MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

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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 - PowerPoint PPT Presentation

Text of MICROBIOLOGY MIMM 386 (Laboratory Course in Microbiology and Immunology)

  • MICROBIOLOGY MIMM 386(Laboratory Course in Microbiology and Immunology)Dr. Benoit CousineauDepartment of Microbiology & ImmunologyMcGill UniversityExercise 3

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

  • Psychotrophs (24C):Facultative psychrophilesSpoilage of refrigerated foods (bacteria and fungi)

  • Mesophiles (37C):Most micro-organismsAll human pathogens (37C)

  • Thermophiles (68C):Most are procaryotesFound in composts, hot water lines, hot springs

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

  • Polymerase Chain Reaction (PCR)Technique to amplify specific DNA regionsFrom one copy to millions of copies30 cycles of amplification1st 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)

  • Polymerase Chain Reaction (PCR)The reaction mix:Template DNATwo primersdNTPs (dATP, dCTP, dGTP, dTTP)Enzyme bufferThermostable DNA polymerase

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

  • PolymeraseSource

    Activities

    half-life at 95OC

    Taq

    Thermus aquaticus

    5 ---> 3 DNA polymerase 1.6 h

    Vent

    Thermococcus litoralis

    5 ---> 3 DNA polymerase 6.7 h

    3 ---> 5 DNA exonuclease*

    Vent exo-Thermococcus litoralis

    5 ---> 3 DNA polymerase 6.7 h

    Deep VentThermococcus litoralis

    5 ---> 3 DNA polymerase 23 h

    3 ---> 5 DNA exonuclease

    Tfl

    Thermus flavus

    5 ---> 3 DNA polymerase 1.5 h

    Tth

    Thermus thermophilus HB85 ---> 3 DNA polymerase (MgCl2)

    5 ---> 3 Reverse transcriptase (MnCl2)

    Tli

    Thermococcus litoralis

    5 ---> 3 DNA polymerase 6.0 h

    3 ---> 5 DNA exonuclease

    Pfu

    Pyrococcus furiosus

    5 ---> 3 DNA polymerase

    3 ---> 5 DNA exonuclease

    * Proofreading activity

  • PCR amplification clip and graph

  • The versatility and power of PCRReverse Transcriptase PCR: RT-PCR (amplify RNA)Inverted or reverse PCR (deletions in plasmids)Rapid Amplification of cDNA Ends: RACEIn situ PCRSemi-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.)