Iwahana H, Itakura M
Otsuka Department of Clinical and Molecular Nutrition, University of Tokushima, Japan.
Methods Mol Med. 1998;16:51-60. doi: 10.1385/0-89603-499-2:51.
Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis is a simple and sensitive method to detect DNA alterations including even one point mutation. A disadvantage of PCR-SSCP, despite its sensitivity, is the necessity to use radioisotopes. To avoid radioisotopes, silver staining was introduced for band detection (1). In the fluorescent amplification refractory mutation system (ARMS)-SSCP using two different fluorescence-labeled primers, bands were visualized by UV-transtilumination (2). Hayashi et al. (3) and Takahashi-Fujii et al. (4) developed F-SSCP using an automated DNA sequencer and a fluorescence-based image analyzer, respectively. In their systems, however, band detection was dependent on a single species of fluorescence. Ellison et al. (5) reported a method to detect multiple fluorescence using Applied Biosystems model 373A DNA sequencer (ABI373A) (Perkin Elmer, Applied Biosystems Division [PE-ABD], Foster City, CA), which can detect four different fluorescent colors in the same lane without controlling gel-temperature. We designed and attached a gel temperature-controlling system to ABI373 A and developed a sensitive method of multiple fluorescence-based PCR-SSCP (MF-PCR-SSCP) analysis in which we introduced three different methods to fluorescently label PCR-amplified DNA fragments 6-8.
聚合酶链反应-单链构象多态性（PCR-SSCP）分析是一种简单且灵敏的检测DNA改变的方法，甚至能检测到单点突变。尽管PCR-SSCP具有灵敏性，但其缺点是需要使用放射性同位素。为避免使用放射性同位素，引入了银染法进行条带检测（1）。在使用两种不同荧光标记引物的荧光扩增阻滞突变系统（ARMS）-SSCP中，通过紫外透射照明观察条带（2）。林等人（3）和高桥-藤井等人（4）分别利用自动DNA测序仪和基于荧光的图像分析仪开发了F-SSCP。然而，在他们的系统中，条带检测依赖于单一荧光种类。埃里森等人（5）报道了一种使用应用生物系统公司373A DNA测序仪（ABI373A）（珀金埃尔默公司，应用生物系统部[PE-ABD]，加利福尼亚州福斯特城）检测多种荧光的方法，该仪器无需控制凝胶温度就能在同一泳道中检测四种不同荧光颜色。我们设计并给ABI373A连接了一个凝胶温度控制系统，开发了一种基于多重荧光的灵敏的PCR-SSCP（MF-PCR-SSCP）分析方法，其中我们引入了三种不同方法对PCR扩增的DNA片段进行荧光标记6 - 8。
