Genetic fingerprinting

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Genetic fingerprinting

• Everyones DNA is unique
• One way of distinguishing individuals sequence
  their genome
• Impractical
• Alternatively, exploit differences that are
  unique, but easily detected

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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)

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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

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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 fathers alleles

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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
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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

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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)

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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

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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

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Transgenic plants

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

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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 plants genome without causing tumours

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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)

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Flavr Savr tomato

• Ripening of tomatoes is caused by enzyme
  polygalacturonase
• Which breaks down the cell wall
• Overripe tomatoes are more easily damaged and
  dont 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

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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

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Future

• Nitrogen fixation into non-leguminous plants
• Difficult as most useful crop species are
  monoctoyledonous (grasses), so Ti plasmid cant
  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)

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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

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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)

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Web Site

• Access Excellence web site
• www.accessexcellence.org

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• 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)