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MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology). Exercise 3. Dr. Benoit Cousineau Department of Microbiology & Immunology McGill University. Psychrophiles (9°C): Large habitat (90% of the ocean is 5°C or colder) Widespread among bacterial taxa

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MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

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

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

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

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

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

  6. Hyperthermophiles (95°C): • Procaryotes (Thermus aquaticus, Thermococcus litoralis) • Along rifts and ridges on the ocean floor • Sulfide chimneys, black smokers, hot vents (300°C) • 121°C (at 265 atmospheres seawater boils at 460°C) • 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 (94°C) • 2nd step: Anneal two primers on both sides of the fragment to amplify (40-60°C) • 3rd step: Copy the DNA with a thermostable DNA polymerase starting from the primers (72°C) • 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)

  10. PCR amplification clip and graph

  11. 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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