Oto M, Miyake S, Yuasa Y
Department of Biotechnology, Tokyo Technical College, Japan.
Anal Biochem. 1993 Aug 15;213(1):19-22. doi: 10.1006/abio.1993.1379.
We have developed a means of nonradioisotopic single strand conformation polymorphism (nonRI-SSCP) analysis and applied it to the detection of a point mutation in the human tumor suppressor gene, p53. The method does not require any particular facilities or apparatus, such as a laboratory for radioactive materials, a large gel unit for sequencing, or a semiautomated electrophoresis system. This technique comprises amplification of DNA fragments by the PCR technique with specific oligonucleotide primers, denaturation, and electrophoresis on neutral polyacrylamide gels in a conventional minislab apparatus. The SSCP patterns on electrophoresis were detected with a commercially available silver stain method. We also evaluated various electrophoretic conditions for nonRI-SSCP analysis, such as the gel concentration and buffer components. A tris/glycine buffer system gave better resolution of SSCP bands. The SSCP patterns of different sized DNAs could be analyzed in a gradient polyacrylamide gel. Thus, nonRI-SSCP analysis with a conventional minislab gel electrophoresis apparatus can be satisfactorily substituted for a commonly used RI-SSCP technique.
我们开发了一种非放射性单链构象多态性（nonRI-SSCP）分析方法，并将其应用于检测人类肿瘤抑制基因p53中的点突变。该方法不需要任何特殊的设施或仪器，如放射性物质实验室、大型测序凝胶装置或半自动电泳系统。该技术包括用特异性寡核苷酸引物通过PCR技术扩增DNA片段、变性，并在传统的小型平板装置中在中性聚丙烯酰胺凝胶上进行电泳。电泳时的SSCP图谱用市售的银染法检测。我们还评估了非RI-SSCP分析的各种电泳条件，如凝胶浓度和缓冲液成分。Tris/甘氨酸缓冲系统能更好地分辨SSCP条带。不同大小DNA的SSCP图谱可在梯度聚丙烯酰胺凝胶中进行分析。因此，用传统的小型平板凝胶电泳装置进行的非RI-SSCP分析可以令人满意地替代常用的RI-SSCP技术。
