Teacher notes: DNA sequencing
What is the lesson about?
There are three components to this lesson:
1.
2.
3.
Explanation of DNA sequencing using fluorescent tagging of
nucleotides to identify the base sequence. This footage can be played in
the classroom or be recreated by the teacher.
A practical analysis of sequencing data (chromatogram) to identify the
base sequence.
Ethical implications: students will consider who should have access to
an individual’s genetic information.
Learning outcomes
Higher Biology (revised), Unit 1
3. Genome d) Genomic sequencing
Aims
Students will:
 be able to describe how DNA nucleotide sequences are determined
 be able to ‘read’ the DNA code from sequence data (chromatogram)
 be able to contribute to an ethical discussion .
Getting started
View the ‘DNA Sequencing’ footage with students or recreate the activity in
the classroom.
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
© Learning and Teaching Scotland 2011
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Background teacher notes: DNA sequencing (supporting video footage
can also be viewed)
Dideoxy sequencing is the most widely used method of d etermining the
sequence of fragments of DNA. In this method, stretches of 500–800 bases
can be read at a time. These short sequences can be aligned to unravel
genomes consisting of billions of bases. Frederick Sanger was awarded the
Nobel Prize in 1980 for developing this technique.
Sequencing can tell us the exact nucleotide sequence of a sample of DNA.
DNA is normally synthesised from four deoxynucleotides, each of which has
a basic structure like this:
5’
O
Base
C
C
P
P
P
C
A, T, C or G
C
C
H
OH
3’
The OH group (attached to the 3′ carbon) is needed to bond to the
neighbouring nucleotide’s phosphate group (attached to the 5 ′ carbon) in the
DNA backbone.
Thus, if we look at what happens in a standard polymerase chain reaction
(PCR) reaction using deoxynucleotides like those shown above:


The DNA is denatured (the two strands are separated) at near -boiling
temperature.
The primers anneal (attach) to their complementary sequence on one of
the strands of denatured DNA.
3’
5’ Primer
1
3’
2
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
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Primer
2
5’

The Taq polymerase then extends the primer to create a new
complementary strand of DNA. Newly synthesised DNA strands therefore
span the area between the primers as shown below:
New DNA strand
+
New DNA strand
Now consider a PCR reaction with only one primer in the mix. In tha t case
only one of the two strands would be extended:
5’
3’
3’
Primer
1
5’
First amplification
cycle, ie denaturation,
annealing, extension
3’
5’
+
New DNA strand
This is what happens in a sequencing reaction. There is only one primer
present in the sequencing mix and so only one of the two strands of DNA is
extended and subsequently amplified.
However, also included in a sequencing reaction are a limited number of
extra synthetic nucleotides, which that lack the hydroxyl group attached to
the 3′ carbon. These are called dideoxynucleotides:
Base
O
C
C
C
C
H
C
H
P
P
P
In deoxynucleotides
this is an OH group
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
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When these nucleotides that lack the OH group are added to a replicating
DNA chain, the chain elongation stops because there is no OH group for the
next nucleotide to attach to.
Example
Imagine that we have isolated a section of DNA (shown below) and we w ant
to check the sequence. The DNA that we wish to sequence is termed the
‘DNA template’:
5′-ATGCGCCATTGCCATACAAGCT-3′
3′-TACGCGGTAACGGTATGTTCGA-5′
The sequencing reaction mix we use contains:





the DNA template
Taq polymerase (the enzyme to replicate DNA)
a single primer
normal nucleotides: deoxy-adenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxy-cytidine triphosphate (dCTP),
deoxy thymidine triphosphate (dTTP)
a limited number of fluorescently tagged dideoxynucleotides (T, C, G, A),
which lack the OH group at the 3′ carbon.

Initially the DNA is denatured at high temperature to separate the strands,
after which the temperature is reduced and the primer (shown below in bold
italics) anneals to its complementary strand of DNA. The temperature is
increased again and the Taq polymerase extends the primer along the
complementary strand, as indicated by the arrow:
5′-ATGCGCCA-3′
3′-TACGCGGTAACGGTATGTTCGA-5′
Each time the Taq polymerase adds a nucleotide it is likely to be a normal
nucleotide, as there are lots of these in the reaction. However, eventually a
dideoxynucleotide will be added. The result of this is that the DNA extension
is terminated and the DNA polymerase falls off and starts another extension.
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TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
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Each time the Taq polymerase terminates a reaction it starts over again so
there is a gradual build-up of differently sized fragments, resulting in many
copies of each of the following products:
5′-ATGCGCCATTGCCATACAAGCT-3′
5′-ATGCGCCATTGCCATACAAGC-3′
5′-ATGCGCCATTGCCATACAAG-3′
5′-ATGCGCCATTGCCATACAA-3′
5′-ATGCGCCATTGCCATACA-3′
5′-ATGCGCCATTGCCATAC-3′
5′-ATGCGCCATTGCCATA-3′
5′-ATGCGCCATTGCCAT-3′
5′-ATGCGCCATTGCCA-3′
5′-ATGCGCCATTGCC-3′
5′-ATGCGCCATTGC-3′
5′-ATGCGCCATTG-3′
5′-ATGCGCCATT-3′
5′-ATGCGCCAT-3′
5′-ATGCGCCA-3′
The differently sized DNA fragments can then be size -separated by a
technique called gel electrophoresis. This is a commonly used tool in
biological science laboratories and involves loading the sequencing reaction
products onto a gel matrix. When an electric current is applied to the gel , the
negatively charged DNA molecules move through the gel at various rates
depending on the size of the fragments. Essentially the gel acts as a mo lecular
sieve: the smaller the DNA fragment, the quicker the passage through the gel.
Each type of dideoxynucleotide used in the sequencing reaction is tagged
with a fluorescent marker of a different colour. Thus, as each DNA fragment
comes out of the bottom of the gel the colour is detected and plotted as a
coloured peak on a graph called a chromatogram. We know which colour
corresponds to which dideoxynucleotide so by looking at the sequence of
coloured peaks on the chromatogram we can ‘read’ the DNA sequ ence from
5′ → 3′. This whole process is usually automated and produces a
chromatogram (see page 10).
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
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Teacher’s notes for student activity: Chromatogram
Suggested introduction
After 13 years of research, the entire human genome was sequenced in 2003.
The genome code is 3.5 billion bases and if it was written out it would fill
500,000 pages of a telephone directory. Only 0.01% of the human genome
differs from individual to individual. Scattered within the genome are our
genes – these are translated into proteins that make up everything from our
fingers to our hormones, our brains to our muscles. Genes also dictate how
and when, for example, our cells divide. The list of functions served by our
genes is lengthy and complex.
Students are asked to solve the missing sequence on a chromatogram. Please
refer to ‘Student activity: chromatogram’ for details.
Solution to student activity: Chromatogram
[ATGGACTCGCTATCTGTCAACCA]
Student extension question: Why might you want to sequence DNA?
Suggested answers:
1.
4.
To check that you are working with the correct fragment of DNA that
you want to study in the laboratory, eg for cloning and experimentation.
To identify sequences that correspond to genes that may help in medical
diagnosis, eg if a patient carries the sequence for a genetic disease.
To compare sequences of DNA and analyse the differences and
similarities between species and across species to broaden our
understanding of evolution.
To better understand the relationship between genotype and phenotype.
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TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
2.
3.
© Learning and Teaching Scotland 2011
Teacher notes for student activity: Ethics and DNA sequencing
Students should investigate diseases that can be determined through genetic
testing.
Our genetic make-up can affect our susceptibility to many diseases. Although
the situation is complex, genetic health scans are now offered to calculate
your genetic risk of developing heart and circulatory diseases , digestive and
metabolic disorders, and various cancers and conditions affecting the brain
and nerves. Although we cannot endorse this company’s products, an example
of a genetic health scan can be found at www.decodeme.com.
In the future we may be able to link an individual’s genetic profile to other
characteristics, such as physical strength, memory or tendency to addiction
(eg alcoholism) etc.
Activity
In groups, consider what kind of information would you like to know if your
genome was sequenced.
When considering this question, students should use their knowledge of what
is possible just now, and what may be possible in the future. For example:



you may be susceptible to diabetes and could change your eating habits to
prevent this
you may develop a degenerative condition but there is nothing you can do
and so you should enjoy life to the full
you may have a predisposition for a talent, eg music, that you should
work on.
Why might the following people want to have access to your genetic profile?
(Starter points to help stimulate discussion with students are listed below).
1.
Research scientists would find it very useful for researching genetic
disease to determine which alleles or mutations are responsible for
causing certain diseases. This type of information could help lead to
cures. Would this open up abuse, creating a ‘super -race’?
2.
Drug companies already create drugs that could help to cure disease or
alleviate symptoms for some people but not others. Bespoke targeted
medicines could be developed for individuals that would prove to be
more effective. What about the cost and the drug companies ’ profits?
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
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3.
Your school, in the future, could use genetic information to see if you
had a tendency to excel in one field, eg athletics, and target your
education accordingly. Does this limit choice?
4.
Your parents or guardians are carers pre-16. Do they have the right to
have access to all your genetic code as a child to help in your care?
Encourage students to imagine themselves in a parent’s role.
5.
You. Think of the cost. To test each member of society would cost
billions. What about counselling individuals about the risk factors?
Could you/should you change the way you live your life in light of the
information?
Ask the students to rate these groups (research scientists, drug companies,
your school, your parents) according to whether they agree or d isagree that
these groups should have access to their genetic profile. There are no right or
wrong answers, therefore each group should be encouraged to explain their
decision, allowing differing viewpoints to be heard. Each group should reach
a consensus opinion on whether to allow the interested parties access to their
genetic code. A spectrum should be created on their table with ‘agree’ at one
end and ‘disagree’ at the opposite end. Each card should be placed along the
spectrum (perhaps some in the middle) to reflect how strongly the group
agrees or disagrees with the right for the interested party to have access to
their genetic code.
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TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
© Learning and Teaching Scotland 2011
Student activity: Chromatogram
Look at the ‘graph’ with coloured peaks on it (page 10). This is called a
chromatogram. It shows data from a sequencing reaction produced by
research scientists working in Glasgow at the Centre for Virus Research. The
peaks in the sequence represent nucleotides on a strand of DNA, the left -hand
side being the 5′ end of the DNA, and the right-hand side the 3′ end of the
DNA.
The key to the colours used in the chromatogram is:
red = T, black = G, blue = C, green = A
Using this colour key your activity is to fill in the blank areas on the fifth and
sixth lines of sequence on the chromatogram. There are 23 blank nucleotides.
Note that the nucleotide letters are written above the coloured peaks.
The entire human genome was sequenced using an automated version of this
process. The genome code is 3.5 billion bases long.
Extension
Why might you want to sequence DNA?
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
_______________________________________________________________
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
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Student activity: Chromatogram (continued)
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TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
© Learning and Teaching Scotland 2011
Student activity: Ethics and DNA sequencing
1.
Our genetic make-up can affect our susceptibility to many diseases.
Although the situation is complex, genetic health scans are now offered
to calculate your genetic risk of developing various conditions.
Research three such conditions and list them:
1.
2.
3.
2.
In groups, consider what kind of information would you like to know if
your genome was sequenced.
3.
Note down the reasons why the following interested parties might want
to have access to your genetic profile:





research scientists
drug companies
your school
your parents or guardians
you.
Cut out the cards (research scientists, drug companies, your school,
your parents or guardians, you, agree, disagree).
Place the ‘agree’ and ‘disagree’ cards at opposite ends of your table.
As a group, consider whether you agree or disagree that the interested
parties should have access to your genetic profi le. Only place the cards
once your group has reached a consensus and is able to explain why the
card was placed in this position.
Remember – you can put the cards anywhere on the spectrum between
‘agree’ and ‘disagree’.
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
© Learning and Teaching Scotland 2011
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Student activity: Ethics and DNA sequencing cards
12
Research
scientists
Your school
Drug
companies
Your parents
or guardians
AGREE
DISAGREE
TEACHER’S NOTES: DNA SEQUENCING (H, BIOLOGY)
© Learning and Teaching Scotland 2011
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