添加引物法改善PCR-SSCP分析
Optimization of PCR-SSCP analysis by PCR primers addition
背景：PCR产物中的引物量显著影响SSCP带型。目的：探讨添加引物法改善PCR-SSCP重复性和灵敏性的机制，并确定最佳引物浓度。方法：HLA-DRB1引物或非DRB1引物与DRB1基因的PCR产物分别以摩尔数比0:1、3:1、6:1、12:1、25:1、50:1、100:1和200:1混合，变性，行聚丙烯酰胺凝胶电泳后银染显色。结果：HLA-DRB1引物可以明显改变相应PCR产物的SSCP带型，增加其灵敏性。当引物与PCR产物的摩尔数比达到200:1时，PCR-SSCP分析的重复性和灵敏性最佳。结论：通过添加适量引物可排除剩余引物对PCR-SSCP中间带型的干扰，明显改善其重复性和灵敏性。
Background. The sensitivity of PCR-SSCP is influenced by the primer concentration in PCR product. Objective. This study is to evaluate the influences of different primer concentrations on mobility of sequence variants in PCR-SSCP analysis. Methods. HLA-DRB1 primers or non-DRB1 primers were mixed with PCR products at the molar ratio of 0:1, 3:1, 6:1, 12:1, 25:1, 50:1, 100:1 and 200:1 before electrophoresis. Mobility shift of banding patterns was detected and analyzed by silver staining. Results. PCR-SSCP banding patterns of HLA-DRB1 PCR fragment were found to be “shifted” when mixed with the specific primers at different molar ratio. The primers and PCR products at the molar ratio of 200:1 had a relatively high resolution for PCR-SSCP banding patterns. Conclusion. Optimization of primer concentration is valuable to exclude the interference of intermediate SSCP patterns and to improve sensitivity and resolution in PCR-SSCP analysis.
陈冬梅、刘英、牛妮芳、都特、刘杨、王心、朱席琳
分子生物学
PCR-SSCP 引物 突变筛查
PCR-SSCP primer mutation screening
陈冬梅,刘英,牛妮芳,都特,刘杨,王心,朱席琳.添加引物法改善PCR-SSCP分析[EB/OL].(2005-12-14)[2025-04-26].http://www.paper.edu.cn/releasepaper/content/200512-351.点此复制
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