A SNaPshot assay for the rapid and simple detection of four common hotspot codon mutations in the PIK3CA gene.
A SNaPshot assay for the rapid and simple detection of four common hotspot codon mutations in the PIK3CA gene.
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DOI：
10.1186/1756-0500-2-66
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发表时间：
2009-04-29
影响因子：
1.8
通讯作者：
Knowles MA
中科院分区：
文献类型：
--
作者：
Hurst CD;Zuiverloon TC;Hafner C;Zwarthoff EC;Knowles MA
PIK 3CA基因中的激活突变已在多种人类恶性肿瘤中鉴定，并且通常在位于外显子9和20中的螺旋和激酶结构域中的热点密码子中检测到。用于检测PIK 3CA突变的现有方法是耗时和/或昂贵的。在本研究中，我们描述了PIK 3CA SNaPshot检测在这些外显子中的频繁突变的筛选的第一个应用。已经开发并评价了用于同时检测四种常见PIK 3CA热点突变（E542 K、E545 G、E545 K和H1047 R）的SNaPshot测定。该检测试剂盒结合了多重PCR扩增和多重引物延伸检测，可在一个反应中靶向检测所有四种突变。使用先前通过高分辨率解链分析和测序分析突变的样品测试该方法。检测到的所有突变均一致，未获得假阳性结果。灵敏度测试表明，SNaPshot检测可以检测突变DNA时，它代表5-10%的总DNA存在。该方法的应用，从福尔马林固定石蜡包埋的样品中提取的DNA的分析也被证明。本文所述的SNaPshot检测提供了一种快速、灵敏、廉价和特异性的方法，用于分析新鲜和存档患者样本中常见的PIK 3CA突变。
Activating mutations in the PIK3CA gene have been identified in a variety of human malignancies and are commonly detected in hotspot codons located in the helical and kinase domains in exons 9 and 20. Existing methodologies for the detection of PIK3CA mutations are time-consuming and/or expensive. In the present study we describe the first application of a PIK3CA SNaPshot assay to the screening of frequent mutations in these exons. A SNaPshot assay for the simultaneous detection of four frequent PIK3CA hotspot mutations (E542K, E545G, E545K and H1047R) has been developed and evaluated. The assay combines multiplex PCR amplification with a multiplex primer extension assay to allow targeted detection of all four mutations in one reaction. The method was tested using samples that had previously been analysed for mutations by high-resolution melting analysis and sequencing. All mutations detected were concordant and no false positive results were obtained. Sensitivity tests showed that the SNaPshot assay could detect mutant DNA when it represents 5–10% of the total DNA present. The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated. The SNaPshot assay described here offers a fast, sensitive, inexpensive and specific approach to the analysis of frequent PIK3CA mutations in both fresh and archival patient samples.