Molecular biology


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  • DNA/RNA overview
  • Figure 20.9 Gel electrophoresis
  • Molecular biology

    1. 1. Patricia Linton Manchester Metropolitan University
    2. 2. It’s just so amazing – that’s what!
    3. 3. It’s not science fiction – it’s science fact! <ul><li>DNA - Encodes all of the genetic information needed for the development and functioning of all cells. </li></ul>Discipline is full of amazing facts
    4. 4. TRANSCRIPTION RNA PROCESSING DNA RNA transcript 3  5  RNA polymerase Poly-A Poly-A RNA transcript (pre-mRNA) Intron Exon NUCLEUS Aminoacyl-tRNA synthetase AMINO ACID ACTIVATION Amino acid tRNA CYTOPLASM Poly-A Growing polypeptide 3  Activated amino acid mRNA TRANSLATION Cap Ribosomal subunits Cap 5  E P A A Anticodon Ribosome Codon E The central dogma DNA RNA PROTEIN
    5. 5. TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide (a) Bacterial cell Nuclear envelope TRANSCRIPTION RNA PROCESSING Pre-mRNA DNA mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell
    6. 6. Uses of molecular biology – endless – revolutionised science <ul><li>Medical </li></ul><ul><li>Production of recombinant proteins for treatment of disease </li></ul><ul><li>Determining the genetic basis of cancer – thereby improving treatment and prognosis </li></ul><ul><li>Diagnosis of disease – fast diagnosis of HIV/TB – infectious diseases </li></ul><ul><li>Agricultural </li></ul><ul><li>pest resistance/ drought resistance/increased productivity </li></ul><ul><li>Industrial </li></ul><ul><li>Manufacture of proteins, strain improvement </li></ul><ul><li>Forensic </li></ul><ul><li>Used to place criminals at scene of crime or rule out suspects – important for conviction </li></ul><ul><li>DNA fingerprinting </li></ul><ul><li>Archaeology </li></ul><ul><li>Analysis of ancient DNA in ceramic food vessels </li></ul><ul><li>Environmental Science </li></ul><ul><li>Identification of species </li></ul><ul><li>Clean-up of oil spills </li></ul>
    7. 7. (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene
    8. 8. Kary Mullis – Genius or eccentric? ‘ Science grows like a weed every year’ ‘ Each of us have things and thoughts and descriptions of an amazing universe in our possession that kings in the 17th Century would have gone to war to possess’ ‘ Science consistently produces a new crop of miraculous truths and dazzling devices every year’ ‘ I have had an encounter with an extraterrestrial in the form of a fluorescent raccoon’ ??????? You can read his Nobel lecture here:
    9. 9. PCR – in the dark ages 8 BORING hours per PCR! 95º C 5 min 35 times 55º C 3 min 72º C 5 min
    10. 10. <ul><li>Individual rows of receptacles can be heated – can try lots of reaction temperatures in one ‘run’ </li></ul>
    11. 12. The different steps of PCR <ul><li>Water </li></ul><ul><li>Buffer </li></ul><ul><li>DNA template </li></ul><ul><li>Primers </li></ul><ul><li>Nucleotides </li></ul><ul><li>Mg 2+ ions </li></ul><ul><li>DNA Polymerase </li></ul>PCR recipe
    12. 13. The temperature profile of a PCR cycle is controlled by the thermal cycler program which results in a near exponential increase in PCR product accumulation for about the first 30 cycles. The 3 Os!!!!!
    13. 14. <ul><li>• Used to separate molecules based on their charge and size </li></ul><ul><li>• Agarose or acrylamide gels can be used to separate DNA fragments </li></ul><ul><li>• DNA is acidic; it migrates from the negative to the positive </li></ul><ul><li>end of the gel </li></ul><ul><li>each fragment’s migration rate is inversely proportional to </li></ul><ul><li>the log of its molecular </li></ul><ul><li>weight </li></ul>
    14. 15.
    15. 16. Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode 1 2 Power source – + + –
    16. 18. DNA Fingerprinting <ul><li>Steps in DNA fingerprinting: </li></ul><ul><ul><li>DNA isolated from tissue sample or PCR’d up </li></ul></ul><ul><ul><li>DNA cut into fragments with enzymes </li></ul></ul><ul><ul><ul><li>DNA with different sequences produce fragments of different sizes </li></ul></ul></ul><ul><ul><ul><li>Can use restriction enzymes to cut either side of VNTRs for genetic typing </li></ul></ul></ul><ul><ul><li>Fragments separated on basis of size and visualized </li></ul></ul><ul><ul><li>Each person’s set of fragments is unique </li></ul></ul>
    17. 22. DNA Fingerprinting – identification of the remains of the Russian Royal Family <ul><li>DNA fingerprinting showed that 9 different people were buried in the Ekaterinburg grave. </li></ul><ul><li>Romanovs would be more similar in pattern to each other than to non-relatives. </li></ul><ul><li>All of a child’s bands must be present in one or both of the parents. </li></ul>
    18. 23. 7.6 DNA Fingerprinting Adult 1 Adult 2 Adult 3 Adult 4 Adult 5 Adult 6 Child 1 Child 2 Child 3
    19. 24. DNA Fingerprinting <ul><li>To see if parents and their children were Romanovs, DNA fingerprints were prepared for relatives of tsar and tsarina. </li></ul><ul><li>Adult male skeleton (related to the children) was related to George, the tsar’s brother. </li></ul><ul><li>Adult female skeleton (related to the children) was related to Prince Philip, the tsarina’s grand-nephew. </li></ul><ul><li>Conclusion : the grave contained the tsar, tsarina, three of their children, and four servants. </li></ul>
    20. 25. Making your own equipment