site-directed mutagenesis - (Jul/04/2005 )

Hi,
I have a favor and question to ask regarding site-directed mutagenesis.
Has anybody ever made a site-directed mutagenesis clone without using a commercial kit? We have been making site-directed mutagenesis clone by using kit (QuikChange) from Strategene. However, we are now trying to make it on our won using the same reagent, but without success. I will appreciate if you could lend me a hand with this regard. Any suggestion will be highly appreciated.

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Hi Wenni,

Shouldn't be any problem, we just buy in the components ie. proof reading polymerase and Dpn1 and haven't had anyproblems. Which part is failing ie. no colonies or colonies are not mutated?

Scott

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I've never used a kit for SDM but I've successfully introduced single base mutations with close to 100% efficiency using the "megaprimer" method. Search for it in pubmed. It's very simple and cheap. If you fancy using it let me know and I'll post some more info about my experiences.

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When you say "same reagent" you need to be a bit more specific. It matters a great deal, for example, which PCR enzyme you are using. In particular, Taq and its friends will not work with the Quikchange protocol, nor will mixtures containing Taq, due to its 5' strand displacement ability. Quikchange relies on the PCR extension reaction stopping when it has gone all the way around the plasmid and encountered the primer. Have you tried the control reaction with your reagents?

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Use the PCR overpal technique !

Design 4 primers, 2 primers for the 5' and 3' ends of your cDNA and 2 other containing the mutated base in your gene ("A" and "V" in the scheme). These 2 ones must overlap. Also be careful to respect Condon usage, the codon mutated must be an abondant one in you host expression system.


Here is the scheme of your PCR :

(a)---> <--A-(
----------------------------------------------------------------------------------------
©--V-> <---(d)

Dilute your plasmid miniprep 1/20 and use 1µL for PCR.

Make 1st PCR with the primer (a) and (.
Make the second PCR with primers © and (d).
You should amplify 2 parts of your gene with the uncluded mutation given by your primers "b" and "c" that include the correct mutation.

Purify yours PCRs.

Then use dilution of these two PCR to make the overlap with the Tm given by the overlap sequence of your 2 primers "b" and "c". To recreate your entire gene with the mutation. Of course you don't need primers for this PCR.

Don't forget to include restriction site to the primers a and d, suitable for PCR digestion and ligation into your expression vector. Or use Zero blunt TOPO system for PCR recovery...

Use high fidelity polymerase like Phusion or Platinum HF.

The primers ( and © should overlap in an Tm like 60°C...

Good luck

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[FONT=Courier]
Sorry my PCR scheme is better here :

(a)---> <--A-(b
----------------------------------------------------------------------------------------
c--V-> <---(d)

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which company do you buy the pfu polymerase ?
and is this the nonstrand-displacing actions of Pfu polymerase?
thank you by Wenni

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I have created several point mutations using the 'megaprimer' method. I used Pfu Polynerase. I also have sucessfully created mutants with 3, 4, and 5 consecutive residues changed using this method without modifying any steps in the procedures as done for single point mutations.

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Hi Wenni,

I think I normally got my Pfu from Roche, it has been a year or two since I was doing that.

Pfu polymerse is non-strand displacing meaning that when it hits the primer it will automatically stop. This is especially handy if your site directed primer has significant secondary structure, a way around it is to design you primers on either side of the mutation with the mutation on the 5'end of your sense primer - the antisense primer is then the next base and do a standard PCR reaction amplifying the plasmid. Pfu will then create a perfect blunt end which can easily be phosphorylated and ligated and the mutation will be incorporated.

Cheers,

Scott

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I will preface this answer by saying that this is my paper but you might find the following paper helpful.

http://www.ncbi.nlm.nih.gov/entrez/query.f...5660&query_hl=1

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