PCR Additives & Buffer Selection

An efficient polymerase chain reaction (PCR) experiment would not be complete without the optimization of necessary reagents. A variety of buffers are available for PCR applications and can come as pre-made solutions containing the key ingredients of the reaction. The purpose of the PCR buffer is to resist pH changes by chemically neutralizing any acidic or basic compounds in the sample. Generally, a PCR buffer consists of Tris-HCl (around pH 8.3), potassium chloride (KCL), and magnesium chloride (MgCl2), though the presence and concentration of each component may vary depending on the needs of the experiment.

PCR buffers may also come premixed with Taq polymerase which cuts out the need for the addition of this reagent, thus limiting handling steps. Alternatively, PCR additives and enhancers are reagents that can help optimize an experiment, and typically benefit amplification in one of two ways. PCR additives and enhancers can either reduce secondary DNA structures to increase target amplification or can reduce non-specific priming to limit off-target amplification.

Basis Of Differentiation	Taq polymerase	DNA polymerase
Definition	Is a thermostable DNA polymerase extracted from thermophiles that creates DNA	Is an enzyme that facilitates DNA replication in the form of nucleic acid molecules
Occurs naturally in	Thermophilic bacteria Thermus aquaticus	Prokaryotic organisms
Optimal temperature	72°C	37°C
High temperatures	Active and does not degrade at high temperatures	Not active at high temperatures because of degradation
Residues	The cytosine residues get methylated	The lysine residues get acetylated
Processivity	Adds 2-4 kilobases / minute	Adds ~15-20 nucleotides/ second
Application	Used in PCR	Used to synthesize DNA in prokaryotic cells

Common Additives

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Magnesium Salt

PCR requires the presence of magnesium (typically MgCl2) to act as a cofactor for Taq polymerase. In general, the PCR product yield will increase with the addition of greater concentrations of MgCl2, though this will also decrease the specificity and fidelity of the DNA polymerase. Too much MgCl2 may prevent complete denaturization of a DNA template and may also prohibit the spurious annealing of primers to incorrect template sites resulting in undesired PCR products. Alas, without enough MgCl2 Taq will remain inactive, and the reaction will not proceed. MgCl2 is usually present in a concentration between 0.5 - 5.0 mM.

Potassium Salt

A potassium salt (typically KCl) is typically added to a reaction when dealing with longer PCR products. The final reaction concentration of KCl should generally remain between 35 - 100 mM, and it is often used alongside the addition of dimethylsulfoxide (DMSO) and/or glycerol.

Deoxynucleotide 5'-triphosphates (dNTPs)

dNTPs may cause issues in a PCR if they're not in equivalent concentrations ([A] = [T] = [C] = [G]) and/or due to their instability from repeated freeze-thaw cycles. dNTPs are generally effective in concentrations between 20 - 200 μM of each dNTP. Lower concentrations of dNTPs may increase the specificity of the reaction while excessive dNTP concentrations may inhibit a reaction. For longer PCR-fragments, a higher dNTP concentration may be ideal.

DMSO

Adding DMSO may enhance the reaction by disrupting base pairing, reducing secondary DNA structures, and effectively lowering the melting time (Tm). DMSO is especially beneficial when the DNA template is particularly GC rich (>60%). Though some research has used variable amounts of DMSO (1 - 10%), a concentration >2% may inhibit Taq polymerase.

Formamide

Formamide also disrupts base pairing and destabilizes the double-helix nature of DNA to lower the Tm of the reaction. The addition of formamide can increase the stringency of primer annealing, increasing the efficiency of amplification. Formamide can be used in concentrations between 1 - 10%, though generally is present <5%.

7-deaza-2'-deoxyguanosine 5'-triphosphate (dc7GTP)

The incorporation of dc7GTP into a PCR experiment has been shown to prohibit the formation of secondary structures from forming in the template DNA, like hairpin loops. It is usually used in a 3:1 ratio with dGTP, as it can weaken the signal of ethidium bromide staining that may be used in an experiment downstream.

Betaine

Betaine is often used in tandem with DMSO, can reduce the presence of secondary structures, and can significantly enhance amplification of a GC rich DNA template. Betaine has been shown to significantly reduce, and in some cases even eliminate, the DNA Tm dependence on the concentration of dNTPs in the template. Generally, the final concentration is between 0.5 - 2.5 M.

Nonionic Detergents

Nonionic detergents (like Triton X-100 , Tween 20 , or NP-40) also prevent the formation of secondary structures and help stabilize DNA polymerase. Nonionic detergents can help neutralize sodium dodecyl sulfate (SDS) , a notorious contaminant from DNA extraction. Concentrations are normally present in the range between 0.1 - 1%, though higher concentrations may inhibit the reaction completely.

Bovine Serum Albumin (BSA)

BSA is a restriction enzyme and can be particularly helpful in eliminating the effect of inhibitors in the sample. Notably, BSA has been used to inhibit FeCl3, hemin, acidic compounds, or contaminants from feces, fresh, and marine water. Up to 0.8 mg/ml of BSA is commonly used.

Tetramethylammonium Chloride (TMAC)

TMAC has the ability to increase hybridization specificity, increase Tm, thereby eliminating non-specific priming and potential DNA-RNA mismatch. TMAC is primarily added to PCR mixes that use degenerate primers (as opposed to specific primers), in a concentration between 15 - 100 mM.

Resources:

Buffer Preparations and Recipes

Products

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References

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Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
PCR Buffer Optimization with Uniform Temperature Regimen to Facilitate Automation