Product Class: Other

Q5U® Hot Start High-Fidelity DNA Polymerase

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 (NEB #N0459) and Uracil DNA Glycosylase (UDG) (NEB #M0372) 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
  • Reads and incorporates uracil and inosine bases

 

 

 

Catalog # Size Concentration
M0515S 100.0 units 2000 units/ml
M0515L 500.0 units 2000 units/ml

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 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.)

This product is related to the following categories:
Analysis of DNA Methylation by Bisulfite Conversion,
DNA Methylation Analysis,
Q5® High-Fidelity DNA Polymerases Products,
PCR, qPCR & Amplification Technologies Products,
This product can be used in the following applications:
Applications of USER® and Thermolabile USER II Enzymes,
Bisulfite Sequencing,
USER® Cloning,
DNA Methylation Analysis,
PCR,
Epigenetics,
High-Fidelity PCR,
Multiplex PCR,
DNA Amplification, PCR & qPCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0515S     -20    
      Q5U® Hot Start High-Fidelity DNA Polymerase M0515SVIAL -20 1 x 0.05 ml 2,000 units/ml
      Q5U®  Reaction Buffer B9037SVIAL -20 1 x 1.5 ml 5 X
  • M0515L     -20    
      Q5U® Hot Start High-Fidelity DNA Polymerase M0515LVIAL -20 1 x 0.25 ml 2,000 units/ml
      Q5U®  Reaction Buffer B9037SVIAL -20 4 x 1.5 ml 5 X

Properties & Usage

Protocols, Manuals & Usage

Protocols

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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. How do I enable carryover contamination prevention with Q5U?
  2. Can I use Q5U Hot Start High-Fidelity DNA Polymerase to amplify bisulfite-treated DNA? 
  3. What are the general recommendations for designing primers for bisulfite-treated/deaminated DNA?
  4. Can I use Q5U Hot Start High-Fidelity DNA Polymerase for USER® cloning methods?
  5. Can I use Q5U Hot Start High-Fidelity DNA Polymerase to amplify FFPE DNA? 
  6. What ends will my PCR products have?
  7. What are Hot Start and WarmStart® polymerases and when would I use them?
  8. How should I determine the appropriate annealing temperature for my reaction?
  9. What are the properties of this polymerase (fidelity, product ends, max amplicon, modified base incorporation, etc.)?
  10. My results are not as expected. Where can I find troubleshooting help?
  11. What are the differences between the numerous Q5® Polymerase products available?
  12. When and how should I use the Q5® High GC Enhancer?
  13. Can I use Q5 Reaction Buffer with this enzyme?
  14. How can I optimize my product yield using Q5U Hot Start High-Fidelity DNA Polymerase?

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

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

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.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

This product is licensed for research and commercial use 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. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

This product is licensed from University of Newcastle Upon Tyne under EPO Patent No. 1463809B1 and Australian Patent No. AU2003226543.    

Nucleic acid-based aptamers for use with thermophilic DNA polymerases are licensed exclusively by New England Biolabs, Inc. from SomaLogic, Inc. New England Biolabs, Inc. gives the Buyer/User a non-exclusive license to use the Q5U® Hot Start High-Fidelity DNA Polymerase for Research Use Only (RUO). Commercial use of this product may require a license from New England Biolabs, Inc. For additional information or to inquire about commercial use, please contact busdev@neb.com.

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