1. © SSER Ltd.

2. Genetic Fingerprinting
Genetic fingerprinting is a technique that was developed in 1984 by
Alec Jeffreys and his colleagues at the University of Leicester
The human genome is made up of approximately genes that
code for the diversity of proteins found in the species
Despite the large number of functioning genes within the human
genome, about 90% of our DNA is non-coding and has no known function
Jeffreys and his co-workers found that, within these non-coding DNA regions,
there were sequences of nucleotides that repeated many times
These nucleotide sequences were found throughout the genome but, in certain
locations, they repeated one after another many times – they repeated
in tandem and became known as satellite DNA
Each nucleotide sequence varies in the number of times it is repeated
such that these satellite regions are sometimes known as
VNTRs (variable number of tandem repeats)
The number of repeats of these nucleotide sequences varies from person to
person as does their location within an individual’s DNA
The pattern of VNTRs within an individual’s DNA is unique (except in the case
of identical twins) and as such are like ‘fingerprints’ of a person’s identity
The genetic fingerprinting technique analyses the lengths of the VNTRs
of a given individual and provides a unique profile of their DNA

3. Making a Genetic Fingerprint three different VNTRs or mini-satellites
In this example, we will use an imaginary source of DNA within which are located
three different VNTRs or mini-satellites
mini-satellite with three
repeating nucleotide
sequences
mini-satellite with eleven
mini-satellite with seven
In this case, the DNA has three sets of repeated regions,
containing three, eleven and seven repeats
The first step in the fingerprinting procedure is to ‘cut’
the DNA under study with a restriction enzyme
Jeffreys used the restriction enzyme HaeIII because this enzyme ‘cuts’
on either side of the mini-satellite regions and not within them
Fragments of different sizes are produced when
the DNA is ‘cut’ with the restriction enzyme

4. C A B Restriction enzyme ‘cuts’ the DNA at specific restriction sites
Fragments of DNA of different sizes are obtained of which three
contain the mini-satellites or VNTRs (A, B and C)
The fragments are now separated from one another
by the technique of electrophoresis

5. Electrophoresis is a technique for separating molecules
from a mixture according to their charge and size
A solution containing the DNA fragments is placed in a
well in a supporting medium of agarose gel
The pH of the sample and the gel are carefully controlled using buffer solutions
porous
agarose gel
solution of
DNA fragments
buffer solution
anode
cathode
A direct electric current is then passed through the gel and the
negatively charged DNA molecules move towards the anode
The length of the fragments determines their speed of movement such that the smaller DNA fragments move further through the gel than the larger fragments

6. C A B In our example, there are eight DNA fragments and Direction of
these move through the gel
according to their size
Direction of
electrophoresis
Once transferred to
the nylon membrane or
nitrocellulose filter, gene
probes will be used
to seek out the fragments
containing the
‘mini-satellites’ A B C
The bands that we wish
to visualise are those
containing the
‘mini-satellites’ or
VNTRs (A, B and C)
The DNA in the gel must
first be denatured in order
to create single-stranded
DNA that will hybridise with
the probe – this is achieved
either by heating the DNA
or by treatment with alkali
In order to locate the
‘mini-satellites’, the DNA
fragments are transferred
to a nylon membrane or
nitrocellulose filter using
a technique called
SOUTHERN BLOTTING

7. Southern Blotting is a technique used for transferring single-stranded
Glass
Block
Blotting paper
soaked in buffer
Southern Blotting is a technique used for transferring single-stranded
fragments of DNA on to a nylon membrane or nitrocellulose filter
The gel containing
the DNA fragments
is placed on wet blotting
paper soaked with buffer
A nylon membrane or nitrocellulose
filter is then laid over the gel
A large weight is placed above
the blotting paper to create
pressure on the gel and hence
to ‘blot’ the DNA fragments
onto the nylon membrane or
nitrocellulose filter
Layers of blotting paper
are placed over the
membrane or filter
Nowadays, the blotting technique used
may be more sophisticated; vacuum
blotting and electroblotting are commonly used
in place of the paper towels and weights
The filter or membrane is
dried and the DNA fragments
are held permanently in place

8. The DNA filter containing the single-stranded fragments of DNA is now
exposed to a solution containing radioactive, single-stranded probes
The probe and its target (the mini-satellites) will hybridise

9. The radioactive probes hybridise with the three fragments that, in our
example, contain mini-satellites
Finally, these bands are visualised by the
technique of AUTORADIOGRAPHY

10. of mini-satellites present
AUTORADIOGRAPHY
A photographic film is
laid over the filter
The three radioactive
bands blacken the
photographic film
revealing the pattern
of mini-satellites present
in our imaginary DNA
sample
Genetic Fingerprint

11. These two fingerprints
show the DNA from
twins with identical
patterns of fragments
Humans have much
more complex genomes
than the simple example
just described
Courtesy of
Lancaster University 8 1 2 3 4 5 6 7 10 11 9
When human DNA
is digested with
restriction enzyme,
numerous fragments
contain mini-satellite
regions that react
with the DNA probe
The ‘mini-satellite’
fragments for eleven
unrelated individuals
are shown in this
photograph
These are the individuals’
unique DNA fingerprints

12. Picture reproduced with The Forensic Science Service
kind permission of
The Forensic Science Service
© Crown Copyright 2002
The complexity of the
‘mini-satellite’ patterns
can be seen in these
human DNA profiles
A technique that creates
coloured bands has been
used for these profiles
to aid identification

13. Applications of Genetic Fingerprinting
Genetic fingerprinting is being used for a variety of purposes and these include:
FORENSIC SCIENCE – matching DNA specimens from the scene of a crime to those of suspects
PATERNITY TESTING – resolving disputes over the paternity of a child
EVOLUTIONARY BIOLOGY – establishing the degree of relatedness between different species
HEALTH CARE – the detection of genetic disease in embryonic cells

14. Paternity is established
Paternity Testing
These DNA fingerprints
are those of a mother (M)
and child (C) together with
the ‘possible’ father (F)
The blue arrows identify
the paternal bands
All of the child’s remaining bands are matched in the
‘possible’ father
Every child receives half
of its DNA from the
mother and the other
half from the father
The mother of the child is
known and so the first
task is to identify which
of the child’s bands were
inherited from its mother (remember that
the mother’s bands are
a mixture of her mother
and father’s DNA)
Paternity is
established
The red arrows identify
the maternal bands
All the remaining bands in the child must have a an exact match in the father’s fingerprint

15. child’s remaining bands
In this example, the
paternal bands
(shown in blue)
do not match the
child’s remaining bands
Paternity is
disproved

16. In forensic science, DNA fingerprinting is used to
match material collected
at the scene of a crime to
that of the suspects
This is a diagram of the
genetic fingerprints of a
rape victim’s blood,
semen (the specimen) and
blood samples taken from
the suspect rapists
The fingerprint results
show an exact match
between the semen sample
obtained from the victim
and the blood sample
of suspect 1
Suspect 1 is confirmed
as the rapist

17. Evolutionary biologists utilise the technique of DNA fingerprinting to
establish the closeness of relationships between different species
Species X
Species Y
Species Z
Which of the species X or Y is most closely related to species Z?

18. The number of DNA bands from species X and species Y
that match those of species Z is determined
Species X
Species Y
Species Z
Nine DNA bands
from species X
match those
found in
species Z
Five DNA bands
from species Y
match those
found in
species Z
The genetic relationship is greatest between species X and Z

19. The Polymerase Chain Reaction (PCR)
In the past, one of the drawbacks in obtaining genetic fingerprints
from material present at a crime scene was the very small
quantities of DNA recoverable for analysis
A technique called the polymerase chain reaction was developed in 1983 by
Kary B. Mullis providing the breakthrough that allowed scientists
to produce multiples copies of a DNA sample within a very short period of time
The polymerase chain reaction (PCR) mimics nature’s way of replicating DNA
and is able to generate billions of copies of a DNA sample within a few hours
- the technology allows for cheap and rapid amplification of DNA
The technique involves heating DNA to high temperatures to separate the strands
and then using the enzyme DNA polymerase to create new strands
Due to the high temperatures required for the technique, a thermostable
DNA polymerase had to be found to avoid the expense of needing to
replenish the enzyme after each round of DNA replication

20. The solution to this problem was to use Taq polymerase, derived from Thermus
aquaticus, a bacterium that is native to hot springs – this enzyme is able to
withstand the high temperatures (up to 95°C) used in the polymerase chain reaction

21. The Technique
The target DNA is first mixed with DNA polymerase and primers
and then heated to 95°C to separate the two strands of DNA
Primers are short, synthetic DNA fragments that are complementary to the DNA
sequences at either end of the
region of DNA to be copied

22. The mixture is now cooled to 55°C to allow the primers to bind
The Technique
The mixture is now cooled to 55°C to allow the primers to bind
to the ends of the separated DNA strands
Polymerase binds to the primers and begins adding bases
to form new complementary strands

23. The mixture is now cooled to 55°C to allow the primers to bind
The Technique
The mixture is now cooled to 55°C to allow the primers to bind
to the ends of the separated DNA strands
Polymerase binds to the primers and begins adding bases
to form new complementary strands

24. Two Identical Copies of the Target DNA Sequence
Result From the First Synthesis Cycle

25. The process is now repeated by first heating the mixture to
separate the strands of the
newly formed DNA molecules
The sample is cooled to allow
the primers to attach to the
ends of the DNA strands so that
polymerase can begin its job of
adding bases to the sequence

26. At the end of the second cycle there are four complete DNA molecules identical to the original target DNA
Cycle 2 Products
The cycle is repeated many times with the number of
DNA molecules doubling with each cycle
This exponential increase creates over a billion
copies of the target DNA within a few hours

27. The number of DNA molecules doubles with each cycle
Cycle 2 Products
Cycle 3 Products
The number of DNA molecules
doubles with each cycle

28. PCR generates billions of copies of target DNA within a few hours
SUMMARY
PCR generates billions of copies of
target DNA within a few hours
Target DNA is heated to separate the strands
Two identical DNA molecules are formed
When the mixture is cooled, primers bind to the
ends of the target strands and polymerase enzymes
add bases to complete the complementary strands
A second cycle is initiated by heating the mixture
once again to separate the strands of the newly
formed DNA molecules
When the mixture is cooled, primers bind to the
ends of the target strands and polymerase enzymes
add bases to complete the complementary strands
Four identical copies of the target DNA are formed at the end of the second cycle
This cycle of heating and cooling continues
for approximately 30 cycles, doubling the
number of DNA molecules with each cycle

29. Acknowledgements
Copyright © 2003 SSER Ltd. and its licensors. All rights reserved. All graphics are for viewing purposes only.