What is the role of magnesium in PCR, and what is the optimal concentration?
What is the role of magnesium in PCR, and what is the optimal concentration?
Magnesium is a required cofactor for thermostable DNA polymerases and is important for successful amplification. Without adequate free Mg2+, PCR polymerases are not active. In contrast, excess free Mg2+ reduces enzyme fidelity and may increase nonspecific amplification. A number of factors can affect the amount of free Mg2+ in a reaction, including DNA template concentration, chelating agents in the sample (e.g., EDTA or citrate), dNTP concentration, and the presence of proteins.
-Some polymerases (e.g., Takara Ex Taq DNA polymerases and Takara LA Taq DNA polymerases) are supplied with a magnesium-free reaction buffer and a separate tube of 25 mM MgCl2. For these enzymes, you can optimize the Mg2+ concentration for each reaction.
-Titanium Taq DNA polymerases and Advantage 2 DNA polymerases are magnesium-tolerant polymerases that are supplied with buffers containing 3.5 mM of MgCl2.
-The final concentration of Mg2+ for PrimeSTAR GXL DNA Polymerase and PrimeSTAR MAX DNA Polymerase reactions is 1 mM; this concentration increases fidelity for these enzymes.