RT-PCR cDNA synthesis - (May/10/2011 )
Hi, I've just started to work on RT-PCR. I don't know if it is an absurd question or not but I wondered that if cDNA, that we have after reverse transcription, is single stranded why do we need the denaturation step in the beginng of the PCR to seperate the double strand? Thank you so much for your help
The RT reaction only gives you the first cDNA strand, not dsDNA. In theory, in the first step of PCR denaturing is not required.
But the cDNA is created on the RNA template. So unless there is RNAse step, it creates double-strand RNA-DNA hybrid.
we have a RNase step, so I really do not know why we need a denaturation step
One more question that I wondered... Is the reason that we synthesized cDNA using mRNA, instead of synthesizing DNA directly, is to get the coding sequence, exons, of the DNA? Will there be any changes, advantages or disadvantages if we use DNA directly for PCR?
If you're talking about initial denaturation, before cycling steps, that can also be used for aktivating hot-start polymerase. If it should be longer than 5 minutes, that's probably the case.
As for your other question, it depends on what exactly do you want and for what reason.
It's clear for me now. Thank you so much for kind help.