If the PCR product needs to be followed by clone expression studies, high-fidelity PCR is needed to reduce the point mutations caused by errors in the PCR process, we usually choose high-fidelity DNA polymerase. The general high-fidelity DNA polymerase is destined to have low efficiency, low yield, weak bands, and slow time due to its own check function. The efficiency of the famous high-fidelity enzyme Pfu is only about 1/5 of that of Taq enzyme. People often sigh "It's expensive and not easy to use (refers to low output)"! In order to improve efficiency, some companies have also introduced mixed enzymes, mixing Taq with DNA polymerase with check function. The result of the mean is that the efficiency is not as good as Taq enzyme, and the fidelity is not as good as Pfu. It is generally three times higher than Taq, and the price is also not cheap.
Finnzymes (now acquired by Thermo Fisher Scientific) has taken a unique approach by fusing double-stranded DNA binding protein (purple) with a new type of thermococcidase (green) with proofreading function to form a unique high Performance polymerase-Phusion® DNA polymerase. At this point, both issues were resolved.
Figure 1. The unique structure of Phusion DNA polymerase
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Due to this unique fusion technology, Phusion DNA polymerase can synthesize DNA at a speed and accuracy that is difficult to match with any single enzyme in the past. Even the most complex template can complete the reaction accurately and quickly. Compared with other enzymes, Phusion DNA polymerase can incorporate more nucleotides in each binding reaction with DNA, and its high processing ability greatly shortens the time of the extension step, thereby speeding up the entire reaction. Its extension speed is 10 times that of Pfu enzyme, which is twice as fast as Taq enzyme, reaching 15-30 seconds / kb. If you want to be faster, you can choose Phusion® Flash high-fidelity polymerase. The extension time per kb base is only 15 seconds or less, the reaction time is the shortest, and the amount of enzyme is significantly lower than other common polymerases. The high speed and efficiency of Phusion DNA polymerase makes it possible to obtain a large number of products in the shortest time.
Finnzymes has done experiments and randomly selected 16 clones from Thermus sp. Genomic library and amplified them with 3 different heat-resistant DNA polymerases. The results show that Phusion® DNA polymerase has strong performance and speed. It can successfully amplify 15 (94%) of 16 randomly selected fragments with high yield. The success rate of Pfu DNA polymerase is only 56%, and the success rate of Taq DNA polymerase is only 62%. The output of the two is significantly lower than that of Phusion® DNA polymerase. Moreover, it can be seen from the figure that the extension time and dosage of Phusion® DNA polymerase are significantly lower than that of Pfu enzyme, which is equivalent to Taq, but the yield is higher. Such strong performance is unmatched.
Figure 2. Phusion DNA polymerase combines ultra-high fidelity, fast and powerful performance
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Due to its unique structure, Phusion DNA polymerase is very efficient. In the PCR reaction, the amount of this enzyme is significantly lower than other common polymerases. Researchers have used three different DNA polymerases to amplify a 3.8kb human beta globulin gene. Using Phusion DNA polymerase can not only successfully amplify this 3.8kb genomic fragment, but the annealing and extension steps only take 1 minute, which is significantly faster than the other two polymerases. The product of one unit of Phusion DNA polymerase is higher than the production of 5 units of Pfu DNA polymerase, which is also much higher than the production of 2.5 units of modified Pfu.
Figure 3. Use less enzyme-get more product
Strong applicability
In addition, Phusion DNA polymerase can tolerate various PCR inhibitors to the utmost extent, so very few amplification steps are required for very efficient amplification. This feature also gave birth to unprecedented direct PCR. For various samples such as blood, tissues, and even FFPE samples, high-fidelity PCR amplification can be carried out without DNA extraction.
Phusion DNA polymerase is available in multiple versions. In addition to the above mentioned Phusion Flash, there is Phusion Hot Start II high-fidelity DNA polymerase. Its high specificity and extremely strong polymerization ability make it an ideal choice for hot start PCR. The characteristics and product forms of several enzymes are as follows:
Phusion high-fidelity DNA polymerase
Phusion Hot Start II High Fidelity DNA Polymerase
Phusion Flash
High-fidelity DNA polymerase
Phire Hot Start II DNA Polymerase
Characteristics of enzymes
Plain end or 3 'A end
Flat end
Flat end
Flat end
Flat end
Target segment length
≤ 20 kb
≤ 20 kb
≤ 20 kb
≤ 7.5 kb
Hot Start
no
Yes
Yes
Yes
Recommended extension time
15-30 s / kb
15-30 s / kb
15 s / kb
10-15 s / kb
Calibration function
Yes
Yes
Yes
general
Fidelity vs. Taq
52x
52x
25x
2x
Product form
Single enzyme 1
√
√
-
√
Master Mix2
√
-
√
-
Kit 3
√
-
-
-
Cheap prices and various options
Phusion DNA polymerase performance is so strong, but the price is quite close to the people. Taking the Phusion high-fidelity PCR kit (including other required reagents in addition to template primers) as an example, the catalog price is only 555 yuan for 125 reactions (20 μl each time), and the single reaction cost is only 4.44 yuan, 1878 yuan. There are 500 reactions, which is simply amazing! This is also a reason why the bio editors strongly recommend it.
Phusion DNA polymerase (containing enzymes, buffers, Mg ions, DMSO) is only 879 yuan / 100U, enough to do 100 50ul reactions. You need to know that the single enzyme (40 reactions) with a price of 100U in the PfuUltra catalog is more than 1900!
Phusion DNA polymerase also offers a variety of other options, hot-start high-fidelity single enzyme (1067 yuan / 100U), as well as premixed enzyme MasterMix series products for GC-rich templates or special HF buffers.
For students who need to perform site-directed mutations for gene function analysis, Thermo Fisher has also launched a 20-site site-directed mutagenesis kit equipped with the ultra-high fidelity enzyme Phusion DNA polymerase. Students who have done this experiment must be very clear that the key to this experiment is a DpnI restriction endonuclease and high-fidelity PCR amplification enzyme. Another brand of this kit can cost 4590 yuan for 10 reactions. This price, hey, only about 1 / 4,20 reactions (with 10 control reactions), the number of reactions is doubled, and the performance of high-fidelity enzymes can be more powerful! However, it is estimated that competent cells are missing.
Less errors
More importantly, Phusion DNA polymerase is the most fidelity thermostable polymerase on the market. Using the improved lacI method, Phusion DNA polymerase has a mismatch rate about 50 times lower than Taq DNA polymerase and 6 times lower than Pfu DNA polymerase, making it ideal for high-fidelity PCR. Phusion DNA polymerase is undoubtedly a better choice for studying gene function, such as site-directed mutation, gene cloning and expression analysis.
The Taq enzyme has a higher probability of error, and the average amplification length does not exceed 3.5 kb. Phusion DNA polymerase can amplify ultra-long fragments of more than 10kb (<20kb), with high fidelity and no error-prone, plus the amplification speed is much faster than ordinary high-fidelity enzyme, so using Phusion DNA polymerase can Effectively save a lot of time.
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