Primers design: how to check for mispriming? - (Jul/06/2005 )

Hi all,
After designing a set of primers to amplify a gene of interest, what is the best way to check whether or not primers may amplify something else besides the gene of interest. I usually work with total DNA (PCR) or total RNA (RT-PCR) extracted from viral infected cell cultures (human or simian derived) and I´m ONLY interested in amplifying the viral genes.

I already did a blast search and the most significant hits were for the viral gene I want to PCR. However, I did get a few hits for human genome sequences, even though the hits are not as strong as the ones for the viral genes. Any idea of how can I tell whether the human genome hit is strong enough for me to give up on that primer (i.e., what should be the cut off for deciding for using or not using the primers?).
Also, should I worry about over-represented seqeunces in transcripts? Is that importante and if yes, is there a way to check that?

Any insight will be appreciated,
-CAS

Hi CAS,

UCSC in-silico PCR is the tool I use for checking mispairing http://genome.ucsc.edu/cgi-bin/hgPcr?db=hg17

"In-Silico PCR searches a sequence database with a pair of PCR primers."

Hi pcrman,
Thanks for the site. It was very helpful. I have one further question though.
According to the UCSC´s site you indicated me, no pCR products would be formed by using my primers. I found however, that each primer individually could anneal to a number of sites within the human genome (by using http://Bisearch.enzim.hu). Even though these sites are way apart and they should not yield a PCR product, should I consider another set of primers or should I just go ahead and try it out with the ones I have (the region I´ve been working on is very diverse and it´s been very hard to find good oligos as those I designed so far).
Thanks,
-CAS