It seems like this task must have been addressed by this point. For example, as described in this publication:

A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array

Using the BLAST program, we matched probes with documented and postulated human transcripts. This resulted in the redefinition of approximately 37% of the probes on the U133 plus 2.0 array. This updated identification specifically points out where the identification is complicated by cross-hybridization from splice variants or closely related genes.

If nothing else, you can probably find some guidance for your own approach in there...

I would something other than BLAST for this purpose. Good options are BLAT and Bowtie.

Basically, what you need to do is:

1. Obtain Refseq human database in a suitable format; e.g. FASTA from the FTP site
2. Obtain file of 25-mers
3. Run either BLAT (just requires query/database sequences to be in FASTA format) or Bowtie (requires more pre-processing of sequences; read the documentation)
4. Parse the output for alignments with length >= 17 bp; again, BLAT is a good choice due to multiple options for output format which are easy to parse

Hi malachig,

I have already gone through that article "A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array" but its confusing So why I was asking help to know how to identify the cross-hybridization of 25mer probes.

Hi neilfws,

         I will surely performed this two softwares. but blat is much similar with BLAST. I am new Bowtie. I will try to solve  hybridization problem.

Thanks a lot to You (malachig and neilfws) for your kind reply and help.

with regards, Nelson