4 replies to this topic

[Xenotoxic]

Hello to all molecular biologists.

I have a question regarding about PCR.

We all know and seem to be common to think of using cDNA as template for PCR. One day, my junior had asked me a very fundamental question and I could not accurately answer to this question that could solve problem

His questions is why DNA must be used for PCR and not RNA???

The question arised from following - I did an experiment which RNA was isolated from liver of experimental mice by Trizol and made cDNA from that RNA. Problem is that I did PCR with RNA and cDNA simultaneously and did gel electrophroesis, I obtained very interesting results which corresponding bands of housekeeping gene were detected when just RNA was used as template but not the target gene. We thought of possibly of genomic DNA contamination but when I designed primer, I intercalated intron between exon so the band product that corresponds to genomic DNA should be very large but the band appeared exactly same as its target size (between 150 ~ 200 bp).

I told junior that you cannot use RNA as template cause 1. RNA will degrade during 95 C denaturation step or 2. DNA polymerase used for PCR is very specific for DNA.

Do you guys have any other answers?

Thank you.

[bob1]

It is due to the polymerases used - RNA pols are not good at amplifying and are usually very temperature sensitive.  Reverse trasncriptases are similar, whereas Taq and other similar polymerases are capable of amplifying DNA over and over, and are not temperature sensitive.  This allows you to have the primers as oligos which are denatured by heat, from the template and then specifically re-natured.

ssRNA is also very unstable and sensitive to RNAses (very very stable, capable of reforming after denaturation, and very common).  ss is also the native form of RNA, so conversion to cDNA, which is much more stable, and less sensitive to degradation makes sense.

[leelee]

What bob1 said.
PCR uses enzymes that are DNA-dependent DNA polymerases (use a DNA template to catalyse the replication of a DNA product).

So the band you got in your RT negative PCR is most likely from contamination of one of your PCR components, or possibly contamination due to error when you were setting up your reactions.

[Xenotoxic]

Thanks for the reply.

Leelee, I thought of contamination however I did not get band of target gene when I did RT negative PCR but only housekeeping gene such as GAPDH or 18S rRNA.

[bob1]

that implies that the contamination is GAPDH or 18s rRNA product...