If you are expecting products of 204 or 259 bp, then it is quite likely that  the product melting temperatures of the two different fragments will be detectably different by melt curve analysis.  One interpretation of your two figures is that the top one is a heterozygous sample (C/T) with both 204 and 259 bp products (81.5 and 85.5 melt temps) while the lower figure is a sample with only the larger "T" allele (85.5 melt only).  You should be able to easily verify that by running an agarose gel.  Did you see any samples that looked like they had a single melt peak at ~81.5?  These would be candidates for "C" allele homozygotes. 

 dear david

thanks for quick reply , i actually ran gel electrophoresis and according to fragment size the samples of C allele homozygosity appeared with tm at 85 C whereas in the  the T allele fragment showed tm at 82C

precisely, my question was about the the shape of the melting curves if they are accurate  or not  ?!

thanks 

Two thoughts:

First, what is the basis of the size difference between the products of the C vs. T allele?  I'm guessing that it must be the lengths of the primers which provide the allele specificity.  The exact sequences of these primers may be more important than the lengths of the final products in determining the melting temps.

With respect to the appearance of the melting curves, it looks like the curve you've shown is the "derivative" curve which shows   -1*slope of the intensity curve (it looks like the software has mislabeled it--it is not the "fluorescence intensity" ).  What does the raw intensity* ("waterfall") curve look like?  It also seems that the resolution of your melt curve is not high.  Can you change the amount that the temperature changes at each step of the melting or can you increase the total time that the melting stage lasts?  This will give you more intensity measures at smaller temperature intervals and give you a smoother-looking curve.  Once you are satisfied that the assay is working, you can change the parameters back if you just want the runs to go faster.

*For the two forms of the melt curve, they are both included in panel 5 at:

http://eng.bioneer.com/products/instrument/Exicycler96-technical.aspx

Dear David

excuse me i couldn't catch it, what do you mean by " Can you change the amount that the temperature changes at each step of the melting or can you increase the total time that the melting stage lasts?"

did you mean change the protocol i'm working with?! increasing the amount of my master mix? or changing the dissociation step number of cycles?

it's very kind of you if you explain 

thanks

Right, I'm referring only to the dissociation ("melt curve') phase of the protocol that occurs after the amplification phase is complete.  During this later part of the protocol, the instrument slowly raises the temperature and measures the fluorescence at each step. If the temperature is raised quickly or in large increments, the fluorescence will also change (decrease) in large increments and the resulting dissociation/melt curve will not look smooth.  I'm not familiar with your software, but it might allow you to raise the temperature in smaller increments or otherwise decrease the rate at which the temperature rises.  The idea is to collect fluorescence measures at more points as the temperature rises and produce a smoother-looking curve--possibly providing more distinct peaks and better detection of  the two peaks that you expect in heterozygotes.

O.K. i'll try to do so and thanks for your advises