host cell, relatively small and easily isolated, unique restriction
sites as cloning sites and a selectable marker.
(25)
(75)
b)
Answer
The gene can be cut with restriction enzyme EcoRI to create sticky
ends. Cutting the plasmid with EcoRI will linearise the plasmid
opening it within the lac Z gene. The plasmid should be treated with
alkaline phosphatase to reduce the chance of vector religation (a
description of how this works should be included). Alternatively
cutting both gene and plasmid with EcoRI and BamHI would allow
directional cloning and the plasmid would be unable to religate due to
the loss of a small section.
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.
After transformation samples from the control (no DNA) are grown on
agar only plate and agar plate containing AMP and X-gal.
Transformed cells onto agar containing AMP and X-gal. Cells
containing the recombinant should grow on the AMP agar as the
plasmid confers resistance to the antibiotic. In the control
transformation cells will grow on agar but not on AMP agar. As the
inserted gene disrupts the lacZ recombinants will be unable to
metabolise X-gal and will remain white. Cells that contain plasmid
only will be capable of metabolising X-gal producing a blue product. A
white colony can be selected from the AMP/X-gal agar 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.
Q2
(100)
Answer
Either technique can be described but should be discussed in detail. PCR
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should describe the technique detailing how the amplification and specificity
are achieved.
Basic components are DNA /cDNA template (Generally double stranded
DNA, no need to be purified, continuous sequence spanning the region to be
amplified), DNA Polymerase (thermostable), Oligonucleotide primers (Two
synthetic oligonucleotides (20–30bp) that hybridise to opposite strands of the
DNA and flank the DNA of interest allowing extension to the 3’ end),
Deoxynucleotide triphosphates, Reaction buffer, Magnesium.
PCR is a repeated cycle of 3 steps each cycle the number of specific DNA
templates doubles.
Denaturation (94oC) – Heat is used to break the H bonds between strands
denaturing the DNA allowing it to be used as a template.
Annealing (40 – 60oC) – the primers anneal to the template, exact
temperature depends on the primer.
Extension (72oC) – DNA Polymerase adds nucleotides to the 3’end of the
primer.
The creation of a genomic library should be described and selection from it
using nucleic acid hybridisation.
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 and transfected
into host cells. A probe is required to isolate a specific DNA sequence this
can be a single stranded DNA sequence, RNA, gene from another species,
cDNA for a genomic library which is complementary to the sequence of
interest. The cells of the library are transferred to nitrocellulose, lysed, the
DNA is made single stranded by treatment with alkali and bound to the
membrane using heat or UV light. The membrane is incubated with the
labelled probe which will bind to the membrane. Non specific binding is
removed by washing. The probe will bind specifically to the complementary
DNA sequence via H bonding between bases. The label allows the correct
cell to be identified. Alternatively the DNA can be expressed and selected
with antibodies or something else specific to the protein.
Q3.
(100)
Answer
The answer should include a description of the different types of mutation
and examples of specific diseases. The answer is fairly open but may cover
some of the points below.
A mutation is a change in a short region of DNA. This can be a change in
one nucleotide, point mutation, or insertion or deletion of a few nucleotides,
frameshift. Mutations can arise from replication errors or environmental
agents such as chemicals or radiation. Some mutations are silent maybe if
they occur in non-coding DNA while others if they occur in coding DNA or
control regions have a significant effect.
A frameshift mutation should be explained (including diagrams) and its
effect on the cell. Examples might include Cystic Fibrosis and loss of
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phenylalanine often by a deletion of nucleotides.
Replication slippage and trinucleotide repeat expansion disease could be
discussed using specific examples e.g. Huntingtons disease.
Point mutations change the sequence of a codon this can be synonymous
–code for the same amino acid, non-synonymous – code for a different
amino acid the effect of this depends on the function of the specific amino
acid it may have no effect, reduce activity of the protein or if the change is
to a key amino acid e.g. at an active site complete loss of function,
nonsense – code for a stop codon which results in a short protein usually
non-functional unless it occurs near the end, readthrough – a stop codon is
changed to an amino acid producing a longer protein most proteins might
tolerate this but it could affect folding and therefore function.
Mutations outwith the coding region can have a variety of effects including
inactivation of promoters or regulatory sequences e.g. if the mutation is to
the sequence recognised by a DNA binding protein. Mutations at the intron
exon boundary might affect splicing e.g. in  thalassemia.
Mutations in germ cells and somatic cells. Recessive mutations and loss of
function e.g. cystic fibrosis or gain of function which may confer an
abnormal activity on a protein but often affects the regulatory region
resulting in altered expression of the protein an example might be a
mutation in the regulatory region of a gene coding for a protein with a role
in cell growth leads to over expression and cancer.
SECTION B
Answer at least ONE Question from this section
Q4
a)
Answer
Within the genome there are short tandem repetitive sequences,
microsatellites or STR e.g. CACACACA. The number of repeats is
unique to individuals and each individual may be different at each
allele as one is inherited from each parent. For example a genotype
7/2, individual has 7 repeats at one allele and 2 at the other.
Either RFLP or the PCR method can be described here as the
technique. Both rely on the difference in product size due to the
different number of repeats.
(50)
b)
Answer
(50)
Proofreading by DNA Polymerase which has a 3’-5’ exonuclease
activity which detects a mismatched base pair and removes it.
General Excision repair – Proteins scan the DNA recognise
mismatch, identify incorrect strand, cut DNA either side of error,
remove strand (may require helicase), fill gap with DNA
Polymerase III and seal with DNA ligase. Direct mechanism may
include repair of thymine dimmers by Photolyase.
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Q5
(100)
Answer
The answer should discuss methods for studying DNA binding proteins.
This should include a description of the methods and the information that
could be gained from the experiment. Methods could include gel
retardation, Footprinting, modification interference, purifying the protein,
studying structure by X-ray crystallography and NMR.
Gel retardation experiment. Based on the fact that naked DNA and DNA
with a protein attached can be separated on agarose gels. The section of
DNA thought to contain a binding site is digested with restriction enzymes
then the digest mixed with an extract of nuclear proteins (the pure protein
will be used if it is available) or not. Banding patterns are then compared.
Diagrams could be used to show how the fragment bound to protein is
held back in the gel. This tells us the general location of a protein-binding
site.
DNase I footprinting is then performed on the identified fragment. DNase
footprinting gives a more localised region of binding. The fragment is
labelled, incubated with nuclear extract or not and digested with DNase I
under limiting conditions. In the absence of nuclear extract in theory each
copy of the fragment should be cut once this results in a ladder of bands
differing by one nucleotide. In the presence of nuclear extract there is a
gap in the ladder, which corresponds to the region the protein, has bound
and protected the DNA from digestion. This identifies the region on the
DNA where the protein binds.
Modification interference identifies nucleotides which are key for protein
binding. Individual nucleotides are modified and the effect on binding is
monitored.
Purifying the protein – the DNA binding protein can be purified by passing
a nuclear extract through a column on which the DNA sequence
immobilsed.
X-ray crystallography and NMR spectroscopy allow the structure of the
protein and its interaction with DNA to be analysed. May also include
some details on the structure of the protein e.g. zinc finger etc.
Q6
In prokaryotes RNA Polymerase (via sigma subunit) binds to promoter
sequence at -35. DNA is opened at -10 (A/T rich). Transcription starts at +1.
First few ribonucleotides are added the sigma leaves. This allows RNA
Polymerase to continue. DNA is opened about 12-14 bp. RNA polymerase
uses the 3’ to 5’ strand as a template. RNA transiently base pairs with the
template DNA. Termination occurs after a hairpin or rho protein.
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In eukaryotes note differences in promoter sequence at 1-25 and initiation
sequence. Binding of TBP to promoter followed by number of
transcription factors and RNA Polymerase to create an initiation complex.
Note the difficulty in accessing the genome due to histones and
packaging. Several elongation factors involved although similar
mechanism. The processing of mRNA should cover
5’ capping,
polyadenylation and intron splicing.
5’ Capping of mRNA. Capping has been completed before RNA is 30
nucleotides in length
Addition of guanosine to 5’ end of mRNA results in 5’ to 5’ bond between
GTP and terminal nucleotide catalysed by guanylyl transferase
Attatchment of methyl group to N-7 on purine ring catalysed by guanine
methyltransferase. Other methyl groups can be added
Polyadenylation is part of the termination process rather than a
posttranscriptional event. Nearly all eukaryote mRNA’s have up to 250
adenosines added to their 3’ end.
Mechanism
PolyA polymerase, which is template independent, is the enzyme which
catalyses the addition of A’s. The polymerase does not act the end of the
RNA but at an internal site, which is cut to create a new 3’ end. There are
specific sequences in the RNA, which direct the enzyme.
Splicing the mechanism for removing introns and joining exons should be
discussed.
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