Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase
- Joint BioEnergy Institute, Emeryville, CA, USA, Sandia National Laboratories, Livermore, CA, USA
- Joint BioEnergy Institute, Emeryville, CA, USA, Physical Biosciences Division, Lawrence Berkeley National Laboratories, Berkeley, CA 94720, USA
- Joint BioEnergy Institute, Emeryville, CA, USA, Physical Biosciences Division, Lawrence Berkeley National Laboratories, Berkeley, CA 94720, USA, Synthetic Biology Program, Space BioSciences Division, NASA AMES Research Center, Mail Stop 239-15, Moffett Field, CA 94035, USA
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.
- Research Organization:
- Sandia National Laboratories (SNL), Albuquerque, NM, and Livermore, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
- Grant/Contract Number:
- AC02-05CH11231
- OSTI ID:
- 1197849
- Alternate ID(s):
- OSTI ID: 1628944
- Journal Information:
- Molecular Biology International, Journal Name: Molecular Biology International Vol. 2014; ISSN 2090-2182
- Publisher:
- Hindawi Publishing CorporationCopyright Statement
- Country of Publication:
- Egypt
- Language:
- English
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