Development and clinical application of highly sensitive detection method (AL-COLD-PCR-SSCP) for somatic mutations in cancer

在癌症中，低水平的突变很难检测到，因为野生型背景的DNA含量要多得多。因此，突变扩增技术是必要的。我们试图建立一种新的检测方法--套接式连接冷-聚合酶链式反应(AL-COLD-PCR)，但未能建立该方法。接下来，我们对COLD-PCR-HRM(高分辨熔融)方法进行了研究。从K-ras突变的癌细胞中提纯的基因组DNA被来自其他细胞系的野生型DNA稀释。通过以下HRM分析，我们比较了冷-聚合酶链式反应和常规聚合酶链式反应检测K-ras基因突变的敏感性。全冷聚合酶链式反应与HRM分析相结合，具有比常规聚合酶链式反应更高的敏感性。将该方法应用于结直肠癌和肺癌临床标本的体细胞突变检测，可检测到大部分突变等位基因。综上所述，COLD-PCR-HRM可用于肿瘤中低水平突变或突变体的突变浓缩和检测。

In cancer, low level mutations cannot easily detected because of the much more DNA amounts of a wild-type background. Therefore, mutation enrichment technique is necessary. We tried to develop a novel detection method, Adapter ligation COLD-PCR (AL-COLD-PCR), however, we could not establish the method. Next, we investigated COLD-PCR-HRM (high resolution melting) method. Genomic DNA purified from cancer cell lines with K-ras mutation was diluted by wild-type DNA from other cell lines. We compared the detection sensitivity of K-ras mutations by COLD-PCR and conventional PCR, by use of the following HRM analysis. Full COLD-PCR can detect with higher sensitivity than conventional PCR, in coupling with HRM analysis. We applied this COLD -PCR-HRM analysis on somatic mutation detection in clinical specimens of colorectal cancer and lung cancer, and most of the mutation alleles could be detected. In conclusion, COLD-PCR-HRM could be utilized in mutation enrichment and detection of low-level mutations or mutants in cancer.