PCR additives play a crucial role in the PCR process, acting on various stages of the PCR reaction through different mechanisms, affecting amplification efficiency, specificity, and yield. The mechanisms of action of several common PCR additives are described in detail below.
Lowers the secondary structure of DNA
Dimethyl sulfoxide (DMSO)
Mechanism of action: DMSO plays a role in PCR reactions mainly by reducing the secondary structural stability of DNA. Specifically, DMSO is able to interact with water molecules on the DNA strand, reducing the hydrogen bonding of the water molecules to the DNA strand, thereby reducing the melting temperature (Tm) of the DNA. This effect allows the DNA strand to thaw at lower temperatures, facilitating primer binding to template DNA and DNA polymerase elongation. However, DMSO also reduces the activity of Taq polymerase. Therefore, a balance needs to be found between template proximity and polymerase activity when using DMSO. Too high a concentration of DMSO may inhibit the PCR reaction, while a concentration too low may not fully exert its effect on reducing the secondary structure of DNA.
Optimization: Try different DMSO concentrations (e.g., 2% to 10%) to find the best one for your experiment. Note: Observe the specificity and yield changes of the PCR reaction while adjusting the DMSO concentration. Consider the potential effects of DMSO on other components of the PCR reaction (e.g., dNTPs, primers, etc.) and optimize accordingly.
Betaine
Mechanism of action: Betaine is an osmoprotective agent that improves amplification efficiency by reducing the formation of DNA secondary structures in PCR reactions. Specifically, betaine is able to interact with negatively charged groups on the DNA strand, reducing the electrostatic repulsion between the DNA strands, thereby reducing the formation of DNA secondary structure. This effect makes it easier for the DNA strand to be primer-bound and polymerase-extended during the PCR reaction. In addition, betaine increases the specificity of PCR reactions. It reduces the occurrence of non-specific amplification by eliminating the dependence on base pair composition when DNA is melted/denatured. This makes betaine particularly effective in amplifying GC-rich DNA sequences.
Suggested use: Use betaine or betaine monohydrate as PCR additives instead of betaine hydrochloride. Because betaine hydrochloride may affect the pH of the PCR reaction and thus the activity of the enzyme. The recommended concentration range of betaine is 1-1.7M, and the specific concentration should be optimized according to the experimental conditions. Care should be taken to control the concentration of betaine when adding it to avoid negative effects on PCR reaction.
Non-ionic detergent
Mechanism of action: Nonionic detergents mainly play a role in PCR reactions by reducing the secondary structure stability of DNA. They are able to interact with water and lipid molecules on the DNA strand, disrupting the hydrophobic interaction and hydrogen bonding between the DNA strands, thereby reducing the melting temperature (Tm) of DNA. This effect makes it easier for DNA strands to unmelt and bind primers during PCR reactions. However, non-ionic detergents can also present problems with non-specific amplification. Because they may bind non-specifically to DNA or primers, interfering with the specificity of the PCR reaction. Therefore, when using non-ionic detergents, their concentrations need to be carefully controlled to avoid non-specific amplification.
Suggested use: Commonly used non-ionic detergents include Triton X-100, Tween 20 and NP-40. They are typically used at concentrations of around 0.1-1%, which need to be optimized according to the experimental conditions. When using non-ionic detergents, take care to observe the specificity and yield changes of the PCR reaction and adjust as needed.
Reduces non-specific priming
Formamide
Mechanism of action: Formamide is an organic solvent that acts in PCR reactions by reducing the stability of the DNA double helix. Formamide is able to bind to the large and minor grooves in DNA, disrupting hydrogen bonds and hydrophobic interactions between DNA strands, thereby reducing the melting temperature (Tm) of DNA. This effect allows the DNA strand to untangle and bind primers at lower temperatures, which facilitates the PCR reaction. Formamide also promotes the specific binding of primers to template DNA, reducing the occurrence of non-specific amplification. In addition, formamide can also improve the amplification efficiency of PCR reactions, so that the reaction can obtain sufficient product amounts in a short time.
Suggestion: The concentration of formamide used in PCR experiments is usually about 1%-5%, and the specific concentration needs to be optimized according to the experimental conditions. Be aware of the potential effects of formamide on other components of the PCR reaction, such as competitive binding to dNTPs and binding to template DNA and primers, and optimize accordingly.
Tetramethylammonium chloride (TMAC)
Mechanism of action: TMAC works in PCR reactions primarily by increasing the specificity of hybridization. It interacts with the negatively charged groups on the DNA strand to form a charge shield, which reduces the electrostatic repulsion between the DNA strands and makes the binding of primers to the template DNA more stable. This allows the PCR reaction to maintain specific binding of primers to the template even at higher annealing temperatures, reducing the occurrence of non-specific amplification. In addition, TMAC can increase the melting temperature of DNA, allowing DNA strands to melt at higher temperatures, which helps to reduce non-specific amplification due to temperature fluctuations during PCR reactions.
Suggested use: The usual concentration of TMAC in PCR reactions is around 15-100 mM, and the specific concentration needs to be optimized according to the experimental conditions. When using degenerate primers for PCR reactions, it is recommended to add TMAC to increase the specificity of the reaction. Be aware of the potential effects of TMAC on other components of the PCR reaction, such as competitive binding to dNTPs and binding to template DNA and primers, and optimize accordingly.
Mechanism of action: DMSO plays a role in PCR reactions mainly by reducing the secondary structural stability of DNA. Specifically, DMSO is able to interact with water molecules on the DNA strand, reducing the hydrogen bonding of the water molecules to the DNA strand, thereby reducing the melting temperature (Tm) of the DNA. This effect allows the DNA strand to thaw at lower temperatures, facilitating primer binding to template DNA and DNA polymerase elongation. However, DMSO also reduces the activity of Taq polymerase. Therefore, a balance needs to be found between template proximity and polymerase activity when using DMSO. Too high a concentration of DMSO may inhibit the PCR reaction, while a concentration too low may not fully exert its effect on reducing the secondary structure of DNA.
Optimization: Try different DMSO concentrations (e.g., 2% to 10%) to find the best one for your experiment. Note: Observe the specificity and yield changes of the PCR reaction while adjusting the DMSO concentration. Consider the potential effects of DMSO on other components of the PCR reaction (e.g., dNTPs, primers, etc.) and optimize accordingly.
Provide the necessary cofactors
Magnesium ions
Mechanism of action: Magnesium ions are a cofactor in DNA polymerases (e.g., Taq polymerase) and are essential for maintaining enzyme activity and stability. In PCR reactions, magnesium ions are mainly involved in the following aspects.
Enzyme activity is maintained. Magnesium ions bind to the active center of DNA polymerase, helping the enzyme maintain its catalytic function. Without the presence of magnesium ions, DNA polymerase will be inactive and unable to carry out DNA strand extension.
dNTPs binding. Magnesium ions are also involved in the binding of dNTPs (deoxyribonucleoside triphosphates) to DNA strands. In the process of DNA synthesis, dNTPs undergo nucleophilic attack with the 3′ hydroxyl group of the DNA strand under the action of magnesium ions, forming phosphodiester bonds, thereby realizing the extension of the DNA strand.
Specific regulation. The concentration of magnesium ions has a significant effect on the specificity of the PCR reaction. Proper magnesium ion concentration contributes to the specific binding of primers to template DNA, reducing non-specific amplification and primer-dimer formation. However, too high a magnesium concentration may increase the risk of non-specific amplification, while too low a concentration may lead to an incomplete reaction.
Optimization suggestion: Optimize the concentration of magnesium ions according to experimental conditions (e.g., template DNA concentration, primer concentration, dNTPs concentration, etc.). Common magnesium concentrations range from 1.0 to 4.0 mM with 0.5-1 mM intervals. Note the interaction of magnesium ions with other PCR components, such as competitive binding to dNTPs, and binding ability to template DNA and primers.
Reduce pollutants
Bovine serum albumin (BSA)
Mechanism of action: BSA mainly plays a role in reducing pollutants in PCR reactions. It binds and removes inhibitors and impurities such as phenolic compounds from the reaction system, thereby protecting the activity and stability of the polymerase. In addition, BSA reduces the adhesion of reactants to the walls of the tube, increasing PCR efficiency and yield. These mechanisms of action of BSA may be related to its abundance of amino acid residues and hydrophobic groups, which are able to interact with a variety of compounds and alter their physicochemical properties.
Suggested use: BSA is usually added to the PCR reaction at a concentration of about 0.8 mg/ml, and the specific concentration needs to be optimized according to the experimental conditions. It is important to note that BSA itself may also have some influence on the PCR reaction, so it is necessary to weigh the pros and cons and choose the appropriate concentration when using it.