Multiple unknown amplification products - (Mar/17/2006 )
Hello! I´m doing Real-Time RT-PCR for viral detection ( not quantification). I designed my own primers ( with Primer 3) and did an essay to define which their ideal concentration is.
After defining this issue, I started doing essays with known positive controls but when I run my gel, I find lots of amplification bands. I thought they were contamination but I´m being really careful so now I doubt between contamination and unspecific primer binding ( although I did an alignment on clustal W and primers bind specifically in their correct position). My band size is 187 bp.
Here I send you two of my essays and their corresponding gel.
The first essay: The blue line is my best positive control and look what happens to it. Really strange!
At the gel, my negative control seems to be contaminated because it has lots of bands but I was really careful.
The second essay: the purple line is my negative, the other three are positive controls. On the gel, you can see the 3 positive controls with lots of bands and my negative control.
I don´t know what to think.
Should I design new primers?
looks like a non-specific binding problem, try optimising the Mg2+ concentration and the annealing temperature, if you still get multiple bands then you may have desigend primers that bind more than once to your target... use a program that will search a defined sequence for your primer sequences to check this.
Not sure which virus you're looking for, but presumably your 'known positive controls' came from either clinical specimens or virus grown in cell culture. In either case, your extracts are likely to contain *lots* of human genomic DNA/RNA, so the problem is almost certainly inadequate primer specificity. Do a quick BLAST search with your primers using the 'Search for short, nearly exact matches' tool at http://www.ncbi.nih.gov/BLAST/. If you get high-scoring hits on human sequences (even if you also get high-scoring hits for your target viral sequence) then you need to select more specific primers. It's always worth checking for non-target homology after designing a new set of primers, after doing the usual checks for dimerisation, hairpins etc. For maximum specificity and sensitivity, I would also recommend that you consider using nested PCR. Using nested RT-PCR, we routinely see 2-3 logs more sensitivity than single round RT-PCR for viral detection in extracts from clinical samples.
also, try UV-irradiating your (open) PCR tubes for 15 minutes - often helps with contamination problems.
hope that helps,