The function of MgCl2 in PCR reaction.
Polymerase chain reaction is one of the important experimental procedure in downstream genomic research. It generates millions of amplicon for the gene of our interest. The process is as similar to replication but it is a temperature dependent process in which each step is governed by different temperature.
Taq DNA polymerase, dNTPs, primers and PCR buffer are used as raw material for amplifying the gene of interest.
Primer is bind to its complementary sequence and Taq DNA polymerase add dNTPs to the growing DNA strand, the entire process is governed by three different steps of temperature: denaturation at 94°C (denature DNA), annealing at 55°C to 65°C (primer binding) and extension at 72°C ( elongation of DNA ).
PCR buffer is one of the important ingredients of PCR reaction. Though the composition of PCR buffer varies from manufacturer to manufacturer, some of the basic chemicals remain same in all buffers.
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One of the important and most common ingredients in the PCR buffer is MgCl2.
MgCl2 is a major component of lysis buffer in DNA extraction. Here MgCl2 breaks cell membrane with the help of Tris. After the lysis of the cell membrane, DNase can easily attack DNA and can break it. MgCl2 binds with DNA and protect it from DNase activity.
However, in PCR reaction it performs a totally different function. Each enzyme requires a cofactor for performing the catalytic reaction. Mostly the cofactor is the metal ion. Mg+ is the main cofactor for Taq DNA polymerase.
Mg+ ions bind to the catalytic site of the enzyme and increase its power to perform the reaction. Hence the ability of Taq DNA polymerase of adding dNTPs is increased.
It is very important to mention polymerase as Taq DNA polymerase because DNA polymerase used in PCR reaction is not same as used during replication.
Taq DNA polymerase is isolated from a bacteria and it can work even at a higher temperature which is not possible by our normal DNA polymerase.
Additionally, MgCl2 increases the Tm of reaction. The Mg2+ ions of MgCl2 bind to the PO3– of DNA backbone temporarily and protects the negatively charged phosphate of DNA backbone by decreasing electrostatic repulsion between DNA strands.
The electrostatic repulsion between two DNA strands hinders the binding of primer to its specific location. Here the addition of MgCl2 helps in a proper binding of primer to its complementary sequence.
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So far MgCl2 involves into two important activity of PCR reaction: increasing Taq polymerase activity by working as a cofactor and helps primers to bind at a specific location.
The standard PCR buffer contains MgCl2 which is sufficient for a normal PCR reaction. However, in some conditions (high GC content of the template, inappropriate forward and reverse primer and for other PCR modifications) more amount of MgCl2 is required.
During the PCR reaction, deoxynucleotide triphosphates convert into deoxynucleotide monophosphate to form a phosphodiester bond between 3’-OH and 5’-P of the adjacent nucleotide.
Here Mg2+ ions bind to the alpha phosphate of dNTPs which helps in removal of beta and gamma phosphate from dNTPs (allows only alpha phosphate to form a phosphodiester bond with 3’-OH).
Generally, 1mM to 5mM concentration can be used for PCR reaction but the standard concentration is 2mM which will give the best result.
Concentration more than 2mM will result in non-specific binding. As the concentration of MgCl2 increases, the chance of non-specific binding will increase.
Higher the concentration of MgCl2, more DNA bands will be observed because primers can bind other than its complementary sequences and amplifies more fragments.
Let us understand how MgCl2 is working:
Suppose we run an experiment with a standard PCR protocol with six different conditions.
- A standard protocol without adding PCR buffer.
- Addition of PCR buffer
- Addition of 2mM MgCl2 in the reaction
- Addition of 3mM MgCl2 in the reaction
- Addition of 4mM MgCl2 in the reaction
- Addition of 5mM MgCl2 in the reaction
We prepared all 6 tubes with one negative control. For positive control, we added a primer which will amplify 800bp fragment in each tube.
Samples are loaded into the agarose gel with a 1000bp DNA ladder and the loading pattern is,
- DNA ladder
- PCR reaction without Buffer
- PCR reaction with buffer
- PCR reaction with 2mM MgCl2
- PCR reaction with 3mM MgCl2
- PCR reaction with 4mM MgCl2
- PCR reaction with 5mM MgCl2
- Negative control
The results indicate that the fragment of our interest (400bp fragment) is amplified very less without a PCR buffer but it gives the best result after addition of 2mM MgCl2 along with PCR buffer ( lane 4).
As the concentration of MgCl2 increases non-specific binding results in more than one bands which indicates that as the concentration of MgCl2 increase the primer binding specificity is decreased.
Conclusively, if you want to modify your PCR protocol, an addition of the appropriate amount of MgCl2 can decrease the annealing temperature of primer. you can run more than two protocol at a single temperature (However, the quality of result may be decreased).
An appropriate amount of MgCl2 increases the specificity of the PCR reaction but more amount of MgCl2 results in nonspecific binding, decreasing the fidelity and yield of the reaction.
It boosts, the activity of Taq DNA polymerase but at higher concentration of MgCl2 polymerase incorporates non-specific dNTPs. it can also form primer dimer.
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Article written by: Tushar Chauhan
Article reviewed by: Ravi Parmar