Question: Tool to design Q-PCR primers with exon skipping?
0
gravatar for gabriel.monteiro233
13 months ago by
gabriel.monteiro2330 wrote:

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"?

primer exon splicing • 389 views
ADD COMMENTlink written 13 months ago by gabriel.monteiro2330

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.

ADD REPLYlink written 13 months ago by ATpoint46k
1
gravatar for mbk0asis
13 months ago by
mbk0asis610
Korea, Republic Of
mbk0asis610 wrote:

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.

ADD COMMENTlink modified 13 months ago • written 13 months ago by mbk0asis610
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