Abstract：
Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.
摘要：
通过DNA解链分析(DMA)扫描突变是基于不对称PCR,然后用TaqMan探针解链由单链扩增子形成的双链体。该方法最适合临床基因检测；它易于执行，高通量，和敏感。DMA法对突变等位基因的检测限约为3%,远远高于Sanger测序的灵敏度。此外,DMA方法以封闭管格式实现，而在没有任何中间或额外程序的单管中进行2小时测定，从而最大限度地减少样品交叉污染的风险。通过扫描具有临床意义的基因KRAS的突变来进行DMA方法的验证，NRAS,BRAF,和PIK3CA来自黑色素瘤患者肿瘤的324个DNA样本，结直肠癌和肺癌。DNA直接从肿瘤组织中分离，或福尔马林固定石蜡包埋的肿瘤组织。通过Sanger测序验证检测到的突变。在每种肿瘤类型中鉴定的突变光谱与文献数据相对应，因此，验证DMA的使用。
