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Genetic fingerprinting is a technique that was developed in 1984 byAlec Jeffreys and his colleagues at the University of Leicester

The human genome is made up of approximately 40 000 genes thatcode 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 certainlocations, they repeated one after another many times – they repeated

in tandem and became known as satellite DNAEach nucleotide sequence varies in the number of times it is repeated

such that these satellite regions are sometimes known asVNTRs (variable number of tandem repeats)

The number of repeats of these nucleotide sequences varies from person toperson as does their location within an individual’s DNA

The pattern of VNTRs within an individual’s DNA is unique (except in the caseof identical twins) and as such are like ‘fingerprints’ of a person’s identity

The genetic fingerprinting technique analyses the lengths of the VNTRsof a given individual and provides a unique profile of their DNA

Genetic FingerprintingGenetic FingerprintingGenetic FingerprintingGenetic Fingerprinting

In this example, we will use an imaginary source of DNA within which are locatedthree different VNTRs or mini-satellites

In this case, the DNA has three sets of repeated regions,containing three, eleven and seven repeats

mini-satellite with threerepeating nucleotide

sequences

mini-satellite with elevenrepeating nucleotide

sequences

mini-satellite with sevenrepeating nucleotide

sequences

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 whenthe DNA is ‘cut’ with the restriction enzyme

Making a Genetic FingerprintMaking a Genetic FingerprintMaking a Genetic FingerprintMaking a Genetic Fingerprint

Restriction enzyme ‘cuts’ the DNA at specific restriction sites

C A B

Fragments of DNA of different sizes are obtained of which threecontain the mini-satellites or VNTRs (A, B and C)

The fragments are now separated from one anotherby the technique of electrophoresis

Electrophoresis is a technique for separating moleculesfrom a mixture according to their charge and size

A solution containing the DNA fragments is placed in awell in a supporting medium of agarose gel

porousagarose gel

solution ofDNA fragments

buffer solution buffer solution

anodecathode

The pH of the sample and the gel are carefully controlled using buffer solutions

A direct electric current is then passed through the gel and thenegatively 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

Direction ofelectrophoresis

AB

C

C A B

In our example, there areeight DNA fragments and these move through the gel

according to their size

The bands that we wishto visualise are those

containing the ‘mini-satellites’ or

VNTRs (A, B and C)

In order to locate the‘mini-satellites’, the DNA fragments are transferredto a nylon membrane ornitrocellulose filter using

a technique calledSOUTHERN BLOTTING

Once transferred tothe nylon membrane ornitrocellulose filter, gene

probes will be usedto seek out the fragments

containing the‘mini-satellites’

The DNA in the gel mustfirst be denatured in orderto create single-stranded

DNA that will hybridise withthe probe – this is achievedeither by heating the DNAor by treatment with alkali

Southern Blotting is a technique used for transferring single-strandedfragments of DNA on to a nylon membrane or nitrocellulose filter

The gel containingthe DNA fragments

is placed on wet blottingpaper soaked with buffer

SouthernBlotting

GlassBlock

Blotting papersoaked in buffer

A nylon membrane or nitrocellulosefilter is then laid over the gel

Layers of blotting paperare placed over the membrane or filter

A large weight is placed abovethe blotting paper to create

pressure on the gel and henceto ‘blot’ the DNA fragmentsonto the nylon membrane or

nitrocellulose filter

The filter or membrane isdried and the DNA fragmentsare held permanently in place

Nowadays, the blotting technique usedmay be more sophisticated; vacuum

blotting and electroblotting are commonly usedin place of the paper towels and weights

The DNA filter containing the single-stranded fragments of DNA is nowexposed to a solution containing radioactive, single-stranded probes

The probe and its target (the mini-satellites) will hybridise

The radioactive probes hybridise with the three fragments that, in ourexample, contain mini-satellites

Finally, these bands are visualised by thetechnique of AUTORADIOGRAPHY

AUTORADIOGRAPHY

A photographic film islaid over the filter

The three radioactivebands blacken thephotographic film

revealing the patternof mini-satellites presentin our imaginary DNA

sample

Genetic Fingerprint

Humans have muchmore complex genomes

than the simple example just described

When human DNAis digested with

restriction enzyme,numerous fragmentscontain mini-satellite

regions that react with the DNA probe

The ‘mini-satellite’ fragments for elevenunrelated individuals

are shown in this photograph

These are the individuals’ unique DNA fingerprints

Courtesy ofLancaster University

81 2 3 4 5 6 7 10119 These two fingerprintsshow the DNA from twins with identical

patterns of fragments

The complexity of the‘mini-satellite’ patterns

can be seen in thesehuman DNA profiles

A technique that createscoloured bands has been

used for these profilesto aid identification

Picture reproduced withkind permission of

The Forensic Science Service© Crown Copyright 2002

Genetic fingerprinting is being used for a variety of purposes and these include:

EVOLUTIONARY BIOLOGY – establishing the degree of relatedness between different species

Applications of Genetic FingerprintingApplications of Genetic FingerprintingApplications of Genetic FingerprintingApplications of Genetic Fingerprinting

FORENSIC SCIENCE – matching DNA specimensfrom the scene of a crime to those of suspects

PATERNITY TESTING – resolving disputesover the paternity of a child

HEALTH CARE – the detection of genetic disease in embryonic cells

M C FPaternity Testing

These DNA fingerprintsare those of a mother (M)

and child (C) together with the ‘possible’ father (F)

Every child receives halfof its DNA from the

mother and the otherhalf from the father

The mother of the child isknown and so the first

task is to identify whichof the child’s bands wereinherited from its mother

(remember thatthe mother’s bands area mixture of her mother

and father’s DNA)

The red arrows identifythe maternal bands

All the remaining bands in the child must have a

an exact match in the father’s fingerprint

The blue arrows identifythe paternal bands

All of the child’s remaining bands are

matched in the ‘possible’ father

Paternity isestablished

M C F

In this example, thepaternal bands(shown in blue)

do not match thechild’s remaining bands

Paternity isdisproved

VICTIM

SPECIMEN

SUSPECTS

2 31

In forensic science, DNAfingerprinting is used tomatch material collectedat the scene of a crime to

that of the suspects

This is a diagram of thegenetic fingerprints of a

rape victim’s blood, semen (the specimen) and blood samples taken from

the suspect rapists

The fingerprint resultsshow an exact match

between the semen sampleobtained from the victim

and the blood sampleof suspect 1

Suspect 1 is confirmedas the rapist

Species X Species Y Species Z

Evolutionary biologists utilise the technique of DNA fingerprinting toestablish the closeness of relationships between different species

Which of the species X or Y is most closely related to species Z?

Species X Species Y Species Z

The number of DNA bands from species X and species Ythat match those of species Z is determined

Nine DNA bandsfrom species Xmatch those

found in species Z

Five DNA bandsfrom species Ymatch those

found in species Z

The genetic relationship is greatest between species X and Z

In the past, one of the drawbacks in obtaining genetic fingerprintsfrom 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 byKary 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 DNAand 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 strandsand then using the enzyme DNA polymerase to create new strands

Due to the high temperatures required for the technique, a thermostableDNA polymerase had to be found to avoid the expense of needing to

replenish the enzyme after each round of DNA replication

The Polymerase Chain Reaction (PCR)The Polymerase Chain Reaction (PCR)The Polymerase Chain Reaction (PCR)The Polymerase Chain Reaction (PCR)

The solution to this problem was to use Taq polymerase, derived from Thermusaquaticus, 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

C CCTAACA AG G G C CG TATC C CGA C G G AT TTGG T

TC C CGA C G G AT TTGG T

C CCTAACA AG G G C CG TA

The target DNA is first mixed with DNA polymerase and primersand then heated to 95°C to separate the two strands of DNA

The Technique

Primers are short, synthetic DNA fragments that are complementary to the DNA

sequences at either end of theregion of DNA to be copied

TGG T

C CG T

C CCTAACA AG G G C CG TATC C CGA C G G AT TTGG T

TGG T

C CG T

The Technique

The mixture is now cooled to 55°C to allow the primers to bindto the ends of the separated DNA strands

Polymerase binds to the primers and begins adding basesto form new complementary strands

C CCTAACA AG G G C CG TATC C CGA C G G AT TTGG T

TGG T

C CG T

T

A

A

G

G

G

C

AT

G

C

C

TC

TA

C

A

G

A

G

C

A

C

The Technique

The mixture is now cooled to 55°C to allow the primers to bindto the ends of the separated DNA strands

Polymerase binds to the primers and begins adding basesto form new complementary strands

C

C CCTAACA AG G G C CG TA

TGG T T A G C T C T C GG A

T

TC C CGA C G G AT TTGG T

C CG TAGGAGCAAA CC

Two Identical Copies of the Target DNA SequenceResult From the First Synthesis Cycle

C

C CCTAACA AG G G C CG TA

TGG T T A G C T C T C GG A

TC C CGA C G G AT TTGG T

C CG TAGGAGCTAAA CCC CCTAACA AG G G TC CGA

C C C CG G GA AT T TTGG T

C CG TCCC T G GG AAAAA

TC C CGA C G G AT TTGG T

TGG T

C CG T

TGG T

C CG TThe process is now repeated by

first heating the mixture toseparate 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 thatpolymerase can begin its job ofadding bases to the sequence

C CCTAACA AG G G TC CGA

C C C CG G GA AT T TTGG T

C CG TCCC T G GG AAAAA

TC C CGA C G G AT TTGG T

TGG T

C CG T

TGG T

C CG TAt 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 ofDNA molecules doubling with each cycle

This exponential increase creates over a billioncopies of the target DNA within a few hours

Cycle 2 Products

Cycle 3 Products

The number of DNA moleculesdoubles with each cycle

C CCTAACA AG G G C CG TATC C CGA C G G AT TTGG T

C CCTAACA AG G G TC CGA

C C C CG G GA AT T TTGG T

C CG TCCC T G GG AAAAA

TC C CGA C G G AT TTGG T

TGG T

C CG T

TGG T

C CG T

TGG T

C CG T

TC C CGA C G G AT TTGG T

C CCTAACA AG G G C CG TA

Target DNA is heated to separate the strands

When the mixture is cooled, primers bind to the ends of the target strands and polymerase enzymes add bases to complete the complementary strands

C

C CCTAACA AG G G C CG TA

TGG T T A G C T C T C GG A

T

TC C CGA C G G AT TTGG T

C CG TAGGAGCAAA CCTwo identical DNA molecules are formed

A second cycle is initiated by heating the mixtureonce again to separate the strands of the newly

formed DNA moleculesWhen 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 continuesfor approximately 30 cycles, doubling thenumber of DNA molecules with each cycle

SUMMARY PCR generates billions of copies oftarget DNA within a few hours

Acknowledgements

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