Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
Clin Chem Lab Med. 2012 Jan 31;50(6):1035-40. doi: 10.1515/cclm-2011-0601.
The aim of our study was to establish an unlabeled-probe high-resolution melting (HRM) approach to the detection of Kirsten RAS (KRAS) codon 12 and 13 mutations in pancreatic adenocarcinoma (PA) tissues as a novel and effective diagnostic technique.
We tested the sensitivity and specificity of this genotyping approach in cell lines with known KRAS mutations using 166 bp amplicons and 37 bp wild-type probe to detect KRAS codon 12 and 13 mutations. We screened 49 PA tissues to be subsequently sequenced to confirm the mutations. Simultaneously, we tested the specimens using Sanger sequencing and then used target-DNA cloning and sequencing for verification.
It was found that unlabeled-probe HRM was reliable in detecting 3% of mutant cell lines DNA diluted with that of the wild-type, whereas Sanger sequencing could only discriminate 20% mutant cell ratios. In detecting 49 specimens, the former was capable of detecting 23 mutations (46.9%); and the latter could observe 15 (30.6%). For further verification, T-A DNA cloning and sequencing was applied to the differences, with the results matching those of the unlabeled-probe HRM.
It was concluded that the unlabeled-probe HRM approach can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in diagnosing and treating PA.
本研究旨在建立一种无标记探针高分辨率熔解（HRM）方法，用于检测胰腺腺癌（PA）组织中的 Kirsten RAS（KRAS）密码子 12 和 13 突变，作为一种新的有效的诊断技术。
我们使用 166 bp 扩增子和 37 bp 野生型探针来检测 KRAS 密码子 12 和 13 突变，在具有已知 KRAS 突变的细胞系中测试了这种基因分型方法的灵敏度和特异性。我们筛选了 49 份 PA 组织，随后进行测序以确认突变。同时，我们使用 Sanger 测序测试了这些标本，然后使用靶向 DNA 克隆和测序进行验证。
结果发现，无标记探针 HRM 可可靠地检测到用野生型 DNA 稀释的 3%突变细胞系 DNA，而 Sanger 测序只能区分 20%的突变细胞比例。在检测 49 个标本时，前者能够检测到 23 个突变（46.9%）；后者只能观察到 15 个（30.6%）。为了进一步验证，应用 T-A DNA 克隆和测序来检测差异，结果与无标记探针 HRM 相符。
无标记探针 HRM 方法可以作为一种敏感和准确的筛选技术，用于检测 KRAS 密码子 12 和 13 突变，以诊断和治疗 PA。
