Analysing p53-gene mutations: Comparison of capillary electrophoresis Single Strand Conformation Polymorphism method with Denaturing Gradient Gel Electrophoresis method and direct sequencing
Finnish Institute of Occupational Health, Helsinki, Finland
AIM: Denaturant Gradient Gel Electrophoresis (DGGE) and Single Strand Conformation Polymorphism (SSCP) are both widely used mutation detection methods. DGGE is often taken as a more laborious but also more sensitive method than traditional slab gel SSCP, whereas SSCP is simpler and as such more commonly used. In this work, traditional DGGE, automated capillary electrophoresis SSCP (CE-SSCP) and direct sequencing were compared to resolve the benefits and sensitivity of each of the methods for detection of unknown p53 mutations. METHODS: DGGE, CE-SSCP and direct sequencing. RESULTS: Twenty previously analysed lung cancer samples were analysed under dummy laboratory codes for mutations of p53 gene in exons 5-9. 17 out of the 20 samples were found to contain a mutation. From these mutations 15 (88 %) were detected by DGGE, 16 (94 %) by CE-SSCP and 12 (71 %) by direct sequencing. One mutation that remained undetected by the DGGE was located in an intronic sequence and not inside the effectively screened area. CONCLUSIONS: Therefore, we conclude that CE-SSCP and DGGE can be considered methods with approximately equal sensitivity, although the sensitivity of SSCP could probably be further improved by using different analysis temperatures. The direct sequencing was clearly less sensitive of the three analysis methods. According to the results of this work, CE-SSCP is a fast, reliable and reproducible method for screening of p53 mutations in human tumour samples.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in poster session 896 (Tumor suppressor genes).