title: Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii.
abstract: An inorganic pyrophosphatase (PPases) was cloned from the hyperthermophilic archaeon Pyrococcus horikoshii and was expressed in and purified from Escherichia coli. The recombinant inorganic pyrophosphatase (PhPPase) exhibited robust catalytic activity of the hydrolysis of pyrophosphate into two orthophosphates at high temperatures (70 degrees C to 95 degrees C). Thermostable pyrophosphatase activity was applied into polymerase chain reaction (PCR) due to its ability to push chemical equilibrium toward the synthesis of DNA by removing pyrophosphate from the reaction. A colorimetric method using molybdate and reducing agents was used to measure PCR progress by detecting and quantifying inorganic phosphate in the PhPPase-coupled PCR mixture. Compared to PCR mixtures without PhPPase, the thermostable PhPPase enhanced the amount of PCR product in the same number of cycles. Thus, thermostable PPase may overcome the limitations of thermodynamically unfavorable DNA polymerization in PCR by yielding more products.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, Republic of Korea.
authors.forename: Sun Young
authors.initials: SY
authors.lastname: Park
authors.forename: Bokhui
authors.initials: B
authors.lastname: Lee
authors.forename: Kwang-Su
authors.initials: KS
authors.lastname: Park
authors.forename: Youhoon
authors.initials: Y
authors.lastname: Chong
authors.forename: Moon-Young
authors.initials: MY
authors.lastname: Yoon
authors.forename: Sung-Jong
authors.initials: SJ
authors.lastname: Jeon
authors.forename: Dong-Eun
authors.initials: DE
authors.lastname: Kim
mesh_terms: D019843:Archaeal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004795:Enzyme Stability / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; D004259:DNA-Directed DNA Polymerase; D043564:Inorganic Pyrophosphatase