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Site-directed mutagenesis protocol

Site-directed mutagenesis protocol

Group’s members:Tran Thi Anh Thuy BTIU08114

Tran Thi Dieu Thanh BTIU08110

Than Thi My Linh BTIU08127

Vietnam National University-HCMCInternational University

Lecturer : Dr. Tran Ngoc Duc

Introduction

What is Site-directed mutagenesis?What is Site-directed mutagenesis?

A molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known

as a plasmid. Simply done by designing a “mutated” primer, followed by PCR.

General procedure General procedure

General procedure General procedure

• Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all

GATC sites are methylated for later digestion with DpnI).

• Design forward and reverse primers that will bind to the region of DNA you want to

mutate but that contain the modifications you wish to make.

• Run a primer-extension reaction with a proof-reading, non-displacing polymerase

such as Pfu DNA polymerase. This results in nicked circular strands of the plasmid.

• Cut up the template DNA with DpnI.

• Transform the circular nicked DNA into a highly competent strain such as XL1-Blue.

These cells will repair the nicks and not restrict the unmodified product DNA.

• Select colonies with the correct DNA.

Site-directed mutagenesis protocol

1. Pick target amino acid to be changed2. Design a synthetic oligonucleotide to mutate the

target amino acid3. Use this primer to synthesize double stranded DNA

4. Verify production of the mutation

5. Characterize the new enzyme

Experimental protocolExperimental protocol

Stratagene protocolStratagene protocolThis is the protocol for site-directed mutagenesis based on the

stratagene kit

• Materials:

– Pfu turbo

– 10X Pfu turbo buffer

– dNTPs (10mM)

– Forward and reverse primers (0.1ug/µl, see methods section for design tips)

– dH2O

– Dpn1

– Competent cells

MethodsMethods

For oligo-design or Primer Design:

• Both primers must contain the mutation.

• The mutation should be in the middle of the primer.

• Primers should be 25-45 nucleotides long and have a GC content of at least 40%.

• The melting temperature (Tm) should be ≥ 78˚C.

• The 3’-end of the primer has to end on an C or a G.

5’5’3’

3’*

*

ReactionReaction

• Set up as follows:Components

AmountTemplate DNA (50ng/ µl) 1 µl10X Buffer 5 µlForward Primer (0.1 µg/ µl) 1µl Reverse Primer (0.1 µg/ µl) 1 µldNTPs (10mM) 1 µlPfu turbo 1 µldH2O 40 µl

PCR program PCR program

• Depending on the length of the plasmid, this program can become very long, so it may be best to turn overnight

1. 950C for 1 minute

2. 950C for 50 seconds, 600C for 50 seconds, 680C for 1 minute/kb of plasmid length – repeat this step 17 times, or 18 cycles total

3. 680C for 7 minutes

4. 40C hold

Following PCRFollowing PCR

DpnI Digest:

• Add 1 µl of DpnI to the reaction. Incubate at 37◦C for 1-2 hour to digest parental DNA

• Run 5µL of the digested reaction on a gel and compare to the undigested parental plasmid – there should be some difference in band pattern.

Transformation:

• Transform the final reaction into competent cells

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Discussion

Trouble ShootingTrouble Shooting

• If no product is seen, try repeating the protocol with 5% DMSO in the reaction mix.

• DMSO disrupts base pairing, facilitating strand separation in GC rich regions of DNA and reducing the propensity of the DNA to form secondary structure.

• The end effect, is a little DMSO will often get you past issues with poor primer design and/or difficult templates.

Verifying the mutationVerifying the mutation

Gene sequencingsequence the DNA to verify that the base changes have been introduced

Restriction mapping

use the creation of a new restriction site or the elimination of an existing one to verify the mutation

Restriction screeningRestriction screening

Allows selection of mutant enzymes through the creation or elimination of a restriction endonuclease site

The creation or elimination of a site changes the size of the DNA fragments obtained

ReferencesReferences

• L Zheng, U Baumann, and JL Reymond. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucl. Acids Res., 32:e115, 2004

• QuikChange® Site-Directed Mutagenesis Kit

• Retrieved May. 18, 20010 from http://www.jove.com/details.php?id=1135

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Thank You for your kind attention!

Thank You for your kind attention!

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