Last updated on April 19, 2022 by
Polymerase Chain Reaction (PCR) Basics
Did you know Mullis and Michael Smith won the Nobel Prize for discovering the Polymerase Chain Reaction (PCR) in 1983?
This groundbreaking technology-enabled scientists to multiply a single strand of DNA hundreds of billions of times in just a few hours. All that was needed for this experiment was just test tubes, a few chemicals, and heating. Without PCR, the molecular and genetic analysis of an organism’s genomes would have been an impossible event.
This in vitro tool is essential in a range of biology labs, including molecular biology, plant biology, zoology, and biotechnology, to amplify a small segment of DNA. The resulting PCR products are used in other lab workflows, such as DNA fingerprinting or genetic analysis. (Did you also know that the majority of mapping techniques used in the Human Genome Project involved the use of PCR?)
PCR is often called “molecular phenotyping,” and today, several PCR methods are available serving different purposes, including RT-PCR, Real-Time PCR, Multiplex PCR, and Hot-start PCR.
Stages of PCR
Irrespective of the different types of PCR, all of them involve three basic stages:
- Denaturing stage: This stage is also known as nucleic acid denaturation. Here, heating the DNA template at 95 °C denatures its helix into two single strands by breaking the hydrogen bonds.
- Annealing stage: At 50 – 65°C (called the annealing temperature), the primers bind to the single-stranded template DNA.
- Elongation/Extension stage: At this stage, the synthesis of a new DNA strand begins. The temperature is set in between 72°C-74°C at which the Taq DNA polymerase (mostly used DNA polymerase in PCR reactions) binds to the primers and uses dNTPs (deoxynucleotide triphosphate) to form new DNA.
Required Reagents for PCR
The reagents required to run a PCR assay include a DNA template (it can be genomic DNA, plasmid, or cDNA), DNA polymerase enzymes, primers, dNTPs (dATP, dGTP, dCTP, dTTP), and PCR buffers, which include tris-HCL, KCL, and MgCl2.
All these components of the reaction mixture affect a PCR reaction in different ways, as all of them work in harmony to reproduce millions of amplicons for the gene of interest.
In this article, you will learn what the role of MgCl2 in PCR reaction is, how it facilitates the process, and how varying its concentration may disturb your reactions.
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What is the Role of MgCl2 in PCR Reactions?
MgCl2 (Magnesium chloride) is an essential ingredient of the PCR master mix. Acting as a cofactor, it enhances the enzymatic activity of DNA polymerase, thereby boosting DNA amplification.
Cofactors are non-protein ions or molecules that help enzymes perform their functions. Thus, the absence of these cofactors will make the enzymes dead—not performing any function at all. These cofactors can either be positively charged metal ions (such as zinc, iron, or magnesium ions) or small carbon-based molecules (for example, vitamin B12).
Look at these examples to better understand the role of cofactors:
- The enzyme pyruvate dehydrogenase, involved in glycolysis and the citric acid cycle, can’t work without its cofactors, which include TPP, FAD, NAD+, CoA, and Mg2+.
- Carbonic anhydrase, an enzyme having a role in maintaining the pH of our body, can’t function in the absence of its cofactor, the zinc ion.
Other than enhancing the DNA polymerase activity, MgCl2 also facilitates the primer binding at specific sites during PCR reaction.
How Does MgCl2 Work?
MgCl2 has functions in both facilitating Taq DNA polymerase activity and primer annealing specificity on the template DNA/RNA strand.
Molecular mechanism of MgCl2 in enhancing Taq DNA polymerases activity
The Mg2+ ion (or magnesium ion) of the magnesium chloride is utilized in the process of PCR amplification to promote the catalytic activity of the Taq DNA polymerase enzyme.
But how does it do it?
During PCR amplification, Mg2+ ion binds to a dNTP at its alpha phosphate group and facilitates the removal of beta and gamma phosphates. The resulting dNMP forms a phosphodiester bond, through its phosphate group, with 3’ OH (hydroxyl) of the adjacent nucleotide.
MgCl2‘s role in facilitating primer binding
Mgcl2 helps in the binding of primers at specific locations by influencing the primer melting temperature (Tm).
Tm is defined as the temperature at which one half of a DNA duplex is dissociated into a single strand, indicating the stability of the duplex.
The MgCl2 increases the Tm of the PCR reaction for better interactions between primer and template DNA. The magnesium ion of MgCl2 binds to the negatively-charged phosphate ion of the DNA and reduces electrostatic repulsion between two DNA strands. This leads to proper annealing of the primers with their complementary DNA strands.
Effects of too much or too little magnesium
MgCl2 is essential for the optimization of a PCR protocol. An optimal amount of the reagent is required to create appropriate PCR conditions for the proper amplification of the desired fragments of DNA/RNA templates.
The PCR buffer contains an adequate amount of magnesium chloride, which supports a normal PCR reaction. However, what’s an “adequate amount” of magnesium chloride refer to?
For standard PCR reactions, 1 mM to 5 mM MgCl2 is used. However, 2 mM MgCl2 is the most commonly used concentration for buffer preparation.
Even after knowing the optimal amount, it’s necessary to optimize the final concentration of MgCl2 for your reaction since it varies depending on the PCR conditions. For example, for DNA templates with high GC content or while working with unsuitable primers, a concentration higher than 2 mM of MgCl2 may be required.
It is also necessary to increase MgCl2 concentrations when trying to compensate for DNA extracts containing PCR inhibitors since they also bind to Mg2+ ions and reduce their availability.
Moreover, you must note that too much or too little MgCl2 can also create problems in your reactions.
- Too much MgCl2: Excessive amounts of Magnesium Chloride in PCR reactions lead to non-specific binding of primers, resulting in errors in DNA replication. Thus, agarose gel electrophoresis will show more DNA bands.
In some situations, a higher MgCl2 concentration might also lead to primer dimer formation.
- Too little MgCl2: In this situation, primers will fail to base pair with the DNA template, which results in a weak amplification or complete PCR failure.
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MgCl2 is one of the essential raw materials for preparing PCR buffers. It’s involved in enhancing the catalytic activity of Taq DNA polymerase and facilitating primer binding.
Furthermore, the optimal concentration of MgCl2 is necessary when it comes to running normal PCR, as too much or too little of it downgrades PCR yield.
To achieve better results and get better output, it’s essential to use high-throughput equipment in combination with a high-quality reagent.
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