The Experts below are selected from a list of 129 Experts worldwide ranked by ideXlab platform
Susan C. Bock - One of the best experts on this subject based on the ideXlab platform.
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A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA Polymerase
Nucleic acids research, 1994Co-Authors: Veronique Picard, Eva Ersdal-badju, Susan C. BockAbstract:A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA Polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.
Holly H Hogrefe - One of the best experts on this subject based on the ideXlab platform.
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engineered split in Pfu DNA Polymerase fingers domain improves incorporation of nucleotide γ phosphate derivative
Nucleic Acids Research, 2011Co-Authors: Connie J Hansen, Lydia Wu, Bahram Arezi, Holly H HogrefeAbstract:Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA Polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (−1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA Polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA Polymerase further illustrates the modular nature of the Family B DNA Polymerase structure.
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pcr fidelity of Pfu DNA Polymerase and other thermostable DNA Polymerases
Nucleic Acids Research, 1996Co-Authors: Janice Marie Cline, Jeffery C Braman, Holly H HogrefeAbstract: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 · 10 ‐6 ) < Deep Vent (2.7 · 10 ‐6 ) < Vent (2.8 · 10 ‐6 ) < Taq (8.0 · 10 ‐6 ) << exo ‐ Pfu and UlTma (~5 · 10 ‐5 ). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2‐3 mM MgSO4 and 100‐300 μM each dNTP and at pH 8.5‐9.1. Under these conditions, the error rate of exo ‐ Pfu was ~40-fold higher (5 · 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 ~2-fold, while the error rate of exo ‐ Pfu increased ~9-fold. An increase in error rate with pH has also been noted for the exonucleasedeficient DNA Polymerases Taq and exo ‐ Klenow, suggesting that the parameters which influence replication error rates may be similar in pol I- and α-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 ~3‐4-fold higher than the error rate of Pfu DNA Polymerase.
Hyun Soo Cho - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of Pfu the high fidelity DNA Polymerase from pyrococcus furiosus
International Journal of Biological Macromolecules, 2008Co-Authors: Suhng Wook Kim, Donguk Kim, Jinkwang Kim, Linwoo Kang, Hyun Soo ChoAbstract:Abstract We have determined a 2.6 A resolution crystal structure of Pfu DNA Polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus . Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The interaction of these two domains is known to coordinate the proofreading and polymerization activity of DNA Polymerases, especially via H147 that is present within the loop (residues 144–158) of the exonuclease domain. In our structure of Pfu , however, E148 rather than H147 is located at better position to interact with the thumb domain. In addition, the structural analysis of Pfu and KOD1 shows that both the Y-GG/A and β-hairpin motifs of Pfu are found to differ with that of KOD1, and may explain differences in processivity. This information enables us to better understand the mechanisms of polymerization and proofreading of DNA Polymerases.
Suhng Wook Kim - One of the best experts on this subject based on the ideXlab platform.
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cloning the Pfu DNA Polymerase from DNA contaminants in preparations of commercial Pfu DNA Polymerase
African Journal of Microbiology Research, 2013Co-Authors: Ji Hye Heo, Suhng Wook KimAbstract:The Pfu DNA Polymerase gene was cloned from commercial Pfu DNA Polymerase by semi-nested Polymerase chain reaction (PCR) using a contaminant template in the commercial Pfu DNA Polymerase and the activity of the Pfu DNA Polymerase itself. We confirmed the occurrence of residual bacterial DNA in standard Pfu DNA Polymerase preparations and demonstrated that the commercial enzyme can be used directly as a PCR template for cloning, thus eliminating the laborious and time-consuming steps of cell recovery and DNA extraction. A simple method was developed for the purification of PfuDNA Polymerase by the heat-mediated (100°C, 10 min) lysis of Escherichia coli and denaturation of E. coli proteins, exploiting using the extremely thermostable properties ofPfu DNA Polymerase, followed by chromatography on heparin columns. The whole purification process could be achieved within 2 h using one type of buffer, with no need for sonication or the use of toxic reagents. This advantageous alternative method is convenient and faster than previously reported methods. Key words: Pfu DNA Polymerase, DNA contamination, cloning, purification
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crystal structure of Pfu the high fidelity DNA Polymerase from pyrococcus furiosus
International Journal of Biological Macromolecules, 2008Co-Authors: Suhng Wook Kim, Donguk Kim, Jinkwang Kim, Linwoo Kang, Hyun Soo ChoAbstract:Abstract We have determined a 2.6 A resolution crystal structure of Pfu DNA Polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus . Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The interaction of these two domains is known to coordinate the proofreading and polymerization activity of DNA Polymerases, especially via H147 that is present within the loop (residues 144–158) of the exonuclease domain. In our structure of Pfu , however, E148 rather than H147 is located at better position to interact with the thumb domain. In addition, the structural analysis of Pfu and KOD1 shows that both the Y-GG/A and β-hairpin motifs of Pfu are found to differ with that of KOD1, and may explain differences in processivity. This information enables us to better understand the mechanisms of polymerization and proofreading of DNA Polymerases.
Veronique Picard - One of the best experts on this subject based on the ideXlab platform.
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A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA Polymerase
Nucleic acids research, 1994Co-Authors: Veronique Picard, Eva Ersdal-badju, Susan C. BockAbstract:A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA Polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.