How can I compare error rates of different high-fidelity polymerases?

Error rates reported by vendors for polymerases cannot always be directly compared, as different methods are used to measure fidelity. These methods include: 1. Blue-white screening This approach is based on phenotypic changes and is widely used since it is fast, relatively simple, and cost effective. The original method for blue-white screening, known as the Kunkel method (Kunkel and Tindall 1987), is based on ?-complementation of the lacZ? gene that restores ?-galactosidase enzyme activity and allows production of a blue color. With this method, colonies derived from lacZ? PCR products containing single-nucleotide errors or frameshift mutations typically have a white color, while clones derived from error-free amplicons generate blue colonies. 2. Sequencing approach This approach utilizes Sanger sequencing of individual colonies after PCR. The blue-white screening approach can quickly measure polymerase fidelity, however it is not as accurate as the sequencing approach. The blue-white method will not detect silent mutations, single-nucleotide substitutions that do not affect translation. The sequencing method can detect all mutations, and thus is more accurate. References Kunkel, T. A. and Tindall, K. R., Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27, 6008–6013 (1987)

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