When do you need a high-fidelity DNA polymerase?

When do you need a high-fidelity DNA polymerase?

Using the right polymerase for your PCR experiment will ensure optimal yield and specificity, but with the overwhelming number of polymerases on the market, it can be difficult to know where to start. Of all the considerations to keep in mind when choosing which DNA polymerase to use, fidelity can be of the most critical.  Polymerase fidelity can be one of the most important for the success of your experiment. Simplify this decision by learning more about the fidelity of DNA polymerases.

What is polymerase fidelity?

The fidelity of a DNA polymerase is the result of accurate replication of a desired template. Specifically, this involves multiple steps, including the ability to read a template strand, select the appropriate nucleoside triphosphate and insert the correct nucleotide at the 3’ primer terminus, such that Watson-Crick base pairing is maintained. To effectively discriminate correct vs. incorrect nucleotide incorporation, some DNA polymerases possess a 3’ to 5’ exonuclease activity. This activity, known as “proofreading,” is used to excise incorrectly incorporated mononucleotides, which are then replaced with the correct nucleotides. High-fidelity PCR uses DNA polymerases that couple low misincorporation rates with proofreading to give faithful replication of the target DNA of interest.

When is fidelity important?

When designing your PCR experiment, the first question you should ask is whether or not your application requires a high-fidelity polymerase. If the outcome of your experiment depends on the correct DNA sequence (e.g., cloning or next-generation sequencing applications), you’ll want to minimize the incorporation of mismatched nucleotides by using a high-fidelity polymerase. Fidelity is less important for standard PCR or colony PCR to determine the presence or absence of an amplicon or to confirm that your plasmid has an insert. Because of the robust nature of certain high-fidelity polymerases, some researchers use them for all their amplifications, regardless of the PCR product’s downstream use.

How do you measure fidelity?

Vendors use a variety of different methods to determine the fidelity of their DNA polymerases. One assay, first described by Thomas Kunkel, uses portions of the lacZα gene in M13 bacteriophage to correlate host bacterial colony color changes with errors in DNA synthesis [1]. Building on the Kunkel assay, Wayne Barnes’ assay is a common permutation found in labs, in which PCR is used to copy the entire lacZ gene and portions of two drug-resistance genes, with subsequent ligation, cloning, transformation and blue/white-colony color determination [2]. The readout of both assays is a white-colony phenotype caused by the disruption of β-galactosidase activity that results from errors in the lacZ gene. With these lacZ-based experimental approaches, the percentage of white colonies must be converted to the number of errors per base incorporated. For a more direct readout of fidelity, Sanger sequencing of individual cloned PCR products also can be used to score DNA polymerase fidelity and offers the advantage of detecting all mutations. Using this method, the entire mutational spectrum of a polymerase can be determined, and there is no need to correct for nonphenotypic changes.

What is “high fidelity”?

Although there is no universal definition for “high fidelity” with respect to DNA polymerases, the measuring stick by which all other polymerases are judged is also the oldest known thermophilic polymerase: Taq DNA polymerase. For this reason, Taq is commonly tested side by side with other polymerases in fidelity measurements. For example, using the blue/white method and correcting for nonphenotypic changes and error propagation during PCR (2) scientists at New England Biolabs measured an error value for Taq at 2.7x10-4±0.8x10-4, or 1 per 3,700 bases. Translating blue/white error rates into practical use, these data suggest that after using 25 PCR cycles to amplify a 400 bp fragment with Taq, several isolates should be screened, because about half the clones are predicted to have an error. For larger fragments of approximately 1,000 bp, each clone amplified with Taq is likely to have an undesired mutation. In contrast, the ultra-high-fidelity polymerase Q5® exhibits error rates 100 X lower than Taq. Based on this value for Q5, one can predict that 199/200 clones amplified will be correct.

There are a number of high-fidelity polymerases on the market, and choosing the correct enzyme can be overwhelming. Understanding the fidelity requirements of the experiment is a critical starting place. Understanding how fidelity measurements are performed and reported by the vendor is another important consideration. This type of evaluation will help ensure optimal results from your DNA amplification experiments.

  1. Kunkel, T.A. and Tindall, K.R. (1988) Biochemistry, 27, 6008–6013.
  2. Barnes, W.M. (1992) Gene, 112, 29–35.

Q5® is a registered trademark of New England Biolabs, Inc.

 

John Pezza, Ph.D., Rebecca Kucera, M.S., Elizabeth Young, Ph.D. and Luo Sun, Ph.D., New England Biolabs, Inc.

Related Products from: New England Biolabs

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