(by novalidaddress from nurfuerspam.de)
Sun Jul 10 05:26:20 EST 2011
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
HTH, good luck and have fun!
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