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Mutagenesis PCR - (Aug/09/2014 )

Hi, I have two questions about mutagenesis pcr. I want to delete 14 bp from a gene inserted into a plasmid, i found the primerX tool which will design the desired primers to do this. However i do not understand when would i like the mutation to be at the center of the primer versus not so, and when to use complementary versus overlapping primers. Thanks and hope someone can help me.

 

 would i obtain a linear product? and if so is it better to add a bsai restriction site or not to the ends of the primers or do blunt end ligation?

-Raygoza-

I have performed similar mutagenesis experiments quite often. The technique I used was first described by Ho, et al (Ho, S.N., Hunt H.D., Horton R.M., Pullen J.K., Pease L.R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene.  77, 51-9.). The basic method is as follows:

1) Design 2 sets of primers (Call them F1 + R1, and F2 and R2). There should be unique restriction sites between F1 and R1, and between  F2 and R2, so that the amplicon can be cloned into the plasmid containing the original gene/cDNA. Primers R1 and F2 should overlap, and should have the final sequence for you wish to include in your gene, etc. You will need at least 12 bases on either side of the desired mutation in this case, since you will be trying to make a deletion. 

2) Set up 2 different PCR reactions, using F1 and R1 in the first and F2 and R2 in the second. 

3) Purify the PCR products (usually just removing the old enzyme, nucleotides and primers is sufficient). 

4) Set up a second PCR with F1 and R2 as the primers, and the amplicons from the first as the template. It may take some adjusting of conditions to get this PCR to work. 

5) Purify the product as above, and cut with the unique restriction enzyme(s). At the same time, cut your original plasmid with the same 2 enzymes and phosphatase treat it to minimize any self ligation. 

6) Gel purify the cut amplicon and the cut vector and ligate them together. 

7) Transform your E. coli strain with the ligation mix, and select as usual. 

8) Ascertain that you have the plasmid you want by restriction enzyme analysis.

9) Sequence the insert to make sure there haven't been any unwanted PCR-induced mutations. 

 

Hope this helps. 

-dfritzin01-

No, this overlap method is too complicated and doesn't work. Thanks though. 

-Raygoza-

Did you look into a technique called "restriction free cloning" ?

-pito-

Yes this works like gibson assembly, thanks though. although it's not part of the question.

-Raygoza-

No, this overlap method is too complicated and doesn't work. Thanks though. 

 

If I am ever fortunate enough to have a lab of my own and my student refused to do some kind of cloning because it was "too complicated"....I'd be showing you the door because molecular cloning work like this is about as easy as it gets in comparison to some of the experiments you'll have to do down the road. wacko.png

 

Could you explain why it "doesn't work"? That would help us help you figure this out.

 

The reality is that overlap extension is really simple, and very effective. Two PCR's, a couple gel purifications, a ligation and you're done. Two days work on the side while doing other experiments.

 

Just buy two complementary primers where the primer pair has the desire sequence of your mutated protein (as stated already by dfritzin). If your original sequence is abcdLMNwxyz, and you want to delete the LMN sequence, just make your primer abcdwxyz. Make a second primer that is complementary to that. And if you don't have primers that flank the ends of your insert where your cloning restriction sites are, get two of those. Easy peasy.

 

Or just buy the mutated gene if your lab has the $$'s.

-labtastic-

Raygoza I think you have two options.

Since it's not really clear what you are asking, I suppose you originaly planned to do some kind of "quickchange" style PCR mutagenesis on a plasmid. This works if the plasmid is not too large and the deletion is not too big.
I know nothing about your plasmid, but 14bp deletion is a pretty big one.

 

As labtastic suggested, for "quickchange" (I call it after the probably most know commercial kit from Agilent, that contains everything needed AND the have online tool to design mutagenesis primers, probably the same thing primerX does) you just design primers that bind the sequence around, missing those 14bp that you want to delete. Then you continue with the quickchange protocol, dozen cycles of PCR with proofreading polymerase, DpnI treatment to digest original plasmid, and transfornation.

BUT

The primers will bing but create a "loop" on the original sequence, where you are deleting. 14bp loop is quite big and the efficiency of such deletion primers will be probably much lower than with shorter deletions. It may work, but i may not work at all.

In that case the overlap PCR, using four primers and combining the result will be a better choice, with those you may design deletion of any length. I would personaly consider it in this case. There is really not much difference. You may threat the OE-PCR product with DpnI too, to make sure no original template will interfere.

The resulting product from a quickchange method is also linear, proofread polymerase used for quickchange doesn't have 5' exo activity or strand displaycement, they stop at the beggining of the primer, and the only sequence where the new plasmid holds is the primer "overhang" itself. 
In OE-PCR you need to blunt ligate the ends, but that is not that difficult.

You may try quickchange version first, but if it doesn't work, you will need to try OE-PCR approach.

-Trof-

Overlap pcr doesn't sound difficult and is not, but there too many details that are not being considered, since i've been doing it i know what i'm talking about. The pcr mutagenesis with the plasmid is a better option for many reasons.

-Raygoza-

I've been doing both methods and even considering quickchange to be quicker and more direct when it works, it's much more difficult to optimize, when it doesn't (unlike OE-PCR). And more specifically when the primers doesn't work.

But when I was thinking about it, maybe the deletion legth may not be that important, since you don't need the primers to align on all parts of it, just the 3' end. The 5' may overhang and not bind at all. The same for the reverse. 
But unlike the internal mismatch primer design, you need to camculate with the fact, the 5' end will not bind in Tm calculation and that the primer length has to be also considered, because only at those sites the new plasmid will align.
And this is probably what were you asking in the first place. But I think even that considered, mutagenesis primer with similar paramaters and lengths as usual mismatch primers would work, only that the binding part of primer should have a length (Tm) of a "classic" primer, to be specific enough.

-Trof-

Thanks for the answer, my initial intent was to understand the plasmid based mutagenesis, not to t on a discussion for which was better. that's why i threw me off when someone came with e overlap pcr since it was not part of the question.

-Raygoza-