Document details
Single-molecule PCR using water-in-oil emulsion(Article)
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- Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
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Abstract
Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule. © 2003 Elsevier Science B.V. All rights reserved.
Indexed keywords
| Engineering controlled terms: | CloningDNAHigh temperature effects |
|---|---|
| Engineering uncontrolled terms | Micro-reactors |
| Engineering main heading: | Emulsions |
| EMTREE drug terms: | water oil cream |
| EMTREE medical terms: | articleconcentration (parameters)DNA templategene amplificationhigh temperaturemethodologypolymerase chain reactionpriority journalprotein stability |
Funding details
| Funding number | Funding sponsor | Acronym | Funding opportunities |
|---|---|---|---|
| Ministry of Education, Culture, Sports, Science and Technology | MEXT | ||
| RIKEN | Toyota Physical and Chemical Research Institute | TPCRI | |
| New Energy and Industrial Technology Development Organization | NEDO | ||
| 10638 | Japan Society for the Promotion of Science | JSPS | See opportunities by JSPS |
| Nagoya University | |||
| New Energy and Industrial Technology Development Organization | NEDO | ||
| Japan Society for the Promotion of Science | JSPS | See opportunities by JSPS | |
| 12450165 | Ministry of Education, Culture, Sports, Science and Technology | MEXT |
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1
The authors would like to thank Assoc. Prof. H. Nakano and Prof. T. Yamane of Nagoya University and Dr M. Ishikawa of the Joint Research Center for Atom Technology (JRCAT) for their valuable discussions. Template DNA (pSV515 DNA) was constructed in collaboration with Prof. Y. Murakami of the Science University of Tokyo and Dr T. Eki of the Institute of Physical and Chemical Research (RIKEN). This work was partially supported by the New Energy and Industrial Technology Development Organization (NEDO), the Angstrom Technology Partnership of JRCAT and by Grants-in-Aid for Scientific Research from the Ministry of Education Science and Culture Japan (No. 12450165), the Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists (No. 10638) and The 21st Century COE Program ‘Ecological Engineering for Homeostatic Human Activities’ from the ministry of Education, Culture, Sports, Science and Technology.
- ISSN: 01681656
- CODEN: JBITD
- Source Type: Journal
- Original language: English
- DOI: 10.1016/S0168-1656(03)00023-3
- PubMed ID: 12697388
- Document Type: Article
- Publisher: Elsevier
Katsura, S.; Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Japan;
© Copyright 2017 Elsevier B.V., All rights reserved.
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