computer exercise design of pcr and pcr-rflp experiments this presentation shows all steps of a...
TRANSCRIPT
![Page 1: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/1.jpg)
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
![Page 2: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/2.jpg)
To perform a PCR-RFLP experiment, we need a DNA sample.
![Page 3: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/3.jpg)
Grow the problem cells
![Page 4: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/4.jpg)
Pick up some bacteria
![Page 5: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/5.jpg)
Re-suspend the cells in the suitable buffer
![Page 6: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/6.jpg)
Re-suspend the cells in the suitable buffer
![Page 7: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/7.jpg)
Apply the desired DNA extraction procedure
![Page 8: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/8.jpg)
The purified DNA is the problem sample
![Page 9: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/9.jpg)
In this presentation, we will consider two samples obtained from two different bacterial strains
![Page 10: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/10.jpg)
The reagents for the PCR reaction must be mixed in a new tube
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 11: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/11.jpg)
Add the sterile double-deionized water
dd H
2O
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 12: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/12.jpg)
Buffe
r
Add the 10X PCR buffer
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 13: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/13.jpg)
Add the MgCl2+ Magnesium ion serves as cofactor for Taq polymerase.
MgC
l 2
MgMg
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 14: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/14.jpg)
dNTP
Add the dNTPs mix (dATP, dTTP, dGTP and dCTP)
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 15: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/15.jpg)
Prim
ers
Add the primers (forward and reverse primers)
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 16: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/16.jpg)
Taq
pol.
Add the Taq DNA polimerase
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 17: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/17.jpg)
Sam
ple
Add the sample (template DNA)
H2O Buffer MgCl2 dNTP Primers Taq pol. Sample
![Page 18: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/18.jpg)
The tube will contain all reagents required for PCR reaction
5´
3´
3´
5´
MgCl2
Taq pol.
Sample
MgMg
dNTP
PrimersG
A CT
![Page 19: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/19.jpg)
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´
![Page 20: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/20.jpg)
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´
![Page 21: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/21.jpg)
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´
![Page 22: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/22.jpg)
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
![Page 23: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/23.jpg)
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
![Page 24: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/24.jpg)
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
![Page 25: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/25.jpg)
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
![Page 26: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/26.jpg)
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
![Page 27: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/27.jpg)
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
![Page 28: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/28.jpg)
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
![Page 29: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/29.jpg)
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
![Page 30: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/30.jpg)
Prior to RFLP, it is convenient to purify the DNA sample to avoid inhibition of the restriction endonuclease activity
![Page 31: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/31.jpg)
Many copies of the purified amplicons will be obtained. They will be the sample for therestriction step
![Page 32: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/32.jpg)
For digestion of the sample DNA (amplicons) the reagents must be mixed in a new tube
H2O Buffer Enzyme Sample
![Page 33: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/33.jpg)
Add the sterile doble-deionized water
ddH
2O
H2O Buffer Enzyme Sample
![Page 34: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/34.jpg)
Buffe
r
Add the 10X buffer
H2O Buffer Enzyme Sample
![Page 35: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/35.jpg)
Add the restriction endonuclease
Enzy
me
H2O Buffer Enzyme Sample
![Page 36: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/36.jpg)
Sam
ple
Add the sample DNA (the purified amplicons)
H2O Buffer Enzyme Sample
![Page 37: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/37.jpg)
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.
![Page 38: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/38.jpg)
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.
![Page 39: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/39.jpg)
For visualization of the PCR-RFLP experiment DNA samples will be electrophoretically separated in an agarose gel.
![Page 40: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/40.jpg)
Ladder
Molecular weight standards will be added to one line.
![Page 41: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/41.jpg)
Sample A
In the second line, sample A will be added. In this example, amplicons in this sample were not cleaved by the endonuclease.
![Page 42: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/42.jpg)
Sample B
In the third line, sample B will be added. In this example, amplicons were cleaved by the endonuclease.
![Page 43: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/43.jpg)
During electrophoresis, the ladder and de samples will migrate within the agarose gel.
![Page 44: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/44.jpg)
L A B
For visualization, the gel will be stained.
![Page 45: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/45.jpg)
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.
![Page 46: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/46.jpg)
L A B
Sample A contains a unique band of approximately 300 bp. This band was not cleaved by the endonuclease.
![Page 47: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/47.jpg)
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
![Page 48: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/48.jpg)
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.
![Page 49: 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](https://reader038.vdocuments.mx/reader038/viewer/2022110321/56649d015503460f949d3b18/html5/thumbnails/49.jpg)
You may try to solve the online PCR-RFLP exercise available at
http://insilico.ehu.es/edu
Thanks