Hairpin template in a PCR

Peter Ellis pjie2 at cam.ac.uk
Tue Oct 3 03:29:09 EST 2006


TR wrote:
>
>I do not want to construct the inverted repeat by cloning because
> in the future I want to try to apply the same strategy to an
> heterogeneous mixture of fragments.

Aha!  Enlightenment dawns!  That looks like a fun project.

I had a thought earlier, which is that your original PCR strategy should
work as long as you can avoid the suppression effect.    Suppression occurs
when the hairpin extends all the way to the end of the molecule, thus
preventing primer access and quenching the PCR.

I wonder if it would be worth trying a "forked" adapter which would avoid
this.

abcdefg\
              stuvwxyz|---------amplicon----------|--hairpinoligogoeshere---\
              stuvwxyz|---------amplicon----------|--hairpinoligogoeshere---/
jklmnop/


Apologies if it doesn't line up, but it should be clear - you ligate your 
magic hairpin onto one end, and a Y-shaped adaptor with two free ends onto 
the other.  That should then give you enough leeway to PCR it up, given some 
playing round with the conditions.  You can then chop off the free ends once 
you're done to leave a pure hairpin.

Peter 




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