Single-molecule PCR using water-in-oil emulsion

被引：175

作者：

Nakano, M ^{[1
]}

Komatsu, J ^{[1
]}

Matsuura, S ^{[1
]}

Takashima, K ^{[1
]}

Katsura, S ^{[1
]}

Mizuno, A ^{[1
]}

机构：

[1] Toyohashi Univ Technol, Dept Ecol Engn, Aichi 4118580, Japan

来源：

JOURNAL OF BIOTECHNOLOGY | 2003年 / 102卷 / 02期

基金：

日本学术振兴会;

关键词：

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

D O I：

10.1016/S0168-1656(03)00023-3

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

页码：117 / 124

页数：8

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