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Amplifying a 1700bp fragment using a mismatched primer pair - (Mar/20/2007 )

I'm trying to amplify about 1700bp fragment of mitochondrial COI gene of several fly species.
Good bands were shown in some fly species, but not in some.
Gene order of mitochondrial genome is as follows:
tRNA-Trp -> tRNA-Cys -> tRNA-Tyr -> COI -> tRNA-Leu -> COII
My primers were designed from multiple sequence alignment of closely related fly species(highly conserved regions),
but sequences of COI and its flanking regions of most fly species I'm trying to amplify are not known.
So some degree of mismatch between primres and template DNA is expected.
Forward and reverse primers bind to tRNA-Cys and COII, respectively.
Primers seemed to bind well to the template DNA in short-length PCR and showed thick bands in all species.
My PCR condition is as follows.

ddH2O 5.32ul
10x gold buffer 1.0 ul
MgCl2(25mM) 1.0 ul
dNTP(10mM each) 0.2 ul
primer(10uM) F 0.2 ul
R 0.2 ul
AmplitaqGold 0.08ul
Template(10ng/ul)2.0 ul

95 - 11min

95 - 30sec
50 - 1min ->35 cycles
72 - 2min

72- 15min
4 - forever

I heard that Pfu polymerase could be helpful in this situation, is it right?


If you have mismatched primers, your Tms will suffer a bit. Try a gradient PCR to see if you can pick up some of the missing/failed reactions. If you don't have access to a gradient machine, you cold try a touchdown reaction, but I can't guarantee you'll get a successful result, but that's why they call it research, isn't it? Sometime,s you've just gotta do the expt and see what comes out the other side.
The risk with these reactions is that you'll start getting some mispriming. If so, I think you could improve things with a MgCl titration. Another possibility is if you extend your primers a bit to give a higher Tm, that'll give you more room to move with touchdown PCR (I think that 50 degrees for the annealing is on the low side).


Several suggestions:

* Try lowering the extension temperature to 68 or lower. Increase extension time a little to compensate. Mitochondrial DNA has low GC and this can be an issue.

* Use primer mixtures if you are uncertain of the sequence. Any oligo manufacturer will insert a mix of bases at particular locations if you use the mixed IUPAC code (R for both A and G, for example). Try to do this as few times as possible and at the 5' end.

* Make sure the 3' ends of your primers are in regions which you are most certain of. Binding of the 3' end is most critical to extension, and even a single base mismatch at the 3' end will fail, whereas a single base near the 5' end is almost a don't care.

* I would try to raise the annealing temperature to 55 if possible.

* Mg++ titration might be called for, or switching to a premix PCR mixture. The ratio of Mg++ to dNTPs is important, and either can be subject to pipetting error.

* How certain are you of the gene order? These are regions which do vary a fair amount.

* An alternative would be to use a primer pair which is working (it sounds like you have some) to sequence a short region within thiis area. Then, you can design exact primers to sequence outward, and do inverse PCR to capture the unknown region.