genomic DNA PCR - is it the same as a cDNA PCR (Aug/28/2008 )
Hi
We wish to detect a DNA virus in samples in paraffin sections. To do this we have isolated genomic DNA are planned to do a PCR. I have a PCR protocol that works perfectly for amplifying cDNA, but when I have used it for amplifying similar housekeeping genes from the genomic PCR I don't get an bands.
Can I just use the same PCR protocol that I used for cDNA for amplification for genomic DNA amplification? If not what parameters are the most important to look at?
Thanks in advance for any advice
Brett
cDNA is DNA reverse transcribed from RNA. As such, it has all the introns removed (if you're dealing with a sample from a eukaryote). The introns, however, are still present in the genomic DNA, thus if either of your primers crosses a cDNA exon-exon boundry, it will not find its target on a genomic DNA template, and the PCR will fail.
Are you working with eukaryotic or prokaryotic DNA?
Hi,
Thanks for the reply. I am using Eukaryotic DNA. We are looking at tumors from humans FFPE samples to see if a virus infection is correlated to prognosis.
May understanding would be that if my primers span an intron then I would get a band much larger than originally expected (including the whole exon-intron-exon amplification), rather than no band at all.
Is this assumption wrong? Is so why is that, I am rather new to working with DNA/RNA.
Is there a good website that provides information on where intron sites are located in each gene?
To give you an update (if your interested ), I tried dropping the annealling temperature right down to 45 C, which the paper I am following used but I thought was too low, and I seemed to get some very faint bands at the expected size. I also however got a band in my -RT lane.
So are these band promising, and if so, which parameters can be adjusted to increase expression? Or is it likely to just be a contamination problem and I need to think again?
Ok, thats it.
Sorry if its too much for one post, but I have so many questions and no one to ask!
Thanks,
Brett
Hi
Do you know the size of the amplicon in your genomic DNA PCR?
For the amplification of DNA extracted from FFPE samples, amplicon size should be around 300bp or smaller.
Hi,
Yes the housekeeping gene (Ubiquitin C) I am using to optimize the PCR should be 132 bp, and eventually when I use the virus primers the amplicon will be 60 bp, which I know is very small so I think I need to have the PCR working well to be able to confirm such a small size.
Thanks,
Brett
This is my point -- if either of your primers spans an exon-intron boundry (in other words, either one anneals to part of an exon and also to part of the adjacent intron), then they were designed for use with a eukaryotic DNA template, and would fail using a cDNA template (because in cDNA, the introns are gone).
On the other hand, if either of your primers crosses an exon-exon boundry, they were designed for use with a cDNA template, and will fail using a genomic DNA template, because in genomic DNA, exon-exon boundries are still separated by intervening introns, and therfore the contiguous sequence to which your primer is designed does not exist.
Ahhhhhh ofcourse, I see now. Thanks for helping with that.
Are the any good websites you know of that define the intron exon sites of genes?
NCBI has this information available, though I find it a bit hard to find. For example, the entry for Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is here. There is a graphical representation of the intron-exon pattern there, in the "Genomic regions, transcripts, and products" panel. If you click on the "CCDS8549.1" link (it's to the right of the schematic map, and the tooltip balloon says "Link to Consensus CDS Report"), it takes you here, which has the cDNA nucleotide sequence in the "CCDS Sequence Data" panel, shaded as to alternate exons.
If you go back to the original page (here, hit the back button), and scroll down to the "NCBI Reference Sequences (RefSeq)" panel, then click on the GenBank link in the Genomic section, you'll get this, the sequence of the genomic DNA in GenBank format. If you scroll down to where it says
CDS join(343..371,2004..2103,2194..2300,2430..2520,2611..2726,
2819..2900,3094..3506,3611..3680)
The "join" instructions show you how to go from the genomic DNA (with introns) to the cDNA (exons only) by defining the exons. For example, the first exon is 343..371, and the second is 2004..2103, thus the intervening intron is 372..2003.
I'm a bacterial guy, and I'm sure there must be an easier way to get this information, but (coincidently) I had to design some primers for the human GAPDH gene, and this is the way I found this info.
Hope this helps!
Hi,
Thanks very much Homebrew, that has been extremely useful, and very well explained.
I appreciate the time you put into that reply.
Thanks,
Brett
Working with DNA and cDNA is very different. Remember that the cDNA is a copy of the genes that were active at the time of the sample collection. PCR of the housekeeping genes is easier because they are more active so more more cDNA you got if compare with genomic or viral DNA. For the reaction you will need a higher concentration of template. And a primer set with a high annealing (60C at least) will be better so you avoid that they anneal to anything. If you'r amplicon is so small (60bp) use metaphor agarose (2-3%) for a better resolution or acrylamide gel. Many persons use the one step RT,but I prefer the 2 step so you can have a cDNA library useful for another assays.