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MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

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MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and ... Department of Microbiology & Immunology. McGill University. Exercise 3. Psychrophiles (9 C) ... – PowerPoint PPT presentation

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Title: MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)


1
MICROBIOLOGY MIMM 386(Laboratory Course in
Microbiology and Immunology)
Exercise 3
  • Dr. Benoit Cousineau
  • Department of Microbiology Immunology
  • McGill University

2
  • Psychrophiles (9C)
  • Large habitat (90 of the ocean is 5C or colder)
  • Widespread among bacterial taxa
  • E.g., Chlamydomonas nivalis (pink spores)

3
  • Psychotrophs (24C)
  • Facultative psychrophiles
  • Spoilage of refrigerated foods (bacteria and
    fungi)

4
  • Mesophiles (37C)
  • Most micro-organisms
  • All human pathogens (37C)

5
  • Thermophiles (68C)
  • Most are procaryotes
  • Found in composts, hot water lines, hot springs

6
  • Hyperthermophiles (95C)
  • Procaryotes (Thermus aquaticus, Thermococcus
    litoralis)
  • Along rifts and ridges on the ocean floor
  • Sulfide chimneys, black smokers, hot vents
    (300C)
  • 121C (at 265 atmospheres seawater boils at
    460C)
  • More stable (Memb., DNA, Proteins e.g., DNA
    polymerase)

7
Polymerase Chain Reaction (PCR)
  • Technique to amplify specific DNA regions
  • From one copy to millions of copies
  • 30 cycles of amplification
  • 1st step Denature the two DNA strands (94C)
  • 2nd step Anneal two primers on both sides of the
    fragment to amplify (40-60C)
  • 3rd step Copy the DNA with a thermostable DNA
    polymerase starting from the primers (72C)
  • Nobel prize in chemistry (1993)
  • Dr. Kary B. Mullis (PCR, 1984)
  • Dr. Michael Smith (SD mutagenesis)

8
Polymerase Chain Reaction (PCR)
  • The reaction mix
  • Template DNA
  • Two primers
  • dNTPs (dATP, dCTP, dGTP, dTTP)
  • Enzyme buffer
  • Thermostable DNA polymerase

9
Polymerase Chain Reaction (PCR)
  • Primer design
  • Primer length (20 to 30 base pairs)
  • Orientation 5 to 3
  • Prevent folding and pairing
  • Primer tails (restriction sites for cloning)

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PCR amplification clip and graph

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The versatility and power of PCR
  • Reverse Transcriptase PCR RT-PCR (amplify RNA)
  • Inverted or reverse PCR (deletions in plasmids)
  • Rapid Amplification of cDNA Ends RACE
  • In situ PCR
  • Semi-quantitative PCR (need internal control)
  • Real time PCR (quantitative)
  • Create random mutations (change ions, dNTPs)
  • Cloning (homologous regions)
  • PCR sequencing (sequence small amounts of DNA)
  • Amplify traces of ancient DNA (mummies,
    dinosaurs, etc.)
  • Disease diagnostic (HIV, HBV, etc.)
  • Forensic science (blood, hair, sperm, skin, etc.)
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