PCR Range

The primer melting temperature (Tm) is the estimate of DNA-DNA hybrid stability. Knowing the Tm is critical for determining an appropriate annealing temperature (Ta). A Ta that is too high will result in insufficient primer-template hybridization, leading to low PCR product yield. A Ta that is too low may lead to non-specific product amplification.

Calculation of the Tm of primers shorter than 20 bases can be performed using the Wallace rule:

Tm = 2°C (A+T) + 4°C (G+C)

For accurate estimation of the Tm of primers longer than 20 bases, we recommend using free primer design software such as Primer3.

The final concentration of each primer should be between 0.1 and 0.5 µM. A stock solution of each primer is typically 10–20 µM.

Primer concentrations that are too high increase the chance of mispriming, which may result in nonspecific amplification. Primer concentrations that are limiting can result in extremely inefficient amplification.

Standard desalted primers are satisfactory for most PCR applications.

Three-step PCR includes denaturation, annealing, and extension steps. This type of protocol should be used when the Tm of the primers is lower than the extension temperature or is less than 68°C.

If the melting temperature of the primer (Tm) is close to the extension temperature (72°C) or a few degrees lower, consider using a two-step PCR protocol that includes a denaturation step and a combined annealing/extension step. With this protocol, the annealing temperature should not exceed the extension temperature.

A 68°C extension temperature is preferred for two-step PCR and when amplifying longer templates (>4 kb). This lower extension temperature dramatically improves yields of longer amplification products by reducing the depurination rate that influences amplification.

72°C should be used as the extension temperature when performing three-step standard PCR and for amplification of short fragments (<4 kb).

PCR conditions:

• Use higher denaturation temperatures (e.g., 98°C as opposed to 94°C or 95°C) to allow complete denaturation of the template.

• Keep annealing times for GC-rich templates as short as possible.

• Use primers with a higher Tm (>68°C), because annealing can occur at a higher temperature.

Template quality:

DNA integrity is critical for amplification of long targets. DNA damage—such as DNA breakage during DNA isolation or DNA depurination at elevated temperatures and low pH—results in a greater amount of partial products and decreased overall yield. DNA damage can also occur in acidic conditions; therefore, avoid using water for resuspending DNA templates. DNA is most stable at pH 7–8 or in buffered solutions.

PCR conditions:

• Denaturation time should be kept to a minimum to decrease depurination events.

• Use touchdown PCR; start at a higher annealing temperature and reduce by two degrees per cycle for several cycles.

• Design primers with melting temperatures (Tm) above 68°C.

The GC ratio varies across the genome. Templates with >65% GC content are considered GC rich. GC-rich regions of the genome are mostly concentrated in regulatory regions, including promoters, enhancers, and cis-regulatory elements. GC-rich tracts tend to form inverted repeats, or hairpin structures, that may not melt during the annealing step of PCR. Therefore, amplification of GC-rich templates is hindered by inefficient separation of the two DNA strands. This results in truncated amplicons due to premature termination of polymerase extension.