• Personalised medicine is based on an
individual’s genome. Analysis of an
individual’s genome may lead to
personalised medicine through
understanding the genetic component of
risk of disease.
4 pictures 1
word!
Personalised Medicine-The future
Personal Genome Sequence
• Complete sequencing of person’s DNA
bases – called personal geonomics
• Why has this become more and more
possibly?
• Faster and cheaper due to techniques like
PCR (next lesson!!)
• Analysis of an individual’s genome may lead
to personalised medicine through
understanding the genetic component of risk
of disease eg. BRCA 1 and 2 genes for
breast cancer means 45 to 65% chance of
developing breast cancer by the age of 70!
Pharmacogenetics
• Pharmacogenetics is the study of how
people's genetic makeup affects their
responses to drugs.
• If a drug is known to produce side
effects in some people,
pharmacogenetic testing may help
identify at-risk patients.
• Once DNA sequencing
has identified the
genes involved in a
specific disease and
established the
structure of the
protein expressed,
pharmacogenetisists
try to synthesise a
specific effective
drug.
• The drug produced
will
– bind to proteins
involved or
– prevent their synthesis
by binding to a specific
region of the DNA
preventing
transcription of
abnormal mRNA
or
– by binding to the
abnormal mRNA
preventing translation
eg interfering RNA
(RNAi)
Making a diagnosis of disease
status or risk of disease onset
•A cell sample from a patient can be screened
for the presence or absence of a particular
sequence, eg a mutation in a gene.
•This information can be used to diagnose a
condition or describe the likelihood of a
condition developing.
•For example, women with a family history of
cancer can be tested for mutations in the BRCA
1 and BRCA 2 genes.
•Mutations in these genes can increase the risk
of developing breast or ovarian cancer.
Personalised medicine
• Studying an individual’s genome may enable
doctors to more accurately diagnose a disease
and then prescribe the correct drug, at the
correct dosage (level) and at the correct
time. It may reduce the possibility of the
patient suffering adverse reactions.
• It may further help to identify an individual’s
susceptibility to genetic diseases and enable
steps to be taken to avoid or reduce the risk
of developing that disease.
• Now do the pharmacogenetic diamond 9
activity
Ethical issues
If a person’s genome
contains genetic ‘markers’
indicating a high risk who should have access to
this information?
Employer?
• Arrays of DNA probes are used to detect
the presence of specific sequences in
samples of DNA. The probes are short
single stranded fragments of DNA that are
complementary to a specific sequence.
Fluorescent labelling allows detection.
What is the link?
Basically a DNA photocopier!
A technique for the
amplification of DNA in vitro
(outside the body – like IVF).
•
Allows selective amplification of any
fragment of DNA providing the nucleotide
sequences flanking the fragment are known
Finds a needle in the haystack and then
produces a haystack of needles by
selective amplification
•
Kary Mullis: Nobel Prize for Chemistry 1993
Polymerase Chain Reaction (PCR)
• PCR has made it possible to analyse and
identify DNA fragments found in minute
quantities in places like a drop of blood
at a crime scene or a cell from an
extinct dinosaur.
Genetic testing
So it uses are ….
Forensics!
The problem!
Maisy the
research meerkat
is pregnant, but
who’s the daddy?
How can we find
out?
A real problem for a real scientist
What did she do?
Johanna Nielsen, a PhD student at
the University of Edinburgh and the
Institute of Zoology at the University
of Cambridge
She knew that DNA is the genetic material of living things
Therefore the DNA sequence of the foetus would have
greater similarity with the father than with a male meerkat
who was not the father
She had two candidate fathers and decided to compare
the DNA from the foetus with that of the two possible
fathers
She decided to use DNA profiling (fingerprinting)
techniques, which involve the use of the polymerase
chain reaction, or PCR for short
The DNA profiling
technique:
compares the size
and sequence of
DNA at a specific
site between
individuals
DNA
Johanna knows that
this section of DNA
can be of different
sizes between
meerkats. She wants
to compare the DNA at
this site between the
three samples.
Firstly, she needed to extract the DNA
from each potential father and the
foetus.
What would she need to do to
get a DNA sample?
PCR process
• If you wanted to amplify DNA what
things would you need?
DNA
polymerase
By using PCR, Johanna can amplify
this section of DNA many times.
• http://courses.scholar.hw.ac.uk/vle/scholar/
session.controller?action=viewContent&ba
ck=topic&contentGUID=0dbebd90-147267b0-663e-8061550d5f73
•PCR uses the same mechanism for
copying the DNA as our cells use for
DNA replication.
•In pairs, remind yourselves of what
happens during DNA replication.
•What is the main enzyme involved?
PCR uses the enzyme DNA polymerase to
replicate or ‘amplify’ the DNA strand.
Film on PCR procedure
• http://www.edvotek.com/Resources
PCR animations
• http://www.dnalc.org/view/15924-Makingmany-copies-of-DNA.html
• http://www.dnalc.org/resources/3d/19polymerase-chain-reaction.html
• http://www.dnalc.org/resources/animations
/gelelectrophoresis.html
The polymerase chain reaction
The DNA is heated to
94°C to denature the
double helix so
amplification can take
place.
Step 1: DNA denatured
Primers are used to start the
replication process of just the
section needed.
Primers anneal
Step 2: Primers anneal
Primers are short single strands
of DNA that bond or ‘anneal’ to
the sections of DNA on either
side of the section of DNA that
is wanted. The temperature is
reduced for this step
DNA polymerase
Step 2: DNA
is copied
(Temperature is
increased to 72°C)
Annealed primers
+
Two strands of DNA are produced.
And then start the three steps all over again!
...but this time the amplified DNA is also used as a
template...
To give four...
PCR Process Summary –
Learn!
DNA is heated 94-96oC
Hydrogen bonds between chains
break
Separate into 2 strands
Mixture cooled to 50-65oC
Allows primers to anneal/attach to each 3’ end of each strand
Heated to 72oC for DNA polymerase to attach nucleotides
Heat tolerant DNA polymerase
then replicates the region of DNA.
Takes longer for polymerisation of
nucleotides
Repeated cycles of heating and cooling amplify this region
of DNA by thermal cycler (~30 times)
A recap...
30-40 cycles
Put the following steps involved in
PCR into the correct order.
1. DNA replication occurs using DNA polymerase.
2. Primers introduced and temperature reduced to allow bonding.
3. Temperature increased again to separate strands and the cycle begins
again.
4. Temperature increased to 95°C to separate strands.
5. Double-stranded DNA produced, consisting of a template and a new strand
of DNA.
Draw an annotated diagram to
accompany each stage.
Share your finished note with a partner
and check the steps are in the correct
sequence
• Temperature increased to 95°C to separate
strands.
• Primers introduced and temperature reduced to
allow bonding.
• DNA replication occurs using DNA polymerase.
• Double-stranded DNA produced, consisting of a
template and a new strand of DNA.
• Temperature increased again to separate
strands and the cycle begins again.
After PCR what will be in the
sample tubes?
Maisy’s
baby
Malcolm
Martin
Millions of copies of the DNA section/fragment
selected by Johanna.
Remind yourself why Johanna wanted to
amplify this fragment.
Johanna needs to find out the size of the DNA fragments inside each
sample.
She uses a technique called DNA gel electrophoresis, which separates
DNA fragments based on their size.
In the past this was done in tanks, using agarose gel electrophoresis,
but laboratories now use an automated machine
that can process many samples quickly using
capillary gel electrophoresis.
DNA probes
DNA probes are pieces of DNA used to
detect the presence of specific
sequences in DNA.
•Made of DNA
•Short
•Single stranded
•Complementary to the sequence being
searched for
• http://courses.scholar.hw.ac.uk/vle/scholar/
session.controller?action=viewContent&ba
ck=topic&contentGUID=3c1030b1-0642a554-5484-24646a7561a2
DNA probes
• DNA probes are short, single stranded fragment of DNA that is
complementary to a specific sequence (called target)
•
DNA Fluorescent labelling allows detection.
How do we find where the probes
have bound?
Before the DNA probe
is introduced to the
DNA it is fluorescently
labelled. This involves
attaching a fluorescent
dye to the probe. For
example, ethidium
bromide fluoresces
orange when bound to
DNA and exposed to UV
light.
Sequencing DNA
A proportion of DNA is chosen and many copies are
synthesised.
DNA polymerase, primer, the four DNA nucleotides
and modified nucleotides are added
When a modified nucleotide binds to the DNA strand
it halts the process
As this is carried out on many copies, eventually all the
strands will have stopped at every possible position
http://highered.mcgrawhill.com/sites/0072556781/student_view0/chapter15/
animation_quiz_1.html
The results are then processed by a computer, producing data like that
shown above. This example shows the results of two different PCR
samples. One of the DNA fragments is 121 base pairs in length, the other
is 159 base pairs.
If the PCR sample contains millions of DNA fragments, why would
one sample only produce one data peak?
Here are Johanna’s data for three different DNA
fragments from each of the meerkats
Martin!
Who is the daddy?
Malcolm
Martin
Polymerase Chain Reaction (PCR)
•
•
•
•
•
Many copies of a specific segment of DNA can be produced using a
technique called polymerase chain reaction (PCR for short). The
copying of this DNA segment is done in vitro – outside the body.
DNA is initially heated to break the hydrogen bonds between base
pairs, separating the two strands.
It is then cooled to allow primers to bind to target DNA. Primers
are pieces of single stranded DNA, complementary to a specific
DNA sequence that bond or ‘anneal’ to the start and end of the DNA
strand to be replicated.
DNA polymerase enzymes then add free DNA nucleotides to the
primers at the 3’ end of the original DNA strand.
This heating and cooling is repeated to create more copies of the
DNA.
DNA probes are finally used. These are short fragments of single
stranded DNA, complementary to a specific sequence, used to
detect the presence of specific sequences in the original DNA.
Fluorescent labelling of these probes allows visible detection of the
strands under UV light.
• Applications of DNA profiling allow the
identification of individuals through
comparison of regions of the genome with
highly variable numbers of repetitive
sequences of DNA.
Medical and forensic applications
In groups discuss how DNA probes along
with PCR can be used to:
• make a diagnosis of disease status or risk
of disease onset
• identify if a particular individual left blood at
a crime scene.
Crime Scene
Forensic
scientists make
use of PCR to
amplify DNA
samples from a
crime scene.
This can then be
compared against
samples of the
victim and
suspects
Identifying if a particular individual
left blood at a crime scene
•DNA profiling allows the identification
of individuals through comparison of
regions of the genome with highly variable
numbers of repetitive sequences of DNA.
•So if a tiny sample of blood is left at a
crime scene, the DNA it contains can be
amplified using PCR.
•A probe can then be created, which binds
to one of these highly variable sequences.
•If a suspect is apprehended their DNA
can be tested with the same probe to look
for the sequence in question.
Electrophoresis animation
• http://learn.genetics.utah.edu/content/labs/
gel/
Paternity testing
Is he the father?
Every band in a
person’s ‘genetic
fingerprint’ must
match one in that
of their father or
mother!
Mother
Child Father
DNA profiling
• Complete the alcoholism profiling scenario
Applications of DNA profiling
• By comparing regions of the genome with
highly variable numbers of repetitive
sequences of DNA, profiling allows individuals
to be identified.
• E.g. blood from a crime scene to a suspect
• E.g. paternity testing.
• E.g. a diagnosis of disease or risk of disease
onset can be made.