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      Modified DNA polymerases for PCR troubleshooting

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          Abstract

          PCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase used, is shown. In addition, the reasons that might justify the need for modification of DNA polymerases, type of modifications, and links between modified DNA polymerases and PCR efficiency are described.

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          Most cited references37

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          PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

          The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.
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            A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro.

            Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalized to others. Here we report a protein engineering-based approach to significantly improve the processivity of DNA polymerases by covalently linking the polymerase domain to a sequence non-specific dsDNA binding protein. Using Sso7d from Sulfolobus solfataricus as the DNA binding protein, we demonstrate that the processivity of both family A and family B polymerases can be significantly enhanced. By introducing point mutations in Sso7d, we show that the dsDNA binding property of Sso7d is essential for the enhancement. We present evidence supporting two novel conclusions. First, the fusion of a heterologous dsDNA binding protein to a polymerase can increase processivity without compromising catalytic activity and enzyme stability. Second, polymerase processivity is limiting for the efficiency of PCR, such that the fusion enzymes exhibit profound advantages over unmodified enzymes in PCR applications. This technology has the potential to broadly improve the performance of nucleic acid modifying enzymes.
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              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. 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.
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                Author and article information

                Contributors
                +48 58347 23 83 , beakrawc@pg.gda.pl
                Journal
                J Appl Genet
                J. Appl. Genet
                Journal of Applied Genetics
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                1234-1983
                2190-3883
                28 October 2016
                28 October 2016
                2017
                : 58
                : 1
                : 133-142
                Affiliations
                ISNI 0000 0001 2187 838X, GRID grid.6868.0, Department of Molecular Biotechnology and Microbiology, , Gdańsk University of Technology, ; ul. G. Narutowicza 11/12, 80-233 Gdańsk, Poland
                Author notes

                Communicated by: Agnieszka Szalewska-Palasz, PhD

                Article
                371
                10.1007/s13353-016-0371-4
                5243913
                27796943
                1ebf78e6-6ab9-4bdd-a09b-bc5d1f9191e9
                © The Author(s) 2016

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 7 June 2016
                : 11 October 2016
                Categories
                Microbial Genetics • Review
                Custom metadata
                © Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2017

                Genetics
                pcr,native dna polymerase,fusion dna polymerases,chimeric dna polymerases,mutagenesis
                Genetics
                pcr, native dna polymerase, fusion dna polymerases, chimeric dna polymerases, mutagenesis

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