Efficiency of making ssDNA by asymmetric PCR?

Andrew Doherty A.Doherty at Bris.ac.uk
Thu Nov 14 03:41:06 EST 1996


Peter Wang wrote:
> 
> I have never done asymmetric PCR (where you use an excess of one primer
> to preferentially generate one DNA strand of the template, which in my
> case will be a normal PCR product) and have some questions.
> 
> Is there a limit to how much asymmetry one can achieve? I would guess
> doing a 50-cycle reaction will not give a 50-fold ratio of one strand
> over the other.  As the (linear) amplification proceeds, it will be more
> and more difficult to prime new synthesis because the primer has to
> compete with increasing amounts of product strands for annealing to the
> template strand, and product strands will be able to anneal at much
> higher temperature than the oligo primer.
> - What kind of ratios have you achieved?
> - Is the ratio that one can achieve dependent on the length of the
> product?
> - Is the ratio dependent on the concentration of starting template?
> - Is it useful to have in the reaction some oligo that primes on the
> non-desired strand?  (I can't see why this would improve the ratio
> achieved except via altering the concentration of template.)
> - Would it be better to use a longer primer so it will be more
> competitive at annealing?
> - Is Pfu as good as (or better than?) Taq polymerase for this?
> - Any other important parameters?
> 
> I'd appreciate any protocols and tips.  Thanks in advance!
> 
> - Peter
> 
> ---------------------------------------------------------
> Peter Wang, M.D., Ph.D.
> MRC Centre for Protein Engineering,
> Hills Road, Cambridge, CB2 2QH, England
> 
> Tel (01223) 402104  (international calls +44-1223-402104)
> Fax (01223) 402140  (     "          "   +44-1223-402140)
> ---------------------------------------------------------
Hi Peter,

I've tried making ssDNA by a one step asymmetric PCR and found it VERY
difficult. One of the main problems seems to be not having a consistent
point at which the true amplification ends and the ss production begins.
I actually found it much better to amplify the product you want as a ds
template, gel purify and then re-amplify but with only one primer to run
off ssDNA. The primer can be present in HUGE excess, so competition with
the template should not be a problem. I didn't measure exactly how much
ss I actually produced, but there was sufficient for it to act as a
megaprimer (~200nts) in a further PCR.

Hope it helps
Andy D

-- 
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Dr Andrew Doherty		email -  a.doherty at bris.ac.uk
Dept. Anatomy			Tel (0117)9287421
School of Medical Sciences	Fax (0117)9287402
University of Bristol
University Walk
Bristol UK
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