Optimizing your PCR

Initial denaturation step

Preheating is sometimes required to denature complex templates (e.g., genomic DNA); 94°C for 1 min is sufficient for denaturation. Excessive heat treatment may lead to enzyme inactivation.

• For Terra PCR Direct Polymerase Mix, which is used for direct PCR amplification from tissue without DNA extraction and purification, preheating at 98°C for 2 min is required.
• For Takara LA Taq DNA polymerases and Advantage GC2 DNA polymerases, an initial denaturation step is required.
• PrimeSTAR enzymes do not require preheating for enzyme activation.

Denaturing conditions

Denaturing conditions should be selected by considering the thermal cycler model that will be used. A general guideline is 94–95°C for 30 sec or 98°C for 10 sec.

If using a heat-resistant enzyme, such as one of the PrimeSTAR polymerases, we recommend a denaturation step of short duration and high temperature (i.e., 5–10 sec at 98°C).

Denaturation at an excessively high temperature or for too long may result in loss of enzyme activity and/or damage to long templates.

Annealing conditions

The annealing step should be adjusted for each primer set; the annealing temperature depends directly on the T_m of primers. Using annealing temperatures that are too low may result in mispriming and nonspecific amplification, leading to low yields of the desired product.

Amplification efficiency and specificity can be improved by adjusting the annealing temperature according to the primer's T_m or by performing two-step PCR.

• For Taq enzymes, the recommended annealing time is 30 sec.
• Enzymes in the PrimeSTAR series have excellent priming efficiency. Therefore, it is important to use a short annealing time of 5–15 sec. Excessively long annealing times may lead to mispriming-induced nonspecific amplification.
• When amplifying short sequences smaller than 1 kb, a three-step PCR protocol is recommended. For GC-rich targets or amplifications of long sequences (>10 kb), a two-step PCR protocol is recommended.

Extension step

In general, an extension time of 1 min/kb is recommended. When using the high-speed enzymes SpeedSTAR HS DNA Polymerase or SapphireAmp Fast PCR Master Mix, use a reaction rate of 10 sec/kb of amplified product (i.e., 10 sec for a 1-kb product, 20 sec for a 2-kb product, etc.).

PrimeSTAR Max DNA Polymerase and PrimeSTAR GXL DNA Polymerase contain a proprietary elongation factor and allow for high-speed reactions at 5–20 sec/kb. If using these enzymes with samples containing excess template, an elongation time of 1 min/kb should be used.

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

If the melting temperature of the primer (T_m) 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).

The optimal amount of template required depends on the complexity of the template and the copy number of the target sequence. Approximately 10⁴ copies of the target DNA sequence are required to detect the amplification product in 25–30 PCR cycles.

• Typically, 1 µg of human genomic DNA contains 3.04 x 10⁵ molecules of DNA. For most PCR applications, 30–100 ng of human genomic DNA is sufficient. High-copy targets, such as housekeeping genes, require only 10 ng of template. Template amounts for higher-complexity templates range between 10 ng and 500 ng.
• Typically, 1 µg of E. coli genomic DNA contains 2 x 10⁸ molecules of DNA; therefore, the recommended amount of template is between 100 pg and 1 ng.
• Typically, 1 µg of lambda DNA contains 1.9 x 10¹⁰ molecules of DNA; therefore, the template input can be as little as 100 pg.
• The amount of cDNA template depends on the copy number of the target. cDNA input is typically described in terms of equivalent RNA input. The amount of cDNA in a PCR reaction can be as little as 10 pg (RNA equivalent).

It is important to note that not all polymerases can tolerate excessive amounts of template. For samples containing excess template (up to 1 µg), we recommend PrimeSTAR GXL DNA Polymerase.

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 (T_m) above 68°C.

PCR polymerases

We offer several PCR polymerases optimized for long-range PCR. Takara LA Taq DNA polymerase, TaKaRa LA Taq Polymerase with GC Buffer, and PrimeSTAR GXL DNA Polymerase are recommended depending on the GC content and size of the target(s).

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.

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 T_m (>68°C), because annealing can occur at a higher temperature.

PCR polymerases

Use a polymerase optimized for amplification of GC-rich sequences. To find an enzyme, visit our selection guide.

We have heard from customers that improved amplification of GC-rich templates was obtained by adding DMSO to reactions using PrimeSTAR MAX DNA Polymerase or CloneAmp HiFi PCR Premix. The recommended concentration of DMSO is between 2.5% and 5%.

Some templates may have long AT-rich stretches that are hard to amplify under standard reaction conditions. The Plasmodium falciparum genome is about 80% AT, and regions flanking genes are often AT rich.

Polymerases recommended for GC-rich templates, such as EmeraldAmp GT PCR Master Mix, EmeraldAmp Max PCR master mixes, and PrimeSTAR GXL DNA Polymerase, are also suitable for AT-rich templates.

The advantage of having AT-rich templates is that a lower extension temperature can be used. For certain templates with AT content >80–85%, the extension temperature can be lowered from 72°C to 65–60°C. DNA replication at this reduced temperature appears to be reliable (Su et al. 1996).

References

Su, X. Z., et al. Reduced Extension Temperatures Required for PCR Amplification of Extremely A+T-rich DNA. Nucl Acids Res. 24, 1574–1575 (1996).

Magnesium is a required cofactor for thermostable DNA polymerases and is important for successful amplification. Without adequate free Mg²⁺, PCR polymerases are not active. In contrast, excess free Mg²⁺ reduces enzyme fidelity and may increase nonspecific amplification. A number of factors can affect the amount of free Mg²⁺ in a reaction, including DNA template concentration, chelating agents in the sample (e.g., EDTA or citrate), dNTP concentration, and the presence of proteins.

• Some polymerases (e.g., Takara Ex Taq DNA polymerases and Takara LA Taq DNA polymerases) are supplied with a magnesium-free reaction buffer and a separate tube of 25 mM MgCl₂. For these enzymes, you can optimize the Mg²⁺ concentration for each reaction.
• Titanium Taq DNA polymerases and Advantage 2 DNA polymerases are magnesium-tolerant polymerases that are supplied with buffers containing 3.5 mM of MgCl₂.
• The final concentration of Mg²⁺ for PrimeSTAR GXL DNA Polymerase and PrimeSTAR MAX DNA Polymerase reactions is 1 mM; this concentration increases fidelity for these enzymes.

Successful PCR requires that the DNA duplex separates during the denaturation step and that primers anneal to the denatured DNA. Salt neutralizes the negative charges on the phosphate backbone of DNA, stabilizing double-stranded DNA by offsetting negative charges that would otherwise repel one another. Potassium chloride (KCl) is normally used in PCR amplifications at a final concentration of 50 mM. To improve amplification of DNA fragments, especially fragments between 100 and 1,000 bp, a KCl concentration of 70–100 mM is recommended. For amplification of longer products, a lower salt concentration appears to be more effective, whereas amplification of shorter products occurs optimally with higher salt concentrations. This effect is likely because high salt concentration preferentially permits denaturation of short DNA molecules over long DNA molecules.

It is important to note that a salt concentration above 50 mM can inhibit Taq polymerases.