computer exercise design of pcr and pcr-rflp experiments this presentation shows all steps of a...
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COMPUTER EXERCISE Design of PCR and PCR-RFLP experiments
This presentation shows all steps of a PCR-RFLP experiment and is a companion of the computer exercise at
http://insilico.ehu.es/edu
To perform a PCR-RFLP experiment, we need a DNA sample.
Grow the problem cells
Pick up some bacteria
Re-suspend the cells in the suitable buffer
Re-suspend the cells in the suitable buffer
Apply the desired DNA extraction procedure
The purified DNA is the problem sample
In this presentation, we will consider two samples obtained from two different bacterial strains
The reagents for the PCR reaction must be mixed in a new tube
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
Add the sterile double-deionized water
dd H
2O
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
Buffe
r
Add the 10X PCR buffer
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
Add the MgCl2+ Magnesium ion serves as cofactor for Taq polymerase.
MgC
l 2
MgMg
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
dNTP
Add the dNTPs mix (dATP, dTTP, dGTP and dCTP)
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
Prim
ers
Add the primers (forward and reverse primers)
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
Taq
pol.
Add the Taq DNA polimerase
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
Sam
ple
Add the sample (template DNA)
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
The tube will contain all reagents required for PCR reaction
5´
3´
3´
5´
MgCl2
Taq pol.
Sample
MgMg
dNTP
PrimersG
A CT
During PCR reaction the following steps will be repeated20 to 40 times: denaturation, annealing and extension
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
5´ 3´3´ 5´
First cycle, denaturation step: The DNA strands are separated
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
5´ 3´3´ 5´
5´ 3´
First cycle, annealing step: Forward and reverse primers will bind to their target sequences.
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
Primer 1 recognition site
Primer 2 recognition site3´ 5´
5´ 3´
3´ 5´
First cycle, extension step: Polymerization of DNA by Taq polymerase
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
C GGAC TG ATCAT
A
C
G
T
A
G C ATMg
During extension, nucleotides complementary to target sequence are incorporated in the new DNA strand
5´ 3´
3´ 5´
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
5´
5´
3´
3´
First cycle, extension step: Polymerization of DNA by Taq polymerase
5´ 3´
3´ 5´
5´ 3´
3´ 5´
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
Second cycle, denaturation step
3´ 5´
5´ 3´
5´ 3´
3´ 5´
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
Second cycle, annealing step
5´ 3´
3´ 5´
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
3´ 5´
5´ 3´
Second cycle, annealing step
5´ 3´
3´ 5´
Denaturation: ~ 1 min 90°CAnnealing: ~ 1 min 45-60°C Extension: ~ 1 min 72°C
3´
3´5´
5´
5´
5´
3´ 5´
5´ 3´
3´
3´
Second cycle, extension step
In each cycle, the number of target DNA copies will double
1st cicle 2nd cicle 3rd cicle 4rd cicle n cycles
21 copies 22 copies 23 copies 24 copies2n copies
Original DNA
Prior to RFLP, it is convenient to purify the DNA sample to avoid inhibition of the restriction endonuclease activity
Many copies of the purified amplicons will be obtained. They will be the sample for therestriction step
For digestion of the sample DNA (amplicons) the reagents must be mixed in a new tube
H2O Buffer Enzyme Sample
Add the sterile doble-deionized water
ddH
2O
H2O Buffer Enzyme Sample
Buffe
r
Add the 10X buffer
H2O Buffer Enzyme Sample
Add the restriction endonuclease
Enzy
me
H2O Buffer Enzyme Sample
Sam
ple
Add the sample DNA (the purified amplicons)
H2O Buffer Enzyme Sample
C C
G
T
AC
G
C
G
T
AT
AGCTA T
C
G
T AT AC
G
T
A C
G
T
A
C
G
T
A G
A
During incubation, the restriction endonuclease will specifically recognize the target sequence.
C C
G
T
AC
G
C
G
T
AT
AGC
G
A TA T
C
G
T AT AC
G
T
A C
G
T
A
C
G
T
A
And it will be cleaved.
For visualization of the PCR-RFLP experiment DNA samples will be electrophoretically separated in an agarose gel.
Ladder
Molecular weight standards will be added to one line.
Sample A
In the second line, sample A will be added. In this example, amplicons in this sample were not cleaved by the endonuclease.
Sample B
In the third line, sample B will be added. In this example, amplicons were cleaved by the endonuclease.
During electrophoresis, the ladder and de samples will migrate within the agarose gel.
L A B
For visualization, the gel will be stained.
1000
500400300
200
100
50
Ladder(pb)
L A B
The molecular weight standards will be used to compute the weight of the bands in samples A and B.
L A B
Sample A contains a unique band of approximately 300 bp. This band was not cleaved by the endonuclease.
L A B
Sample B contains two bands. Cleavage of a 300 bp band containing the target for the endonuclease yielded these 100 and 200 bp bands
L A B
The PCR-RFLP experiment was able to discern the two samples due to the presence of a target sequence for the endonuclease in sample B.
You may try to solve the online PCR-RFLP exercise available at
http://insilico.ehu.es/edu
Thanks