scholarly journals Predominant mutations induced by the Thermococcus litoralis, vent DNA polymerase during DNA amplification in vitro.

1993 ◽  
Vol 2 (4) ◽  
pp. 288-292 ◽  
Author(s):  
P Keohavong ◽  
L Ling ◽  
C Dias ◽  
W G Thilly
Keyword(s):  
Dna Polymerase ◽  
Dna Amplification ◽  
Thermococcus Litoralis

scholarly journals Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

1991 ◽  
Vol 19 (18) ◽  
pp. 4967-4973 ◽  
Author(s):  
P. Mattila ◽  
J. Korpela ◽  
T. Tenkanen ◽  
K. Pitkämem
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Total Concentration ◽  
Dna Amplification ◽  
Base Substitution ◽  
Thermococcus Litoralis ◽  
Heat Stable ◽  
Original Target

Abstract We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3′—5′ proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30×10−6. These values were 5–10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15−35×10−4 being 2 – 4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis In vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

DNA Amplification In Vitro Using T4 DNA Polymerase

DNA ◽  
1988 ◽  
Vol 7 (1) ◽  
pp. 63-70 ◽  
Author(s):  
PHOUTHONE KEOHAVONG ◽  
ALEXANDRA G. KAT ◽  
NEAL F. CARIELLO ◽  
WILLIAM G. THILLY
Keyword(s):  
Dna Polymerase ◽  
Dna Amplification

scholarly journals Impact of metal oxide nanoparticles on in vitro DNA amplification

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7228 ◽  
Author(s):  
Chun-Hui Gao ◽  
Monika Mortimer ◽  
Ming Zhang ◽  
Patricia A. Holden ◽  
Peng Cai ◽  
...  
Keyword(s):  
Metal Oxide ◽  
Dna Polymerase ◽  
Metal Oxide Nanoparticles ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Amplicon Sequencing ◽  
High Fidelity ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide

Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (Fe2O3, ZnO, CeO2, Fe3O4, Al2O3, CuO, and TiO2) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs (p > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.

scholarly journals A fusion of Taq DNA polymerase with the CL7 protein from Escherichia coli remarkably improves DNA amplification

2020 ◽  
Author(s):  
Wang Fei ◽  
Rao Ben ◽  
Ni Jing ◽  
Ma Lixin ◽  
Wang Yaping
Keyword(s):  
Escherichia Coli ◽  
Molecular Biology ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Rate Sensitivity ◽  
Dna Amplification ◽  
Extension Rate ◽  
Essential Components ◽  
Improved Performance

Abstract Background DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a double-stranded DNA binding-like protein from Escherichia coli. Results The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 aM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Conclusions Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

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