Single-molecule PCR using water-in-oil emulsion.
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.
Nakano
Michihiko
M
Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan.
Komatsu
Jun
J
Matsuura
Shun-ichi
S
Takashima
Kazunori
K
Katsura
Shinji
S
Mizuno
Akira
A
D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D004655:Emulsions / Q000737:chemistry; D004867:Equipment Design; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012827:Silicone Oils / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
D004655:Emulsions; D012827:Silicone Oils; D014867:Water; D004247:DNA