help with negative strand specific primer design needed - Primers for Sindbis virus (-) strand (Apr/05/2011 )
I have a real basic question, for which I am ashamed of, but here it is.
I'm trying to design a primer set for qPCR to specifically the negative strand of the Sindbis (SINV) RNA genome. (The virus comes as a single stranded + oriented genome, but it needs a negative strand intermediate to replicate. I need to detect that intermediate, and ONLY that intermediate.)
It seems quite straightforward: negative complement the SINV genome, and design primers that are specific for that.
Indeed, with primer blast, it does not find anything complementary on the + genome, or anything for that matter, only if I use the reverse complemented one.
Yet after discussing it with someone I got confused (we probably confused each other).
Would these primers actually be specific for the (-) genome? (After all, in the second step of amplification there might be a double stranded DNA, with both the + and - strands, but we would not get there without the first step, right?)
but PCR doesn't work with just one primer. you would also need the second primer that binds to the +strand.
Or is the +strand only RNA and the -strand DNA?
tea-test on Tue Apr 5 19:36:32 2011 said:
but PCR doesn't work with just one primer. you would also need the second primer that binds to the +strand.
Or is the +strand only RNA and the -strand DNA?
I know that I do have a Fw and a Rev primer pair -my question is, is it, in theory, possible to design orientation specific primers for the (-) stranded version of the RNA? The cell would have both positive and negative strands -making an RT reaction that would mean... what exactly? Do I have (-) and (+) oriented cDNA molecules, to which my (-) strand specific primer is specific, or at this point it is impossible to tell the difference?
I can do by Northern, I think - after all, I just need a probe that hybridizes to the (-) strand. But I'd love to do it on PCR as well. As a plan B.
Thank you.
(Here's the life cycle: http://www.freepatentsonline.com/6592874-0-large.jpg
I would like to amplify the negative strand only.)
If I understand it well and the virus goes from (+)RNA -> (-)RNA -> new (+)RNA -> packing and you need to detect the (-)RNA, then what you need is a specific RT primer (single for a single amplicon) and you design it from (+)RNA sequence in programs like primer3 ("left primer" or "forward primer" will be complementary to (-)RNA strand, that's what you need ). Later you can do normal PCR with pair of primers on the cDNA you get this way, which only contains reverse complement of your (-)strand (the rest is RNA and isn't amplified).
IMHO.
Trof on Wed Apr 6 12:47:50 2011 said:
If I understand it well and the virus goes from (+)RNA -> (-)RNA -> new (+)RNA -> packing and you need to detect the (-)RNA, then what you need is a specific RT primer (single for a single amplicon) and you design it from (+)RNA sequence in programs like primer3 ("left primer" or "forward primer" will be complementary to (-)RNA strand, that's what you need ). Later you can do normal PCR with pair of primers on the cDNA you get this way, which only contains reverse complement of your (-)strand (the rest is RNA and isn't amplified).
IMHO.
Thank you very much. I think this is what I'll need to do. (Haven't thought of the possibility of using specific primers for RT...) The only thing left is to think about how I can compare -RNA levels with +RNA levels if the cDNA is not the same... Oh, man. This is going to be painful