The easiest answer is yes, you should order a new primer. They are cheap, and turnaround time to acquire a new primer is usually just a couple days.

Now with that said, how will it affect your PCR? It really depends on what you are performing the PCR for. In quantitative RT-PCR, you would be in big trouble because by having a mismatch you change the primer efficiency, and thus your quantification may be off compared to an exact matching primer. If you are performing PCR to clone, well then you have now essentially created a point mutation in your PCR product which will carry through to the plasmid and downstream assays.... which you do not want. Last, if you are just performing a PCR to see if your product is "there or not," it in THEORY could work. The problem once again is that the efficiency is most likely going to be hindered because of the mismatch, and may even predispose the primer to bind elsewhere in the genome amplifying non-specific products.

Probably not the answer you wanted to hear, but rigorous science is always better science. Put the order in today, and hopefully you will have a new primer by the end of the week and will not have to worry. Good luck!

~Adam

The easiest answer is yes, you should order a new primer. They are cheap, and turnaround time to acquire a new primer is usually just a couple days.

Now with that said, how will it affect your PCR? It really depends on what you are performing the PCR for. In quantitative RT-PCR, you would be in big trouble because by having a mismatch you change the primer efficiency, and thus your quantification may be off compared to an exact matching primer. If you are performing PCR to clone, well then you have now essentially created a point mutation in your PCR product which will carry through to the plasmid and downstream assays.... which you do not want. Last, if you are just performing a PCR to see if your product is "there or not," it in THEORY could work. The problem once again is that the efficiency is most likely going to be hindered because of the mismatch, and may even predispose the primer to bind elsewhere in the genome amplifying non-specific products.

Probably not the answer you wanted to hear, but rigorous science is always better science. Put the order in today, and hopefully you will have a new primer by the end of the week and will not have to worry. Good luck!

~Adam

The best thing you could do is to order a new correct primer as Adam Case suggested. In theory you could use this primer for some applications but you will never be sure of your results. Also, as the mismatch is one nucleotide before the 3' end I doubt that it will work at all.

Hi Javad!

I have used intentionally primers with a mismatch at the second last nucleotide at 3' and the PCR works. However, I agree with the others that you have less risks ordering a primer with no mismatches, if you are going to use it for quantification or sequencing. For an unbiased quantification the ptimal choice is to have a reaction with an efficiency >1.8 and a mismatch reduces the efficiency. For sequencing it can always happened an unexpected second mismatch that can lead to monoallelic amplification.

Wishes,

Paraskevas

https://www.researchgate.net/publication/233538525_Differential_haplotype_amplification_leads_to_misgenotyping_of_heterozygote_as_homozygote_when_using_single_nucleotide_mismatch_primer

Please read this paper, this would give you some idea how this mismatch primer can affect your PCR....

Article Differential haplotype amplification leads to misgenotyping ...

i did the same thing when cloning for protein expression. Although the affected nucleotide is negligible, the protein turned out to be unstable. Best if you order new, correct primer.

It'll affect a lot. Rather, you design a primer with wobble bases on that mismatch nucleotides.

General PCR axion: put the mismatch in the middle of primer.

You should better order a new primer without mismatch.

With you primer, you may have no amplification at all, our non specific amplifications or allele drop-out if you do PCr for genotyping , and so on !

Good luck !

You should order a new primer. If you are not sure of what is he right base in that posoition, you could design a degenerate primer. On the other hand, you PCR with the mismatched primer could work well or not, you have to try to know it, but what it is for sure is that you will be introducing a mutation in your sequence and that could affect the subsequent steps in your research.

If this is just to see if your sequence is there or not, the primer may work. I've done PCRs with intentional mismatches to get specificity if there are two sequences that are similar. However, you need to use a non-proofreading polymerase, such as AmpliTaq.

Javad: If needed to work with this mismatch, probably the annealing temperature has to be adapted accordingly. Also an increase of annealing/elongation time is worth to try for PCR with mismatched primers. Good luck, Roland.

That depends on your goals. If you want to introduce a mutation, use it. Otherwise you may want to order a new primer.

If you want/have to use this primer, use a lower annealing temperature for the first 5 cycles with longer enlongation time. Then go back to normal for the rest of the cycles.

Mismatch is always bad for the efficiency of PCR and it would be lethal if a mismatch is on 3' end of the primer because if DNA polymerase can not find dsDNA then there would be no amplification. So its better to order new primer with no mismatch at least on 3' end. Best of luck with your experiments

Khadim sir PCR would be there but the efficiency or quantity yield would be much less. Cause, the said mismatch is not at the last position in the 3énd but a nucleotide before so in his case its not true that there would be no PCR.

Christian Praetorius : thhe way you have said, he would definitely get successful PCR. But he may end up with strand bias. I have a publication very recently just on this issue.

Try hotstart with pyrophosphatase to have less aspecific bands

http://www.antibody-antibodies.com/us/product1090957-Bioneer-AccuPower_HotStart_RT_PCR_PreMix_NEW_0.2_ml_thin_wall_8_strip_tubes_with_attached_cap___96_tubes.html

If you introduce a mismatch on purpose in your primer, you should have a long primer to make sure that it will still bind to your template. Thus, you should design a pair of primers with a high melting temperature.

@Navonil: Thats why I wrote it depends on his goals. If he want to test for the presence of a transcript its fine. If he wants to mutagenize, its ok as well. If you are looking for the transcript you have to sequence anyway.

As per my experience it would be safe if you insert the mismatch at 15th position or later from 3' end.

A 3' mismatch in a primer (within 3nt of the end) will really hurt your priming efficiency by inefficient polymerase read-through. Assuming you're not happy to use a degeneracy, a neutral base such as deoxyinosine (dI) will neutralise the mismatch without harming Tm or priming effuciency too much.

Mark Laverick.. Does it form bond with its complementary bases???

dI will base pair with any other nucleotide, although it is 'neutral' it does have a slight bias for dC pairing. It's a useful alternative to having degenerate base positions as it means you only have a single molecule in the synthesis rather than mixed oligo populations (as obtained with N,Y,R degeneracy etc).

Yes, Adam and Bastian have answered the query very well put by Javad. It will lead to bind elsewhere in the genome leading to non-specific amplification and will give false results. secondly it will depend upon length if the primer and objective of using the primer. Best option- get new primer synthesized to avoid any confusion and authenticity of the results

A mismatch one nt before the 3' end is bad news, but it doesn't need to be the end of the world. It depends on the type of the mismatch. Purine:purine and pyrimidine:pyrimidine mismatches are highly destabilising, but mismatched purine:pyrimidine (A:C, G:T) much less. I have used primers that form a G:T basepair at nt 3' -1 and they work just fine. So you can use G as a universal purine and T as a universal pyrimidine.

Alternatively, as Mark suggested, you can use inosine as a wildcard base. It probably won't do much for your specificity or the efficiency of the reaction, but you may be able to get away with it.

Thank you all for your good answers. The truth is that I want to perform a PCR-RFLP and I use this mismatch purposefully, because I have to introduce a point for my restriction enzyme. my reverse primer is 31 bases long. as I just want to see if the pcr product is present or not, based on your answers I think I can do it.