Hi,
I'm looking for a gene by qPCR method but there are 2 isoforms et their gene sequences are very close to each other.
if i don't want to amplify the second isoform, how can I do ?
I tried to design with Roche and by myself but one of the primers matches everytime with second isoform and I found only 2 papers (but primers target both isoforms)
thank you very much
just see the differences between isoforms and design the primers on these differences.
fred
I agree with Frederic Lepretre . It does not matter if one primer is common to both isoforms. Design the other primer with as many bases at the 3' end of the oligos as different as possible different from the 2 isoforms. Even one mismatched base at high stringency ( high annealing temperature ) can be enough to stop amplification and if you can get 2-4 mismatched bases it should be fairly easy to design pcr conditions that disable amplification of the wrong isoform
Thanks Paul, as always the best...
you can also use differences between those 2 isoforms to amplify them separately since isoforms have selected expression and roles.
fred
I agree with Frederic Lepretre
Design your own primers based on the difference region with idt primer quest ( https://www.idtdna.com/Primerquest/Home/Index ). Remember to set for qPCR primers with intercalating dyes. Just 1 primer in the difference region is enough to garantee specific amplification.
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