qPCR on gDNA and digested gDNA

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kakera
kakera's picture
qPCR on gDNA and digested gDNA

Hi, I am trying to set up a qPCR using genomic DNA and digested genomic DNA. Essentially, I have two identical conditions (concentration, buffer, etc.), except one has enzyme. From the concentration of gDNA, I can figure out how many copies of my gene should be there and how much I am putting into my PCR reaction for the intact genomic. However, I was wondering if the same applied for the digested genomic DNA? Since they both started with the same amount of genomic DNA, there should be the same number of gene copies in both conditions (and the amount of sample I add to my PCR is based on volume, rather than re-measuring the concentration after digesting). Would the PCR conditions be the same for both? Or does the digestion mess with the kinetics of the qPCR, such that I couldn't compare the two samples directly?

Thank you.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi kakera,

If the only difference between the two reactions is that in one you have intact genomic DNA and in the other you have the same amount of genomic DNA, but digested using a restriction enzyme, then very likely you will get a higher signal for the digested genomic DNA than for the undigested one. The reason for this is that when you digest genomic DNA you are releasing most of the secondary structure of genomic DNA, which results in the PCR reaction being more efficient (it is easier for the Taq to reach the DNA template). This of course assumes that the genomic DNA will not be degraded during the restriction digest. 

In my opinion  you cannot really compare these two kinds of genomic DNA directly since the efficiency of the PCR reaction is affected significantly by the status of the genomic DNA. 

kakera
kakera's picture
Those were along the lines I

Those were along the lines I was thinking as well. Thank you. I was just wondering because someone once mentioned to me that if you begin with an initial denaturing of 95 for 10 min, it should break down the secondary structure. I didn't know if that was the case.

Ivan Delgado
Ivan Delgado's picture
 

 
The initial 95 for 10 minutes does help in denaturing the secondary structure of genomic DNA, but ultimately you are comparing very large complementary molecules to smaller complementary molecules, so the smaller molecules (digested genomic DNA) will always melt faster than the larger ones, thus makes PCR more efficient.