PCR specificity

Landry Pierre-Alexandre landryp at ERE.UMontreal.CA
Tue Jan 28 10:37:19 EST 1997


Els Van Geldre <Els.VanGeldre at rug.ac.be> writes:

>Hi there,

>Lately, I performed some PCR reactions with different MgCl2 
>concentrations (1.5mM, 3mM and 4.5 mM) in order to optimize my PCR 
>conditions. What surprised me was that with the same primercombination 
>and template, but different MgCl2 concentration, I got products of 
>diffent lengths. In other cases the MgCl2 concentration influenced the 
>number of PCR products I obtained (from 1 PCR product to 5 PCR products 
>for the same primercombination and template, but other MgCl2 
>concentration). I am aware of the fact that MgCl2 plays a very important 
>role in PCR succes, but is it normal that it also influences the number 
>of PCR products and thus the specificity of the PCR reaction ?

>Thanks for answering,
>Els

The answer is yes, it is absolutely normal (see Park and Russel, Effect 
of concentration of MgCl2 on random amplified DNA polymorphism, 
biotechniques, vol 16 no 4, 1994). The specificity of a primer, 
if I understand correctly, depends on the stability of the complex 
template-primer, which in turn calls for the polymerase to start its 
work. The complex of replication is in a dynamic equilibrium.The Mg++ will 
stabilize the replication complex because it 
will facilitate the attachment of the polymerase to a less stable 
template-primer complex and begin the replication, which will contribute 
to the stability. And as long as this non-specific annealing happens in 
the first cycles, it will produce undesirable bands. That's how I see it ;)

Ax



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