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CSI: SNAB DNA Fingerprinting. Lesson Objectives Describe the uses for DNA Fingerprinting Explain the process of DNA Fingerprinting Discuss the ethics.

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Presentation on theme: "CSI: SNAB DNA Fingerprinting. Lesson Objectives Describe the uses for DNA Fingerprinting Explain the process of DNA Fingerprinting Discuss the ethics."— Presentation transcript:

1 CSI: SNAB DNA Fingerprinting

2 Lesson Objectives Describe the uses for DNA Fingerprinting Explain the process of DNA Fingerprinting Discuss the ethics behind DNA Figerprinting

3 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

4 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

5 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!

6 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

7 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)

8 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 Satellites Mini-satellites contain base pairs and are repeated 50 to several hundred times Micro-satellites contain 2-4 base pairs and are repeated between 5 and 10 times

9 Satellites and Loci Satellites occur at particular places in the human genome These 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

10 Example of Satellites at a Locus

11 Mini-satellite repeat (VNTR – Variable Number Tandem Repeat)

12 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

13 DNA Primers and Polymerase DNA Primers are short sequences of DNA which bind to the the sequence at the start of a micro- satellite repeat The enzyme DNA Polymerase then copies the section of DNA at the locus using the free nucleotides in solution This 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

14 Hot water bacteria: Thermus aquaticus Taq DNA polymerase Life at High Temperatures by Thomas D. Brock Biotechnology in Yellowstone © 1994 Yellowstone Association for Natural Science

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17 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!)

18 DNA Restriction Enzyme Site

19 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.

20 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

21 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

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23 Making the gel

24 Adding samples

25 Electrophoresis

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27 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 sequence The membrane is then placed in a bag and placed on a photographic film which is exposed where the radioactive probes are attached The resulting pattern of bands is called the DNA fingerprint A single band occurs where the maternal and paternal chromosomes have the same number of satellite repeats, if not there will be two bands

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29 Summary

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31 Automation This shows the result of a modern automated technique using fluorescent DNA to make a computer generated graph

32 Amelogenin The genes for amelogenin can be used in sex determination of samples from unknown human origin through the Polymerase Chain Reaction (PCR). 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. primersintronX chromosomeAMELXY chromosomeAMELYprimersintronX chromosomeAMELXY chromosomeAMELY 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. agarose gelagarose gel Thus, this process allows for sex determination of unknown samples.

33 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

34 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

35 The defendant stated that the blood on his clothing was his.

36 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.

37 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.

38 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.

39 DNA and Paternity The DNA fingerprints of children share loci with both parents

40 DNA and paternity testing A B C D E A mother B male 1 C male 2 D child E standards

41 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

42 Problems with DNA Fingerprinting Statistics Population genetics Technical difficulties

43 DNA Databases In 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. Sweden 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. NorwayGermanyNorwayGermany All 50 states in the USA keep profiles of violent offenders, and a few keep profiles of suspects. USA Portugal has plans to introduce a DNA database of its entire population


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