Single-molecule PCR using water-in-oil emulsion

Michihiko Nakano, Jun Komatsu, Shun Ichi Matsuura, Kazunori Takashima, Shinji Katsura, Akira Mizuno

Research output: Contribution to journalArticle

164 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)117-124
Number of pages8
JournalJournal of Biotechnology
Volume102
Issue number2
DOIs
Publication statusPublished - Apr 24 2003
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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    Nakano, M., Komatsu, J., Matsuura, S. I., Takashima, K., Katsura, S., & Mizuno, A. (2003). Single-molecule PCR using water-in-oil emulsion. Journal of Biotechnology, 102(2), 117-124. https://doi.org/10.1016/S0168-1656(03)00023-3