Post on 16-Dec-2015
Lesson Objectives
Describe the uses for DNA Fingerprinting
Explain the process of DNA Fingerprinting
Discuss the ethics behind DNA Figerprinting
What is DNA Fingerprinting?
DNA Fingerprinting (or DNA Profiling) is a technique using DNA to create a unique pattern of DNA fragments to identify an individual
DNA fingerprinting is not DNA sequencing and the exact sequence of the DNA is not discovered
Applications of DNA Fingerprinting
DNA fingerprinting has a variety of uses:Matching unknown biological samples to known samples. This is used in:– Forensic Science– Pet Recovery services
Identifying a family relationship (mother/father/siblings etc)Identifying a species e.g. Tiger DNA from a pelt, useful in conservation
Forensic DNA Fingerprinting
This technique can be used to match a biological specimen to an individual/ suspect
Blood
Skin (Epithelial Cells)
Semen
Saliva (as it often contained Epithelial Cells)
Hair (From cells in the Root Sac)
Any biological sample which contains cells!
Steps to DNA Fingerprinting
Isolate a sample of DNA
Use Polymerase Chain Reaction to amplify DNA
Treat DNA with Restriction Enzymes
Separate DNA Gel Electrophoresis
Probe DNA using Southern Blotting
Interpretation
Why are People’s DNA different
The difference between the coding DNA (that’s the DNA in genes) is very small!
There is however a great difference in the non-coding DNA between genes and in Introns (non coding DNA within a gene)
Mini and Micro Satellites
In the non coding DNA (introns) there are short sequences of DNA which are repeated many times. These are known as SatellitesMini-satellites contain 20-50 base pairs and are repeated 50 to several hundred timesMicro-satellites contain 2-4 base pairs and are repeated between 5 and 10 times
Satellites and Loci
Satellites occur at particular places in the human genomeThese places are called Loci (or a Locus)The number of repeats at each locus varies from person to person and even vary between a person’s two chromosomes (Maternal and Paternal)This variation is caused by the process of Crossing Over during meiosis
Mini-satellite repeat (VNTR – Variable Number Tandem Repeat)
Polymerase Chain Reaction
PCR is a very modern technique designed to amplify (make large numbers of copies) a small amount of DNA
PCR requires the following:– A DNA Sample– DNA Primers– DNA Polymerase– Solution of free nucleotides
DNA Primers and Polymerase
DNA Primers are short sequences of DNA which bind to the the sequence at the start of a micro-satellite repeatThe enzyme DNA Polymerase then copies the section of DNA at the locus using the free nucleotides in solutionThis DNA polymerase is very special as it has been obtained from thermophillus (heat loving) bacteria which works even at 95 degrees!In the UK ten primers are used to amplify ten loci
Hot water bacteria: Thermus aquaticusTaq DNA polymerase
Life at High Temperaturesby Thomas D. BrockBiotechnology in Yellowstone© 1994 Yellowstone Association for Natural Sciencehttp://www.bact.wisc.edu/Bact303/b27
Restriction Enzymes
The DNA sample is treated with Restriction Enzymes
These Enzymes cut DNA at specific sites dictated by the sequence at that site
The DNA is cut up into a series of lengths
These lengths vary in size between individuals (even animals!)
Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences
The enzyme EcoRI cutting DNA at its recognition sequence
Different restriction enzymes have different recognition sequences.
Gel Electrophoresis
Gel Electrophoresis is a technique used to separate molecules by their size (and even by charge)
It can be used to separate different sizes of lengths of DNA
How it works
A gel is made from a chemical called agarose (made from seaweed!)
Samples of unknown DNA and DNA of known lengths are loaded at one end of the gel
An electrical current is passed along the gel
As DNA is mainly negative it is attracted to the positive electrode
The smaller the DNA fragment the quicker and further it travels along the gel
Southern Blotting
The fragments of DNA within the gel are transferred to a nylon membrane and washed over with a DNA probe that binds to the repeated sequenceThe membrane is then placed in a bag and placed on a photographic film which is exposed where the radioactive probes are attachedThe resulting pattern of bands is called the DNA fingerprintA single band occurs where the maternal and paternal chromosomes have the same number of satellite repeats, if not there will be two bands
Automation
This shows the result of a modern automated technique using fluorescent DNA to make a computer generated graph
AmelogeninThe genes for amelogenin can be used in sex determination of samples from unknown human origin through the Polymerase Chain Reaction (PCR).Using primers specific for intron 1 of the gene, the gene sequence for the intron can be amplified. The X chromosome gene, AMELX, gives rise to a 106 bp amplification product (amplicon) and the Y chromosome gene, AMELY, a 112 bp amplicon. Hence, the AMELX contains a 6 bp deletion in the intron 1.When the amplicons are run on an agarose gel, samples from male sources (XY) will show two bands on an agarose gel (one for the 106 bp fragment and one for the 112 bp fragment), while females (XX) will show only one band.Thus, this process allows for sex determination of unknown samples.
Interpretation
In the UK ten loci are copied during DNA fingerprinting
In the USA thirteen loci (they call them alleles) are copied, why?
Because the USA has a larger population and so there is more chance of a similar/identical DNA fingerprint
Even so this is still very unlikely
Matching Loci
To match a sample of DNA all ten loci must be of the same length!
If even one is different there is no match
The defendant stated that the blood on his clothing was his.
The First Case
22/11/83 - Lynda Mann was found murdered
No clues but the murderer/rapist had left a semen sample.
Four years later - 31/7/87 - Dawn Ashcroft was found murdered and raped and there were enough similarities between the cases for the police to link them.
The First Case
A massive man hunt was launched resulting in the arrest of a young dishwasher.He confessed to the murder of Dawn but would not admit to the murder of Lynne.DNA from the suspect was compared to that from the samples found at both crime scenes.The results were surprising.
The First Case
The suspects DNA did not match either of the samples and therefore the dishwasher was innocent of both murders.As a result a huge DNA screening of the local population was undertaken without success.The killer and rapist was eventually identified as Colin Pitchfork after a tip-off from a work colleague.
DNA and Paternity
The DNA fingerprints of children share loci with both parents
DNA and paternity testing
A B C D EA mother
B male 1
C male 2
D child
E standards
DNA and paternity testing
Which is the father of child D?
Male 1 because the child has one allele matching male 1 and one matching the mother
Problems with DNA Fingerprinting
Statistics
Population genetics
Technical difficulties
DNA DatabasesIn England and Wales, anyone arrested on suspicion of a recordable offence must submit a DNA sample to the database, which is then kept on permanent record.In Scotland, the law is different and most people are removed from the database if they are acquitted. In Sweden, only criminals who have spent more than two years in prison are recorded.In Norway and Germany, court orders are required, and are only available, respectively, for serious offenders and for those convicted of certain offences and likely to reoffend.All 50 states in the USA keep profiles of violent offenders, and a few keep profiles of suspects.Portugal has plans to introduce a DNA database of its entire population