Hi Lech,

My understanding is that you are half right.

The first annealing will give two products that can be extended by the Taq.

A)

 5-------                                           5--------------------------------------A

A--------------------------------------------5                                 --------5

and

B)

5--------------------------------------------A

                                                        A--------------------------------------5

In A) the Taq will extend from the end primers, and as it has 5' - 3' Exonuclease activity will join to the annealed fragment, thus creating a correct full length template.

With B) it will be as you have suggested that there will be an A introduced to the new full length template. Of course with careful primer design this A can be made to be the correct base.

So further rounds of amplification will produce a mix of correct and mutant fragments.

It is too long ago for me to remember if I ever ran a SOE PCR without the presence of a proof-reading enzyme in the mix. I suspect that I have, and that it worked but it is lost in the mists of time for me to be certain :)

-Richard

Hi Richard

"A" can´t work, even if the polymerase has an exonuclease activity to digest one strang of the ds DNA in the overlapping region, without a template it will amplify nothing.

Lech, you are right with the assumption that the additional A may create a problem. If you design your overlapping primer clever (let it end before an "T" in the complementary strang and the additional A will pair correctly) you won´t get an additional base.

Hi Michael,

Good point, you could be right, after giving it a bit more thought I think 'A' is a bit more complicated than at first glance. There will be also be a template strand, which is generated from the taq that started from the lower strand primer.

5------->                                          5--------------------------------------A

A--------------------------------------------5                                      5--------------------------------------A

A--------------------------------------------5     5----------------------------------A

A-----------------------------------------5

Dear Richard,

My understanding is that Taq polymerases will never meet, as they go in the same direction on the template strand (3'-->5').

Dear Michael,

So far I always used T/A cloning-compatible polymerase blends, and never run into problems.

I guess that the following is true:

1) in the reaction mix, you usually have excess of primers over overlapping PCR products. Therefore, you quickly produce an excess of correct products (with the correct overlap, without As), and eventually the amount of incorrect products will be negligible.

2) commercial Taq blends for cloning contain proofreading polymerases too, which would remove the non-template As

Thank a lot for a comprehensive explanation!

Cheers,

Lech

Straight answer, I have used taq polymerase and performed gene knockout by SOE PCR.

1) You need to perform two different reaction. First to amplify up stream and down stream fragments and then, in second PCR these fragments would serve as template. I guess in this way, you do not need exonuclease activity of polymerase.

2) In my opinion, you need to add restriction sites within your primers. So after TA cloning and digestion from T-vector the problem of A tail would be solved.