Peng H, Du M, Ji J, Isaacson P G, Pan L
Department of Histopathology, University College London Medical School, UK.
Biotechniques. 1995 Sep;19(3):410-4.
We describe here an improved methodology for single-strand conformation polymorphism (SSCP) analysis of PCR products. The method utilizes a polyacrylamide agarose composite gel and background-free silver staining. In comparison with conventional pure polyacrylamide gel for PCR-SSCP analysis, the composite gels have a much greater mechanical strength and improved resolution. The background staining commonly seen in many silver staining protocols has been eliminated by incorporation of thiosulfate, which can prevent nonspecific deposits of silver salts. As shown in our titration tests, the composite gel and background-free silver staining together have allowed clear identification of point mutations in samples containing as little as 5% of the target sequences, the same sensitivity achieved by radioactive labeling methods.
我们在此描述一种用于PCR产物单链构象多态性（SSCP）分析的改进方法。该方法采用聚丙烯酰胺琼脂糖复合凝胶和无背景银染技术。与用于PCR-SSCP分析的传统纯聚丙烯酰胺凝胶相比，复合凝胶具有更高的机械强度和更好的分辨率。通过加入硫代硫酸盐消除了许多银染方案中常见的背景染色，硫代硫酸盐可防止银盐的非特异性沉积。如我们的滴定试验所示，复合凝胶和无背景银染技术相结合能够清晰鉴定出目标序列含量低至5%的样品中的点突变，其灵敏度与放射性标记方法相同。
