Enhancement of PCR efficiency using mono- and disaccharides

Сахабутдинова, А. Р.; Чемерис, А. В.; Гарафутдинов, Р.Р.

doi:10.1016/j.ab.2020.113858

Cited by 6 publications

(2 citation statements)

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“…Current research on PCR enhancers is mostly focused on inorganic molecules, such as DMSO [ 42 ], tetraalkylammonium salts [ 43 ], saccharides [ 16 ], and nanomaterials [ 40 ], while there are very few reports on PCR enhancement by PPases. To date, only Park has studied the effect of thermophilic Ph PPase from the archaeon Pyrococcus horikoshii on PCR [ 9 ], and the results showed that Ph PPase could increase the PCR yield with a target gene of 1.54 kbp by approximately 25%.…”

Section: Discussionmentioning

confidence: 99%

“…Accelerating the effective collision between the reactants by increasing the temperature quickly (graphene nanoflakes) [ 13 ]; and 3. Safeguarding and maintaining the DNA polymerases and template DNA structures (detergents, DMSO) [ 14 , 15 , 16 ]. PPases have been shown to be helpful for driving the equilibrium of the DNA elongation reaction because they hydrolyze PP i to relieve the inhibition of PCR.…”

Section: Introductionmentioning

confidence: 99%

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Thermophilic Inorganic Pyrophosphatase Ton1914 from Thermococcus onnurineus NA1 Removes the Inhibitory Effect of Pyrophosphate

Yang

Gao

2022

IJMS

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Pyrophosphate (PPi) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PPi can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg2+ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32–41%, while that of short-chain DNA was only improved by 9.5–15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.

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