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针对传统电泳检测方法存在操作复杂、费时等缺点,提出一种用于检测K-ras癌基因点突变的实时荧光等位基因特异性扩增(Allele specific amplification,ASA)方法。该法采用突变型引物对结肠癌基因组中的K-ras基因进行等位基因特异性扩增,只有突变型样品能被顺利扩增出双链DNA产物,该产物能与双链DNA染料SYBR GreenⅠ结合,产生荧光信号从而被检测到。通过对荧光域值和溶解曲线分析来区分不同的基因突变类型。该法可以检测到野生型DNA中含量为1/1 000的突变型DNA,整个检测时间小于1 h。我们用该法检测31例结肠癌样品中K-ras基因密码子12发生的点突变,其中有15例检出为阳性。此外,还采用等位基因特异性扩增结合电泳分析对样品进行了检测,并对两种方法进行了比较。结果显示:实时荧光等位基因特异性扩增方法具有操作简便、快速、检测成本低等优点,为临床诊断基因突变引起的疾病提供了一种可行的手段。
Aiming at the disadvantages of complex and time-consuming traditional electrophoresis detection methods, a real-time fluorescence allele-specific amplification (ASA) method for detecting point mutation of K-ras oncogene was proposed. The method uses a mutant primer for allele-specific amplification of K-ras gene in colon cancer genome. Only the mutant samples can be successfully amplified double-stranded DNA product, which can be double-stranded DNA dye SYBR Green Ⅰ In combination, fluorescent signals are generated and detected. Different types of gene mutations are distinguished by analysis of fluorescence domain values and dissolution curves. The method can detect wild-type DNA in the content of 1/1 000 mutant DNA, the entire detection time is less than 1 h. We used this method to detect point mutations in codon 12 of K-ras gene in 31 colon cancer samples, of which 15 were positive. In addition, the samples were also tested using allele-specific amplification combined with electrophoretic analysis and the two methods were compared. The results showed that the real-time fluorescent allele-specific amplification method has the advantages of simple, rapid, low cost and so on, which provided a feasible method for the clinical diagnosis of diseases caused by gene mutation.