1 / 25

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.

vern
Télécharger la présentation

Genetic fingerprinting

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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)

More Related