FailSafe(tm) PCR: A New System For Reliable and Consistent Amplification

Successful PCR depends on a variety of factors including the quality of the template, choice of enzyme, and primer design, as well as the amplification conditions used. The ideal PCR system would be able to: 1) consistently amplify a wide variety of templates including difficult (e.g., high GC content or secondary structure) and long sequences, 2) amplify with high fidelity, and 3) achieve amplification with little optimization. While enzymes for high fidelity PCR exist, as do methods for difficult and long PCR, no one system exists that addresses all of these factors in an easy-to-use format.

Here, we introduce FailSafe™ PCR. The FailSafe PCR System ensures successful and consistent PCR results with both routine and challenging templates, including long templates (up to approximately 20 kb in length) and templates with high GC content (>80% GC). The FailSafe PCR System consists of two components. The FailSafe PCR Enzyme Mix is a unique enzyme blend containing a 3'to 5' proofreading enzyme for high fidelity. PCR products generated by the FailSafe PCR Enzyme Mix are readily cloned with high efficiency in TA or blunt-end vectors. The second component of the FailSafe PCR System is a set of FailSafe PCR PreMixes. The FailSafe PCR PreMixes contain buffer, dNTPs, and various amounts of MgCl2 and FailSafe PCR Enhancer (with betaine).* The user simply adds template, primers, and the FailSafe PCR Enzyme Mix to the FailSafe PCR PreMixes and amplifies. This single-step protocol is used for all templates, no tedious optimization is required. The FailSafe PCR Enhancer included in the PreMixes increases PCR specificity, sensitivity, and consistency.

In this article, we compare the fidelity of the FailSafe PCR System to other commercially available PCR enzymes and enzyme blends. We also demonstrate amplification of long and difficult templates and multiplex PCR using the FailSafe PCR System.

Methods and Results

Fidelity of the FailSafe PCR Enzyme Mix

Applications of PCR such as cloning, expression, mutation analysis, and long amplification require the use of enzymes with low error rates. We compared the fidelity of the FailSafe PCR Enzyme Mix with enzymes and enzyme blends from other suppliers using a PCR-based forward mutation assay. The method is similar to that used by Cline et al.1 and measures PCR fidelity by amplifying the alpha - complementing portion of the lacZ gene and assessing its sequence integrity using a blue/white colony screening assay. Amplification reactions were performed using the reagents and protocols supplied by each respective manufacturer and fidelity assays were performed side by side. As shown in Figure 1, the fidelity of FailSafe PCR Enzyme Mix was at least three times better than both standard Taq polymerases tested and was equivalent to or better than the other high fidelity enzyme blends tested. Although the FailSafe PCR Enzyme Mix exhibits slightly lower fidelity than has been reported for Pfu DNA polymerase, the FailSafe PCR System is much more robust and is able to achieve more specific and consistent amplification of difficult templates (e.g., with high GC content) and long templates on which Pfu fails.