Please check about effect of humic acid during DNA extraction from environmental samples and also extraction kit. Read articles such as Article Effects of humic substances on fluorometric DNA quantificati...

https://dx.doi.org/10.1002%2Fmbo3.453

The melt-curve is the least informative piece of information from qPCR. Run out your samples on a gel and see if the products are the expected sizes. My guess is that you are not getting amplification from your unknowns and the melt curve is showing the left-over primers.

What at the Ct values for your unknowns?

Hi Termeh Hesam Mahmoudinejad

melt curves depend on many things but the first is the sequence. While trying to amplify different species, differences in their sequence (in the amplicon position but also the primers), can led to loss or decrease of amplification and deviated melt curves.

fred

Do you get poper amplification curves for the unknown?

And as Katie said: check the gel! If the do show the correct product, the weird melting curves might be caused by residing substances from sample purification, which may be different between the standard prep and the sample preps.

Katie A Burnette Thank you very much for taking the time and answering my question.

The Ct values for unknown samples are between 20-30. Actually, I ran the gel with the qPCR products (picture is attached). The lanes 8-13 are the unknowns.

Thanks.

Jochen Wilhelm Thank you so much for your reply.

The Ct values are between 20-30 in 40 cycles qpcr.

Amplification curves look ok*. The gel seems to show that you do get the correct product, without any recognizeable unspecific products -- if the length of the product is correct. The resolution of the gel is a bit poor. You could try a 10-15% PAGE to get sharper bands with a higher resolution of small PCR products (I suggest to try that!).

So far, my guess is that your unknowns contain some residual chemicals/components that interfere with melting. Consider a different purification protocol or just second round of purification to see if this resolves the problem.

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* Just an aside - it's not relevant for your current problem: the threshold shown is selected quite too high, as it cuts the curves already in the transition (between exponential and plateau). It should be not above 100-200 for there data, I would guess. Look at the curves using a logarithmic scale for the vertical axis; the threshold should cut the straight segments there in their lower 3rd.

Jochen Wilhelm

I quantified IntI1 gene in the same samples, which is the marker for antibiotic resistances.

I'm wondering if the 16s result is like this, does it mean the IntI1 quantification is invalid?

Thanks for your kind attention and help.

I don't think so, but your strange melting curves do cast a little doubt.

Termeh Hesam Mahmoudinejad Thanks for uploading the picture of your gel. You'll need to run out the samples longer to get better separation of your products. I can't quite tell the expected product size since the size ladder is still a bit bunched up. Did you include a no template control?

Hi Termeh. The different melting temperature for your samples can be explained by sequence variation in the PCR products. Have your tried melt curve simulation of your target sequences by uMelt BATCH https://www.dna.utah.edu/umelt/umelt.html?

If your target sequence is well conserved, then different salt concentration in the PCR reaction mixture can explain the different melting temperatures. If so, you might consider purify your DNA further.

On the agarose picture, the sample in lane 9 shows two bands, perhaps also the sample in lane 10. Are these two the same samples that give the most broad curves in the melting? You have two amplicons made with the same primers – i.e. same flanking sequences - lots of homology between them - heteroduplexformation - broad melting temperature. Double bands, I think you have an issue with primer(s) not matching well to the template.

Katie A Burnette the NTC is in lane 7 (unfortunately it seems the target sequence is inside).

Einar S Berg Thank you very much for answering my question.

yes, the primers can be the problem I guess.

I also further diluted the samples, but the result is almost the same.

I'm trying to understand what influences can hypervariable region in 16s have on amplification process.

Terneh, if I were you I would do an experiment where the annealing of the primers are done at different temperatures, read in a temperature gradient for just one of the controls, one problematic sample and importantly, a water control. Then the melt analysis after the PCR become valuable showing the temperature where primer artefacts occur, including the temperature where mismatch primer hybridization occur and finally, and hopefully, the optimum annealing temperature for PCR of unknowns and/or diluted samples.

Please check about effect of humic acid during DNA extraction from environmental samples and also extraction kit. Read articles such as Article Effects of humic substances on fluorometric DNA quantificati...

https://dx.doi.org/10.1002%2Fmbo3.453

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