Homework Prepare for 6x task 2 and 6x written assessment June 2012 June 2011 June 2010 Bring marked corrected papers to tues 23 rd april Test DNA technol tues 9 th april (revise) Exam style q p

Genetic fingerprinting Aqa p.276-9

Minisatellites

Genetic fingerprinting

Syllabus Genetic fingerprinting - Extraction - Digestion - Separation - Hybridisation - Development Interpreting the results Uses of DNA fingerprinting Application - Other uses of genetic fingerprinting - Forensic science - Medical diagnosis - Plant and animal breeding Application - Locating DNA fragments An organism’s genome contains many repetitive, non-coding base sequences. The probability of two individuals having the same repetitive sequences is very low. The technique of genetic fingerprinting in analysing DNA fragments, that have been cloned by PCR, and its use in determining genetic relationships and in determining the genetic variability within a population. Candidates should be able to explain the biological principles that underpin genetic fingerprinting techniques interpret data showing the results of gel electrophoresis to separate DNA fragments explain why scientists might use genetic fingerprints

Objectives What is genetic fingerprinting? How is genetic fingerprinting carried out? How are the results interpreted? For what purposes is it used? Poster then bingo/card sort

Homework Extension - How science works: Breeding better bananas worksheet How science works: Saviour sibling

Electronic resources Animation: Genetic fingerprinting

© SSER Ltd.

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 Genetic Fingerprinting

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 Genetic Fingerprinting

In this example, we will use an imaginary source of DNA within which are located three 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 three repeating nucleotide sequences mini-satellite with eleven repeating nucleotide sequences mini-satellite with seven repeating 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 when the DNA is ‘cut’ with the restriction enzyme Making 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 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 porous agarose gel solution of DNA fragments buffer solution anode cathode 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 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

Direction of electrophoresis A B C C A B In our example, there are eight DNA fragments and these move through the gel according to their size The bands that we wish to visualise are those containing the ‘mini-satellites’ or VNTRs (A, B and C) 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 Once transferred to the nylon membrane or nitrocellulose filter, gene probes will be used to seek out the fragments containing the ‘mini-satellites’ 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

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 Southern Blotting Glass Block Blotting paper soaked in buffer A nylon membrane or nitrocellulose filter is then laid over the gel Layers of blotting paper are placed over the membrane or filter 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 The filter or membrane is dried and the DNA fragments are held permanently in place 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 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

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

Humans have much more complex genomes than the simple example just described 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 Courtesy of Lancaster University These two fingerprints show the DNA from twins with identical patterns of fragments

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 Picture reproduced with kind permission of The Forensic Science Service © Crown Copyright 2002

Make a sequencing exercise - genetic fingerprinting - Extraction - Digestion - Separation - Hybridisation - Development Interpreting the results Uses of DNA fingerprinting 3

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 Fingerprinting 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 Health care – the detection of genetic disease e.g. Huntingdons more common in people with >38 repeats >50 = early onset p.279 Plant and animal breeding – determines genetic variability/relatedness Can target specific (undesirable) allelles

Paternity Testing These DNA fingerprints are those of a mother (M) and child (C) together with the ‘possible’ father (F) 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) 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 The blue arrows identify the paternal bands All of the child’s remaining bands are matched in the ‘possible’ father Paternity is established

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

The number of DNA bands from species X and species Y that match those of species Z is determined 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 Species Y Species X Species Z Species XSpecies YSpecies Z

Teacher definitions Clones Genetically identical cells DNA polymerase Enzyme that joins nucleotides in DNA synthesis DNA ligase Enzyme that joins lengths of DNA in gene technology Electrophoresis Separation technique Gene probe Locates a specific gene Genome The genetic make up of an organism Germ line gene therapy Altering DNA in gametes Marker gene Used to identify transformed cells Mutation Change in DNA in a cell Oncogene A gene that stimulates cells to divide too quickly PCR Process used to amplify DNA outside the body Plasmid A vector in gene technology Primer Marks region of DNA to be amplified Recombinant DNA DNA from more than one organism Restriction enzyme Enzyme that cuts at specific base sequences Reverse transcriptase Enzyme that produces RNA from DNA Semi-conservative replication Method by which DNA is copied Somatic gene therapy Altering DNA in non-sex cells Sticky end Single-stranded section of DNA Tumour suppressor gene Gene that prevents cells dividing too rapidly