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real time PCR primer design - (Aug/06/2014 )


I am new to primer design, so my question might sound kind of silly!!

I need to detect expression of a target gene in my cells. the gene has several variants and I want to detect expression of all of them with one pair of primers (otherwise I will need to design variant specific primers). so I need to find identical sequences in all variant. my question is do I need to use the CDS (start ATG to stop codon) of the mRNAs or I can use the UTR containing sequences?


If I use the immature mRNA I might have a better chance of finding identical regions.




Try using PrimerBLAST with setting "Allow primer to amplify mRNA splice variants" for the design. Also some of the option should allow you to find a specific exon-exon boundary where to design primers (exons that are common for all variants).

And yes, you can design in UTRs, but mostly that is where the splice variants differ (different start/ends).