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Q5® High-Fidelity DNA Polymerases


Comparison of High-Fidelity Polymerases

1 PCR-based mutation screening in lacZ (NEB), lacI (Agilent) or rpsL (Life)
2 Due to the very low frequency of misincorporation events being measured, the error rate of high-fidelity enzymes like Q5 is difficult to measure in a statistically significant manner. Although measurements from assays done side-by-side with Taq yield a Q5 fidelity values of approximately 200X Taq, we report “>100X Taq” as a conservative value.
3 Takagi et al (1997) Appl. Env. Microbiol. 63, 4504-4510.

Advantages

  • Highest fidelity amplification (>100X higher than Taq)
  • Ultra-low error rates
  • Superior performance for a broad range of amplicons (from high AT to high GC)
  • Hot start and master mix formats available

The Q5 buffer system is designed to provide superior performance with minimal optimization across a broad range of amplicons, regardless of GC content. For routine or complex amplicons up to ~65% GC content, Q5 Reaction Buffer (NEB #B9027) provides reliable and robust amplification. For amplicons with high GC content (>65% GC), addition of the Q5 High GC Enhancer ensures continued maximum performance. Q5 and Q5 Hot Start DNA Polymerases are available as standalone enzymes, or in a master mix format for added convenience. Master mix formulations include dNTPs, Mg++ and all necessary buffer components.

Robust Amplification with Q5 (A) and Q5 Hot Start (B) High-Fidelity DNA Polymerases

Amplification of a variety of human genomic amplicons from low to high GC content using either Q5 or Q5 Hot Start High-Fidelity DNA Polymerase. Reactions using Q5 Hot Start were set up at room temperature. All reactions were conducted using 30 cycles of amplification and visualized by microfluidic LabChip® analysis.

In contrast to chemically modified or antibody-based hot start polymerases, NEB's Q5 Hot Start (NEB #M0493) utilizes a unique synthetic aptamer. This molecule binds to the polymerase through non-covalent interactions, blocking activity during the reaction setup. The polymerase is activated during normal cycling conditions, allowing reactions to be set up at room temperature. Q5 Hot Start does not require a separate high temperature activation step, shortening reaction times and increasing ease-of-use. Q5 Hot Start Polymerase is an ideal choice for high specificity amplification and provides robust amplification of a wide variety of amplicons, regardless of GC content.

Amplification Performance Across a Wide Range of Genomic Targets

PCR was performed with a variety of amplicons, with GC content ranging from high AT to high GC, with Q5 and several other commercially available polymerases. All polymerases were cycled according to manufacturer's recommendations, including use of GC Buffers and enhancers when recommended. Yield and purity of reaction products were quantitated and represented, as shown in the figure key, by dot color and size. A large dark green dot represents the most successful performance. Q5 provides superior performance across the range of GC content.

Master Mix and Stand-Alone Formats Provide Convenience and Flexibility

Q5® is a registered trademark of New England Biolabs, Inc.
LabChip® is a registered trademark of Caliper Life Sciences, part of Perkin Elmer, Inc.