site directed mutagenesis
TRANSCRIPT
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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
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Introduction
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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.
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General procedure General procedure
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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.
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Site-directed mutagenesis protocol
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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
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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
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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’*
*
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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
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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
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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
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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.
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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
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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
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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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L/O/G/O
Thank You for your kind attention!
Thank You for your kind attention!
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