Hi! Based on your description, it seems that the second lower peak appearing after your main species-specific peak in the melt curve analysis could be related to non-specific amplification products or primer-dimers with higher GC content or more stable secondary structures than your target amplicon.

Although it is more common to observe non-specific peaks or primer-dimers melting before the target amplicon (because they are typically shorter and less stable), it is also possible for certain non-specific products or alternative secondary structures to exhibit higher melting temperatures. This can occur if the non-specific product has a higher GC content, is of similar or longer length than the target, or forms stable hairpins or homodimers, all of which increase thermal stability and consequently shift the melting peak to a higher temperature.

Another possibility is the formation of heteroduplexes during the late PCR cycles, especially if your reactions contain multiple similar sequences, such as in environmental DNA (eDNA) samples. These heteroduplexes can melt at unusual temperatures, sometimes higher than the target amplicon, depending on their composition and stability.

Additionally, if you are using SYBR Green or other non-specific intercalating dyes, these will detect all double-stranded DNA, regardless of specificity, leading to the appearance of additional peaks in the melt curve corresponding to non-target products.

To better understand the nature of this secondary peak, I would recommend:

Also, running no-template controls (NTCs) can help verify whether the extra peak is related to primer-dimer formation or reagent contamination.

In summary, although less common, secondary peaks appearing after the specific melt peak can occur due to non-specific products with higher thermal stability, secondary structures, or heteroduplexes. Careful optimization and product verification can help resolve this.

Hope this helps! Let us know how it turns out.

Dear Katerina Tasiouli,

This may be due to

1. non specific amplification due to primer binding in non target region

2. Primer dimer

So must be improving primer design using tools such as Primer-BLAST.

The melt curve is the LEAST informative part of a qPCR experiment. You don't need any of that information to publish.

You need your primer efficiency (95-105% is acceptable) and Ct values & controls to all be successful.

So, what does your NTC look like on the melt curve? I'm guessing it's basically a flatline.

I've seen this before a few times when using melt curve-based genotyping. The secondary tiny peak is due to either your product being a bit too long for efficient qPCR and/or it has variable GC content in different regions. AT has 2 hydrogen bonds vs 3 for GC pairs. Regions with higher GC will melt at a higher temp & this can be by region.

Hope this helps

Try increasing the annealing temp 1-2 degrees and/or adding something like 2% DMSO (final concentration)

A second peak after your main melt could be a heteroduplex, a GC-rich off-target product, or a melt curve artifact from fast rampingtry slowing to 0.1 °C/sec. Also check for dye or salt effects using a no-template control, and confirm with a gel or sequencing.