I would agree that a 0.1 degree change in the annealing temperature would not cause more stringent PCR conditions particularly when it is a decrease in temperature. This is supported by the fact that you generated a non-specific band at 58.5 degrees and 59 degrees. As the others have stated, this simply could be a slight difference in temperature throughout the PCR block. To eliminate a non-specific band in PCR optimization you would look at variables such as annealing temperature, primer concentration, template concentration and the length of time for annealing. Did you try to duplicate the conditions of the PCR reaction using 58.9 degrees and see if you end up with a single band again?

What is the size of the product you are amplifying versus the size of the non-specific band with a size 50 bases larger?

Another option is touchdown PCR. Start with an annealing temperature above 59 degrees and decrease 0.5 to 1 degree each cycle until you reach the target annealing temperature of 59 degrees.

Interestingly, you describe obtaining just one specific band at the (slightly) lower annealing temperature at 58.9 vs. 59 degree. I usually would expect higher specificity at higher annealing temperature. Did you or could you measure the temperature within the different areas in the PCR block? Sometimes there could be differences as well.

I expect you use good primers...

Have you verified with BLAST the specifity of your primer? I would rather suggest to perform gradient PRC, maybe you will try to make your primer work at higher temperature to avoid non-specific amplification?

May i know how many samples did you test at each of the annealing temperatures (Ta)? Usually you should get higher specificity when increasing Ta. But sometimes, because of variation in equipment or efficiency of the enzymes, you might get inconsistency in banding patterns even with no Ta variation. In your case, i think that the primer you are using might not be stringent enough in terms of the annealing site, and you were just lucky to get specific band in one case. I would suggest that you run multiple samples using the same PCR machine and the same reagents at different Ta just to test the optimum Ta. But the best way would still be that you cut out both bands and sequence them separately and redesign your primers.

Hope it helps :)

I would agree that a 0.1 degree change in the annealing temperature would not cause more stringent PCR conditions particularly when it is a decrease in temperature. This is supported by the fact that you generated a non-specific band at 58.5 degrees and 59 degrees. As the others have stated, this simply could be a slight difference in temperature throughout the PCR block. To eliminate a non-specific band in PCR optimization you would look at variables such as annealing temperature, primer concentration, template concentration and the length of time for annealing. Did you try to duplicate the conditions of the PCR reaction using 58.9 degrees and see if you end up with a single band again?

What is the size of the product you are amplifying versus the size of the non-specific band with a size 50 bases larger?

Another option is touchdown PCR. Start with an annealing temperature above 59 degrees and decrease 0.5 to 1 degree each cycle until you reach the target annealing temperature of 59 degrees.

@ Robert. Thanks. really good point! I wanna test that.

@Wei Lung, I tested three samples in 5 different temps in gradient PCR. all sample gave single band in 58.9 and one of them also in 61. But none of them showed band from 63 to 68. I used the same PCR machine and the same reagents at different Ta just to test the optimum Ta.

@Doug, I wanna explain somthing. when I used 59 degrees I just had one specific and one no specific band at the size of 740bp and about 800-850bp respectively. But when I used 58,5 degrees lots of no specific bands appeared. Today I have started experiment with 58.9 and as Robert suggested, I have used the same position.

I will inform you about the result.

If you have done many PCR and get the same result, I think you should check you DNA sample. It may contaminated. Good luck

@ Trung, Thanks. But the problem is that when I tested the DNA samples with another primer sets with the same amplicon size, I have obtained perfect single band. So, the problem would be the optimization of the Primer set in question not from DNA samples.

Try decreasing your MgCl concentration for better specificity. Also, what region are you amplifying? Is it a single bacterial species or a community? If more than one species then there can be differences in amplicon size, depending on the 16S rRNA gene variable region, If they are universal primers you may even be amplifying Archaea or Eukaryotic plastids.

@ Barbara, V3-V6 16s gene. The samples are DNA from one bacterium not complex samples.

Hi Mahdi,

Thank you for the clarification. It would make more sense that you have more non-specific bands with a lower annealing temperature. I look forward to hearing what the additional testing shows.

Change your MgCl concentration. Magnesium concentration is critical to the success of PCR amplification because it may affect DNA polymerase activity and fidelity, Excess magnesium results in accumulation of nonspecific amplification products seen

as multiple bands on an agarose gel, whereas insufficient magnesium results in reduced yield of the desired PCR product.

Finally I got very interesting result. I obtained four single specific bands when I tested the samples with 58.9 degrees centigrade. One of the samples, however, showed one more no specific band at the size of 2 kb!. What is that high size band?

You really need to optimize your PCR protocol for the DNA you want to amplify. Too many unspecific bands indicate wrong conditions, could be temperature, buffer, primer...

Unspecific PCR conditions and annealing temperature are most probably the reason for your problem. Try to work on your protocol by testing all PCR reaction components and your annealing temperature.

Eventually, you have got dimers of your PCR product in the sample. Please check if the length of the second band is exactely twice the length of the first one.

If this is the case, use less template and Taq Polymerase. You sometimes get this phenomenon in PCRs that produce a very high yield in PCR product.

You can also try to reduce the extension time.

reduce the DNA concentration, set up a touch down PCR

I think its a very important that you pointed the samples which you worked? Means the isolated bacteria or clinical samples?

Increase your annealing temperature. I think first you apply the gradient programming and select the specific one..

At first check your sample DNA concentration by spectrometric method and Standard MgCl concentration following PCR reaction.

All The Best..

Hello.check your PCR products because the artifact substance or pollution

If you are using a generic 16s PCR test, it may be your reagents that are the problem. Many polymerases are prepared from transgenic E. coli and contain traces of E. coli DNA. Water often contains traces of Legionella, which is a ubiquitous bacterium in water systems. Sequencing your non-specific product could be informative if you can't get it to go away by simple means.

Dear Aparna,

I am going through non-specific band problem in PCR results. So, I want to try with Touch down PCR. I also have condition for that. But the issue is that I do not know how to set those condition, particularly decrease in temperature in each cycle.

Could you please help me to solve this problem?

Regards,

Sailesh