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Biotechnology. 4 major areas Human Genome Project Gene Therapy Forensic science Agriculture. Human Genome Project. Aim Identify sequence of bases on all 23 human chromosomes (3 billion bases) Identify genes within those sequences (~30 000 genes)

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  1. Biotechnology • 4 major areas • Human Genome Project • Gene Therapy • Forensic science • Agriculture

  2. Human Genome Project • Aim • Identify sequence of bases on all 23 human chromosomes (3 billion bases) • Identify genes within those sequences (~30 000 genes) • Locate the position of the genes on the chromosomes • $6 billion dollars, 1000 scientists, 50 countries, completed 2000! • Only ~3% of genome codes for protein • Remainder is regulatory or of unknown function (junk) e.g repetitive sequence, possibly viral DNA

  3. 3. Chop chromosome - small, overlapping fragments • Sequence • Computers align overlapping sequences Human Genome Project – Approach Used • Concept • Produce ever more detailed maps of chromosomes • 1. Genetic linkage map (low resolution) • Relative order & spacing of disease linked genes (not physical map) • 2. Combine with STS/EST (sequence tag site/ expressed sequence tag) maps • Position of unique DNA sequences (physical map) • Linkage data to disease genes • Pain stakingly slow, but links to useful disease information • Alternatively SHOTGUN sequencing

  4. Human Genome Project Methods

  5. Restriction Enzymes - Summary • Variety of enzymes • Isolated from bacteria • Cut DNA at specific sequences • Used to produce DNA fragments • Blunt or sticky ended • DNA Ligase (not a RE), used to LIGATE (joins DNA) fragment into a plasmid • Animation

  6. DNA Amplification • To increase the concentration of specific pieces of DNA • PCR (polymerse chain reaction) • Thermostable Taq DNA polymerase • Nucleotides (AGCT) • template DNA • Primers (bind to DNA, initiate DNA replication) • Either side of area of genome to be amplified • Repeated cycles of heating and cooling • Heating – breaks apart DNA template • DNA primers anneal (hydrogen bond) as cools • DNA polymerase synthesises complementary strand • VideoVideo 2

  7. What is electrophoresis? • Separation of charged molecules. • DNA is negatively charged; attracted to the positive terminal • Small molecules easily pass through spaces in gel, so travel faster. • Larger molecules have difficulty travelling through spaces in agarose. so in DNA agarose gel electrophoresis the fragments are separated by size.

  8. Electrophoresis Gel Preparation Molten agarose 55 - 60°C During polymerisation the sugar molecules all cross link with each other causing the solution to ‘gel’ into a semi-solid matrix; a bit like jelly in a trifle! Comb Tray

  9. DNA SIZE MARKERS/STANDARDS -ve • Smaller fragments travel faster. • The sizes of bands are known (in base pairs). largest smallest +ve

  10. Typical DNA gel showing bands of DNA of different sizes. First and last lanes contain DNA size markers

  11. DNA sequencing • 4 tubes with DNA polymerase, template DNA • DNA nucleotides • 1 Dideoxynucleotide (e.g. ddATP, terminates DNA synthesis where A is located) labelled (radioactive / 4 fluorescent colours) • Produces strands of DNA terminated at different points • Fragments separated by electrophoresis • Labels visualised by autoradiography or computer (fluorescence) • VIDEO

  12. DNA probes • Short sequences of DNA complementary to specific sequences in the genome • Labelled (radioactive/ fluorescent) • Binds to complementary sequence • Used extensively • Search for genes • Locate genes (FiSH – fluorescence immunohistochemistry) • DNA fingerprinting

  13. Using a Probe to Find Sequences on a Gel Usually a nitrocellulose membrane DNA on the gel is double stranded & needs to be single- stranded for probe to bind: gel treated with sodium hydroxide to do this

  14. Chromosome Walking • Marker sequence identified • Target gene is some distance from marker • 2 Restriction enzymes digest DNA • Probe to find fragments containing marker DNA • Sequence 3’ ends • Probe for these sequences, repeat above • Use overlaps in digests to identify fragment order • Gradually move towards gene (Fig. 8.3 P157)

  15. Human Genome Project Methodology - FiSH • Fluorescence in-situ hybridisation • Use metaphase chromosomes • Probes fluorescently labelled • Highlight chromosome on which a specific sequence or gene is located • (antibody technology used allows labelling of more than one site on the same sample ) • Use of interphase chromosomes gives 50kbp resolution

  16. Human Genome Project Methodology - Linkage Studies • Find linkages between genes • Linkage mapping from genetic studies • Recombination studies • Crossover at meiosis – frequency indicates distance between the genes

  17. Human Genome Project Methodology – EST maps

  18. Human Genome Project Methodology – EST maps • Expressed sequence tag (EST) maps • Partial gene sequence data of a cDNA clone, which provides a sequence from which to generate a probe. • Extract mRNA • Reverse transcribe it (RNA  complementary DNA (cDNA)) • Use cDNA sequence to probe genome • Finds the location of expressed genes

  19. Human Genome Project Methodology – STS maps • Sequence tagged site (STS) maps • STS- PCR primer based on known sequence (randomly found) • Can be used to link the genetic maps to the physical map

  20. Applications of Gene technology • Genetic testing • Identify gene defects • Human therapeutics • Replace defective genes with corrected sequence in affected tissues • Useful single gene defect disorders (monogenic) • E.g cystic fibrosis • E.g. Duchenne muscular dystrophy • E.g. Huntingdon’s disease • More difficult for multiple gene defect disorders (polygenic) e.g heart disease • Introduce antisense DNA to produce mRNA complementary to e.g cancer causing genes and so prevent their translation

  21. Cystic Fibrosis • Single gene defect • Gene encoding a chloride ion channel protein is incorrect sequence • Leads to reduction in secretion of water with mucus – sticky, thick mucus produced • Coats airways, gut • Prone to respiratory infection, recurrent cough • Malnutrition due to poor secretion of digestive enzymes • Reduced life expectancy • Genetic disorder established 1946, • Gene isolated 1989

  22. Cystic Fibrosis • Possible treatment • Introduce good copy of gene into airways cells • Use aerosol technology • Delivery methods: • Aerosol • Viral vector or Liposome containing DNA • Animal trials show good reversal • Human trials less encouraging

  23. Duchenne Muscular Dystrophy • Defect in gene for Dystrophin (muscle protein) • Onset of symptoms age 2-6 • Falling, difficulty getting up from sitting/lying • Waddling gait • Large calf muscles (fat deposition) • X linked gene (1987)

  24. Duchenne Muscular Dystrophy • Treatment • Injection of liposomes into bloodstream • Good copy of gene introduced into muscles • Targeting/ control of tissue specific expression • Alternative antisense technology

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