Hi,
Say I want to design primers for Q-PCR for a gene with exons A, B, and C. My cells are grown in conditions in which skipping of exon B should be increased, so I want to do Q-PCR to confirm. I thought of designing a primer set like this:
Forward primer: spanning the junction of exons A and C.
Reverse primer: somewhere downstream of exon C.
Is there a tool like Primer Blast or Primer3 that automatically allows me to "skip" an exon when designing primers? Or I will have to make the sequence of the alternative RNA product "by hand"?
1 answer
Primer3 has an option "Excluded Regions" to skip the regions you want.
You can specify the length and positions of the Excluded regions. Read following instruction!
E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<cccc>TCAT.. forbids primers in the central CCCC.
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Can't you construct a dummy cDNA and feed this into PrimerBLAST? If exonB is skipped then make a sequence that does not contain it. This still would have all the benefits from PrimerBLAST which you need for a reliable qPCR primer.