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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 40 000 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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?

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

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

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

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

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

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

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

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

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

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

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

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