Abstract: Mutation analysis is of great importance in molecular genetics. However, conventional electrophoresis-based methods have many shortcomings, such as time-consuming, multi-step, and using radioactive isotopes or other hazardous materials. In this work, a one-step real-time fluorescence allele specific amplification(ASA) method was developed for rapid detection of K-ras oncogene point mutation at codon 12. Thirty-one colon cancer samples were analyzed by the assay. Genome DNA was amplified by a pair of mutant specific primers, only the mutant sample could be amplified, producing double-stranded DNA product, which can be detected by the fluorescence of SYBR Green Ⅰ, a double-stranded DNA-selective fluorescent dye. The results show that the sensitivity of the assay was 1/1000 of mutant/wild-type DNAs. The positive rate for K-ras oncogene point mutation was 48.4%. The real-time fluorescence ASA method is rapid, simple, sensitive, safe, high throughput, and low cost. It can be used for screening a large amount of clinical samples.

Key words: Real-time fluorescence allele-specific amplification, Point mutation, K-ras oncogene, Colon cancer