High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene

University of Georgia, Атина, Georgia, United States
Gene (Impact Factor: 2.14). 01/1992; 108(1):1-6. DOI: 10.1016/0378-1119(91)90480-Y
Source: PubMed


A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.

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    • "Evolution in these extreme environments has resulted in archaeal proteins that have properties of value to biotechnologists, including stability and activity under a range of comparatively harsh in vitro conditions. A familiar example is the widespread use of the Pyrococcus furiosus DNA polymerase in the Polymerase Chain Reaction (PCR) [7], in which its thermostability and processivity also make it valuable for related protocols such as QuikChange mutagenesis [8] [9]. In this review, we turn the attention to archaeal nucleic acid ligases. "
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    ABSTRACT: With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus , thus demonstrating the potential for further work in this area.
    Full-text · Article · Oct 2015 · Archaea (Vancouver, B.C.)
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    • "Improving fidelity with the goal of producing error-free PCR products has been a substantial challenge and has many bioscience companies still looking for a solution. This has, however, led to the discovery of a number of new thermostable enzymes that have error rates improved from the original Taq polymerase [17] [18] [19]. Yet with all these improvements the commercially marketed polymerases with the highest fidelity still misincorporate 1 base out of 16,000 up from 1 base out of 1600 seen with Taq polymerase [18]. "
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    ABSTRACT: DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the í µí»¼ polymerase, the í µí»½ sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of í µí»¼, í µí»½ and DnaX (í µí¼ and í µí»¾), í µí»¿ and í µí»¿ í® í° components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the í µí»¼ polymerase was found to be unstable at temperatures above 65 ∘ C. We were able to increase the thermostability of the pol III HE to 98 ∘ C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.
    Full-text · Article · Jan 2015
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    • "The fidelity of DNA synthesis in vitro is markedly affected by the reaction condition. However, the archaeal family B enzymes generally perform more accurate DNA synthesis as compared with Taq polymerase (Cariello et al., 1991; Ling et al., 1991; Lundberg et al., 1991; Mattila et al., 1991), suggesting that the strong 3′–5′ exonuclease activities of the hyperthermophilic family B polymerase in vitro affect the fidelity of PCR. "
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    ABSTRACT: DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the template DNA in living cells. Multiple enzymes have been identified from each organism, and the shared functions of these enzymes have been investigated. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labeling, mutagenesis, and other purposes. The fundamental ability of DNA polymerases to synthesize a deoxyribonucleotide chain is conserved. However, the more specific properties, including processivity, fidelity (synthesis accuracy), and substrate nucleotide selectivity, differ among the enzymes. The distinctive properties of each DNA polymerase may lead to the potential development of unique reagents, and therefore searching for novel DNA polymerase has been one of the major focuses in this research field. In addition, protein engineering techniques to create mutant or artificial DNA polymerases have been successfully developing powerful DNA polymerases, suitable for specific purposes among the many kinds of DNA manipulations. Thermostable DNA polymerases are especially important for PCR-related techniques in molecular biology. In this review, we summarize the history of the research on developing thermostable DNA polymerases as reagents for genetic manipulation and discuss the future of this research field.
    Full-text · Article · Aug 2014 · Frontiers in Microbiology
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