两种等位基因特异性扩增方法在结肠癌K-ras癌基因点突变检测中的应用比较

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。

K-ras Point Mutation Detection in Colorectal Cancer:Comparison of Two Approaches Using Allele Specific Amplification

Traditional electrophoresis-based methods for detection of K-ras oncogene mutations are complicated and time-consuming.We aimed to develop a rapid real-time fluorescence allele-specific amplification(ASA) approach using SYBR Green Ⅰ to detect K-ras point mutation in colorectal cancer.Genome DNA from colorectal cancer samples were extracted and subjected to K-ras point mutation analysis by real-time fluorescence ASA with a pair of mutant primer.Only the mutant samples can be amplified,producing double-stranded DNA(ds-DNA) products which can be detected by the fluorescence of a ds-DNA dye,SYBR Green Ⅰ.Genotypes were discriminated according to the different threshold cycles of the samples and the different melting temperatures of their amplicons.The real-time fluorescence ASA assay allowed the detection of mutation in a 1 000-fold excess of wild-type DNA.The analysis time was <1 h.In colorectal cancer samples,K-ras codon 12 mutations were detected in 15 of 31 malignant cases.As a comparison,the sample were also detected by ASA followed by gel electrophoresis analysis.The results show that the real-time fluorescence ASA assay is simple,rapid and low cost.It may hold great promise in practical clinical diagnosis of gene-mutant diseases.