2 replies to this topic

[Nephrite]

Hello.

I posted this question elsewhere but nobody answered. It is stupid basic question but I need to know.

I do two-step RT-PCR, i.e. first I do the RT reaction, and then - basic PCR.

I use recombinant RevertAid M-MuLV Reverse transcriptase from Fermentas - in datasheet they say that the enzyme has RNA- and DNA-dependent polymerase activity and an RNase H activity specific to RNA in RNA-DNA hybrids. As I imagine it is something like 3 in 1 - RNase activity cleaves the RNA, and DNA-dependent polymerase builds the second strand.

Thus, I think that in the end of reaction I have double stranded cDNA.

It that wrong?

Thank you.

[phage434]

This is mostly wrong.  In step one of an RT-PCR reaction, you have only a single primer, and don't cycle.  If this is a gene-specific primer, then you will create ss-DNA starting with that primer.  If you have a random primer, you will also prime the RNA and create ss-DNA.  These products are a duplex of RNA and DNA, and won't dissociate, since you aren't cycling to denature them.  It might be possible for some dsDNA to be made, but the amounts and location of the dsDNA would be small and unpredictable.

[Nephrite]

phage434, on 19 March 2011 - 02:10 PM, said:

This is mostly wrong.  In step one of an RT-PCR reaction, you have only a single primer, and don't cycle.  If this is a gene-specific primer, then you will create ss-DNA starting with that primer.  If you have a random primer, you will also prime the RNA and create ss-DNA.  These products are a duplex of RNA and DNA, and won't dissociate, since you aren't cycling to denature them.  It might be possible for some dsDNA to be made, but the amounts and location of the dsDNA would be small and unpredictable.

Thank you very much.
Now I understand it.