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.)
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