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Genetic fingerprinting

Genetic fingerprinting. Everyone’s DNA is unique One way of distinguishing individuals – sequence their genome Impractical Alternatively, exploit differences that are unique, but easily detected. Genetic fingerprinting.

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Genetic fingerprinting

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  1. Genetic fingerprinting • Everyone’s DNA is unique • One way of distinguishing individuals – sequence their genome • Impractical • Alternatively, exploit differences that are unique, but easily detected

  2. Genetic fingerprinting • Within the genome are repeated core sequences (minisatellites; 12 to 100 bp, up to 3000 repeats) • The number of repeats varies – they are called variable number tandem repeats (VNTRs – equivalent to alleles for genes) • Digestion of DNA with specific restriction enzymes produces lengths of DNA (fragments). The enzymes do not cut in the minisatellites. • Size of fragments containing VNTRs will vary between individuals due to variation in the number of times the sequence is repeated (Restriction fragment length polymorphisms – RFLPs)

  3. Genetic fingerprinting • Detection of VNTRs • Extract DNA • Digest DNA using restriction enzyme • Separate fragments by gel electrophoresis • Southern blot (transfer DNA onto nylon membrane) • Hybridise with labelled probe which recognises the particular repeated sequence

  4. Genetic Fingerprinting • VNTRs can occur only once in the genome (single locus) or can occur in a number of places in the genome (multilocus). • Single locus probes are fine for paternity cases (each individual has two VNTR “alleles” – one from mother/ father) • Analysis with a single locus probe will indicate if baby has one of father’s alleles

  5. Genetic fingerprinting • For criminal investigation VNTRs located at a variety of loci are used • Initially 6 loci used, now 14 loci • Consequently a complex series of bands is produced reflecting a variety of RFLPs • Statistically identification on the order of one in 100 million. • Cross checking can be done using different VNTRs Animation

  6. DNA Profiling • PCR based technqiue • Simple tandem repeats (STRs) • Similar to VNTRs, but shorter sequences are repeated so can be “PCRed” • Advantages: • Automated analysis using the laser detector on a DNA sequencer to indicate lengths of STRs • Smaller sequences less sensitive to degradation • PCR means exceptionally small amounts of DNA can give a result (e.g DNA left by touching an object) • Colour labelling of probes means multiple probes can be used simultaneously speeding up the process greatly whilst maintaining certainty

  7. Probability • E.g VNTR (17bp) repeated 70-450 times • Chance of two individuals being the same? • 1 in 380 = 0.003 • If VNTR is located at 4 loci • Chance of two individuals being the same? • (1 in 380)4 = 1 in 20,000,000,000 • In practice less (fewer than 380 variants)

  8. DNA Database • Established 1995: 700,000 , by April 2000; by July, 2005 • 2,900,000 (~ 5%) profiles held on the database in UK ( 5,000,000 by 2010) • 630,000 matches made between crime scene and suspect • 40,000 leads as a result of profile • 50% of UK crime scenes now yield DNA on NDNAD • Family relationships can also be detected ( and have been) • Computer analysis now allows mixed DNA samples to be analysed • Proposed that eye colour, hair colour of suspects can be determined from DNA, surname? • 52% of innocent DNA is from black people; 77% young black men are on NDNAD • Transplants

  9. Agriculture & Biotechnology • Gene modification/ insertion to improve • Crop plants • Yield • Disease/ pest resistance • Herbicide resistance • Crop properties • Vitamins • Shelf life • Medically useful products • Industrially useful products (biodegradable plastics) • Animal • Faster growth rate • Higher yield • Medically useful products • Quicker results than with selective breeding • Introduce foreign species DNA

  10. Transgenic plants • Production: • Introduce DNA • Requires vector • Regenerate whole plants (clones) • Needs to ensure all cells contain transgene

  11. Introducing DNA (non grasses) • Dicotolydenous plants (i.e not grasses) • Use Agrobacterium tumefaciens • Causes crown gall disease in plants • Contains a Ti plasmid (Ti = tumour inducing) • Use a modified Ti plasmid which does not produce tumour • or • The Ti plasmid contains a region T-DNA that integrates into plant genome • T-DNA can be used by itself to carry useful genes into a plant’s genome without causing tumours

  12. Technique • Use restriction enzymes to excise T-DNA • Insert gene of interest (sticky ends, ligase) • Transform plant cells in tissue culture • Grow calluses • Manipulate hormones to grow fully functional plants (clone more using conventional methods)

  13. Flavr Savr tomato • Ripening of tomatoes is caused by enzyme polygalacturonase • Which breaks down the cell wall • Overripe tomatoes are more easily damaged and don’t sell well. • An antisense copy of PG was introduced into the tomato • It prevents production of PG (the two mRNAs base pair and cancel each other out) • No PG, no rotting

  14. Other examples • Monsanto Roundup resistant soybean • Can apply large amounts of herbicide • Improves productivity of crop • Pest resistance genes • Bacillus thuringiensis produces a protein, toxic to insects • Gene for protein inserted into tomato

  15. Future • Nitrogen fixation into non-leguminous plants • Difficult as most useful crop species are monoctoyledonous (grasses), so Ti plasmid can’t be used • Alternatively, use DNA gun (gold, DNA coated pellets shot directly into cells) • Arabidopsis thaliana (thale cress) R genes confer pesticide resistance • Possible to insert them into crop species • Stress (heat/ drought) tolerant genes • Modification of structure to improve harvesting • Nutritional improvement (added protein/ amino acids/vitamins) • Manufacture of biodegradable plastics (monomer polyhydroxybutyrate)

  16. Animals • Less advanced than plants • Greater ethical concerns • Currently use of biotech produced growth hormone (Bovine somatotrophin – BST) in cows to improve milk yield (USA) • Produced by transformed E.Coli. Containing BST gene

  17. Future • Replace selective breeding • Directly introduce desirable genes into animals • Tried in pigs – multiple copies of GH • Increase growth rate and ultimate size • Pigs collapsed under their own weight • Introduce genes for pharmaceutically useful proteins into animals • Vaccines/ antibodies/ organ production • e.g. PPL therapeutics (Edinburgh) • Sheep producing -1-antitrypsin in their milk (treats emphysema)

  18. Web Site • Access Excellence web site • www.accessexcellence.org

  19. E.g VNTR (17bp) repeated 70-450 times • Chance of two individuals being the same? • 1 in 380 = 0.003 • If VNTR is located at 4 loci • Chance of two individuals being the same? • (1 in 380)4 = 1 in 20,000,000,000 • In practice less (fewer than 380 variants)

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