Dear Mohammad, Cathal is right, You'll need to check the primers for their suitability to discriminate the species you want to detect from those which will (most likely) interfere. For the PCR reaction, I would not rely on in silico calculations only. They give you a good idea about the Tm range, but in reality, you should test the rigidness of the system by variation of Tm, i f possible with a gradient cycler. First, do this experiment just for positive detection (to minimize false negative results), then also together with interfering material, to exclude false positives. Of course, then use a Tm as high a possible in the downstream experiments. .
Another option of increasing specificity is to use a qPCR system with a sequence specific third oligo. Third, sequencing of the PCR product (i.e. least some representative samples from the above experiments) is a must. I'd even do that when there are published primer sequences that are said to work. But you don't know about your specific environment, of course. Remember that, when you BLAST your primer sequences against a database, you'll only get hits which are already listed in the database. As you'll probably know the species you want to discriminate, it might be a good starting point to get all their 18S sequences and make an alignment (eg. with MULTALIN, see http://www-archbac.u-psud.fr/genomics/multalin.html). HTH, good luck and have fun! Wo _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
