The Role of KCl and MgCl₂ in PCR

The Role of KCl:

The KCl salt in the PCR buffer acts by neutralizing the charge present on the backbone of DNA. During the elongation step of the PCR, the primer has to anneal or stick properly to the template and this is facilitated by the KCl. Thus, it functions by reducing the repulsion between the negatively charged DNA strands i.e. the primer and template, thereby stabilizing the primer-template binding. The temperature that is used during the annealing step of the PCR is less definitive and the salt helps in the annealing of the primer so the polymerase can start adding nucleotides from the bound primer properly.

Increasing the KCl concentration have shown to result in longer double stranded DNA to be reluctant in separating i.e. they denature at a slower rate than short length DNA. It is demonstrated that longer products are more efficiently amplified at lower concentrations of K⁺ ions. Cheng et al., 1995 showed that 10- 40% reduced concentration of KCl helped in increasing the efficiency of long DNA segment amplifications. Increasing the KCl concentration to 70mM - 100mM from the normal concentration in the PCR buffer of 50mM was shown to result in the improved yield of small sized products. It is to be noted that the polymerase enzyme’s storage buffer generally contains 100 mM KCl and this should be kept in mind since it can result in variation of final concentration of KCl.

The second generation of PCR buffers has the combinatorial effect of KCl and (NH₄)2SO₄. The K⁺ ions bind to the phosphate backbone whereareas the NH4+ exists both in its ionic form and as ammonia. It interacts with by means of hydrogen bonds. These interactions can help destabilize the weak hydrogen bonds between mismatched bases during annealing. This combined role helps in specificity of primer annealing in a wide range of temperatures.

The role of MgCl₂:

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.


References:

1. Cheng S., Cheng Y., and Monforte J.A. (1995). Template integrity is essential for PCR amplification 20- 30kb sequences from genomic DNA. PCR Methods Appl. 4: 294-298.

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