PCR conditions

The primer and Mg2+ concentration in the PCR buffer and annealing temperature of the reaction may need to be optimized for each primer pair for efficient PCR. In addition, PCR efficiency can be improved by additives that promote DNA polymerase stability and processivity or increase hybridization stringency, and by using strategies that reduce nonspecific primer–template interactions (1). Use of high-purity reagents is also essential for successful PCR, especially for amplification of rare templates, for example, single copy genes in genomic DNA or pathogenic viral DNA sequences in genomic DNA isolated from an infected organism.

Inclusion of control reactions is essential for monitoring the success of PCR reactions. Wherever possible, a positive control should be included to check that the PCR conditions used can successfully amplify the target sequence. As PCR is extremely sensitive, requiring only a few copies of target template, a negative control containing no template DNA should always be included to ensure that the solutions used for PCR have not become contaminated with the template DNA.

PCR setup should be performed in a separate area from PCR analysis to ensure that reagents used for PCR do not become contaminated with PCR products. Similarly, pipets used for analysis of PCR products should never be used for setting up PCR.

In PCR, annealing occurs between the primers and complementary DNA sequences in the template. Primer annealing must be specific for successful amplification. Due to the high concentration of primers necessary for efficient hybridization during short annealing times, primers can anneal to non-complementary sequences. Amplification of products from nonspecific annealing competes with specific amplification and may drastically reduce the yield of the specific product.

The success of PCR largely depends on maintaining a high ratio of specific to nonspecific annealing of the primer molecules. Annealing is primarily influenced by the components of the PCR buffer (in particular the cations) and annealing temperature. Special cation combinations can maintain high primer annealing specificity over a broad range of annealing temperatures. This eliminates the need for optimization of annealing temperatures for each individual primer–template system and also allows the use of non-ideal PCR assays with different primer annealing temperatures.

The annealing temperature in PCR is crucial for achieving specific primer-to-template binding, essential for successful DNA amplification. In the dynamic environment of a PCR reaction, primers are used at high concentrations to ensure efficient and rapid hybridization during the relatively short annealing phase. However, this high primer concentration also increases the likelihood of primers binding to non-complementary sequences. Such nonspecific binding can result in the amplification of unwanted products, which compete with the amplification of the target sequence and can significantly decrease the desired product's purity and yield

The optimal primer annealing temperature is dependent on the base composition (i.e., the proportion of A, T, G and C nucleotides), primer concentration and ionic reaction environment.

Magnesium ion concentration in PCR is a pivotal factor that significantly influences the enzymatic activity and the overall specificity of the reaction. As a crucial cofactor for DNA polymerase, Mg²+ ions are essential not only for the enzyme's catalytic activity but also play a role in stabilizing the interaction between DNA, primers, and nucleotides. Typically, the concentration of magnesium ions in a PCR setup is kept higher than that of the deoxynucleotide triphosphates (dNTPs) and the primers to ensure efficient enzyme function and DNA synthesis.

However, the appropriate magnesium ion concentration can vary depending on the specific requirements of the template and primer sequences used. Each PCR reaction may require some degree of optimization to find the balance that maximizes the yield of specific products while minimizing nonspecific bindings and artifacts such as smears on gel electrophoresis. A concentration of Mg²+ that is too high can inadvertently stabilize nonspecific primer-to-template bindings, leading to a lower yield of the intended product and the emergence of nonspecific products and PCR artifacts like smears or background noise.

Therefore, careful titration of Mg²+ is necessary to tailor the PCR conditions to the specific assay requirements. This optimization usually involves experimenting with different Mg²+ concentrations to identify the level that provides a clear, specific amplification product without unwanted byproducts. This step is critical for ensuring the reliability and specificity of PCR results, particularly in sensitive applications such as diagnostic testing, where the accuracy of PCR amplification can directly impact clinical outcomes.