Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification

Kim, Yun-Jae; Ryu, Yong-Gu; Lee, Hyun Sook; Cho, Yona; Kwon, Suk‐Tae; Lee, Jung-Hyun; Kang, Sung Gyun

doi:10.1007/s00253-008-1467-5

Cited by 8 publications

(8 citation statements)

References 33 publications

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“…People have found different types of PCR enhancers, including formamide [28], betaine [29], DMSO [30], gold nanoparticles [12,13], carbon nanotubes [14][15][16], carbon nanopowder [17], proteins [31][32][33], ethylene glycol/propanediol [34], dendrimers [35,36], nucleotide analogs [37], and other chemicals [38][39][40]. In this report, the enhancing effects of CdTe quantum dots in the context of hot-start performance for Taq DNA polymerasebased polymerase chain reaction were demonstrated, Figure 7.…”

Section: Resultsmentioning

confidence: 83%

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

Sang¹,

Yang²,

Wang³

et al. 2012

JBiSE

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Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques

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Section: Resultsmentioning

confidence: 83%

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

Sang¹,

Yang²,

Wang³

et al. 2012

JBiSE

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“…Literature reports of increased PCR yield when a thermostable inorganic pyrophosphatase (IPPase) was included in the reactions inspired us to test this enzyme in a qPCR series to see if we could drive the reaction forward by degrading the pyrophosphate, one of the two products of the chain reaction [17], [18]. Unexpectedly, the addition of IPPase reduced the apparent reaction yield ( Figure 5A ).…”

Section: Resultsmentioning

confidence: 99%

Robust Quantification of Polymerase Chain Reactions Using Global Fitting

Carr

Moore

2012

PLoS ONE

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BackgroundQuantitative polymerase chain reactions (qPCR) are used to monitor relative changes in very small amounts of DNA. One drawback to qPCR is reproducibility: measuring the same sample multiple times can yield data that is so noisy that important differences can be dismissed. Numerous analytical methods have been employed that can extract the relative template abundance between samples. However, each method is sensitive to baseline assignment and to the unique shape profiles of individual reactions, which gives rise to increased variance stemming from the analytical procedure itself.Principal FindingsWe developed a simple mathematical model that accurately describes the entire PCR reaction profile using only two reaction variables that depict the maximum capacity of the reaction and feedback inhibition. This model allows quantification that is more accurate than existing methods and takes advantage of the brighter fluorescence signals from later cycles. Because the model describes the entire reaction, the influences of baseline adjustment errors, reaction efficiencies, template abundance, and signal loss per cycle could be formalized. We determined that the common cycle-threshold method of data analysis introduces unnecessary variance because of inappropriate baseline adjustments, a dynamic reaction efficiency, and also a reliance on data with a low signal-to-noise ratio.SignificanceUsing our model, fits to raw data can be used to determine template abundance with high precision, even when the data contains baseline and signal loss defects. This improvement reduces the time and cost associated with qPCR and should be applicable in a variety of academic, clinical, and biotechnological settings.

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“…T. onnurineus NA1 is a recently identified hyperthermophilic archaeon that was isolated from geothermal sediments from a black smoker on the ocean floor. 16 Several enzymes such as PPase17 (80°C), dITPase18 (75°C), histidinol phosphate phosphatase19 (80°C), and fructose‐1,6‐bisphosphatase20 (95°C) from T. onnurineus have been reported to have unique thermophilic enzymatic activities. A major interest in thermophilic enzymes concerns the development of specific physicochemical regulatory functions under harsh conditions while maintaining enzyme stability.…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel ligand binding site in phosphoserine phosphatase from the hyperthermophilic archaeon Thermococcus onnurineus

Jung

Kim

et al. 2013

Proteins

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Phosphoserine phosphatase (PSP) catalyzes the final and irreversible step of L-serine synthesis by hydrolyzing phosphoserine to produce L-serine and inorganic phosphate. Developing a therapeutic drug that interferes with serine production is of great interest to regulate the pathogenicity of some bacteria and control D-serine levels in neurological diseases. We determined the crystal structure of PSP from the hyperthermophilic archaeon Thermococcus onnurineus at 1.8 Å resolution, revealing an NDSB ligand bound to a novel site that is located in a fissure between the catalytic domain and the CAP module. The structure shows a half-open conformation of the CAP 1 module with a unique protruding loop of residues 150-155 that possesses a helical conformation in other structures of homologous PSPs. Activity assays indicate that the enzyme exhibits marginal PSP activity at low temperature but a sharp increase in the k(cat)/K(M) value, approximately 22 fold, when the temperature is increased. Structural and biochemical analyses suggest that the protruding loop in the active site might be an essential component for the regulation of the activity of PSP from hyperthermophilic T. onnurineus. Identification of this novel binding site distantly located from the catalytic site may be exploited for the development of effective therapeutic allosteric inhibitors against PSP activity.

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Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification

Cited by 8 publications

References 33 publications

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

Robust Quantification of Polymerase Chain Reactions Using Global Fitting

Identification of a novel ligand binding site in phosphoserine phosphatase from the hyperthermophilic archaeon Thermococcus onnurineus

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