Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes
Detection of Low-Level KRAS Mutations Using PNA-Mediated Asymmetric PCR Clamping and Melting Curve Analysis with Unlabeled Probes
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DOI：
10.2353/jmoldx.2010.090146
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发表时间：
2010-07-01
影响因子：
4.1
通讯作者：
Yoo, Nam Jin
中科院分区：
文献类型：
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作者：
Oh, Ji Eun;Lim, Hee Sun;Yoo, Nam Jin
临床癌症标本中体细胞突变的检测常常受到过量野生型DNA的阻碍。本研究的目的是开发一种简单而经济的方案，而不使用荧光探针来检测低水平的突变。在这项研究中，我们结合肽核酸（PNA）夹PCR与不对称引物和熔解曲线分析使用未标记的检测探针。在5种不同的突变细胞系中，抑制野生型等位基因扩增的PNA夹持PCR对KRAS密码子12突变检测比非夹持PCR更敏感。测试了三种检测探针（完全匹配的反义、错配的反义和错配的有义），错配的有义检测探针在夹持条件下显示出最高的灵敏度（0.1%突变体检测）。使用该探针，我们不仅能够检测完全匹配的KRAS突变，而且还能够检测其他4种KRAS错配突变。然后，我们将该方案应用于10个具有KRAS密码子12突变的人结肠癌组织，成功地检测了所有这些组织中的突变。我们的数据表明，完全匹配的反义PNA和错配的正义检测探针的组合可以在细胞系和人体组织中以高灵敏度检测KRAS突变。此外，这项研究可能会证明一个容易适用的协议，用于检测其他癌症基因的低水平突变。（J Mol Diagn 2010，12：418-424; DOI：10.2353/jmoldx.2010.090146）
Detection of somatic mutations in clinical cancer specimens is often hampered by excess wild-type DNA. The aim of this study was to develop a simple and economical protocol without using fluorescent probes to detect low-level mutations. In this study, we combined peptide nucleic acid (PNA)-clamping PCR with asymmetric primers and a melting curve analysis using an unlabeled detection probe. PNA-clamping PCR, which suppressed amplification of the wild-type allele, was more sensitive for KRAS codon 12 mutation detection than nonclamping PCR in 5 different mutant cell lines. Three detection probes were tested (a perfectly matched antisense, a mismatched antisense, and a mismatched sense), and the mismatched sense detection probe showed the highest sensitivity (0.1% mutant detection) under clamping conditions. With this probe, we were able to detect not only the perfectly matched KRAS mutation, but also 4 other mismatched mutations of KRAS. We then applied this protocol to 10 human colon cancer tissues with KRAS codon 12 mutations, successfully detecting the mutations in all of them. Our data indicate that the combination of perfectly matched antisense PNA and a mismatched sense detection probe can detect KRAS mutations with a high sensitivity in both cell lines and human tissues. Moreover, this study might prove an easily applicable protocol for the detection of low-level mutations in other cancer genes. (J Mol Diagn 2010, 12:418-424; DOI: 10.2353/jmoldx.2010.090146)