Hairpin template in a PCR

[TR]

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=3DqNj0karb

Maybe you have guessed it by now, but the inverted repeat will be used for
gene silencing (by RNAi) in my favourite fungus.

Obviously the PCR template presents the problem of having secondary
structure, and PCR has not worked so far. We have tried varying the Mg
concentration, the annealing temperature, and adding additives (DMSO and
formamide), but we have not seen even a faint band. Now we are going to buy
the epicentre FailSafe=99 PCR kit to give it a try.

Any PCR guru out there has any idea what we may try next?

Which do you think is the problem, enough oligo is not binding in first
place, of taq cannot extend the oligo?

We have only used taq so far, any other enzyme can perform better for this
task?

Should we increase the concentration of the oligo in the PCR, to increase
the probability of binding before the hairpin is renatured?

phi29 DNA Polymerase has, according to Fermentas, the highest processivity
and strand displacement activity among known DNA polymerases. We could use
this enzyme to convert the hairpin into the inverted repeat, before the PCR=
.
Would this help?

Please have the smart idea that does not come to my mind, and save my
project!!!

Thank you very much

TR