I just want to know whether the real time PCR will show different denaturation curves for single sample contaminated with different genomic DNA from different organismal sources? Suppose I have mixed 4 different genomic DNA samples in one vial and used it as a template for real time PCR with same primer. Will I get a single dissociation curve or 4 different dissociation curves for 4 kinds of genomic DNA?
If it gives different dissociation curve for this sample then where will I find that in the RT PCR software?
If I get only one dissociation curve and I want to know dissociation curve for all the templates in my sample then which software and technique I should use?
You speak about qPCR. Using 4 different sequences amplified by the same primers, you might have a melting curve with 2 or more peaks which correspond to your different template. The temperature of dissociation is dependent of the sequence but you can not be sure that you will not have the same temperature for two templates. The melting curve is performed in the qPCR after all cycles of amplification. It is an additional step.
You speak about qPCR. Using 4 different sequences amplified by the same primers, you might have a melting curve with 2 or more peaks which correspond to your different template. The temperature of dissociation is dependent of the sequence but you can not be sure that you will not have the same temperature for two templates. The melting curve is performed in the qPCR after all cycles of amplification. It is an additional step.
Thanks stephane for your suggestion. But in this case I have mixed 4 template in one pcr tube and used for qPCR then in this case, will it shows different dissociation curve for all templates in single pcr vial provided that all have different denaturation tempreatures.
Agreed with Stephane,if you have mixed all the 4 template in one PCR tube and analysed the melting curve you may get the single peak if there is no variation in the DNA sequence of Your gene of interest in all the genomic DNA samples.
But you may get the more than 1 melting peak if there is sequence variation in the DNA sequence of your gene of interest present in the genomic DNA.
Hi Sachin, you should get only one peak for your melting curve if your primers are specific for only one gene in one organism. you should check your primers across all speices using primer blast, will give you idea about possible products. if the gene have a mutation or varient or something it should give 2 or more peaks in your melting curve as Ravi and Stephane discribed above. but if your primers are specific and there is no copy varient of the template you should only get one peak
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