> Variable PCR results - Ideas and Suggestions

[Mr. T. Weissensteiner]

In my previous posting to this group I mentioned a paper in the old 
"PCR Methods and Applications" suggesting to increase *both* dNTPs 
and Taq concentration to increase PCR product yield (still haven't 
looked it up, but I'll do ;-) ! ).

The reason why this may not work for all templates is probably 
differences in the internal sequence, i.e. its tendency to form 
stable secondary structure.

An apparently random "all-or-nothing" effect as observed by Warren 
Lushia has been found in such systems under suboptimal (but not yet 
completely inhibitory) PCR conditions.

Although different types of DNA secondary structure have been 
reported to cause amplification problems, all are promoted by 

- domains of high GC-content in amplimer

- suboptimally low denaturation (and probably extension) temperatures

- salt in the PCR mix

The latter point could explain 

- the "inhibitory effect" of high dNTP salt concentrations observed by
  some contributors to this thread

- the fact that increasing MgCl2 does not compensate for increased dNTP 
  (as would be expected if dNTPs would inhibit Taq by complexing Mg++)

- but rather aggravates the problem (MgCl2 is itself a salt and 
  therefore stabilizes dsDNA)

- why "Deep Vent" polymerase tolerates higher dNTP (=salt) 
  concentrations than Taq (the former is less prone to pausing 
  - if I remember it right...)

I wonder if anyone who had experienced both negative and positive 
effects of high dNTP concentrations could comment whether this
is a template-specific effect ?


Bob Steinberg:

>> I don't know where the idea that high dNTPs are inhibitory comes from--
>> the original PCR protocols called for 0.5 mM dNTPs and worked fine-- in
>> our hands, the higher dNTPs generally gave higher PCR efficiencies-- 
>> the reason for decreasing dNTPs to 0.2 mM or lower is that the fidelity 
>> of Taq polymerase is higher at lower dNTP concentrations--

Warren Lushia:

> What do you mean by PCR efficiencies?  More yield of the desired product
> per rxn?  I had a similar problem with inconsistent results, for many
> months, and the trick that solved everything (by that point I thought I
> had tried everything) was to lower the dNTP's to 50uM.  Interestingly,
> when the PCR would work, the yield of the desired product was huge, but
> when it wouldn't work, there was nothing (using the higher dNTP conc.)
> Now it is extremely consistent, although not quite as much product as I
>. know is possible (from my previous experiences.)  BTW, this was for
> Taq.....with a different enzyme (Deep Vent) I found the higher dNTP
> conc. worked better.  I don't have an explanation, but I'm sure that
> dNTP conc. was in fact the major contributor to inconsistencies.
> [ .. ]
> Just wondering .... seems everyone has their own way to do PCR 
> (Pure Chance Reaction)...


Thomas Weissensteiner
Autoimmunity Group
Jenner Institute for Vaccine Research
Compton / Newbury
Berkshire RG20 7NN

T    :  0044 1635 577920
FAX  :  0044 1625 577901

email : tweissen at RETURNMEhgmp.mrc.ac.uk
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