In PCR experiments, apart from Taq DNA polymerase, is the Buffer really that important? How can Buffer be optimized?

Article source: NIRO Research Cat

In PCR(Polymerase Chain reaction) experiments, the Buffer does indeed play a crucial role, and its significance is no less than that of Taq DNA polymerase. The buffer solution not only provides an optimal enzymatic reaction condition for the PCR reaction, but also ensures the efficient and specific progress of the PCR reaction by adjusting the pH value and providing the necessary ionic environment.

The Importance of PCR amplification buffer

1. Maintain pH stability: The PCR reaction is a complex biochemical process involving the interaction of multiple enzymes and substrates. These interactions are highly sensitive to pH values. Any slight pH fluctuation may affect the activity of enzymes and the binding efficiency of substrates. As a "stabilizer" in the reaction system, the buffer solution can resist the interference of external acidity or alkalinity and maintain the reaction system within a relatively stable pH range. This plays a crucial role in ensuring the efficiency and specificity of the PCR reaction.

2. Provide an ionic environment: In addition to maintaining pH stability, the buffer also provides the necessary ionic environment for PCR reactions, such as magnesium ions (Mg)²+) is a necessary cofactor for the catalytic reaction of Taq DNA polymerase, and its concentration has a significant impact on the specificity and yield of PCR reactions. Meanwhile, the concentration of magnesium ions in the buffer needs to be precisely controlled to ensure that the PCR reaction proceeds under the optimal conditions. In addition, other ions such as potassium ions (K+) also play significant roles in processes like primer annealing.

3. Reduce non-specific amplification: Non-specific amplification is a common issue in PCR reactions. This might be caused by impurities in the template DNA, unreasonable primer design or improper control of reaction conditions, etc. Appropriate buffer conditions can reduce the occurrence of non-specific amplification and enhance the specificity of PCR reactions. By optimizing the composition and concentration of the buffer, the PCR reaction can more precisely amplify the target DNA fragment.

4.Protecting enzyme activity and DNA integrity: Enzymes such as Taq DNA polymerase used in PCR reactions are highly sensitive to reaction conditions. Inappropriate buffer conditions may lead to a decrease or even inactivation of enzyme activity, thereby affecting the efficiency of PCR reactions. In addition, DNA templates may be damaged or contaminated during the extraction and purification process. Appropriate buffer conditions can protect the integrity of DNA and reduce the occurrence of non-specific degradation

Optimized components of PCR amplification buffer

The standard buffer for PCR is shown in the PCR operation example. At 72℃, the pH value of the reaction system will drop by 1 unit, approaching 7.2. The presence of divalent cations is of vital importance, affecting the specificity and yield of PCR. Experiments show that Mg²+ Superior to Mn²+, while Ca²It has no effect at all.

1.Mg²+(MgCl₂Concentration: Mg²The optimal concentration of + is 1.5 mmol/L(when the concentration of various DNTPS is 200 mmol/L), but it is not the best for the combination of any template and primer. When using the target sequence and primers for the first time, Mg²The concentration is adjusted to the optimal range, with a variation range of 1 to 10 mmol/L. Mg²Excess is prone to generating non-specific amplification products, Mg²Insufficiency can easily lead to a reduction in output. High concentrations of chelating agents such as EDTA or high concentrations of negatively charged ionic groups such as phosphate in the sample will combine with Mg2+ and reduce Mg²+ Effective concentration. Therefore, the DNA used as the template should be dissolved in 10 mmol/l Tris-HCl(pH7.6) and 0.1 mmol/L EDTA. dNTP contains phosphate ions, and changes in its concentration will affect Mg²The effective concentration of +. The total concentration of 4×dTNPs in the standard reaction system is 0.8 mmol/L, which is lower than 1.5 mmol/L Mg²+ Concentration. Therefore, under conditions of high concentrations of DNA and DNTPS, Mg must be adjusted accordingly²The concentration of +.

2. Tris-HCl buffer: In PCR experiments, Tris-HCl buffer with a concentration of 10 to 50 mmol/L is used, and other types of buffers are rarely employed. Tris buffer is a bipolar ionic buffer with a pKa of 8.3(at 20℃) and a Δ pKa of 0.021/℃. Therefore, when the pH of 20 mmol/L Tris is 8.3(at 20℃), under typical thermal cycling conditions, the true pH value is between 7.8 and 6.8

3. K+(KCl) concentration: At 50 mmol/L, it can promote primer annealing. However, current research indicates that when the NaCl concentration is 50 mmol/L, a KCl concentration higher than 50 mmol/L will inhibit the activity of Taq DNA polymerase. Adding less or no KCl has little impact on PCR results.

4. Gelatin and BSA or nonionic detergents: They have the function of stabilizing enzymes. The general dosage is 100 μg/mL, but current research shows that good PCR results can be achieved whether it is added or not, with little impact.

5. Dimethyl sulfoxide (DMSO): DMSO is useful when using large fragments for PCR. Adding 10%DSO is beneficial for reducing the secondary structure of DNA, making templates with high GC content more prone to complete denaturation. Adding DMSO to the reaction system makes direct sequencing of PCR products easier, but exceeding 10% will inhibit the activity of Taq DNA polymerase. Therefore, DMSO is not used in most cases.

6. Glycerol: Protects Taq DNA polymerase, enhances the stability of the enzyme, and thereby increases yield.

Case: PCR amplification buffer for High GC Content Genes and Its amplification methods and applications

PCR amplification buffer for genes with high GC content

PCR amplification buffer for high GC content genes is a reagent specially designed for amplifying DNA fragments with high GC content. This type of buffer usually contains specific components to optimize PCR reaction conditions, enhance amplification efficiency and specificity. The following are some key components and their functions:

1) Tris-HCl: Provides the pH environment required for the reaction, maintaining the stability and activity of the enzyme.

(2) (NH₄)₂SO₄：It can replace the traditional KCl, increase the specificity of primer and template binding, and be compatible with a wider range of Mg²⁺ concentrations.

(3) Betaine monohydrate：It helps stabilize the double-stranded structure of DNA, reduces the formation of secondary structures, and thereby enhances the specificity of PCR reactions.

(4) Emulsifier Tween 20：It may be used to reduce foam and surface tension in the reaction system, which is conducive to the uniform mixing of all components.

(5) PCR reaction enhancer：Such as PCRx Enhancer Solution, etc., can further enhance the yield and specificity of PCR reactions.

In addition, there are also ready-made products on the market, such as the GC-rich PCR Buffer launched by Beijing Kangrun Chengye Biotechnology Co., LTD. (GenStar), which offers a variety of different buffers for users to choose from. They can be used alone or in combination to optimize the PCR reaction conditions.

2. Amplification method

For PCR amplification of genes with high GC content, in addition to using specific buffers, the following points also need to be noted:

Primer design:

1) The length is usually 18 to 24 nucleosides to avoid a decrease in specificity due to excessive length.

2) The GC content should be controlled between 40% and 60%, or reflect the GC content of the template.

3) Design primers with G or C at the 5' end and the middle region to enhance stability and hybridization stability.

4) Avoid having A high GC content at the 3' end and ensure that there are 3 A or T in the last 5 nucleosides.

For details, you can browse this articlePractical Tips: Three Methods for Designing PCR Primers using NCBI/Oligo 7/primer 5》。

(2) Optimization of reaction conditions

1) Hot-start PCR: By using hot-start PCR techniques, such as Qiagen's Hotstar Taq DNA polymerase, the template DNA is completely denatured by extending the initial denaturation time, thereby enhancing primer-specific renaturation and elongation. 2) Annealing temperature: Set the annealing temperature based on the Tm value of the primers, and you can try to use the drop PCR technique, that is, use a higher annealing temperature at the beginning of the PCR reaction and then gradually reduce it to near the Tm value of the primers.

3) Mg²⁺ concentration: Mg²⁺ is a key cofactor in the activity of Taq DNA polymerase, and its concentration has a significant impact on the PCR reaction. The concentration of Mg²⁺ needs to be adjusted according to specific circumstances to achieve the best amplification effect.

4) dNTP concentration: dNTP serves as the raw material for PCR reactions. Both excessively high and low concentrations of DNTP can affect the specificity and yield of PCR reactions. The concentration of dNTP usually needs to be adjusted according to specific circumstances.

(3) Add auxiliary agents:

Organic solvents such as DMSO can eliminate the influence of the secondary structure of DNA templates and enhance the specificity of PCR reactions.

2) The use of specialized PCR reaction enhancers, such as PCRx Enhancer Solution, can further enhance the yield and specificity of PCR reactions.

PCR amplification buffers for high GC content genes and their amplification methods are widely used in various fields of molecular biology research, such as gene cloning, gene expression analysis, and gene mutation detection. By optimizing PCR reaction conditions and using specific buffers and adjuvants, accurate amplification and efficient detection of genes with high GC content can be achieved.