BLAST of ITS4 ITS5 sequence - (Dec/26/2011 )

Hi, I have a question about BLASTing. Sequencing was done for a fungal strain. The PCR primers were ITS4 (reverse) and ITS5 (forward). I used the ITS4 sequence to BLAST and it gave a different results than the ITS5 BLAST; both results are quite close - results showed strain from the same genus; but the top result was different. Did I use BLAST wrongly? Or to rephrase the question, since I have one reverse sequence and one forward sequence, which should I use to BLAST or will need to do anything to the sequence before BLASTing? Thanks in advance!

I believe those primers are based on consensus and may not be an absolute perfect fit in all situations. Try starting with a search of one of the primers, then do a quick visual inspection for the reverse.

You should do Primer-BLAST (http://www.ncbi.nlm....s/primer-blast/). It will BLAST both of your forward and reverse primers simultaneously and would generate all possible results.