Phusion Plus DNA Polymerase

As a proofreading enzyme with very high fidelity, Phusion Plus DNA Polymerase generates amplicons with very few errors, giving you confidence in PCR sequence accuracy.

Technical note: Measuring fidelity of Phusion Plus DNA Polymerase

Due to its unique buffer formulation, Phusion Plus DNA Polymerase no longer requires a primer melting temperature (T_m ) calculator to determine the annealing temperatures. All targets can be amplified using a universal annealing temperature of 60°C regardless of the calculated primer T_ms .The enzyme also works with the calculated annealing temperatures following the original Phusion protocol.

Figure 2. PCR cycling under two annealing conditions. 12 targets with varying calculated annealing temperatures (indicated above each lane) were amplified from 50 ng of human genomic DNA (gDNA), following a universal annealing temperature of 60°C (left), or the annealing temperatures calculated with the T_m calculator (right). The molecular weight marker is Thermo Scientific ZipRuler Express Ladder DNA 2.

The universal annealing feature of Phusion Plus DNA Polymerase also allows a universal cycling protocol, helping you to circumvent multiple PCR runs and save time. One annealing temperature (60°C) and one extension time based on the longest amplicon can be used for targets of different lengths—i.e., co-cycling different targets on the same block—without compromising PCR yields and specificity.

Figure 3. Different PCR targets can be co-cycled using Phusion Plus DNA polymerase. Five targets of different lengths were amplified from human gDNA using a universal cycling protocol for all targets (up to 7.5 kb), with the extension time of the longest amplicon (3 min 45 sec for 7.5 kb) (left), or following separate cycling protocols with a different extension time calculated for each target (9 sec for 0.3 kb, 21 sec for 0.7 kb, 60 sec for 2 kb, 2 min for 3.9 kb, 3 min 45 sec for 7.5 kb) (right). The molecular weight marker is ZipRuler Express DNA Ladder 2.

Phusion Plus DNA Polymerase improves PCR specificity and yields since its Affibody molecule–mediated hot-start modification prevents enzyme activity and primer degradation during reaction setup.

Phusion Plus DNA Polymerase can detect targets from as little as 0.08 ng of human genomic gDNA.

By combining its fusion protein technology with buffer reformulation, Phusion Plus DNA Polymerase can better tolerate inhibitors from plants (e.g., xylan), soil (e.g., humic acid), and blood (e.g., hemin) during PCR.

Phusion Plus DNA Polymerase includes Phusion GC Enhancer in the package for more efficient amplification of sequences with >65% GC content.

Figure 8. Efficient GC-rich amplification of Phusion Plus DNA Polymerase. Five targets (0.78 kb, 0.74 kb, 0.72 kb, 0.66 kb, and 0.55 kb) with high GC content (their percentages indicated) were amplified from 50 ng of human gDNA using various DNA polymerases according to the manufacturer recommendations for GC-rich PCR. The molecular weight marker is ZipRuler Express Ladder DNA 2.

High processivity of Phusion Plus DNA NA Polymerase enables amplification of long DNA fragments—up to 10 kb from human gDNA and 20 kb from lambda DNA. Its cycling time is considerably short due to a fast extension rate at 15–30 sec/kb.

Figure 9. Amplification ranges of Phusion Plus DNA polymerase. A 9.1 kb target of human gDNA (left) and an 18 kb target of lambda DNA (right) were successfully amplified from two template amounts using Phusion Plus DNA Polymerase. The molecular weight marker is GeneRuler 1 kb Plus DNA Ladder.