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

5. 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

6. 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

7. Human Genome Project Methods

8. 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

13. 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

14. 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.

15. 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

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

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

18. 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

19. 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

20. 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

21. 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)

26. 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

27. 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

28. Human Genome Project Methodology – EST maps

29. 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

30. 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

31. 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

32. 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

33. 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

34. 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)

35. 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