amplicon which species does it belong to? - (Sep/24/2006 )
I have designed primers using the human sequence for a particular gene of interest. but i need to know if this gene is expressed in bovine corneal epithelial cells. I ran real time pcr and got good results and ran it on gel to confirm absence of primer dimers and also the size of the amplicon is what i was expecting around 13bp. now i have a couple of questions.....
one is why should the size of the amplicon be smaller for RT-PCR as oppossed to semi-quantitative???
two how do i confirm that the amplicon that i have with the bovine cDNA is the same as the one for human. i want to knoe if there are methods other than sequencing to do thisconfirmation in bovine cells. i ran no RT sample, NTC and the homology between human , mouse and rat sequences are about 9o%. i chose this region to design my primer.
I can't think of any method easier than sequencing? you can sequence a PCR product directly after some clean-up, and it's fairly inexpensive, especially since you're only going to do it once
I'm guessing you meant 130 bp, not 13??
what do you mean, RT vs semi-q size? is there gDNA in the semi-q experiment, and are your primers intron-spanning?
may be using antibodies on your WB can give u some results...
i don't know if antibodies would recognize bovine and human amplicons differently...just wondering
DNA is DNA...I don't know of an antibody that would show you a difference in species after PCR
The easiest answer is sequence the PCR product to do a BLAST search of the human genome sequence or PUBMED. I am actually very surprised that your primers from human would work for bovine since most homologous genes have changes at the DNA level and more conservation at the protein level. Perhaps I am missing something here but you may want to re-evaluate what you are doing. Most people pick up conserved genes through degenerate PCR using primers that target conserved regions of genes.
I don't have much experience with the design of this assay, but to identify species specific PCR products, the restriction analysis assay would be recommended. If the fragment is cut, this becomes visualized on an agarose gel. Perhaps you are lucky and there are specific SNPs in the fragment that can be cut with restriction enzymes. If you want to adopt this technique, you must perhaps redesign primers to obtain mismatch/mutational situations and insert a restriction site.
Thanks for all those inputs. I have decided to go ahead with sequencing and check out if the amplicon is the gene of interest. I am sorry but i am not familiar with the SNP's and the degenerate primers. If someone can give me a head up on that it would be good. I was not aware that the conservation is at the protein level then why do we even look for the homology of the sequences??? and call them conserved regions. Thankfully most of the primers that i have designed so far are using bovine sequences. Also how reliable is it to use predicted sequences as i know these have not been tested for in the species but is computer generated based on other species.