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 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 ions are a critical DNA polymerase cofactor necessary for enzyme activity. Mg2+ binds to DNA, primers, and nucleotides contained in the amplification reaction. The Mg2+ concentration is generally higher than that of dNTPs and primers, and some optimization may be necessary for different template and primer concentrations. A higher than optimal concentration of Mg2+ can stabilize nonspecific binding and is often indicated by decreased yields of specific PCR products and the appearance of background smear or other PCR artifacts.