Hairpin template in a PCR

Peter Ellis pjie2 at cam.ac.uk
Wed Sep 27 23:10:25 EST 2006


TR wrote:
> Hi everybody,
>
> I am trying a strange PCR designed to transform a short DNA fragment
> (500 bp) into an inverted repeat. Basically the idea is to: 1) ligate
> the fragment to a hairpin oligo and 2) Set up a PCR with a single
> oligo that binds to the opposite end. I have put and image to make
> this clearer in http://www.flypicture.com/bin/?id=qNj0karb

Won't work, can't work.  During the annealing step of the PCR cycle, all 
your single strands form up into neat little hairpins and don't let the 
primer bind, so the PCR fails.  This effect (suppression PCR) is regularly 
used by Clontech in their various selection/subtraction kits to *prevent* 
amplication of unwanted material.

Why don't you just amplify your 500bp using an extended primer to introduce 
a restriction site at one end?  Cut it, ligate it to itself, and that'll 
give you the dimeric inverted-repeat product you're looking for.

Peter 




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