ARMS实时PCR和MALDI-TOF-MS进行K-ras基因分型

Genotyping of K-ras Alleles by ARMS Real-time PCR and MALDI-TOF-MS

[目的]建立K-ras基因12位密码子第一、二位点基因分型的研究模型。[方法]采用ARMS实时PCR和MALDI-TOF-MS两种方法,以自行构建的质粒DNA为模板,建立K-ras基因12位密码子第一、二位点基因分型的研究模型。[结果]在相同的扩增条件下,采用ARMS实时PCR方法,第一个位点的野生型和突变型的ΔCt值至少为,但第8二个位点野生型和突变型的ΔCt值仅为1～2左右。第二个位点改用MALDI鄄TOF-MS分型方法,采用一条公用引物和dATP,ddTTP,ddCTP和ddGTP的混和物,12密码子第二位点基因分型可以准确检测。[结论]ARMS实时PCR方法分析速度快,操作简便,但它需要合适的反应条件及高质量模板。MALDI-TOF-MS分型方法虽然步骤较多,但准确度高,反应体系稳定并具有高通量潜力。

To establish a research model for genotyping the first and second position of codon 12 of K-ras genotype. ARMS real-time PCR and MALDI-TOF-MS methods were used to screen K-ras genotype of the first and second position of codon 12 from the man-made plasmid templates DNAs. ΔCt value between wild-type and the mutants were at least 8 cycles for the first poisition of codon 12,but only 1～2 cycle for the second site by using ARMS real-time PCR with allele-specific primers. The second position was further analyzed by the MALDI-TOF-MS method using one common primer in combination with dATP, ddTTP, ddCTP and ddGTP. Different genotypes at second position of codon 12 were detected accuratly. [Conclusions] ARMS real-time PCR genotyping is faster and more convenient, but it needs a suitable reaction conditions and high -quality templates. MALDI-TOF-MS method is more time-consuming, but it has characteristic of high accuracy, high stability, and high-throughput.