4.5 Article

Single-molecule PCR using water-in-oil emulsion

期刊

JOURNAL OF BIOTECHNOLOGY
卷 102, 期 2, 页码 117-124

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ELSEVIER SCIENCE BV
DOI: 10.1016/S0168-1656(03)00023-3

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single-molecule PCR; W/O emulsion; reverse micelle

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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. (C) 2003 Elsevier Science B.V. All rights reserved.

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