Hi researchers,
the thing is that I've been trying to amplify a PCR product of 700 bp long and when I run the electrophoresis gel to verify the amplification result all I get is a PCR product of 1000 bp, so when I designed the primers I was paying all my attention just to the gene and not all the DNA template, so after obtaining this result (1000 bp) I evaluated the DNA template and it's so funny because there's one primer (Forward) that anneals complementarity to a site where it has only 2 mismatches (it's 22 bp but has an overhang of 4 bp that obviously don't anneal).
So, my concern of all of this is to ask you why it's thermodynamically possible that a primer prefers to anneal in 1 site where it has 2 mismatches and not where it's 100% complementary.
Do you think a Touchdown PCR would be useful? I'm going to try with a Nested PCR.
It is not just a case of annealing to a slightly different sequence. This is pcr so there is great amplification of product so even the wrong product can be produced so long as the other primer anneals. Are you sure that the other primer anneals perfectly to the template?
Another factor is the position of the mismatch. A mismatched base near the 3' end of a primer can stop it working at all but 2 mismatches near the 5' end may make no difference at all. Have you sequenced the amplimer or done a restriction digest on it or are you concerned only on the basis that it is the wrong size on agarose gel. It would be easier to answer accurately with primer sequences,target name and species and pcr conditions . Nested pcr could work well but be careful not to over amplify which produces a smear. use 1ul of 1/100 pcr template and 15-20 cycles should be enough
Try to design new primer with low AT nucleotides specially at the 3 end of primer
Checking again it has 3 mismatches actually...
Paul Rutland Thank you for your answer. I'm sure that the primer is 100% complementary to the other site that is not binding, prefers the site where it has 3 mismatches and it gives me 1000 bp (that I'm not looking for):
This how the primer is:
5' - 3'
(Overhang) CACC TTGA[C]GAAC[C]CCGA[G]GCG
Where I put the box brackets [ ] is where the mismatch is binding to my DNA template.
And it binds in my DNA template 300 bp after the site I want it to bind.
I'll definitely try the Nested PCR. Thank you for the advice!
Another thing, my DNA Template is a Cosmid 34 kb long.
Your primer is quite high GC so may come from a high GC region. In this case maybe the template is reannealing in the region of your primer and forcing it to bind elsewhere or not at all. If The template can be kept single stranded for longer then the correct pcr may work. Try running your pcr in a final concentration of 1 Molar betaine with and without up to 6% DMSO to melt the sequence easier and keep it apart and possibly the primer may find its true position . Relevant to this is that your primer is really inly about 15 bases long so your annealing temperature is very low. This means the other primer can anneal in other places and also that the template can re anneal almost completely by the time the primer can anneal. Can you make the primer longer to let it anneal hotter?
Can you run a temperature gradient to about 6c above the annealing temperature to try to get the correct product. Why are you working with mismatched primers? You can get primers made at no extra cost with redundant bases at each position if you know the correct sequence and it has snp s
Paul Rutland Yes! I actually use DMSO 5% in every tube of PCR reaction. I haven't used Betaine yet but the result in the electrophoresis gel shows a very strong band using 20 ng of DNA template in the tube of reaction. I also digested my Cosmid template to linearize the template with 1 digestion site, and it still was getting the 1000 bp product. I appreciate your point of view because it's true about the reannealing of the template and the annealing temperature for the primers, I'm now ordering the primer to perform the Nested PCR.
I'll try the gradient with the higher temperatures! And well I'm not actually working with mismatched primers, I designed the forward primer to anneal exactly to the start codon of my gene of interest (700 bp) but it anneals (don't know why) 300 bp after my gene of interest and gives me a product of 1000 bp which I'm trying to avoid.
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