Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations
Δ-PCR, A Simple Method to Detect Translocations and Insertion/Deletion Mutations
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DOI：
10.1016/j.jmoldx.2010.11.004
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发表时间：
2011-01-01
影响因子：
4.1
通讯作者：
Gocke, Christopher D.
中科院分区：
文献类型：
--
作者：
Lin, Ming-Tseh;Tseng, Li-Hui;Gocke, Christopher D.
当潜在的扩增子大小变化很大时，染色体易位和小的插入/缺失突变的 PCR 检测具有挑战性。分子诊断实验室面临滤泡性淋巴瘤中的 BCL2-IGH 易位和白血病中 FLT3 基因内部串联重复突变的困难，这些突变点广泛分布。突变可能是多重的，信号强度低，背景噪音升高。我们开发了一种称为 Delta-PCB 的策略，可确保 PCR 特异性并识别各个断点。 Delta-PCR 同时使用两个正向引物（外部和内部）和反向引物。内部引物充当探针，与外部引物具有确定的距离 Delta。对于滤泡性淋巴瘤，我们为潜在断点制备了上游 BCL2 特异性引物，以与常见的下游 VLJH 引物配对。通过毛细管电泳确定多重 PCR 扩增子的大小。每个上游对都有一个定义的间隔来分隔它们，该间隔唯一地标识断点。即使信号强度较低，两个具有确定大小差异的扩增子的存在也证实了重排的有效性和特定断点的身份。通过测试 40 个滤泡性淋巴瘤和 12 个来自福尔马林固定石蜡包埋 (FFPE) 块的对照样本，我们表明多重 Delta-PCR 是一种简单、灵敏的策略，可识别具有多个断点或伴侣的易位。该策略还应用于检测具有内部串联重复突变的小型白血病克隆，并且可以在其他插入/缺失和重复突变方面具有更广泛的应用。 (J Mol Diagn 2011, 13:85-92; DOI: 10.1016/j.jmoldx.2010.11.004)
PCR detection of chromosomal translocations and small insertion/deletion mutations is challenging when potential amplicon size varies greatly. Molecular diagnostic laboratories face such difficulties with the BCL2-IGH translocation in follicular lymphoma and with internal tandem duplication mutation of the FLT3 gene in leukemia, where breakpoints are widely distributed. mutations may be multiple, signal strength is low, and background noise is elevated. We developed a strategy, called Delta-PCB, that ensures PCR specificity and identifies individual breakpoints. Delta-PCR uses two forward primers (external and internal) and a reverse primer simultaneously. The internal primer functions as a probe with a defined distance Delta from the external primer. For follicular lymphoma, we prepared upstream, BCL2-specific primers for potential breakpoints to pair with a common, downstream VLJH primer. Multiplexed PCR amplicons are sized by capillary electrophoresis. Each of the upstream pairs has a defined interval separating them that uniquely identifies the breakpoint. The presence of two amplicons with a defined size difference confirms validity of the rearrangement and identity of the specific breakpoint, even if signal strength is low. By testing 40 follicular lymphoma and 12 control specimens from formalin-fixed, paraffin-embedded (FFPE) blocks, we showed that multiplex Delta-PCR is a simple, sensitive strategy to identify translocations with multiple breakpoints or partners. The strategy was also applied to detect minor leukemic clones with internal tandem duplication mutations and could have broader applications for other insertion/deletion and duplication mutations. (J Mol Diagn 2011, 13:85-92; DOI: 10.1016/j.jmoldx.2010.11.004)