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PyroPol® HiFi DNA polymerase
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PyroPol®  HiFi DNA polymerase

HiFi DNA Polymerase
After multiple rounds of protein engineering and selection to achieve superior performance, NovoHelix introduces PyroPol HiFi DNA polymerase. Our team enriched a mega-library of natural and synthetic DNA polymerase variants to select for those candidates that exhibited enzymatic superiority by evaluating for the following phenotypes:

[1] enhanced processivity for amplification of long DNA targets, i.e. ~20-kb, and of notoriously challenging genomic sequences such as cruciforms, repetitive structures, inverted terminal repeats, direct repeats, short tandem repeats, high GC-content such as the 5’ arm of Rosa26, hairpins, and pro-telomeres;
[2] extreme thermostability at elevated thermocycling temperatures;
[3] ultra-low error rates, high fidelity and elevated proofreading activity (3’->5’ exonuclease activity) to reduce mutational load 100-fold relative to Taq DNA polymerase;
[4] absence of terminal DNA transferase (TdT) activity for blunt product cloning;
[5] increased speed and lack of strand displacement activity;
[6] direct amplification of DNA in crude samples from human or animal tissues, from microbiological colonies, or from the environment like archeological site sediment containing complex mixtures of inhibitors.

So use a formidable molecular workhorse that performs well in almost every wet bench application: PyroPol HiFi DNA polymerase!

Product

Catalog Nr

Size

Concentration

Pricing

PyroPol®  HiFi DNA polymerase 
P2625S
100 Units
2.5 U/μl
PyroPol®  HiFi DNA polymerase 
P2625M
500 Units
2.5 U/μl
PyroPol®  HiFi DNA polymerase 
P2625L
1,000 Units
2.5 U/μl
PyroPol®  HiFi DNA polymerase 
P2625X
2,500 Units
2.5 U/μl
PyroPol®  HiFi DNA polymerase 
P2625B
bulk
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Product Information
Product Literature & Protocol
FAQs & Troubleshooting
References
Supporting Documents
Product Information
Applications:
    • High-fidelity PCR and primer-extension reactions
    • Generation of blunt-end PCR products for cloning and expression
    • Single-cell blastomere PCR for genotyping mammalian preimplantation embryos
    • RT-PCR for cDNA cloning and expression
    • Site-directed mutagenesis
    • Long-range junction PCR to validate CRISPR gene-edited loci


Source:
PyroPol HiFi DNA polymerase was sourced from a hyperthermophilic archaeon and improved through combinatorial protein engineering efforts such as directed evolution and rational design.  The final engineered protein was synthesized as a codon-optimized minigene, was expressed heterologously in a chassis such as Escherichia coli or Vibrio natriegens with the assistance of chaperone proteins and was purified by proprietary chromatographic techniques. This product is manufactured in the USA using chemically-defined components and, therefore, does not contain manufacturing components such as serum or animal-derived raw materials.  PyroPol HiFi DNA polymerase is suitable for sensitive applications such as single-cell blastomere PCR or long-range junction PCR to validate CRISPR-gene-edited loci.

Unit Definition:
One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmol of dNTPs into acid insoluble material in 30 minutes at 72°C under standard DNA polymerase assay conditions.

Residual Nuclease Activity: 
PyroPol HiFi DNA polymerase is free from detectable RNase A, endonuclease (nicking) and non-specific DNase activities as determined by fluorometric assays using cleavable fluorescent-labeled substrates to be less than the limit of quantitation ≤ 0.3 pg/ml. 

Supplied With:

10x PyroPol®HiFi DNA polymerase Reaction Buffer
200 mM Tris pH 8.8
20 mM MgSO
100 mM KCl
80 mM (NH)SO
1% Triton X-100
0.5% IGEPAL CA-630
1 mg/ml rHSA  (recombinant human serum albumin)

Supplied In:
20 mM Tris-HCl (pH 8.0)
40 mM NaCl
0.1 mM EDTA
1 mM TCEP
50% (v/v) glycerol

Heat Inactivation:
This polymerase is extremely thermostabile, and unable to be heat-inactivated. PyroPol HiFi DNA polymerase half-life is ~8 h at 95°C, ~3 h at 98°C.

Recommended Reaction Conditions:
1X PyroPol® buffer, 200 µM each dNTP, 0.1-0.5µM each primer, 5 units PyroPol® DNA polymerase enzyme, 1-100 ng plasmid template DNA, or 100-250 ng genomic template DNA.

Recommended Storage Condition:  -20ºC

Storage/Stability:  Store PyroPol HiFi DNA polymerase, 10x PCR Buffer with Mg2+, 5x GC-enhancer, and dNTPs (if supplied) at -20°C.  Use aseptic technique and work in a PCR workstation to avoid contamination of reagents.

These products may be shipped at room temperature or on blue ice and should be stored immediately upon arrival at -20°C. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt. Approximately 30 minutes before use, thaw the product and its components in an ice bucket and mix thoroughly by gently vortexing at a low speed. After thawing, these products should be kept on ice or a 4˚C cold block before use. 

Avoid repeated freeze-thaw cycles by aliquoting to reduce the loss of enzymatic activity. Thawed aliquots are stable for 1-week if stored at 2-8˚ C. 

Disclaimer & Precautions: 
This product is solely for research and development use only and may be subject to conditional use and licensing restrictions. The product shall not be used as an advanced pharmaceutical intermediate (API) or investigational drug or a biologic. This product is not intended to be used as a therapeutic agent or facilitate clinical diagnosis or be used as an in vitro diagnostic (IVD) product. 

The Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER) define an IVD as: 
“In vitro diagnostic products are those reagents, instruments, and systems intended for use in the diagnosis of disease or other conditions, including a determination of the state of health, in order to cure, mitigate, treat, or prevent disease or its sequelae. Such products are intended for use in the collection, preparation, and examination of specimens taken from the human body. These products are devices as defined in section 201(h)of the Federal Food, Drug, and Cosmetic Act (the act), and may also be biological products subject to section 351 of the Public Health Service Act. Title 21, Code of Federal Regulations (CFR), section 809.3(a).” 

This product shall not be used or formulated in any agricultural, pesticidal, veterinary or animal products, food additives or household chemicals or any other unspecified use.  Please consult the Safety Data Sheet for information regarding hazards and safe handling practices. NovoHelix distributes products for basic and translational research use only. NovoHelix will report any unspecified use to respective regulatory authorities for enforcement to ensure safeguarding of our research products from potential abuse.

Notice to purchaser: 
The purchase price of this product includes a limited, non-transferable license under U.S. and foreign patents or applications owned by NovoHelix to use this product. No other license under these patents or applications is conveyed expressly or by implication by purchase of this product.
Product Literature & Protocol

1.  Please note that protocols with PyroPol®  HiFi DNA polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.


Reaction Setup:
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use. PyroPol®  HiFi DNA polymerase may be diluted in 1X PyroPol Reaction Buffer just prior to use in order to reduce pipetting errors.

COMPONENT

25 µl REACTION

50 µl REACTION

FINAL CONCENTRATION

5X PyroPol
Reaction Buffer
5 µl
10 µl
200 µM
10 mM dNTPs
0.5 µl
1 µl
200 µM
10 µM Forward Primer
1.25 µl
2.5 µl
0.5 µM
10 µM Reverse Primer
1.25 µl
2.5 µl
0.5 µM
Template DNA
variable
variable
< 1,000 ng
PyroPol®  HiFi DNA polymerase
0.25 µl
0.5 µl
0.02 U/µl
5X PyroPol High GC Enhancer (optional)
(5 µl)
(10 µl)
(1X)
Nuclease-Free Water
to 25 µl
to 50 µl

2.  Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.


Transfer PCR tubes to a PCR machine and begin thermocycling.


Thermocycling Conditions for a Routine PCR:

STEP

TEMP

TIME

Initial Denaturation
98°C
30 seconds
25–35 Cycles
98°C
*50–72°C
72°C
5–10 seconds
10–30 seconds
20–30 seconds/kb
Final Extension
72°C
2 minutes
Hold
4–10°C
3.  *Use of the Tm+3 is highly recommended.



4.  General Guidelines:


Template:
Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:

STEP

TEMP

DNA Genomic
1 ng–1 µg
Plasmid or Viral
1 pg–10 ng
5.  Primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.

6.  Mg++ and additives:
Mg++ concentration of 2.0 mM is optimal for most PCR products generated with PyroPol®  HiFi DNA polymerase. When used at a final concentration of 1X, the PyroPol Reaction Buffer provides the optimal Mg++ concentration.

Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1X PyroPol High GC Enhancer. The PyroPol High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the PyroPol Reaction Buffer when other conditions have failed.

7.  Deoxynucleotides:
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. PyroPol®  HiFi DNA polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.

8.  PyroPol®  HiFi DNA polymerase concentration:
We generally recommend using PyroPol®  HiFi DNA polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of PyroPol®  HiFi DNA polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 μl reaction, especially for amplicons longer than 5 kb.

9.  Buffers:
The 5X PyroPol Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the PyroPol High GC Enhancer can improve reaction performance. The 5X PyroPol Reaction Buffer is detergent-free and contains 2.0 mM Mg++ at the final (1X) concentration.

10.  Denaturation:
An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.

During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.

11.  Annealing:
Optimal annealing temperatures for PyroPol®  HiFi DNA polymerase tend to be higher than for other PCR polymerases. The Tm+3 should be used to determine the annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step at 3°C above the Tm of the lower Tm primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.

For high Tm primer pairs, two-step cycling without a separate annealing step can be used (see note 12).

12.  Extension:
The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.

A final extension of 2 minutes at 72°C is recommended.

13.  Cycle number:
Generally, 25–35 cycles yield sufficient product.  For genomic amplicons, 30-35 cycles are recommended.

14.  2-step PCR:
When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.

15.  Amplification of long products:
When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.

16.  PCR product:
The PCR products generated using PyroPol®  HiFi DNA polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as PyroPol®  HiFi DNA polymerase will degrade any overhangs generated.
FAQs & Troubleshooting


References
1: Fogg MJ, Pearl LH, Connolly BA. Structural basis for uracil recognition by archaeal family B DNA polymerases. Nat Struct Biol. 2002 Dec;9(12):922-7. PubMed PMID: 12415291.


1: Connolly BA, Fogg MJ, Shuttleworth G, Wilson BT. Uracil recognition by archaeal family B DNA polymerases. Biochem Soc Trans. 2003 Jun;31(Pt 3):699-702. PubMed PMID: 12773186.


1: Śpibida M, Krawczyk B, Olszewski M, Kur J. Modified DNA polymerases for PCR troubleshooting. J Appl Genet. 2017 Feb;58(1):133-142. doi: 10.1007/s13353-016-0371-4. Epub 2016 Oct 28. Review. PubMed PMID: 27796943; PubMed Central PMCID: PMC5243913.


1: Lahr DJ, Katz LA. Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase. Biotechniques. 2009 Oct;47(4):857-66. doi: 10.2144/000113219. PubMed PMID: 19852769.


1: Packer MS, Liu DR. Methods for the directed evolution of proteins. Nat Rev Genet. 2015 Jul;16(7):379-94. doi: 10.1038/nrg3927. Epub 2015 Jun 9. Review. PubMed PMID: 26055155.
Supporting Documents

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