Using BLAST to check primers - (Mar/06/2012 )
So i am designing a Q-PCR reaction using Taqman style primers/probes (human probe library). I have designed the primers and probes using the Roche assay design site, and i now have a list of the relevant primers/probes for my genes. However, i am designing expeiment for Canines and not humans (Roche have blasted the primers against human genome), so i need to BLAST the primers against the canine geneome to check for specificity to gene of interest only.
Just one snag though - i havent used BLAST before, so i go to blast, and i paste the sequences of the 2 primers into the box and hit run...
The top 3 results are the gene i want, and give max score 40.1, total score 80.3, query coverage 100%, E-value 0.003 and max ident 100% . im presuming that the coverage being 100% is good, but whats the max ident?, as a bit further down the list there are some hits that also have 100% max ident, but the E-value is 10, and the query coverage is 35% with total score of 28.2 Can anyone please help explain this to me! (i should have attached the table of hits)
Wrong way to do it.
You can't just BLAST the primers but probe also. You need 100% coverage and 100% identity on both primers and the probe. I would say that is pretty difficult to obtain, the sequences are different. Also the algorithm checks for specificity in human genome, that is useless for you, the rank of the primers would be skewed for what you need.
You need to select Other organisms on the Roche site and paste there reference sequence of canine MMP-2. It would then design primers for that sequence. You need to choose a probe number that is present in your human library, that's important. You can't design automatically intron spanning assays this way, but you can look up the primers if they span an intron. I would find that way easier than to BLAST human primers.