MICROBIOLOGY MIMM 386(Laboratory Course in Microbiology and Immunology)

Dr. Benoit CousineauDepartment 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

• 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)

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)

Polymerase Source 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 Vent Thermococcus litoralis 5’ ---> 3’ DNA polymerase 23 h

3’ ---> 5’ DNA exonuclease

Tfl Thermus flavus 5’ ---> 3’ DNA polymerase 1.5 h

Tth Thermus thermophilus HB8 5’ ---> 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

C y c l e 2

C y c l e 1

d e n a t u r a t i o n ( 9 5o

C )

a n n e a l i n g ( 4 0 - 6 0o

C )

e l o n g a t i o n ( 7 2o

C )

T a r g e t D N A s e q u e n c e

d e n a t u r a t i o n ( 9 5o

C )

a n n e a l i n g ( 4 0 - 6 0o

C )

e l o n g a t i o n ( 7 2o

C )

5 '3 '

3 '5 '

3 '5 '

5 '3 '

+

3 '5 '

5 '3 '

3 '5 '

5 '3 '

+

C y c l e 2

C y c l e 1

d e n a t u r a t i o n ( 9 5o

C )

a n n e a l i n g ( 4 0 - 6 0o

C )

e l o n g a t i o n ( 7 2o

C )

T a r g e t D N A s e q u e n c e

d e n a t u r a t i o n ( 9 5o

C )

a n n e a l i n g ( 4 0 - 6 0o

C )

e l o n g a t i o n ( 7 2o

C )

5 '3 '

3 '5 '

3 '5 '

5 '3 '

+

3 '5 '

5 '3 '

3 '5 '

5 '3 '

+

C y c l e 3

d e n a t u r a t i o n ( 9 5o

C )

a n n e a l i n g ( 4 0 - 6 0o

C )

e l o n g a t i o n ( 7 2o

C )

C y c l e 4

+

+ +

PCR amplification clip and graph

25

o

C

1

st

hold 25-30 cycles 2nd

hold

Temperature (o

C)

Denaturation

Annealing

Elongation

95

o

C 95o

C

55o

C

72o

C 72o

C

4

o

C

5 min 30 sec/30 sec/30 sec to 30 min 30 secTime

n o i n s e r tw i t h i n s e r t

P l a s m i d P C R p r o d u c t

a )

b )

c )

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.)