L/O/G/O 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-HCMC International University Lecturer : Dr. Tran Ngoc Duc
Nov 27, 2014
L/O/G/O
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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