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