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 Katherine.a.hinchliffe@manchester.ac.uk

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