In this lab class, students amplified the ALU repeat sequence in the PV92 region of chromosome 16 from their own DNA. Controls were used including samples that were homozygous or heterozygous for the repeat to compare results. The precise target sequence is not amplified until the third PCR cycle because primers are needed at both ends of the sequence to prime DNA synthesis. Studying repeat sequences can provide early detection of cancers and insight into drug metabolism for treatment.

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1PCOL305 ANTIVIRAL AND ANTINEOPLASTIC AGENTS
COURSEWORK2009
Amplificationof ALUrepeats inthe chromosome 16 PV92 region
WRITE - UP
In this lab class you have amplified a DNA repeat sequence. The same technology
that you have used is utilised to investigate other repeat sequences which are of greater
significance to cancer formation.
Answer the following questions:
 Explain why in PCR the precise length target DNA sequence doesn’t get amplified
until the third PCR cycle. (10)
The precise length target DNA sequence doesn’t get amplified until the third PCR cycle
because it is at this cycle that the DNA primers serve as the beginning and ends of the
amplified region.
In the first cycle of PCR, after denaturation of the original DNA molecule, the primer
anneals to the unzipped DNA strand and a new complementary DNA strand is
synthesized onto said primer. Taq polymerase extends the new strand way beyond the
target sequence to be amplified to the “flanking region” so the new strand thus contains
forward and reverse primers and is much longer than the precise target sequence

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Fig 1. Cycle 1 of PCR
In the second cycle therefore, there are 2 types of template, the original DNA strand and
the newly synthesised DNA strands that have the target DNA fragment and flanking
region on the 3’ end of the DNA strand but at the beginning of this cycle they are
annealed together and so need to be denatured to separate them, and after this the
primers anneal to the original DNA strand again, as well as the newly synthesized strand
made in the 1st cycle. When new strands are synthesized they make a small number of
target DNA sequences and hybrid sequences
Fig 2. Cycle 3 of PCR
In the third cycle of PCR the target sequence DNA now acts as a template for
polymerisation. The original DNA sequence is still present but the specific target DNA
exhibits dominance when being primed or polymerised. The newly synthesized target
sequence is then reprimed forward and back and Taq polymerase synthesizes copies of
target DNA, copied exponentially for however many cycles the PCR is set to

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Fig 3. Cycle 3 of PCR
 What kind of controls were run in this experiment? Why are they important? Could
others be used? (10)
The controls run in the experiment were an MMR DNA standard, a control that was
(++) homozygous for the ALU PV92 repeat, a homozygous (--) control, and a
heterozygous (+-) control.
Controls are important in laboratory experiments because they give the scientist
working the experiment a standard to compare their sample results to, as well as
showing whether the methods used to conduct the experiment have worked
properly.
For example in this experiment these controls can be used to figure out which
students sample exhibit which genotype e.g. if a student has the homozygous ++
genotype, the band formed on the agarose gel after electrophoresis will be at the
same position as that of the ++ control. The controls shown so far are positive
controls and are used to help find a result
Other controls that could be used in this experiment are negative controls such as to
add a tube with a mix of master mix, nuclease free water and water(instead of cheek
cells) and this should be added to the PCR and ran with the other samples all the way
to the electrophoresis. This should show a negative result and should not show
bands on the gel picture. If a result is shown there is contamination of reagents with
DNA.
 Why in this lab class, using your DNA, have we investigated the number of ALU
repeat sequences in PV92 rather than a disease-associated repeat sequence? What
was your genotype ?(10)
In this lab class we tried to identify the Alu element in the PV92 region of
chromosome 16 within our own cheek cells. The ALU PV92 element is a short

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interspersed element (SINE), making it an intron (region of DNA not serving any
function in the body) this particular Alu element is dimorphic, and is present in some
individuals and not others. Some people have the insert in one copy of chromosome
16 (one allele), others may have the insert in both copies of chromosome 16 (two
alleles), while some may not have the insert on either copy of the chromosome. The
presence or absence of this insert can be detected using PCR followed by agarose gel
electrophoresis.
Genetic testing has developed to a point whereby certain disease states caused by
genes as well as the possibility of screening people for drug metabolism. Certain
disease states can be assessed by doing neonatal tests e.g. in the UK DNA testing is
done in neonates for sickle cell anaemia and cystic fibrosis. In the USA breast cancer
patients are screened for metabolism of tamoxifen and this screening leads to
whether the patient will be prescribed tamoxifen or not.
Although it would have been possible to amplify a disease related DNA fragment,
this was not done because of various ethical issues associated with gene screening
used for diagnosis.
The first reason for this is that if we were testing for a disease related gene it would
be legally necessary to draw up informed consent forms and get all of those in the
lab class to go through them and decide whether they want to give consent. Because
of this some people may not be comfortable giving their consent because of the
results they might get that would tell them their possible risk of contracting cancer.
This could possibly reduce the sample size significantly.
The next issue with screening for a cancer related gene would be that of
confidentiality. This is because if we had been testing for the presence of a cancer
related gene the lab class would essentially making a medical diagnosis and this
would require a medical practitioner to be present, and revealing any results
obtained would be the choice of the “patients” which could also cause problems
with population size as well as running the risk of the results being given out to third
parties and this may cause stigmatization along with discrimination e.g. when
applying for health insurance.
If we were to have done screening for a disease related gene and any patients were
to end up having the disease causing genotype it would cause unnecessary lifelong
psychosocial and psychological issues and would require counselling that the
invigilators in the lab may not be trained for.
In a nutshell, it is very unlikely that people would want to have themselves screened
for a potentially cancer causing gene because of the “ticking time bomb” effect the
results that may show a increased neoplastic risk in later life may give them, and if it
were to be done more formalities would have to be gone through before conducting
the experiment.

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 Describe screening for microsatellite instability as a diagnostic and prognostic marker
for certain cancers, citing hereditary colorectal cancer (HNPCC) as an example (20)
Microsatellite instability is described by the national cancer institute as a change that
occurs in the DNA of certain cells in which the number of repeats of microsatellites
(short repeats of DNA) are not the same number as when the DNA was inherited.
Microsatellite instability is thought to be caused by DNA mismatch repair gene
(MMR) not working properly on certain genes.
Many studies have been conducted to evaluate the role of high levels of MSI as a
prognostic marker and predictor of risk of certain kinds of cancer, as well as an
indicator of the success of chemotherapy in these patients.
An example of this is in hereditary non polyp colorectal cancer. HNPCC is also known
as Lynch syndrome, after Henry T Lynch who first characterised it in 1966. It is known
to cause an early onset of cancers of the lower GI tract as well as those of the
reproductive system. In HNPCC the cause of MSI is a germline mutation in a
mismatch repair enzyme and alterations of the MutS homologue 2 and MutL
homologue 1 mismatch repair genes account for 90%. Microsatellites that had
changed in length are found in all HNPCCs, not only in the critical genetic region but
virtually everywhere in the genome of tumours tested.
Testing for microsatellite instability in colorectal cancer is done using polymerase
chain reaction and it is the most straightforward method of doing so: DNA from a
tumour and from normal tissue are tested. Mutations that alter microsatellite
lengths are visualised as band shifts on electrophoresis gel. The choice of
microsatellite markers is important for MSI testing. The 1997 Bethesda guidelines
proposed a panel of five microsatellite markers for uniform analysis in HNPCC. These
included two mononucleotide (BAT-25 and BAT-26) and three dinucleotide (D5S346,
D2S123 and D17S250) repeats. The Bethesda guidelines have since been revised as
the use of dinucleotide repeats may cause underestimation and overestimation of
instability status. The revision mainly recommends the use of more mononucleotide
markers especially in dinucleotide-unstable cases. Screening using the revised
Bethesda MSI markers is now performed in trials for detection of HNPCC but
diagnostic yield and costs cause an issue so even though screening in this way is
feasible, the option of screening all patients with colorectal cancer with MSI should
wait for more evidence of its clinical value and therapeutic influence of information
gained.
 How can the number of repeat sequences at the ends of chromosomes be
modulated and is this important in neoplastic disease? (20)
Repeat sequences at the end of chromosomes are known as telomeres and their
function is to prevent the end of chromosomes from destruction. Telomeres are

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modulated by the enzyme telomerase, which is a reverse transcriptase enzyme as
DNA polymerase can only prime in the 5’ to 3’ direction and so cannot copy extreme
end sequences of linear DNA.
Telomerase carries its own template of RNA (TERC) which is complementary to
telomere repeat sequences. This allows telomerase to create multiple copies of
telomere repeat sequences without needing a DNA template to direct synthesis. A
defect in telomerase proficiency causes abnormal maintenance of telomeres and this
is associated with some disease states. In somatic cells the telomeres shorten with
every cell division, and this may cause cell aging or cell growth arrest. Disease
associated with this have links to premature aging where there is a major loss of
telomeres whereas in cancer cells there is an excess production of telomerase, which
allows cells to grow indefinitely.
This is important in neoplastic disease as one will be able to test for high levels of
telomerase and check for an increase in telomeres present within a patients DNA
using bioassays. Telomerase inhibitors are a valuable new method used for cancer
therapy. Their effectiveness however may be variable firstly because of slow
response due to time needed for telomeres in cancer cells to undergo apoptosis, and
so long term therapy used in combination with either radiotherapy or surgery would
be necessary. There have also been concerned that inhibiting telomerase may lead
to an increase in malignancy as it would cause genomic instability. These findings
have emerged from studies on gene knockout mice.
When using drugs that target telomeres and telomerase there are several taget sites
that have been studied and can be manipulated. The RNA component can be
targeted by the antisense oligodeoxynucleotides and the hammerhead ribozymes.
The catalytic(hTERT) component can be targeted by reverse transcriptase inhibitors,
and immunotherapy. There have also been studies conducting using small molecules
tested using NCI-COMPARE system. They’re mechanism of action is not fully known
but it is thought that they disrupt the anchoring of the telomerase to the enzyme or
 What impact does CA repeat sequence in intron 1 of the EGFR have on neoplasia?
(20)
Epidermal growth factor(EGF) along with its complementary receptor tyrosine
kinases are important in understanding how signals fromgrowth factors can be used
in a cell in order to modulate gene expression and cause cell proliferation and
subsequently replication. EGFR(epidermal growth factor receptor), the first of its
kind to be discovered is involved in cell growth and gene expression, however gene
expression happens within the nucleus, and EGFR is an extracellular receptor so to
get a signal from it to the nucleus certain pathways must be activated such as
binding of the growth factor by a ligand, receptor demonization,
Autophosphorylation, activation of signal transducers(intracellular), cascading of

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kinases and regulation of transcription factors used for gene expression. All of these
steps can be used as targets by therapeutic agents.
Cancer cells have malfunctioning DNA and so grow and replicate exponentially and
so it is known that growth factors and growth factor receptors play a critical role in
tumour progression, and it is thought that the length of the CA repeat of intron 1 in
the EGFR gene is correlated with the expression of the EGFR receptor in our cells. It
has been found that EGFR is over expressed in a number of solid tumours and is
linked to resistance to clinical strategies including hormone replacement therapy,
chemotherapy and radiotherapy. This over expression is also linked to poor
prognosis of cancer in patients that have it. Recent in vivo and in vitro studies have
shown conclusive evidence that there is a decrease in gene transcription with an
increase in CA repeats on intron 1 and vice versa. In breast cancer shorter CA repeats
have been found and in these tumours EGFR over expression was noted.
EGFR is a good target for neoplastic disease and there are currently several drugs
that affect signal transduction. The anti-EGFR drugs are kinase inhibitor drugs that
have their mode of action directed against kinase activity on members of the EGFR
super family examples of the drugs are Tarceva, and Iressa. Another target used is
the extra-cellular domain of EGFR receptor and monoclonal antibodies are used. An
example is the drug Herceptin which causes receptor degradation, recruitment of
immune cells causing cellular toxicity, as well as inhibition of angiogenesis of cancer
cells.
 What do you see as the clinical benefit to studying DNA repeat sequences in
carcinogenesis? (10).
Carcinogenesis, the formation of cancer, can be caused by spontaneous mutation,
which disrupts the cell cycle and causes uncontrolled cell proliferation.
Carcinogenesis may also be caused by other factors such as chemicals (carcinogens)
which involve DNA damage which is unable to repair itself causing mutated DNA
resulting in carcinogenesis. Radiation (for example UV radiation causing skin cancer)
can also cause carcinogenesis as radiation will penetrate skin and cause mutation of
DNA.
DNA repeated sequences can be found all across the genome and vary in length and
number in different people. In some cases; the number of repeats can help the
development or maybe even cause certain conditions.
Thus the study of these repeated sequences can help in the treatment and
prevention of some disorders, such as cancer. Studying repeat sequences has certain
benefits such as giving an early prognosis of cancer, maybe even years before
cancers start to develop. Early prognosis results in higher survival rates and in the
USA there has been a major decrease in deaths caused by cancer in the past 2 years .
the study of repeat sequences and the genome can also give details of drug

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metabolism and this will give clues of what therapy is the best to use in a certain
patient. There could also be some financial advantages involved as early detection
will allow treatment to start earlier, decreasing the need for more costly treatments
in later stages of disorders. Thus the study and research of DNA repeat sequences
may be highly beneficial in cancer therapy.
Results
+/+ +/- -/- No result
Frequency 1 16 10 11
Percentage(f/38) 2.63% 42.11% 26.32% 28.95%
Fig4. Pie chart of percentage frequencyof differentgene combinationsforALU PV92

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REFERENCES
http://irc.igd.cornell.edu/MolecularMarkers/PCR%20basics.pdf
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http://scientificinquiry.suite101.com/article.cfm/scientific_method_uses_experiments_and_control
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http://www.patient.co.uk/doctor/Newborn-Screening.htm
http://www.nci.nih.gov/dictionary/?CdrID=285933
http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1628&ckt=1
www.cancerresearchuk.org
http://www.ncbi.nlm.nih.gov/pubmed/9563488
http://www.genetichealth.com/CRC_HNPCC_Microsatellite_Instability_Testing.shtml
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