Product Class: Other

Q5U®  Hot Start High-Fidelity DNA Polymerase
Item# Description List Price Web Price Qty
M0515L Q5U Hot Start High-Fidelity DNA Polymerase - 500 units $925.00
$832.50
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M0515S Q5U Hot Start High-Fidelity DNA Polymerase - 100 units $239.00
$215.10
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

Product Introduction

Q5U Hot Start High-Fidelity DNA Polymerase is a modified version of Q5® High-Fidelity DNA Polymerase, a novel thermostable DNA polymerase that possesses 3′ to 5′ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases.

  • Achieve superior amplification of bisulfite-converted, deaminated, or damaged DNA (e.g., FFPE)
  • Prevent carryover contamination when combined with dUTP and Uracil DNA Glycosylase (UDG) treatment
  • Generate higher assembly efficiency and improve accuracy in USER® cloning
  • Enable room temperature setup with aptamer-based hot start formulation
  • Utilize optimized protocols for recommended applications
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Product Information

Description

Many proofreading polymerases derived from archaeal Family B DNA polymerases stall replication in response to uracil bases in DNA templates (Wardle, J. et al. (2008) Nucleic Acids Res. 36 (3), 705-711). Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases. This feature is useful for amplifying bisulfite-converted, deaminated, or damaged DNA, preventing carryover contamination in PCR (when used with dUTP and UDG), and in USER cloning methods.

Q5U is a hot start polymerase containing a unique aptamer selected for polymerase inhibition at room temperature and optimal amplification during typical PCR conditions.

 

Figure 1: Common applications enabled by Q5U Hot Start High-Fidelity DNA Polymerase



Archaeal family B-type polymerases can incorporate/tolerate a variety of modified nucleotides but will stall upon encountering uracil and inosine residues. Q5U Hot Start High-Fidelity DNA Polymerase is a modified Q5 High-Fidelity DNA polymerase which efficiently incorporates dUTP and amplifies uracil-containing templates. Common applications enabled by Q5U Hot Start High-Fidelity DNA Polymerase are illustrated above.

 

Figure 2: Q5U Hot Start High-Fidelity DNA Polymerase provides superior amplification of bisulfite-converted DNA



Amplification of bisulfite-converted human genomic DNA targets using Q5U Hot Start High-Fidelity DNA Polymerase (Q5U), Phusion U Hot Start DNA Polymerase (P) and KAPA Hifi HotStart Uracil+ ReadyMix PCR Kit (K). Amplicon name and sizes are indicated above the gel. Each 25 μl reaction contained 12 ng of bisulfite-converted DNA (Qiagen). All reactions were conducted according to manufacturer’s recommendations using 35 cycles and visualized by microfluidic LabChip® analysis.

 

Figure 3: Q5U Hot Start High-Fidelity DNA Polymerase enables robust amplification of FFPE normal lung DNA



Amplification of FFPE normal lung DNA (Biochain). Amplicon sizes are indicated above the gel. Each 25 μl reaction contained 18 ng of FFPE DNA (Biochain). Cycling conditions were consistent with recommendations for this sample type and visualized by microfluidic LabChip analysis.

 

Figure 4: Q5U Hot Start High-Fidelity DNA Polymerase enables robust amplification of enzymatically deaminated DNA



Amplification of enzymatically deaminated (APOBEC) genomic DNA targets using Q5U Hot Start High-Fidelity DNA Polymerase (Q5U), Phusion U Hot Start DNA Polymerase (P) and KAPA Hifi HotStart Uracil+ ReadyMix PCR Kit (K). Amplicon name and sizes are indicated above the gel. Each 50 μl reaction contained 12 ng of enzymatically deaminated DNA. All reactions were conducted according to manufacturer’s recommendations using 35 cycles and visualized by microfluidic LabChip analysis.

 

Figure 5: USER® cloning workflow



In USER cloning, target DNA molecules and cloning vectors are generated by PCR with 8–12 bases of homology between two fragments. PCR primers start with a 5′ A and contain a single deoxyuracil residue (dU) flanking the 3′ end of the homology region, and can be designed to accommodate multiple-fragment assembly, nucleotide substitutions, insertions and/or deletions. We recommend using the GeneDesign (https://genedesign. thruhere.net/gd/) software to design primers for USER junctions. The best results are typically seen when using each primer at a final concentration of 0.5 μM.

 

Figure 6: Q5U Hot Start High-Fidelity DNA Polymerase enables highly efficient and accurate USER cloning as compared to Hot Start Taq DNA Polymerase



USER cloning was performed to ligate a 3 kb lacZ gene into a pET21a vector backbone with Q5U Hot Start High-Fidelity DNA Polymerase and Hot Start Taq DNA polymerase. Reactions containing Q5U resulted in a higher assembly efficiency and better accuracy as evidenced by the total number of colonies obtained (A) and percentage of blue colonies (B). Accuracy was further examined using Sanger sequencing which revealed a number of point mutations in the blue colonies obtained from the Taq assembly and no mutations from the 4 clones sequenced from the Q5U experiments (C). The “X”s indicate the number of additional mutations found in the clones produced with Taq, consistent with previous results demonstrating the high mutation tolerance of lacZ. (Barnes, W. M. (1992) Gene, 112, 29-35.)

Properties & Usage

    Storage Temperature

    -20°C

    Protocols, Manuals & Usage

    Protocols

    1. PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515)

    Tools & Resources

    Web Tools

    FAQs & Troubleshooting

    FAQs

    1. What is the difference between Q5U Hot Start High-Fidelity DNA Polymerase and NEBNext® Q5U Master Mix?
    2. How do I determine the appropriate annealing temperature for my reaction?
    3. Will Q5U Hot Start High-Fidelity DNA Polymerase incorporate dUTP?
    4. Can I use Q5U Hot Start High-Fidelity DNA Polymerase to amplify bisulfite-treated DNA? 
    5. What are the general recommendations for designing primers for bisulfite-treated/deaminated DNA?
    6. Can I use Q5U Hot Start High-Fidelity DNA Polymerase for USER® cloning methods?
    7. Can I use Q5U Hot Start High-Fidelity DNA Polymerase to amplify FFPE DNA? 
    8. Does Q5U amplify unmodified DNA?
    9. What length of product can be made by Q5U Hot Start High-Fidelity DNA Polymerase?
    10. Will adding the GC enhancer help?
    11. Can I use Q5 Reaction Buffer with this enzyme?
    12. How can I optimize my product yield using Q5U Hot Start High-Fidelity DNA Polymerase?
    13. Does Q5U Hot Start High-Fidelity DNA Polymerase exhibit a strand displacement activity?
    14. Are the DNA fragments produced by Q5U Hot Start High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3′ overhang that Taq DNA Polymerase yields?
    15. Where can I find help troubleshooting my PCR?

    Troubleshooting

    Quality, Safety & Legal

    Quality Assurance Statement

    Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    Certificate Of Analysis

    The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

    Safety DataSheets

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Legal and Disclaimers

    This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

    While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

    For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].

    This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

    Licenses

    This product is licensed from University of Newcastle Upon Tyne under EPO Patent No. 1463809B1 and Australian Patent No. AU2003226543.    This product is licensed from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645 and corresponding patents in other countries for use only in: (a) standard (not real-time) PCR in the research field only, but not real time PCR or digital PCR; (b) real-time PCR for use as a library preparation quantitation tool in Next Generation Sequencing workflows; (c) any in-vitro diagnostics applications, except for applications using real-time PCR or digital PCR; and (d) any non-PCR applications in DNA sequencing, isothermal amplification, and the production of synthetic DNA.  Notice to Purchaser:  Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. (See Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,874,557; and 5,693,502).   New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the aptamer-based Q5U Hot Start High-Fidelity DNA Polymerase for RESEARCH PURPOSES ONLY. Commercial use of the aptamer-based Q5U Hot Start High-Fidelity DNA Polymerase requires a license from New England Biolabs, Inc. Please contact [email protected] for more information.