Facilitation of polymerase chain reaction with thermostable inorganic pyrophosphatase from hyperthermophilic archaeon Pyrococcus horikoshii.
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.
Park
Sun Young
SY
Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, Republic of Korea.
Lee
Bokhui
B
Park
Kwang-Su
KS
Chong
Youhoon
Y
Yoon
Moon-Young
MY
Jeon
Sung-Jong
SJ
Kim
Dong-Eun
DE
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
D019843:Archaeal Proteins; D004259:DNA-Directed DNA Polymerase; D043564:Inorganic Pyrophosphatase