Mutation detection by real-time PCR: a simple, robust and highly selective method.
Mutation detection by real-time PCR: a simple, robust and highly selective method.
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DOI：
10.1371/journal.pone.0004584
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Sinicropi D
中科院分区：
文献类型：
--
作者：
Morlan J;Baker J;Sinicropi D
用于诊断疾病，特别是癌症的分子测试，越来越多地被医生及其患者接受，用于疾病预后和选择治疗方案。基因表达谱和基因突变是肿瘤分子表征的关键参数。突变分析的方法多种多样，但高选择性分析的发展往往需要一个反复试验的过程，很少与实时PCR兼容。我们试图开发一种基于实时pcr的突变测定方法，成功地解决了这些问题。我们描述的方法是基于广泛使用的TaqMan®实时PCR技术，并结合等位基因特异性PCR和阻断试剂（ASB-PCR）来抑制野生型等位基因的扩增。ASB-PCR可用于检测任何类型组织中提取的DNA或RNA的种系或体细胞突变，包括福尔马林固定石蜡包埋的肿瘤标本。开发了一套试剂设计规则，能够灵敏和选择性地检测单点替换、插入或缺失，以对抗野生型等位基因的背景，在千倍或更大的过量。ASB-PCR是一种简单而强大的方法，用于分析广泛使用的TaqMan®实时RT-PCR协议中的单核苷酸突变和多态性。ASB-PCR设计规则始终产生高选择性突变分析，同时避免了重新设计和优化分析试剂的需要。该方法与福尔马林固定组织兼容，可在同一板上同时进行RT-PCR基因表达分析。不需要TaqMan化学以外的专有试剂，因此该方法可以在任何具有实时PCR能力的研究实验室中进行。
Molecular tests for diagnosis of disease, particularly cancer, are gaining increased acceptance by physicians and their patients for disease prognosis and selection of treatment options. Gene expression profiles and genetic mutations are key parameters used for the molecular characterization of tumors. A variety of methods exist for mutation analysis but the development of assays with high selectivity tends to require a process of trial and error, and few are compatible with real-time PCR. We sought to develop a real-time PCR-based mutation assay methodology that successfully addresses these issues. The method we describe is based on the widely used TaqMan® real-time PCR technology, and combines Allele-Specific PCR with a Blocking reagent (ASB-PCR) to suppress amplification of the wildype allele. ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive and selective detection of single point substitutions, insertions, or deletions against a background of wild-type allele in thousand-fold or greater excess. ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqMan® protocol for real time RT-PCR. The ASB-PCR design rules consistently produce highly selective mutation assays while obviating the need for redesign and optimization of the assay reagents. The method is compatible with formalin-fixed tissue and simultaneous analysis of gene expression by RT-PCR on the same plate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any research laboratory with real-time PCR capability.
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