Direct Monitoring of DNA Polymerase Reactions on a Quartz-Crystal Microbalance.
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Accession number;00A0163467
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| Title;Direct Monitoring of DNA Polymerase Reactions on a Quartz-Crystal Microbalance. |
| Author;
OKAHATA YOSHIO
(Tokyo Inst. of Technology, Graduate School)
MATSUNO HISAO
(Tokyo Inst. of Technology, Graduate School)
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Journal Title;Abstracts. Symposium on Biofunctional Chemistry
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Journal Code:L0836A
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ISSN:
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VOL.14th;NO.;PAGE.14-15(1999)
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| Figure&Table&Reference; |
| Pub. Country;Japan |
| Language;Japanese |
| Abstract;DNA polymerase is one of the responsible enzymes for replication and repair of DNA in the cell and for genetic engineering. In this study, a quartz-crystal microbalance(QCM) was employed as a new technique to examine kinetic mechanism of DNA polymerase reactions. Oligonucleotide duplexes were immobilized on a cleaned Au electrode of the QCM, and then it was soaked into a buffered solution for measurement. When Klenow fragment of DNA polymerase I from E. coli was added into the solution, the frequency decrease (mass increase) of the QCM was observed. This change indicates the binding of polymerase on the primer terminus of oligonucleotide duplexes. When as a second injection, complementary monomers were added into the solution, the frequency was rapidly decreased (mass increased) and then increased (mass decreased). The mass increase indicates the elongation reaction along the template, and the mass decrease means the release of the polymerase from the completely polymerized oligonucleotides. Kinetic analyses of three processes, 1) binding of polymerase to DNA, 2) elongation of complementary nucleotides along the template, and 3) release of the enzyme from the polymerized DNA, were performed respectively. Effects of template length and sequence on kinetic values of each step were also investigated. (author abst.) |
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