Is PCR efficiency higher if mutation is in the middle of primer? - (Jun/19/2012 )

I'm trying to make a single nucleotide mutation in my PCR product, so I'm designing primers for it, but because of the Tm issue I can't have the mutation in the middle of my forward primer, it is at 5' area. would this affect efficiency of PCR? could I not get any band at all? I have seen most people have mutations in the middle.

Mutagenesis is usually done with two mutagenesis primers, idealy on a circular template, with destruction of the original (not newly made) molecules.
Other way only half of your product will contain the mutation and the selection is difficult.

But as for efficiency of the primer with mismatch on 5' end, it will be actually better than the missmatch in the middle, see that it's a common practise to put restiction sites that don't match on 5' ends. The closer the mismatch is to the 3' the worse AFAIK, for the efficiency. But mismatches near 5' end could cause the whole 5' end to unbind and then reduce the specifity of the primer, that is I think the reason why mutagenesis mismatches should be in the middle (but mutagenesis primers are quite long). If you have the primer long enough on the 3' part, this may not be serious issue.

awesome reply Trof, many thanks

I have done site directed mutagenesis before, but I don't know why I thought mutagenesis can be done with only one primer on a linear template, silly of me....You also mentioned that the chance of having mutation with only one mutagenesis primer is just half. It makes sense. However, I think I will go ahead with my design and just ligate my PCR product to pJet and then extract plasmid from several colonies and just check which one has the mutated product??! I hope I can select the right one !

But even if I don't select the right one, I will run site directed mutagenesis with the PCR product in pJet later.

Trof, you said that with one mutagenesis primer only half of my PCR products will have the mutation. Now, If gel purify my PCR product and use it in Overlap-Extension PCR with another PCR product, and use the same mutagenesis primer, will I still have only half my final product mutated?

It goes like this:


Product 1 ------------------------- Product 2
==================== + ===================
F primer > -------------------------------------------<mutagenesis R primer

WHat you can do is design two separate PCRs with two internal primers that have tails (red and yellow) containing the mutation. Purify the products, anneal the primer tails together and then do a nested PCR (green primers) to make the completed product. See attached image

Hope you aren't colour blind...

How many bases can the tails be?
I think i finally got a band at 6.7 kb today after oe-pcr. I am ligating it to pjet and will send for sequencing. hope that's the correct band. my only worry is that the more oe-pcr i do,the harder i can purify them after gel extrsction.

The tails can be as long as you like. The protocol I have seen (sorry no ref, it is a colleague's protocol) says to use 15 bp, but my gut instinct says that is too short - I would go for at least 30.

thanks Bob1,

This time I kept my overlap length at 20 bp based on what pcrman suggested last time...I think the longer the overlap size, the higher the Tm. I mean it will be difficult for high Tm regions to bind together. I kept it at 61C this time, to match the reverse and forward primers too. I think that was the trick.