Hairpin template in a PCR

[Austin So]

TR wrote:
> Thank you to those who responded (see question and responses below).
> 
> 
> I am going to try Betaine and the polymerases I can found in the labs
> around, to see what happens, but I am quite worried after reading
> Peter's response (not very optimistic, but quite well informed). I
> jumped to PubMed to look for suppression PCR, and didn't like what I
> found. Indeed it looks that my PCR is going to be a hard one. Well,
> after the initial panic, I have been reading and came up with an
> alternative solution. And again I would like to know your opinion
> about this plan B:

the alternative is to use a primer with a very high Tm (65-70C), and use 
a 2-step protocol (combined annealing-extension and melt)

> A graphical version of the plan B is in
> http://www.flypicture.com/bin/?id=qNj3lKrc
> 
> 1.- Ligate the 500 bp-fragment to TWO different hairpin oligos, one
> binding to one end and the other binding to the opposite end. This
> would result in a single-stranded circle.
> 
> 2.- Amplify this circle by Rolling Circle Amplification with Phi29 DNA
> polymerase, so that it will transform into concatemers of
> double-stranded DNA consisting of repetitions of the whole circle
> generated in 1. In case you are interested, the original paper
> describing the Rolling Circle Amplification is freely available in
> http://www.genome.org/cgi/content/full/11/6/1095?ijkey=86fed9f82a6c8ddb8e90da5a9c4c7897798367ae 
> 
> 
> 3.- Cutting with a RE in the sequence corresponding to one of the two
> hairpin oligos would produce an inverted repeat of the 500-bp
> fragment, with the other oligo in the middle.
> 
> Should I order the second hairpin oligo and the Phi29 polymerase right
> away?, or am I missing something?

That is a good potential solution...and keep in mind that you need not 
limit yourself to phi29, but could just as easily use any polymerase 
with strong strand displacement activity, as long as you are able to use 
an appropriately designed primer in lieu of the random hexamers (the NEB 
catalogue has a nice table that you can look at).


That being said, it is not clear that you will end up with a nice clean 
linear concatenate, since as the pol continues the hairpin will likely 
form immediately before being primed by any hexamers. In other words, I 
don't know if you will ever generate the linear molecule that you 
desire. I suppose if you used a specific primer with its complement, you 
could do it at an elevated temperature...

If you have the resources, just give it a go...it won't cost too 
much...that is part of science...nothing is ever clear cut...

Austin

BTW...the reference I was referring to about strand slippage is a JMB paper.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11554789