I am new to PCR and digging into learning the roles of the different components in a PCR based on the FastStart HF PCR System I am using from Roche. If EDTA is a chelating agent, and we need the MgCL2 in the PCR, then why would it be included in the same mix? Isn't that counter-intuitive? I am assuming not, because I get the PCR product I want when using this kit. I was hoping for some insight from the RG community. Thanks in advance.
This is a very old trick to prevent "poisoning" of magnesium-dependent enzymes with irrelevant metal ions, mostly Pb2+. Usually, the concentration of EDTA is much lower than that of magnesium so that the significant part of magnesium ions remain unbound while EDTA exists as a Mg2+*EDTA complex. The undesirable metal ions have much higher affinity to EDTA than magnesium, often it is higher by many orders of magnitude (f.ex., the formation constant for Mg2+*EDTA complex is 108.79M-1, while for Pb2+*EDTA is 1018.0M-1, for Cd2+*EDTA is 1016.5M-1, and for Hg2+*EDTA is 1021.5M-1). Therefore, even if trace amount of such a contaminant is present in your PCR mixture, it will replace magnesium as EDTA ligand. Thus, the contaminant metal ions will rather be chelated by EDTA instead of binding to the enzyme and inactivating it.
Hi Raven,
I am not very familiar with the kit you use but it seems like the EDTA you are referring to is the one present in the enzyme solution (0.1mM). I assume that EDTA is used as a preservative, to keep the enzyme in the proper conditions. As you mentioned EDTA will chelate magnesium and that could be a problem in the PCR reaction, however EDTA is not a great chelator for magnesium (but much better for calcium) so in your situation it should not be a problem. For example, if you have a 0.1 mM solution of EDTA and you add 1.8 mM MgCl2 only around 0.3% of the magnesium will be chelated while 99.7% will be available. In your case the EDTA concentration is even lower in your final reaction so almost all the MgCl2 you add will be available. If you are interested in calculating the chelating properties of EDTA you can use the free software maxchelator (http://maxchelator.stanford.edu/CaMgATPEGTA-TS.htm). Another important property of EDTA is that for it to bind Mg+2 it needs to release protons so the pH can be affected if a high level of chelation is acquired (again, this would be more common with Ca+2).
Hope this helps,
Ezequiel
Dear Raven Baxter-Christian
Magnesium ion concentration can affect the specifity and efficiency of the reaction and is therefore identified as a critical component. Almost all polymerases require divalent cations for their activity but some tend to function in buffers containing Mn2+ as well, although a little less efficiently. dNTPs and oligonucleotides bind to Mg2+ and therefore its concentration should exceed the molar concentration of phosphate groups that are contributed from both the dNTPs and primers. dNTPs bind in equimolar concentration with Mg2+. Chelating agents (EDTA) and negatively charged ions from the DNA template buffer can also sequester Mg2+ and this to be minimized. The polymerase enzyme functions actively in higher concentrations of Mg2+ and has lower fidelity i.e. the enzyme is more error prone at excess concentration of the Mg2+ ions. Magnesium ion concentration can be lowered, so that the polymerases are barely processive. This will result in higher fidelity of amplification. The concentration of Mg2+ is dependent on the empirical proportions of template and primer DNA and will need to be standardized for an experiment. Polymerase activity when plotted against the Mg2+ gives a bell curve, and the highest activity is seen at 1.2 to 1.3mM. Standard PCR buffers contain 1.5mM of MgCl2.
Primer dimer formation often competes with formation of the DNA fragment of interest, and may be avoided using primers that are designed such that they lack complementarity—especially at the 3' ends—to itself or the other primer used in the reaction. If primer design is constrained by other factors and if primer-dimers do occur, methods to limit their formation may include optimisation of the MgCl2 concentration or increasing the annealing temperature in the PCR concentration
Regards
Prof. Houda Kawas
This is a very old trick to prevent "poisoning" of magnesium-dependent enzymes with irrelevant metal ions, mostly Pb2+. Usually, the concentration of EDTA is much lower than that of magnesium so that the significant part of magnesium ions remain unbound while EDTA exists as a Mg2+*EDTA complex. The undesirable metal ions have much higher affinity to EDTA than magnesium, often it is higher by many orders of magnitude (f.ex., the formation constant for Mg2+*EDTA complex is 108.79M-1, while for Pb2+*EDTA is 1018.0M-1, for Cd2+*EDTA is 1016.5M-1, and for Hg2+*EDTA is 1021.5M-1). Therefore, even if trace amount of such a contaminant is present in your PCR mixture, it will replace magnesium as EDTA ligand. Thus, the contaminant metal ions will rather be chelated by EDTA instead of binding to the enzyme and inactivating it.
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