Hairpin template in a PCR
nobody at nowhere.com
Mon Oct 2 17:50:47 EST 2006
>> Austin So wrote:
>> 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)
> Sounds good. I could even use a 200-bp or so fragment, obtained by PCR
> from the 500-bp fragment, as a primer, and the two-step PCR. Looks
> that even the original 500-bp fragment could be used a primer, and
> would be transformed into the hairpin in the PCR, then the Tm for the
> closing of hairpin and for the binding of the primer would be the
> same. However the other half of the hairpin will still be closer and
> easier to find that the primer.
No...you don't need to (and in fact shouldn't) go to that length in
get a primer that will anneal at 65-70C...just use the simple formula to
figure it out a guess-timate...at some point (determined by sequence),
there is a greater chance that non-specific annealing will offset any
gain in Tm that you get by increasing length...a 2 base mismatch on a
20-mer is gonna have more impact than a 2 base mismatch on a 200-mer...
But yes...even if the Tm of the hairpin and the duplex are the same, the
simple fact that the first is unimolecular versus the bimolecular
association of the latter will always favour the hairpin to form...which
is why you have to add a whack load of primer...
Of course, in context of PCR, that isn't really important, since you are
amplifying the successes and not the failures...
> The problem with this approach is that I have in mind the application
> of the same method to a heterogeneous population of DNA fragments, to
> obtain an equivalent population of inverted repeats. Otherwise the
> inverted repeat would be easier to generate by traditional cloning.
> The heterogeneous population would have known ends:
> I am limited to that ends for the designing of the primers, but it may
> be possible to find a primer with Tm high enough.
Given that you want to preserve the composition of this "library", I
would probably go against the PCR route <it seems that you have come to
that conclusion regardless...>. Too many things can go wrong, and the
combination of secondary structure as well as sequence dependent bias
along with fidelity issues...
I think the RCA method is as good as any (and strand-displacing DNAP can
be used provided that you optimize conditions accordingly)...you are
quite right that the length of your template should minimize problems
with the product immediately forming...if you have restriction sites at
the ends of your monomer construct, then it makes it easy to clean up
your product and get your library, and perhaps clone the library
individually later on.
But I guess it all depends on how you wish to process it in the end as I
don't know what the desired end-product is...
All you can do is give it a go, and take it from there...
P.S. there is no need to be an ass when people are offering suggestions
gratis (does anyone acknowledge the information and help gained in this
listserv?)...helpful or not...
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