How would you create a strand specific PCR?

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Ammie
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How would you create a strand specific PCR?

Post by Ammie » Fri Apr 18, 2008 1:52 pm

Hi! I want to run a strandspecific PCR. I have sense and antisense RNA transcript as control. How would you do it?

The simpliest would be just to use one primer for cDNA synthesis (forward får antisense and reverse for sense) and than do a real time PCR with that primer pair.

In theory that would work but in reality it didnt. It was not strand specific. I have read about others that also had that problem, and they give different explanations.

A common way is to make cDNA with a primer with a tag. Than the a primer with the tag-sequence is used for PCR.

So the question is: How to create the tag? Is there anything to think about, or is it just to take any nt-combination in a good length to complement the other primer? Off cause align it with my sequence, but there is many other RNAs with no possibility to check specificity for.

Or would ju use some other method to detect sense-specific RNA?

Thanks!

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