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Title: Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

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. Mutationmore » 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.« less
Authors:
 [1] ;  [2] ;  [3]
  1. Joint BioEnergy Institute, Emeryville, CA, USA, Sandia National Laboratories, Livermore, CA, USA
  2. Joint BioEnergy Institute, Emeryville, CA, USA, Physical Biosciences Division, Lawrence Berkeley National Laboratories, Berkeley, CA 94720, USA
  3. 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
Publication Date:
OSTI Identifier:
1197849
Grant/Contract Number:
AC02-05CH11231
Type:
Published Article
Journal Name:
Molecular Biology International
Additional Journal Information:
Journal Volume: 2014; Related Information: CHORUS Timestamp: 2016-07-28 15:25:53; Journal ID: ISSN 2090-2182
Publisher:
Hindawi Publishing Corporation
Sponsoring Org:
USDOE
Country of Publication:
Country unknown/Code not available
Language:
English