Hongyo T, Buzard G S, Calvert R J, Weghorst C M
Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, MD.
Nucleic Acids Res. 1993 Aug 11;21(16):3637-42. doi: 10.1093/nar/21.16.3637.
A rapid (< 2.5 hrs) method for single-strand conformation polymorphism (SSCP) analysis of PCR products that allows the use of ethidium bromide staining is described. PCR products ranging in size from 117 to 256 bp were evaluated for point mutations and polymorphisms by 'cold SSCP' in commercially available pre-cast polyacrylamide mini-gels. Several electrophoretic parameters (running temperature, buffers, denaturants, DNA concentration, and gel polyacrylamide concentration) were found to influence the degree of strand separation and appeared to be PCR fragment specific. Use of the 'cold' SSCP technique and the mini-gel format allowed us to readily optimize the electrophoretic conditions for each PCR fragment. This greatly increased our ability to detect polymorphisms compared to conventional, radioisotope-labeled 'hot' SSCP, typically run under two standard temperature conditions. Excellent results have been obtained in resolving mutant PCR fragments from human p53 exons 5 through 8, human HLA-DQA, human K-ras exons 1 and 2, and rat K-ras exon 3. Polymorphisms could be detected when mutant DNA comprised as little as 3% of the total gene copies in a PCR mixture. Compared to standard 'hot' SSCP, this novel non-isotopic method has additional advantages of dramatically increased speed, precise temperature control, reproducibility, and easily and inexpensively obtainable reagents and equipment. This new method also lacks the safety and hazardous waste management concerns associated with radioactive methods.
本文描述了一种用于PCR产物单链构象多态性（SSCP）分析的快速（<2.5小时）方法，该方法允许使用溴化乙锭染色。通过“冷SSCP”在市售预制聚丙烯酰胺迷你凝胶中对大小在117至256 bp之间的PCR产物进行点突变和多态性评估。发现几个电泳参数（运行温度、缓冲液、变性剂、DNA浓度和凝胶聚丙烯酰胺浓度）会影响链分离程度，并且似乎是PCR片段特异性的。使用“冷”SSCP技术和迷你凝胶格式使我们能够轻松优化每个PCR片段的电泳条件。与通常在两种标准温度条件下运行的传统放射性同位素标记的“热”SSCP相比，这大大提高了我们检测多态性的能力。在解析人p53外显子5至8、人HLA - DQA、人K - ras外显子1和2以及大鼠K - ras外显子3的突变PCR片段方面取得了优异结果。当突变DNA在PCR混合物中仅占总基因拷贝数的3%时，就可以检测到多态性。与标准“热”SSCP相比，这种新型非同位素方法具有速度大幅提高、精确温度控制、可重复性以及试剂和设备易于获得且成本低廉等额外优点。这种新方法还不存在与放射性方法相关的安全和危险废物管理问题。
