Hairpin template in a PCR

TR taskan4 at gmail.com
Mon Oct 2 03:37:32 EST 2006


Peter,

I really appreciate all your effort in designing strategies to
construct the inverted repeat by cloning, and also all the effort to
write them so nicely (I especially liked the word "nocilmpa"). Your
strategy is certainly possible, actually so possible that I actually
have used it in the past. HOWEVER, I owe you an apology, and I owe you
a couple of beers. I should have said this before, but the thing is
that 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. Construction by cloning is OK if I
start with a population of identical DNA molecules in the tube, but if
the population is heterogeneous you end up with heterogeneous products
in the ligation reaction, instead binding each molecule with itself to
form a hairpin. The strategy I am trying to design should produce a
hairpin from a single individual molecule, so that when applied to a
population of molecules would result in a population of hairpins. The
starting population would have known ends, including RE sites:

abc-NNNNNNNNNNNN-def
abc-NNNNNNNNNNNN-def

so I can still use PCR, if needed, in the strategy.

TR



> Peter Ellis wrote:
> > TR wrote:
> >>
> >> 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:
> >
> > I don't see why you can't make the two halves of the hairpin
> > separately, with compatible sticky ends, and just ligate them
> > together.
> > Thus:
> >
> >> --------amplicon---------|
> >> --------amplicon---------|
> >
> > Amplify with an extended primer to introduce a restriction site at
> > one end
> >> --------amplicon---------|---RRRR---
> >> --------amplicon---------|---RRRR---
> >
> > Cut to leave a sticky end
> >
> >> --------amplicon---------|---RRRR
> >> --------amplicon---------|---
> >
> > Ligate this back to itself
> >
> >> --------amplicon---------|---RRRR---|---------nocilmpa--------|
> >> --------amplicon---------|---RRRR---|---------nocilmpa--------|
>
>
> Note that doing this leaves you with the "spacer" section also forming an
> inverted repeat - the hairpin continues all the way to the restriction
> site.
> However, if you need the spacer loop to *not* form an inverted repeat, you
> can just do two initial PCRs, thus:
>
>
> |--------amplicon---------|
> |--------amplicon---------|
>
> Do two different PCR amplifications with different extended primer to
> introduce a restriction site at one end
>
> |--------amplicon---------|abcdeRRRR---
> |--------amplicon---------|abcdeRRRR---
>
> |--------amplicon---------|zyxwvutsRRRR---
> |--------amplicon---------|zyxwvutsRRRR---
>
>
> Cut these to leave a sticky end
>
> |--------amplicon---------|abcdeRRRR
> |--------amplicon---------|abcde
>
> |--------amplicon---------|zyxwvutsRRRR
> |--------amplicon---------|zyxwvuts
>
>
>
> Ligate these together, you'll get three products.
>
> 1)
> |--------amplicon---------|abcdeRRRRedcba|---------nocilmpa--------|
> |--------amplicon---------|abcdeRRRRedcba|---------nocilmpa--------|
>
> 2)
> |--------amplicon---------|zyxwvutsRRRRstuvwxyz|---------nocilmpa--------|
> |--------amplicon---------|zyxwvutsRRRRstuvwxyz|---------nocilmpa--------|
>
> 3)
> |--------amplicon---------|abcdeRRRRstuvwxyz|---------nocilmpa--------|
> |--------amplicon---------|abcdeRRRRstuvwxyz|---------nocilmpa--------|
>
> The third one is the one you want.  If you design your initial primers
> properly so they're different lengths, you can then gel-purify the correct
> one.
>
> Peter



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