Hi
I am currently working on a gene that has got many pseudogenes and is located in an MHC region. when I blast the primers I've designed (for qPCR), in the NCBI primer blast I get so many off-target results but with a 3' end base mismatch in about 90% of the results.
So now my question is that do you think placing a dspacer right before the 3' end base will help me to cut the unspecific results I get? since the 3' end makes a mismatch in almost all the off-target results.
example: CTGACGTGCAAC(dspacer)G
Hi Zahra Rezaee
the best thing to do is to design the primers across the mismatch. the longer the primers and the mismatches will be, the better and reliable results you'll have.
increasing specificity will also need use of higher Tm and use of DMSO (not more than 5% of total volume) to unwind the template and reduce charges.
Designing primers for qRT-PCR will also allow you to designing across junctions to avoid junk DNA amplification and increase the specificity of your target.
all the best
fred
Frederic Lepretre
thank you for your answer
I'm targeting a promoter in the genomic DNA with rich CG content and have placed the 3' end of the primer on a mismatch in off-target result, is one mismatch enough or should i increase it to two mismatches by adding a dspacer?
Hi
not sure that adding a dspace at 5'end of the primer will help but for qRT-PCR you won"t have many choices in regards to the designing priority. of course you'll need to balance the TM of the second primer and avoid delta more than 2°c.
good luck
fred
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