PCR reaction components

Template

The quality of the template influences the outcome of the PCR. For instance, large amounts of RNA in a DNA template can chelate Mg2+ and reduce the yield of the PCR. Also, impure templates may contain polymerase inhibitors that decrease the efficiency of the reaction.

Note: To get the purest template, always use a purification product specifically designed to purify DNA such as the High Pure PCR Template Preparation Kit from Roche Applied Science (Cat. No. 1796828).

The integrity of the template is also important. Template DNA should be of high molecular weight. To check the size and quality of the DNA, run an aliquot on an agarose gel. When testing a new template, always include a positive control with primers that amplify a product of known size and produce a good yield.
Example: For testing human genomic DNA, the Human tPA Control Primer Set from Roche Applied Science (Cat. No. 1691104, for amplification of 4.8 kb, 9.3 kb, and 15.0 kb targets) can serve as a positive control.

The amount of template in a reaction strongly influences performance in PCR. The recommended amount of template for standard PCR is:

• a maximum of 500ng of human genomic DNA

• 1–10 ng bacterial DNA

• 0.1–1 ng plasmid DNA

Low amounts of template, e.g. <10 ng human genomic DNA, will require specific reaction modifications, such as changes in cycle number, redesign of primers, use of “Hot Start”, etc.

Primers

In most PCR applications, it is the sequence and the concentration of the primers that determine the overall assay success. For convenience, several primer design software programs are available. These can be used to ensure that the primer sequences have the following general characteristics:

• Are 18–24 bases long.

• Contain no internal secondary structure.

• Contain 40–60% G/C.

• Have a balanced distribution of G/C and A/T rich domains.

• Are not complementary to each other at the 3' ends (so primer-dimers will not form).

• Have a melting temperature (Tm) that allows annealing temperatures of 55°–65°C (for maximum specificity use temperatures of 62°–65°C).

Note: Optimal annealing temperatures are often higher than the Tm of the primers (approx. 5°–10°C) and have to be determined empirically. Caution: For both primers the Tm should be similar.

Bases that do not hybridize to the template may be added at the 5' end of a primer, (e.g. for introducing restriction sites into the amplification product). Primer concentrations between 0.1 and 0.6 µM are generally optimal. Higher primer concentrations may promote mispriming and accumulation of nonspecific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of desired product.

Note: For some systems, a higher primer concentration (up to 1 µM) may improve results. When testing new primers, always include a positive control reaction with a template that has been tested for function in PCR. This control shows whether the primers are working. The Human Genomic DNA from Roche Applied Science (Cat. No. 1691112) is a good control template for evaluation of human primer sequences.

Choice of DNA polymerase

The choice of a DNA polymerase can profoundly affect the outcome of PCR. For most routine PCR, Taq DNA Polymerase has long been the standard PCR enzyme. However, Taq DNA Polymerase has its limitations. Our Product Selection Guide will provide more information on choosing the appropriate DNA polymerase. Here are some general guidelines:

• To increase the fidelity of PCR, use Pwo DNA Polymerase or enzyme blends (e.g. the Expand PCR Systems which contain blends of Taq DNA polymerase and a thermostable polymerase with proofreading activity).

• To improve PCR yield, use the Expand High Fidelity PCR System.

• To amplify GC-rich targets, preferably use the GC-RICH PCR System, or the Expand Long Template PCR System with supplied buffer 3.

• To amplify targets up to 5 kb, use the Expand High Fidelity PCR System, or the High Fidelity PCR Master.

• To amplify targets >5 kb, use Expand Long Template PCR System or Expand 20 kb PLUS PCR System.

For most assays, the optimum amount of thermostable DNA polymerase (or a blend of polymerases) will be between 0.5 and 2.5 units/50 µl reaction volume. Increased enzyme concentrations sometimes lead to decreased specificity.

MgCl₂ concentration

Mg2+ forms soluble complexes with dNTPs to produce the actual substrate that the polymerase recognizes. The concentration of free Mg2+ depends on the concentrations of compounds that bind the ion, including dNTP, free pyrophosphate (PPi) and EDTA. For best results, always determine the optimal Mg2+ concentration empirically. The optimal Mg2+ concentration may vary from approximately 1 mM to 5 mM. The most commonly used Mg2+ concentration is 1.5 mM (with dNTPs at a concentration of 200 µM each). Mg2+ influences enzyme activity and increases the Tm of double-stranded DNA. Excess Mg2+ in the reaction can increase non-specific primer binding and increase the non-specific background of the reaction.

Note: For easy optimization of MgCl₂, see the PCR Optimization Kit from Roche Applied Science (Cat. No. 1636138).

Deoxynucleoside triphosphate (dNTP) concentration

Always use balanced solutions of all four dNTPs to minimize polymerase error rate. Imbalanced dNTP mixtures will reduce Taq DNA Polymerase fidelity.
Note: For maximum convenience, a premixed, balanced mixture of dNTPs such as the PCR Nucleotide Mix, Cat. No. 1581295) may be added to the reaction mixture as a single reagent. In addition, individual PCR Grade dNTPs and a set of PCR Grade dNTPs (Cat. No. 1969064) are available.

If you increase the concentration of dNTPs you must also increase Mg2+ concentration. Increases in dNTP concentration reduce free Mg2+, thus interfering with polymerase activity and decreasing primer annealing. For prevention of carry-over contamination a higher concentration of dUTP is usually used in place of dTTP (for details, see Preventing carry-over contamination with uracil-DNA glycosylase). The final dNTP concentration should be 50–500 µM (each dNTP). The most commonly used dNTP concentration is 200 µM.

pH

Generally, the pH of the reaction buffer supplied with the corresponding thermostable DNA polymerase (pH 8.3–9.0) will give optimal results. However, for some systems, raising the pH may stabilize the template and enhance results.

Note: Determining the optimal reaction pH can be simplified with the PCR Optimization Kit from Roche Applied Science (Cat. No. 1636138).

Reaction additives

In some cases, adding the following compounds can enhance the efficiency or specificity of PCR:

• Betaine (0.5–2 M)

• Bovine serum albumin (BSA; 100 ng/50 µl)

• Detergents

• Dimethylsulfoxide (DMSO; 2–10%) (v/v)

• Gelatine

• Glycerol (1–5%) (v/v)

• Pyrophosphatase (0.001–0.1 units/reaction)

• Spermidine

• T4 Gene 32 protein

The effect of these additives must be determined empirically, e.g. with the PCR Optimization Kit from Roche Applied Science (Cat. No. 1636138).

Reaction overlay

A layer of mineral oil is often placed atop the reaction mixture to prevent evaporation during PCR.

Note: If the thermal cycler has a top heater, oil overlay is not necessary.