Smearing in Overlap Extension PCR

[kamran]

Dear All,

I have to join four fragments of sizes 1.2 (A), 2.5 (B), 1.9(C) and 0.7kb (D) each having an overlapping region of around 50bp with Tm >80°C. I successfully amplified individual fragments ( as a single band) and purified them with the PCR purification kit. This whole exercise didn't take enough time.

Now comes the hard task of joining;

I performed overlap extension PCR for 10 cyces using equimolar conc. of fragment A and B in 25µl reaction volume using phusion high fidelity polymerase. After 10 cycles, I added 25µl fresh master mix containing Forward-A and Reverse-B primers and allowed the amplification to continue for another 30 cycles. Since Tm of overlapping region was >72°C, I used 72°C for annealing as well as extension termperatures.
The result I get every time is "smearing" . I tried different MgCl2 conc. as well as different polymerases however, smearing pattern is consistently reproduced

Same is the case with C and D.

Could you please give me any suggestion that can help me getting rid of this smearing pattern.

Thanks in advance.

MKR

[mchlbrmn]

Might be it's just too many cycles. After all, you hardly need to amplify anything at all, not at all if you put in lots of template.
I never understood the 10 cycles without primers I've seen people mention. Does some protocol recommend that? What's the purpose? even from the second cycle the primers can start creating a full strand, and incomplete strands can complete in later cycles. You could try mixing 1ul from 2 PCR products (or more if you want to try in parallel for fun and see if you can do the whole thing, and the BC one to be thorough.) and the primers and do only 12 cycles (if that's your lucky number) with 1.5 mM Mg++ (if that's the normal minimum for phusion).
Could you see a correct size band under the smear?

[HLper]

What are your concentrations for the fragments? I would go with a low 1.5pmol for the double stranded fragments, which will end up as you template, and about 30pmol for the amplification primers. That's IF I were going to do that with an Assembly PCR approach.

Here is a second suggestion: have you checked out the Gibson Isothermal Assembly master mix from New England Biolabs (NEB)? I haven't used it, but I occasionally write on synthetic biology methods and that one seems well suited to your overlapping, double-stranded fragments.

I hope that's helpful.
-H-

[kentboles]

Smears in overlap reactions are usually from too much of the overlapping fragments (template), badly designed primers or repeats in the template. In the first round, the fragments will hybridize and act like primers extending out to make the full template. PCR cycling is just to recover the whole piece. If you add too much of the overlapping fragments, they will continue to bind to different areas and interact badly with your primers. You want to consume / overwhelm your template early on. I usually use in the 10-50ng range at most for the overlapping templates. Make sure your extension time is long enough to make a full-length template, too. Phusion is very fast so you will only need 60-90sec. To summarize, set up a single 100ul Rx:
Phusion Rx, A_forward and B_reverse primers, 10ng of A, 20ng of B. Cycle 25 times for 60 sec/cycle. Make the annealing temperature appropriate for the portion of A_forward and B_reverse primers that are homologous to the template (+3C for phusion).
Sometimes, less is more