The good news is that you are getting bands and you don't have contamination in your no DNA control. So, you need to figure out which band(s) are from your gene of interest.

Have you tried gel purifying and sequencing to see what you are amplifying?

Did you try using BLAST to make sure your primers don't match to other regions in the genome? It's a common issue if your gene of interest is part of an expanded gene family.

You also need a better set of controls. Can't assume the genotype of cells from a cell culture and they usually have aneuploid genomes. Are you allowed to use your own cheek cells as a source of genomic DNA?

Good luck!

Dear Katie A S Burnette,

Thank you very much for your answer and suggestions.

I have not attempted to sequence the products, as I assume they are nonspecific. The negative control cells (I tested three different cultures) consistently produced the same bands as the DM1 cells. To me it seems highly unlikely they would have expansions of the same size. That said, it might still be interesting to see what is being amplified, but I am not sure how this would help me optimize the PCR.

I tested several tools, including BLAST, and all confirmed the high specificity of both primer pairs.

Regarding the controls, are you referring to the positive or negative controls? I would not expect my cheek cells to serve as a good positive control, as I do not have myotonic dystrophy. Since all three negative control samples yielded the same result, I believe that the issue lies with the PCR amplification rather than the negative control cells.

I really appreciate your help, thank you!

The bands are pretty bright and distinct so I'm assuming at least one is the size you are expecting. What size(s) were you anticipating?

You could have a control of cells that have only the non-expanded allele (e.g. your cheek cells). That will at least help you figure out if the issue is non-specific amplification.

Have you tried designing your own PCR primers? Not all published primers are necessarily accurate. Reviewers tend to trust the authors when they list primer sequences & don't ask folks to verify.

In the original article, they obtained bands of approximately 600 and 1500 bp. However, I usually obtain much larger products. Moreover, when I change the PCR conditions, new bands of various sizes appear.

I assume my negative controls should contain only unexpanded alleles, but it might be worth confirming this with my own DNA.

I did not design my own primers. I checked the two pairs I used against the reference human genome, as well as their melting temperatures and potential for dimer formation—all parameters appeared fine. However, you’re right that designing my own primers might also be worth trying.

Thank you very much for your help.

Your extension time after the cycles is too long (5mins at most should be ok). Also, the extension time within the cycle should depend on the size of your fragment of interest. Usually, I take 30secs/kb. In addition, since you're using genomic sequences, try template starting amounts as high as 1ug, because I think 200ng range is more suitable for plasmids.

Note: if you suspect your targeted sequences have a lot of complex repeats, you can also try 1min/kb for extension within cycle. By the way, your Tm should be within 5 degrees BELOW the Tm of your primers (usually the primer with the lower Tm). That is, If you have a primer pair with F:60°C and R:63°C, your Tm should be according to F, the primer with the lower Tm.

Hope that helps

Finally, your primer concentration is quite low, don't you think. For genomic DNA template, I'd try using 10uM for each primer

Muhammed Boye Jallow, thanks a lot for your suggestions!

I will try using 1 µg of template DNA, increase the primer concentration, and shorten the extension time.

Regarding the primers, some tools estimated the Tm as high as 73 °C, with values ranging from 63°C to 73°C depending on the tool used. When I lower the annealing temperature below 60 °C, I get many (likely nonspecific) bands. However, when I increase it above 65 °C, I mostly see only the unexpanded allele or no amplification at all.

(I believe the rather high Tm of the primers is intended to minimize the formation of secondary structures in TNR expansions)

Lemme know of any improvement in results!

By the way, while some biotools cannhelpnyou predict Tm of primers, it is best to based your Tm modifications based on the actual Tm from the synthesizing company. That is, your final Tm modifications should be based on what is written on the primer tube from your order.

Good luck mate!

I tried what you suggested, but unfortunately, none of the PCR modifications changed the outcome, only the bands became thicker.

Also, thanks for the Tm clarification. I was quite confused by the wide range of temperatures given by different tools.

I appreciate your help!