Hairpin template in a PCR

Domo rimask at yahoo.com
Sat Sep 30 00:35:30 EST 2006


just take right Betaine...
Salt is ok

"TR" <taskan4 at gmail.com> wrote in message 
news:mailman.1242.1159570094.20007.methods at net.bio.net...
> 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:
>
>
> 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?
>
> Well, I'll wait for your comments, and thank you again for the responses.
>
> TR
>
>
> Original question:
>
>
>
>> > 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
>
>
>
> Response 1:
>
>
>
>> 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
>
>
>
> Response 2:
>
>
>
>> I would be surprised to see that you get anything with Taq. When you
>
>> think about it, a 50 mer duplex would melt at 80C or something like
>
>> that, so you really need to find some additive that will split the
>
>> duplex apart and allow a polymerase to plow through.
>
>>
>
>> You could also try 1M betaine, or even single strand binding protein.
>
>> And add a whack load of primer. I think Invitrogen has some DNa binding
>
>> proteins in their Accuprime Taq. You could also try Phusion or
>
>> PfuUltraII to see if they will work since they have a built in DNA
>
>> binding domain. Vent is apparently a strong strand displacer at higher
>
>> temp and is less prone to slippage in the presence of secondary
>
>> structure (I forget the reference).
>
>>
>
>> Cheers
>
>>
>
>> Austin
> 




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