Polymerase Chain Reaction (PCR) relies on thermocycling, consisting of repeated cycles of heating and cooling allowing for DNA-primer annealing and enzymatic synthesis of thousands to billions of copies of a target DNA sequence. Since its invention in 1983, PCR has evolved into an assemblage of methodologies widely used in Life Sciences, Biotechnology, Clinical Diagnostics, Forensics and many other fields.

Thermostable DNA Polymerase

PCR employs a thermostable DNA polymerase that is heat resistant and capable of generating new strands of DNA using a template and primers. The first described thermostable DNA polymerase is Taq DNA polymerase isolated from bacterium Thermis aquaticus. However, the lack in 3’ to 5’ exonuclease proofreading activity of Taq results in a high error rate when replicating DNA by PCR. High fidelity PCR is required for many applications, including cloning and sequencing, where sequence accuracy is crucial. It is recommended that low error rate enzymes, such as Pfu DNA polymerase from Pyrococcus furiosus, should be used in order to reduce spurious mutations introduced during PCR. Although it possesses 3’ to 5’ proofreading activity, Pfu exhibits only moderately 5 to 10-fold lower error rate than Taq. To eliminate spurious mutations in particular for long amplicons, a thermostable DNA polymerase with higher fidelity is required.

G&P HiFi™ PCR DNA Polymerase

G&P HiFi™ is a Thermococcus proofreading DNA polymerase engineered for superior fidelity and performance. The engineered PCR enzyme contains amino acid modifications conferring dramatic improvements through screening a large library of enzyme variants. G&P HiFi™ is the most accurate thermostable DNA polymerase, featuring an error rate over 100-fold lower than Taq, making it a superior choice for applications requiring high fidelity. Combined with the proprietary reaction buffers that facilitate specific primer annealing and accurate DNA replication, successful amplification of a wide range of template types and sizes (up to 15 kb) can be achieved using a single enzyme. G&P HiFi™ requires only 10-20 sec per kb extension time per cycle, making it possible to perform PCR with extreme speed. It also produces higher yields with lower enzyme amounts than other common high fidelity enzymes. Overall G&P HiFi™ delivers industry-leading performance with following unique features that no other system can match:

1. HIGHEST FIDELITY

The improved thermostability, proofreading activity and optimized buffer systems of G&P HiFi™ result in superior accuracy for high fidelity PCR applications. The mutation frequency (%) of G&P HiFi™ DNA polymerase is over 100-fold lower than that of Taq, ~15-fold lower than that of Pfu, 4-5-fold lower than that of KOD+, 2-3-fold lower than that of Phusion (see Figure 1 below), making G&P HiFi™ the most accurate PCR enzyme available for high fidelity PCR.

Figure 1. Fidelity Comparison of Thermostable DNA Polymerases.

The fidelity in DNA replication by commonly used DNA polymerases, Taq, Pfu, KOD+, Phusion, and G&P HiFi™ was measured as the mutation frequency (%) in PCR products using a LacI-based fidelity assay.

2. SUPERIOR ROBUSTNESS

Using as a single enzyme, G&P HiFi™ delivers robust performance across a wide range of amplicons from 0.4 to 12.5 kb in size when using only 0.1 to 1 ng of template DNA per reaction. It outperforms two other commonly used high fidelity enzymes, which are engineered by fusion or other technologies (supplier N and supplier T), when amplifying the large target DNA of 12.5 kb (Figure 2). Supplier N’s enzyme generates predominantly non-specific, smaller PCR products, while supplier T’s product works much less efficiently (compare lane #8 with lanes #9 & #10 in Figure 2). All G&P HiFi™ PCR reactions are performed with a consolidated PCR protocol using a single enzyme, eliminating the need for hot-start in most cases (refer to the product details and protocols for more information). G&P HiFi™ is capable of amplifying extremely long amplicons (up to 15 kb), enabling truly high fidelity PCR-based DNA cloning, assembly and mutagenesis by directly amplifying large vectors and DNA inserts.

Figure 2. Superior Amplification Robustness by G&P HiFi™.

G&P HiFi™ was capable of amplifying of a wide range of amplicons from 0.4 to 12.5 kb in size using only a single enzyme. Each PCR product was amplified from a concentration of template DNA of 0.1 to 1 ng per reaction. G&P HiFi™ PCR reactions were performed using a standard 3-step cycling profile (30 cycles): 5-second denaturation, 15-second annealing, and 10-second per kb extension time. Total reaction time for the 12.5 kb amplicon was less than 2 hours. PCR reactions with supplier N’s and supplier T’s DNA polymerases were conducted using the buffers and protocols provided by the manufacturers. 5 µl of each PCR reaction (50 µl total) was loaded on the agarose gel and molecular size markers in kb were indicated left.

3. EXTREME SENSITIVITY

The engineered G&P HiFi™ DNA Polymerase exhibits dramatic improvements in sensitivity and specificity, outperforming two other commercial DNA polymerase from supplier N and T (Figure 3). G&P HiFi™ shows superior performance in amplifying of a template DNA with concentrations as low as 0.1 pg per reaction using two different reaction buffers (HS and HD buffers) supplied with the enzyme. In contrast, one of commercial DNA polymerases (supplier N) yields much less 3.4-kb PCR product and supplier T’s enzyme fails to generate any 3.4-kb PCR product when using 0.1 pg of the template DNA in the PCR reactions according to the manufacturers’ procedures (compare lane #11 with lanes #12 & #13 in Figure 3).

Figure 3. Extrem Sensitivity by G&P HiFi™.

The 3.4-kb target DNA sequences were amplified from a 10-fold dilution series of plasmid DNA template starting from 1 ng down to only 0.1 pg per reaction using G&P HiFi™ DNA polymerase or two commercial enzymes, supplier N and T. All reactions were performed using G&P HiFi standard protocols or manufacturers’ protocols from supplier N and T, consisting of standard 3-step cycling profiles (30 cycles). 5 µl of each PCR reaction (50 µl total) was loaded on the gel and molecular size markers in kb were indicated left.

G&P HiFi™ also delivers superior product yields with minimal enzyme amounts needed. As shown in Figure 4, it is capable of amplifying a DNA template (7.5 kb in size comprising an entire plasmid vector) using only 1/8 amount of G&P HiFi™ that is used in a standard PCR reaction. In contrast, two commercial PCR enzymes (supplier N’s and T’s) perform very poorly, exhibiting either inefficient amplification or failure to produce any 7.5-kb PCR product with predominantly non-specific, smaller products (compare lane #5 with lanes #6 & #7 in Figure 4). The unique condition and formulation of G&P HiFi™ greatly increases PCR amplification sensitivity and specificity, eliminating spurious amplification resulting from non-specific annealing.

Figure 4. High Specificity Amplification with Minimal Amounts of G&P HiFi™.

The 7.5-kb target DNA sequences (i.e., entire plasmid vector) were amplified using a 2-fold dilution series of G&P HiFi™ DNA polymerase from 1 unit (the amount used in a standard reaction) down to 0.125 unit or using two commercial enzymes (supplier N’s and T’s) at 0.125 unit per reaction. All reactions (50 µl each) were performed using G&P HiFi™ standard protocols or manufacturers’ protocols with standard 3-step cycling profiles (30 cycles). 5 µl of each PCR reaction was loaded on the gel and molecular size markers in kb were indicated left.

Featured Products:

G&P HiFi™ PCR Enzyme, Kit and Master Mix

G&P HiFi™ DNA polymerase is supplied with two proprietary buffers (one standard and another one detergent free) for optimal performance and compatibility with downstream applications (SKU#P01301 & #P01302). Both buffers contain magnesium with PCR conditions optimized, consistently delivering excellent efficiency, specificity and sensitivity for high fidelity PCR. We also offer G&P HiFi PCR kit and ready-to-use 2x master mix. G&P HiFi™ PCR kit (SKU#K01301) contains all reagents for high fidelity PCR and optimization by the user. The master mix (SKU#M01303) contains all ingredients for high fidelity PCR, including G&P HiFi™ DNA polymerase, salts, magnesium, dNTPs, stabilizers, and enhancers in an optimized reaction buffer.