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

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

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

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

Mesophiles (37°C): –Most micro-organisms –All human pathogens (37°C)

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

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)

Polymerase Chain Reaction (PCR) Technique to amplify specific DNA regions –From one copy to millions of copies –30 cycles of amplification 1 st step: Denature the two DNA strands (94°C) 2 nd step: Anneal two primers on both sides of the fragment to amplify (40-60°C) 3 rd 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)

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

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)

PCR amplification clip and graph

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.)