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Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification
Nature Biomedical Engineering ( IF 28.1 ) Pub Date : 2017-09-04 , DOI: 10.1038/s41551-017-0126-5
Lucia R. Wu , Sherry X. Chen , Yalei Wu , Abhijit A. Patel , David Yu Zhang

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.



中文翻译:

通过序列选择和稳健扩增扩增稀有DNA变体的多重富集

罕见的DNA序列变异体具有重要的临床和生物学信息,但是现有的检测技术昂贵,复杂,等位基因特异,或者不允许进行大量的多路复用。在这里,我们报告了一种温度稳健的聚合酶链反应方法,我们将其称为阻断剂置换扩增(BDA),该方法选择性地将大约20个核苷酸窗口内的所有序列变异体(包括单核苷酸变异体(SNV))扩增了1,000个折叠超过野生型序列。这样可以轻松检测和定量最初在等位基因频率中≤0.1%的数百种潜在变体。BDA与廉价的热循环仪仪器兼容,并采用合理设计的竞争性杂交反应,可在56°C至64°C的退火温度范围内实现相当的富集性能。为了显示BDA的序列通用性,我们证明了156个SNV的富集和可靠的单位数拷贝检测。我们还显示,BDA检测从肺癌患者血浆中提取的无细胞DNA样品中罕见的驱动程序突变与使用分子谱系标签的深度测序高度一致,接收器操作员特征精度为95%。

更新日期:2017-09-04
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