All the figures are fully modifiable and can be supplied as Powerpoint presentations on request.
Activity Duration Content related to the GCSE
curriculum
Content related to the Post 16
/ A level curriculum
1.Introduction: the
topics covered by the
resource: reminding
participants of basic
concepts
~5 mins DNA structure DNA structure and function
Gene cloning technologies
2. Agarose gel
electrophoresis
~10 mins DNA structure;
3. Cutting DNA into
fragments
~15 mins DNA structure; the genome
4. Examples Genetic engineering; the
genome
5. DNA gel practical
activity:
demonstration. All
specialist equipment
and consumables are
available to borrow
from the University of
Manchester.
~45 min - 1
hour
electrophoresis
Slow staining
overnight (fast
staining 30min)
Gel drying 2-3
days
Title Teaching Resources for Molecular Genetics and DNA Manipulation
Authors Katherine Hinchliffe, Shazia Chaudhry
Contact [email protected]
Target level Post 16 but suitable for KS4 / GCSE
Publication date July 2014
These worksheets introduce students to the concept of DNA manipulation using restriction enzymes
and to the analysis of different sized DNA fragments using electrophoresis. Several examples of the
ways in which the analysis of DNA fragments based on size is used by scientists are provided.
There is also the opportunity for interested schools to borrow equipment and materials from the
University of Manchester to allow them to run DNA fragments on a gel and use a safe stain to
visualise the DNA bands.
University of Manchester
Faculty of Life Sciences
July 2014
Teaching Resources for Molecular Genetics and DNA
Manipulation
Faculty of Life Sciences
The University of Manchester
Image by ynse from Poland (Dna rendering) [CC-BY-SA-2.0 (http://creativecommons.org/licenses/by-sa/2.0)], via Wikimedia Commons
DNA
DNA is famous as the blueprint of life, and without it life as we know it would not exist. With all
the publicity it has received, we sometimes forget that it’s just a chemical, though admittedly
it’s one with some important and unusual properties. Fortunately, some of these properties can
be exploited in the laboratory, allowing scientists to manipulate genes and to identify DNA from
different sources.
One very important feature of DNA is the fact that it usually consists of 2 strands. The base
adenine (A) on one strand is always paired with thymine (T) on the other, whereas guanine (G)
always binds with cytosine (C). This is known as ‘complementary base pairing’ (Figure 1), and it
means that if we know the sequence of bases on one strand we can always work out the
sequence on the other strand. Due to its double stranded structure, we usually think of DNA as
being made up of many pairs of bases, usually referred to as ‘base pairs’ or ‘bp’.
Base pairing is also really useful for detecting DNA of a particular sequence. Scientists make a
short piece of DNA (known as a ‘probe’) which is complementary to the DNA sequence they
want to locate. The probe is labelled in some way (eg with radioactivity or a fluorescent dye) so
that it can be detected. The DNA sample is then heated, causing the 2 strands to separate, and
the probe is added. When the mixture is cooled again the probe will bind to its complementary
sequence in the DNA sample, allowing it to be located (Figure 2).
Figure 1: DNA Base pairing. Adenine (A) always pairs with Thymine (T);
Guanine (G) always pairs with cytosine (C). The pairs are held together
by hydrogen bonds (dotted lines). The backbone of the DNA molecule
(solid line) is made of covalently linked sugar-phosphates.
Figure 2: Using a probe to find a DNA sequence. In this example we are looking for the
DNA sequence CCCGCCC in the sample DNA (the target). The double stranded sample is
heated, causing the strands to separate. It is then cooled in the presence of the labelled
probe. As the probe has the sequence GGGCGGG it is complementary to the target and
will bind to it, allowing it to be located.
Complementary base pairing is also important for the polymerase chain reaction (PCR), a
method of producing many identical copies of part of a DNA sample (eg part of a single gene).
This process is known as ‘amplification’. The piece of DNA to be amplified is defined by adding
two ‘primers’, very short pieces of DNA that bind to the DNA sample by base pairing either side
of the region to be amplified. If the primers do not bind to the DNA sample, PCR will not work.
For an animation of PCR in action: http://youtu.be/2KoLnIwoZKU [Accessed July 2014]
Although DNA is composed of only 4 bases, the fact that these can be arranged in an almost
unlimited number of different ways means that two pieces of DNA can have a very different
sequence of bases. The human genome contains around 3 billion base pairs. Although the DNA
of every human is 99.9% identical, there are still enough differences for everyone to have their
own unique DNA sequence (unless they have an identical twin).
DNA is negatively charged, due to the many phosphate groups in its backbone. Like any
negatively charged molecule, when placed in an electrical field it will move towards the positive
electrode, and away from the negative electrode. We can use this feature to separate a mixture
of pieces of DNA based on their size, using a technique called Electrophoresis. For DNA we
usually use a technique called ‘agarose gel electrophoresis’.
Agarose gel electrophoresis
Agarose gels, derived from agar-agar (from seaweed), are used for the separation of nucleic acid
molecules. Agarose is a jelly-like substance which contains small pores; DNA can move through
these, but can’t escape from the gel completely. The gel acts as like a molecular sieve through
which smaller DNA fragments can move more easily than larger ones. This means that when
DNA moves through a gel, the speed at which it moves towards the positive electrode depends
on how big the piece of DNA is: small pieces move faster and further than big ones.
The agarose gel slab is placed between two electrodes in a chamber and immersed in a
conductive buffer solution. Individual nucleic acid samples are applied to wells (small holes at
one end of the gel that don’t go all the way through it) (Figure 3). An electrical field is then
applied and the DNA is allowed to migrate through the gel.
Figure 3: Loading DNA onto an
agarose gel
The DNA is added to wells at one
end of the gel using a micro-
pipette. An electric current is then
applied, with the anode (positive
electrode) at the opposite end of
the gel to the wells. The negatively
charged DNA will move through
the gel towards the anode. In a
given period of time, smaller DNA
fragments will travel farther than
larger ones. Fragments of the
same size stay together and
migrate as single bands.
The DNA fragments can only be seen by staining the gel. This is often done using a fluorescent
dye. The dye binds to the DNA, to form a complex that fluoresces under long-wave UV light. The
DNA fragments become visible as bright bands when viewed under UV light, allowing the gel to
be photographed: (Figure 4). The DNA pattern looks a bit like a ladder, with each rung being a
DNA fragment of a different size.
We measure the size of pieces of DNA by the number of base-pairs they contain. If we want to
estimate the size of the DNA fragments, we run a ‘DNA marker’ in one lane of the gel. This
consists of a mixture of DNA fragments of known sizes. We can estimate the sizes of the
unknown DNA fragments based on where they migrate relative to the positions of DNA marker
fragments (see Figure 5).
For a wonderful animation of agarose electrophoresis in action, and for more information
about separating DNA fragments in gels:
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html [Accessed July 2014]
Figure 4: A typical agarose gel
containing DNA of different sizes,
photographed under UV light.
Different samples of DNA were
loaded into the wells of an agarose
gel. The anode was at the bottom
of the picture: when the electrical
field was switched on the DNA
pieces began to move towards it.
Bigger pieces of DNA move more
slowly than smaller ones, so as
time goes by the different sized
DNA fragments become separated
from each other. The DNA marker
is a mixture of fragments of DNA
of known size. It is used to
estimate the size of the unknown
fragments (see Figure 5)
On Figure 4, label the smallest and largest DNA fragments
Figure 5: Estimating DNA Fragment size using gel electrophoresis. The sizes of the different DNA marker
in the left hand lane are indicated. They range from 100 base pairs to 2000 base pairs. We can estimate how
big the DNA fragments in samples 1-7 are, based on where they migrate relative to the samples of known
size in the DNA marker. For example, in sample 1 the DNA fragment runs in roughly the same position as the
900 base pair fragment in the DNA marker, so the DNA here is around 900bp.
Complete the table to give the approximate sizes in base pairs (bp) of the DNA
fragments in samples 2-7 of Figure 5 (sample 1 has been completed for you as
an example)
Sample 1
2 3 4 5 6 7
900bp
-
-
Cutting DNA into fragments – restriction enzymes
Very large pieces of DNA cannot be separated effectively on gels. In order to study large pieces
of DNA on a gel (e.g. bacterial genomes which are typically composed of millions of base-pairs),
the DNA has to be cut into smaller fragments. We use specialised enzymes called ‘restriction
endonucleases’ (also known as ‘restriction enzymes’) to do this. Restriction enzymes do not cut
DNA randomly - they only cut at specific sequences called ‘recognition sites’ which are usually 4
– 8 base-pairs in length. Different enzymes have their own recognition site: they do not cut the
DNA if the recognition site is not there (see Figure 6). They are so specific that if even 1 base
pair doesn’t match their recognition sequence they will not cut the DNA
If a piece of DNA is digested with restriction endonucleases, the number and size of fragments
produced will depend on the base sequence of the DNA (Figure 7). This means that samples of
DNA from different sources produce their own characteristic ladder of fragments when
separated on a gel.
Figure 6: Restriction endonuclease recognition sites. The 2 restriction enzymes
shown, EcoRI and HindIII, will only cut DNA that contains the sequences shown. They
are very specific: EcoRI will not cut DNA at the recognition site of HindIII, and HindIII
does not cut at the EcoRI recognition site, even though the recognition sites contain
the same bases, just in a different order.. When the restriction enzyme finds its own
recognition site in a piece of DNA, it cuts through the DNA backbone at the points
marked by the scissors.
The following pages give examples of just some of the ways in which restriction digests and
agarose gel electrophoresis are used by scientists to analyse DNA.
Figure 7: Restriction Digestion of DNA
The piece of DNA with the sequence shown at the top of the figure contains 1 recognition site for the
restriction enzyme EcoRI (red) and two for the enzyme HindIII (blue). Exposing the enzyme to EcoRI
produces 2 fragments (Digest 1). Exposing to HindIII cuts the DNA into 3 fragments (Digest 2). If both
enymes are used together (Digest 3), 4 fragments are produced. The pattern of bands produced when
each sample of the digested DNA is separated by electrophoresis is shown on the right.
Example 1: Putting DNA to work: cloning and manipulating genes
Scientists often want to introduce a gene they wish to study into a cell or bacterium that doesn’t
normally contain that gene. This is the basis of genetic engineering. To do this, they ‘clone’ the gene
(make a copy of the DNA they want to study). Typically this is done by inserting the DNA of interest
into a circular piece of DNA called a plasmid. When introduced into bacteria, many copies of the
plasmid can be produced.
These days, scientists usually buy a plasmid and then introduce the DNA they are working on into it.
To do this, and also to make sure that the DNA has been successfully introduced into the plasmid,
they often use restriction enzymes and electrophoresis as tools. Commercially available plasmids are
usually designed so that many restriction enzymes cut the plasmid once only, with the different
recognition sites close together. The plasmid is cut with 2 different restriction enzymes, and the
piece of DNA (known as the ‘insert’) is inserted between the two sites (a process called ‘ligation’)
(Figure 8)
Figure 8: Ligation of DNA into a plasmid.
1 A 4,500bp plasmid with 2 recognition sites for
restriction enzymes (red and blue squares),
which are close together.
2. The plasmid is cut with both restriction
enzymes; this converts it from a circular piece
of DNA to a linear piece. The linear plasmid is
mixed with another piece of DNA, the insert (in
this case a 1,200bp long piece of DNA), which
does not contain recognition sites for the
restriction enzymes.
3. By a process called ligation, the insert is
attached to the 2 cut ends of the linear
plasmid. This creates a single, longer DNA
molecule, and restores the circular shape of the
plasmid. Now, however, the 1,200bp of the
insert is between the 2 restriction enzyme
recognition sites. The total size of the DNA
molecule is now 5,700bp (4,500bp + 1,200bp).
The recognition sites are no longer close
together – the insert lies between them.
Ligation doesn’t always work – sometimes the insert is incorporated, but sometimes it isn’t. When
carrying out this procedure scientists often have to check (‘screen’) several clones to make sure they
have a plasmid that contains the insert, rather than an ‘empty’ plasmid. This is again done using
restriction enzymes and electrophoresis. Cutting an empty plasmid with the 2 restriction enzymes
used in cloning will produce 1 piece of DNA (in this case of 4,500bp).Cutting a plasmid that contains
an insert with the restriction enzymes will produce 2 pieces of DNA, one the same size as the empty
plasmid, the other of the size of the insert.
On the gel (left) draw the appearance of the following
DNA samples after electrophoresis:
Lane 1: plasmid + insert cut with the red restriction
enzyme
Lane 2: plasmid + insert cut with the blue restriction
enzyme
Lane 3 plasmid + enzyme cut with both restriction
enzymes
Lane 4: plasmid cut with both restriction enzymes
Lane 1 2 3 4
Example 2: Who’s the Daddy? Paternity testing using DNA fingerprinting
The genomes of higher eukaryotic organisms (including humans) contain a lot of copies of short
DNA sequences. Some of these sequences are repeated a different number of times in the
genomes of different individuals. They are known as ‘variable number tandem repeats’ (VNTRs),
and because we know the base sequence of the VNTRs, we know which restriction enzymes will
and won’t cut the DNA. We can also generate complementary DNA probes to locate them in a
DNA sample that has been run on a gel. We can use this information to produce a pattern of
DNA fragments that is unique to a particular person (unless they have an identical twin, in which
case both twins’ pattern will be the same). People who are not related to each other are likely
to have very different patterns of VNTRs, but people who are related will have similar patterns.
This process of identifying someone by their DNA is popularly known as DNA fingerprinting.
When the technique was first introduced in forensic science, DNA samples were taken from the
person whose DNA fingerprint was to be produced and cut with a restriction enzyme that does
not cut the VNTR sequence (if an enzyme that does cut the VNTR was used, the DNA would be
broken into lots of tiny pieces of the same size in everybody, and we wouldn’t get a unique
fingerprint). The digested fragments were then separated on agarose gels using electrophoresis.
Following electrophoresis, the DNA is transferred from the gel to a nylon membrane in a process
called ‘Southern blotting’. The position and number of VNTRs is then assessed by tagging a piece
of DNA that will bind to the VNTR sequence with a dye or radio-label (a probe) and seeing how
often it sticks (‘hybridises’) to complementary DNA sequences on the membrane (see Figure 2
for a reminder about probes). Several different VNTRs can be probed at the same time to
produce a characteristic DNA fingerprint for the individual.
Although the original approach was an important breakthrough in forensic science, it is quite
slow and not very sensitive. In cases where the amount of DNA available for analysis is low (e.g.
if the only source of the DNA is a single hair left at a crime scene), PCR is now used to amplify
the VNTR sequences directly and these are then separated in agarose gels and probed as
described above. In recent years, VNTR analysis has been replaced by searching for
‘microsatellite’ sequences consisting of short tandem repeats (STRs) of a few nucleotide base-
pairs. The shorter sequences allow better detection in degraded DNA samples and also are more
suitable for automation, speeding up the detection process.
DNA ‘fingerprints’ obtained from such analyses can help to determine if DNA from an individual
contains any matches with a test sample e.g. in a crime investigation or in a paternity dispute
(Figure 9). In this way ‘suspects’ can be easily ruled out of being a genetic match. Note that DNA
fingerprinting can absolutely disprove a genetic relationship between individuals, but it can
only offer a percentage probability of a positive match.
1 2 3 4
Figure 9. DNA paternity test results.
DNA samples are taken from the mother, the child, and the alleged father. Four different STRs
were probed (gels 1 – 4) to reveal whether there is a possible match between the alleged father
and the child. The different sized fragments have been labelled A –O. The band pattern of a child
will be a mixture of the mother’s and real father’s patterns. This means that any fragment in the
child that doesn’t match the pattern from the mother must have come from the child’s real
father. If the child possesses one or more fragments that aren’t present in either the mother or
the alleged father, then the man being tested cannot be the father of the child.
What can you conclude about the alleged father in this case?
Example 3: The Plague Pit: Diagnosis of ancient disease
Under certain conditions, DNA is a very stable molecule and can survive for many centuries after
death if it’s protected from chemicals and micro-organisms. The inside of teeth is a place where DNA
often survives long after it has decayed in other body parts. Not only does the DNA of the person
whose tooth it is get preserved, but that of any bacteria that was present in their blood when they
died is also protected. This fact has been used to diagnose disease in the bodies of people who died
hundreds of years ago.
A bit of history…..
The Black Death, an epidemic of the disease bubonic plague that swept Europe in the 14th Century,
was caused by the bacterium Yersinia pestis. So many people died that the dead had to be buried in
hastily dug mass graves, known as plague pits. Sometimes these are unearthed during construction
work. Molecular genetics has been used to determine whether these bodies contain DNA from
Yersinia pestis, making it likely that they died from the plague.
DNA is extracted from teeth taken from mass graves suspected of containing the bodies of plague
victims.
Using DNA sequences that are unique to the plague bacterium’s genome, PCR is used to amplify
specific sequences of DNA. The amplified DNA is then separated on an agarose gel. The presence of
a DNA band of a particular size in a sample shows that the person was infected with the plague
when he or she died (Figure 10).
Figure 10: Agarose gel of DNA fragments from the analysis of teeth from 15 individuals suspected
of having died of plague. The presence of a DNA band in samples 1, 2, 6, 14 and 15 demonstrates
that these people were infected with the plague bacterium at the time of death.
Plague is still present in some parts of the world today, and PCR can also be used to help produce a
rapid diagnosis in sick people with plague symptoms. Samples are taken from sores on the patient
and analysed by PCR. This is faster than culturing the bacteria themselves.
Figure adapted from image by Rkalendar (Own work) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia
Commons
Practical Activity: Demonstration of DNA electrophoresis and DNA staining
The equipment and consumables necessary to carry out agarose gel electrophoresis of DNA samples
is available to borrow from the Faculty of Life Sciences, the University of Manchester.
The materials are suitable for use in schools – there is no use of toxic materials or UV light. DNA is
visualised using a safe visible stain (FastBlast) developed by Bio-Rad. This causes the DNA bands on
the gel to appear as darker blue lines against a lighter blue background. The stained gel can be
photographed or dried down onto specialised film and kept as the ‘results’ of the practical.
The package contains:
a flat bed electrophoresis system and power-supply
a pre-cast agarose gel (no stain present)
TAE electrophoresis buffer
DNA loading buffer
DNA samples
Micro-pipettes and tips for loading the DNA (single volume pipettes with disposable tips can
be supplied for students to practice with if required; however, most of the gels only have 8 wells so
it isn’t feasible for an entire class to be involved in loading the gel).
FastBlast DNA stain
Gel drying film
To be provided by the school:
Disposable gloves (advisable for the gel staining, and electrophoresis).
Hot tap water for destaining the DNA gel (if using the fast DNA staining protocol)
Disposable paper towels
Abridged protocol
1. Electrophoresis
Briefly, in class the DNA samples provided are loaded onto a prepared gel. Preparation
simply requires removal of the pre-cast gel and the plastic tray in which it is supplied from its
packaging, after which both are placed in the electrophoresis system – do not remove the gel from
the tray. DNA samples are pipetted into the wells on the gel (which should be positioned at the end
of the tank nearest the negative electrode (black)). It is easier to load the gel if the wells are pre-
filled with TAE buffer (see below). Instructions for use of the pipettes will be provided with the kit.
After loading the samples, the electrophoresis tank is filled to its maximal fill line with 250ml
TAE solution (Tris/Acetate/EDTA; a standard solution for DNA electrophoresis). The lid is placed on
the tank, which is then connected to the power supply and 150V placed across it. The DNA will
migrate towards the positive electrode; its progress can be followed by the movement of the blue
dye (bromophenol blue) present in the samples. In fact the samples should have entered the gel
from the wells after 3-4min of turning on the current.
It takes about 45min for the blue dye to run to about 1cm from the bottom of the gel. It is
not necessary to run the sample all the way down, halfway is enough to separate the bands
sufficiently for a clear demonstration of the principle of electrophoresis. .This should mean that the
exercise can be adapted as necessary, depending on the available time.
2. Staining DNA
Two protocols are possible. Personal Protective Equipment should be worn for the staining process
as although the stain is non-toxic, the 100x stock used in protocol A it will stain clothes and skin a
vivid blue. Agarose gels are also fairly fragile and should be handled gently and supported as much
as possible to prevent breakage.
A). Fast staining (takes about 30 min, although DNA should begin to be visible after 10 min)
The gel is removed from the tank (disconnect the power supply and be aware that during
electrophoresis the TAE buffer gets hot: care should be taken in retrieving the gel). The gel is then
carefully slid from its plastic carrying tray into a plastic staining tray (provided). The gel is submerged
in 100x Fast Blast DNA stain (provided; this is re-usable and can be returned with the kit) for 2 min,
then transferred into a bowl containing a litre (or more) of hot tap water for 10s to remove excess
stain. It is then placed in another container of hot tap water and moved gently within it for 5min.
This destaining process should be repeated until the band become visible.
B.) Slow staining (overnight; less messy)
The gel is removed from the tank (disconnect the power supply and be aware that during
electrophoresis the TAE buffer gets hot: care should be taken in retrieving the gel). The gel is then
carefully slid from its plastic carrying tray into a plastic staining tray (provided). The gel should be
submerged in 1x Fast Blast DNA stain (provided; this is re-usable and can be returned with the kit).
After incubation overnight (with the lid on to prevent excessive evaporation) the DNA should be
visible as blue bands (in fact some bands may be visible after about 40min immersion, and will
become clearer as time progresses; however it takes about 8 hours for bands to become fully
visible).
3. Drying the Gel (optional)
After staining the gel should be placed on the hydrophilic side of the supplied piece of
drying film, which should then be placed on top of some paper towels and then left
somewhere where it won’t be disturbed, away from direct sunlight. It takes 2-3 days for
the gel to dry down, during which time it will shrink considerably, but the stained bands
should not fade. To identify which side of the film to use, apply a drop of water – the
drop will be repelled by the hydrophobic side and form a discrete droplet, but will
spread out on the hydrophilic side (the correct side to use).
More detailed instructions will be provided with the kit.
Answers to Worksheet Questions
On Figure 4, label the smallest and largest DNA fragments
Largest fragments
Smallest fragments
Complete the table to give the approximate sizes in base pairs (bp) of the DNA
fragments in samples 2-7 of Figure 5 (sample 1 has been completed for you as
an example)
Sample 1
2 3 4 5 6 7
900bp
900bp
800bp
900bp
700bp
800bp
700bp
-
- 250bp
- 400bp
150bp
400bp
-
- - - - 250bp
Paternity test: the alleged father is likely to be the real father of the child (they share fragments C,
K and O, which the child must have inherited from his/her father). Also, the child does not have
any fragments inherited from the father (ie ones that are not found in the mother) that are not
present in the alleged father. However, it is not possible on the basis of this analysis to be 100%
certain that the alleged father is the real father of the child.
On the gel (left) draw the appearance of the following
DNA samples after electrophoresis:
Lane 1: plasmid + insert cut with the red restriction
enzyme
Lane 2: plasmid + insert cut with the blue restriction
enzyme
Lane 3 plasmid + enzyme cut with both restriction
enzymes
Lane 4: plasmid cut with both restriction enzymes
5700bp
4500bp
1200bp
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