As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. 1. Introduction With the rapid pace of developments in systems biology-based research, for example, genomics, proteomics, and metabolomics, larger-scale biological discovery projects are becoming more common. Put differently, the scope of many projects has changed from the study of one/few targets to the study of hundreds, thousands, or more. An example of research that has been transformed by developments in systems biology is the cloning of expressed open reading frames (ORFs) from cDNA substrates. The traditional path for ORF cloning has usually started with experimental observations driving the identification of one or several genes of interest to a particular pathway. Cloning of target(s) then typically resulted in further refinements of pathway details and often identification of new cloning targets. With the creation and continual refinements of databases of genomic sequences, cloning now often takes place on a much larger scale. Microarray technology and DNA sequencing breakthroughs have led to a vast increase in the number of ORFs
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