PfuTurbo DNA Polymerase

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Introduction

PfuTurbo DNA polymerase is an enhanced version of Pfu DNA polymerase for robust, high-fidelity PCR. PfuTurbo DNA polymerase is a mixture of cloned Pfu DNA polymerase and the exclusive thermostable ArchaeMaxx polymerase-enhancing factor that enhances PCR product yields and increases target length capability without altering DNA replication fidelity. PfuTurbo DNA polymerase can be used to amplify complex genomic DNA targets up to 19 kb and vector targets up to 15 kb in length. In general, PfuTurbo DNA polymerase amplifies complex targets in higher yield than Taq DNA polymerase or other commercially available proofreading PCR enzymes. Like Pfu DNA polymerase, PfuTurbo DNA polymerase has an error rate six-fold lower than Taq DNA polymerase, and significantly lower than the error rates of most other proofreading enzymes or DNA polymerase mixtures. The enhanced performance of PfuTurbo DNA polymerase allows the use of shorter extension times, fewer PCR cycles, and lower DNA template concentrations than are required for Pfu DNA polymerase, making it ideally suited for high-performance PCR applications.

PfuTurbo DNA polymerase provides robust amplification of long, complex genomic targets. A key component of PfuTurbo DNA polymerase is the ArchaeMaxx polymerase-enhancing factor. The ArchaeMaxx factor eliminates a PCR inhibitor and promotes shorter extension times, higher yield, and greater target length capabilities. The ArchaeMaxx factor improves the yield of products by overcoming dUTP poisoning, which is caused by dUTP accumulation during PCR through dCTP deamination. Once incorporated, dU-containing DNA inhibits Pfu and most archaeal proofreading DNA polymerases, such as Vent and Deep Vent DNA polymerases, limiting their efficiency. The ArchaeMaxx factor functions as a dUTPase, converting poisonous dUTP to harmless dUMP and inorganic pyrophosphate, resulting in improved overall PCR performance.

Table I: Comparison of Thermostable DNA Polymerases Using a lacIOZ-Based Fidelity Assay

^a Fidelity is measured using a published PCR forward mutation assay that is based on the lacI target gene.
^b The error rate equals mutation frequency per base pair per duplication.
^c The percentage of mutated PCR products after amplification of a 1-kb target sequence for 20 effective cycles (2²⁰- or 10⁶-fold amplification).

Critical Optimization Parameters for PfuTurbo DNA Polymerase-Based PCR

All PCR amplification reactions require optimization to achieve the highest product yield and specificity. Critical optimization parameters for successful PCR using PfuTurbo DNA polymerase are outlined in Table II and are discussed in the following sections. These parameters include the use of an extension time that is adequate for full-length DNA synthesis, sufficient enzyme concentration, adequate primer-template purity and concentration, optimal primer design, and appropriate nucleotide concentration.

Table II: Optimization Parameters and Suggested Reaction Conditions (50 µl reaction volume)

^a See Primer-Template Purity and Concentration for recommended amounts of other forms of template DNA.

Extension Time

Extension time is one of the most critical parameters affecting the yield of PCR product obtained using PfuTurbo DNA polymerase. For optimal yield, use an extension time of 1.0 minute per kb for vector targets up to 6 kb and genomic targets up to 6 kb. When amplifying vector targets greater than 10 kb or genomic targets greater than 6 kb in length, use an extension time of 2.0 minutes per kb.

Enzyme Concentration

The concentration of PfuTurbo DNA polymerase required for optimal PCR product yield and specificity depends on the individual target system to be amplified. Most amplifications are successful with 2.5 U of enzyme per 50 µl reaction for vector targets up to 19 kb and for genomic targets up to 6 kb.

Primer-Template Purity and Concentration

The most successful PCR results are achieved when the amplification reaction is performed using purified primers and templates that are essentially free of extraneous salts. Gel-purified primers, generally >18 nucleotides in length, are strongly recommended for use in PfuTurbo DNA polymerase-based PCR.

Additionally, an adequate concentration of primers and template should be used to ensure a good yield of the desired PCR products. When DNA of known concentration is available, amounts of 50–100 ng of DNA template per 50-µl reaction are typically used for amplifications of single-copy chromosomal targets. When amplifying genomic targets greater than 6 kb, increase the template amount to 200–250 ng. The amplification of a single-copy target from complex genomic DNA is generally more difficult than amplification of a fragment from a plasmid or phage. Less DNA template can be used for amplification of lambda (1–30 ng) or plasmid (100 pg–10 ng) PCR targets or for amplification of multicopy chromosomal genes (10–100 ng).

Primer Design

We recommend using primers at a final concentration of 0.2–0.5 µM, which is equivalent to ~100–200 ng of an 18- to 25-mer oligonucleotide primer in a 50-µl reaction volume.

Primer pairs that exhibit similar melting temperatures and are completely complementary to the template are recommended. Depending on the primer design and the desired specificity of the PCR amplification reaction, melting temperatures between 55° and 80°C generally yield the best results. The following formula is commonly used for estimating the melting temperature (Tm) of the primers:

Tm (°C) = 2(N_A + N_T) + 4(N_G + N_C)

where N equals the number of primer adenine (A), thymidine (T), guanidine (G), or cytosine (C) bases. Several other articles present additional equations for estimating the melting temperature of primers. Finally, care must be taken when using degenerate primers. Degenerate primers should be designed with the least degeneracy at the 3′ end. Optimization of degenerate primer concentration is necessary.

Note: Because of the unique composition of the Pfu buffer, the actual primer Tm may be 3°–5°C lower than that estimated by this formula.

Deoxynucleoside Triphosphates

For PfuTurbo DNA polymerase-based PCR, use a dNTP concentration range of 100–250 µM each dNTP (0.4–1.0 mM total) for optimal product yield. Supplying dNTPs in this concentration range generally results in the optimal balance of product yield (greatest at high dNTP concentrations) versus specificity and fidelity (highest at low dNTP concentration). The yield produced from genomic targets >6 kb in length can be improved by increasing nucleotide concentration to 500 µM (each dNTP) and the reaction buffer to 1.5× final concentration. The use of a balanced pool of dNTPs (equimolar amounts of each dNTP) ensures the lowest rate of misincorporation errors.

Reaction Buffer

The reaction buffer provided with this enzyme has been formulated for optimal PCR yield and fidelity when performing PCR amplification using PfuTurbo DNA polymerase. Use the reaction buffer provided with PfuTurbo DNA polymerase in standard PCR amplification reactions. If alterations in these buffers are made, significant increases in the error rate of Pfu DNA polymerase can be avoided by maintaining the Mg²⁺ concentration above 1.5 mM, the total dNTP concentration at 0.4–1.0 mM, and the pH of Tris-based buffers above pH 8.0 when measured at 25°C.

To improve yields of genomic targets >6 kb, increase the final concentration of reaction buffer from 1× to 1.5×.

PCR Cycling Parameters

Standard PCR amplification reactions typically require 25–30 cycles to obtain a high yield of PCR product. Thermal cycling parameters should be chosen carefully to ensure (1) the shortest denaturation times to avoid template damage, (2) adequate extension times to achieve full-length target synthesis, and (3) the use of annealing temperatures near the primer melting temperature to improve yield of the desired PCR product.

When performing PCR on a new target system, we suggest using an annealing temperature 5°C below the lowest primer melting temperature.

For best results, PCR primers should be designed with similar melting temperatures ranging from 55° to 80°C. The use of primers with melting temperatures within this range reduces false priming and ensures complete denaturation of unextended primers at 94–95°C (see also Primer-Template Purity and Concentration and Primer Design).

See Table IV for suggested PCR cycling parameters, depending upon template size and complexity. Optimized cycling parameters are not necessarily transferable between thermal cyclers designed by different manufacturers. Therefore, each manufacturer's recommendations for optimal cycling parameters should be consulted.

Amplification of Genomic Targets >6 kb

To improve yields of genomic targets >6 kb, increase the amount of PfuTurbo DNA polymerase from 2.5 U to 5.0 U, and increase the final concentration of reaction buffer from 1× to 1.5×. Use 200–250 ng of genomic template DNA, 200 ng of each primer, and 500 µM each dNTP. Use a denaturing temperature of 92°C, an extension temperature of 68°C, and an extension time of 2.0 minutes per kilobase.

Additional Optimization Parameters

Order of Addition of Reaction Mixture Components

Because PfuTurbo DNA polymerase exhibits 3′- to 5′-exonuclease activity that enables the polymerase to proofread nucleotide misincorporation errors, it is critical that the enzyme is the last component added to the PCR mixture (i.e., after the dNTPs). In the absence of dNTPs, the 3′- to 5′-exonuclease activity of proofreading DNA polymerases may degrade primers. When primers and nucleotides are present in the reaction mixture at recommended levels (i.e., primer concentrations of 0.2–0.5 µM and nucleotide concentrations of 100–250 µM each dNTP), primer degradation is not a concern.

Magnesium Ion Concentration

Magnesium ion concentration affects primer annealing and template denaturation, as well as enzyme activity and fidelity. Generally, excess Mg²⁺ results in accumulation of nonspecific amplification products, whereas insufficient Mg²⁺ results in reduced yield of the desired PCR product. PCR amplification reactions should contain free Mg²⁺ in excess of the total dNTP concentration. For PfuTurbo DNA polymerase-based PCR, yield is optimal when the total Mg²⁺ concentration is ~2 mM in a standard reaction mixture, and ~3 mM for amplification of cDNA. A 2 mM total Mg²⁺ concentration is present in the final 1× dilution of the 10× cloned Pfu DNA polymerase reaction buffer. For the amplification of cDNA, Mg²⁺ should be added to the PCR reaction to a final concentration of 3 mM.

Adjuncts and Cosolvents

The adjuncts or cosolvents listed in the following table may be advantageous with respect to yield when used in the PCR buffer. Fidelity may or may not be affected by the presence of these adjuncts or cosolvents.

Formamide

Formamide facilitates certain primer-template annealing reactions and also lowers the denaturing temperature of melt-resistant DNA.

Dimethylsulfoxide and Glycerol

Cosolvents, such as DMSO and glycerol, improve the denaturation of GC-rich DNA and help overcome the difficulties of polymerase extension through secondary structures. Studies indicate that the presence of 1–10% DMSO in PCR may be essential for the amplification of the retinoblastoma gene and may also enhance amplification of Herpes simplex virus (HSV) sequences. Glycerol is known to improve the yield of amplification products and also serves as an enzyme stabilizer.

Perfect Match PCR Enhancer

Perfect Match PCR enhancer improves the specificity of PCR products. This adjunct performs this function by destabilizing mismatched primer-template complexes and by helping to remove secondary structures that could impede normal extension.

Application Notes

Thermostability

Pfu DNA polymerase is a highly thermostable enzyme, retaining 94–99% of its polymerase activity after 1 hour at 95°C. Unlike Taq DNA polymerase, denaturing temperatures up to 98°C can be used successfully with PfuTurbo DNA polymerase to amplify GC-rich regions.

Inherent "Hot Start" Properties

PfuTurbo DNA polymerase exhibits optimal polymerase activity at ≥75°C and only 2–8% activity between 40–50°C. Taq DNA polymerase, however, exhibits optimal polymerase activity between 60–70°C and 27–70% activity between 40–50°C. The minimal activity of PfuTurbo DNA polymerase at lower temperatures should result in fewer mispaired primer-extension reactions than occur with Taq DNA polymerase during the lower temperatures encountered during PCR cycling (e.g., primer annealing).

Consequently, hot start techniques, which are commonly used with Taq DNA polymerase to improve product yield and specificity, generally are not required for PCR amplifications with PfuTurbo DNA polymerase.

However, PfuTurbo Hotstart DNA polymerase is also available. PfuTurbo Hotstart DNA polymerase provides improved specificity when amplifying systems prone to primer-dimer formation or when performing robotic PCR applications that require prolonged incubations at room temperature prior to temperature cycling.

Terminal Transferase Activity

Studies demonstrate that thermostable DNA polymerases, with the exception of Pfu DNA polymerase, exhibit terminal deoxynucleotidyltransferase (TdT) activity, which is characterized by the addition of nontemplate-directed nucleotide(s) at the 3′ end of PCR-generated fragments. PfuTurbo DNA polymerase, like Pfu polymerase, is devoid of TdT activity and generates blunt-ended PCR products exclusively. Therefore, PfuTurbo DNA polymerase is optimal for use with the PCR-Script Amp SK(+) cloning kit and the PCR-Script Cam SK(+) cloning kit.

In addition, PfuTurbo DNA polymerase can be used to remove 3′ overhangs (polishing) or to fill-in 5′ overhangs with greater efficiencies than either Klenow polymerase or T4 DNA polymerase.

Reverse Transcriptase Activity

PfuTurbo DNA polymerase lacks detectable reverse transcriptase activity.

PCR Protocol for PfuTurbo DNA Polymerase

1. Prepare a reaction mixture for the appropriate number of samples to be amplified. Add the components in order while mixing gently. Table III provides an example of a reaction mixture for the amplification of a typical single-copy chromosomal target. The recipe listed in Table III is for one reaction and must be adjusted for multiple samples. The final volume of each sample reaction is 50 µl.

Table III: Reaction Mixture for a Typical Single-Copy Chromosomal Locus PCR Amplification

^a The 10× buffer provides a final 1× Mg²⁺ concentration of 2 mM. To amplify cDNA, Mg²⁺ may need to be added to a final 1× concentration of 3 mM.
^b The amount of DNA template required varies depending on the type of DNA being amplified. Generally 50–100 ng of genomic DNA template is recommended. Less DNA template (typically 0.1–30 ng) can be used for amplification of lambda or plasmid PCR targets or 10–100 ng for amplification of multicopy chromosomal genes.
^c Primer concentrations between 0.2 and 0.5 µM are recommended (this corresponds to 100–250 ng for typical 18- to 25-mer oligonucleotide primers in a 50-µl reaction volume).
^d The amount of PfuTurbo DNA polymerase varies depending on the length of the template to be amplified. The standard amount for vector targets up to 19 kb and genomic targets up to 6 kb in length is 1 µl (2.5 U).

2. Immediately before thermal cycling, aliquot 50 µl of the reaction mixture into the appropriate number of reaction tubes.

3. Perform PCR using optimized cycling conditions. Suggested cycling parameters are indicated in Table IV (see also PCR Cycling Parameters).

4. Analyze the PCR amplification products on a 0.7–1.0% (w/v) agarose gel.

Table IV: PCR Cycling Parameters for PfuTurbo DNA Polymerase with Single-Block Temperature Cyclers

A. Targets <10 kb (vector DNA) or <6 kb (genomic DNA)

B. Targets >10 kb (vector DNA) or >6 kb (genomic DNA)

^a Optimized cycling parameters are not necessarily transferable between thermal cyclers designed by different manufacturers; therefore, each manufacturer's recommendations for optimal cycling parameters should be consulted.
^b Denaturing temperatures above 95°C are recommended only for GC-rich templates.
^c The annealing temperature may require optimization. Typically annealing temperatures will range between 55° and 72°C.
^d The annealing temperature may require optimization. Typical annealing temperatures will range between 60 and 65°C.

Troubleshooting

Preparation of Media and Reagents

10× Cloned Pfu DNA Polymerase Reaction Buffer

200 mM Tris-HCl (pH 8.8)
20 mM MgSO₄
100 mM KCl
100 mM (NH₄)₂SO₄
1% Triton X-100
1 mg/ml nuclease-free BSA

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MSDS Information

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