实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.

Rapid Detection of K-ras Oncogene Point Mutation by Real-time Fluorescence Allele-specific Amplification

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.