Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.
Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.
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DOI：
10.1038/s41551-017-0126-5
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发表时间：
2017
影响因子：
28.1
通讯作者：
Zhang DY
中科院分区：
文献类型：
--
作者：
Wu LR;Chen SX;Wu Y;Patel AA;Zhang DY
罕见的dna序列变异具有重要的临床和生物学信息，但现有的检测技术昂贵，复杂，等位基因特异性，或者不允许显著的多路复用。在这里，我们报告了一种温度稳定的聚合酶链反应方法，我们称之为阻滞剂位移扩增（BDA），它可以在大约20个核苷酸窗口内选择性地扩增所有序列变体，包括单核苷酸变体（snv），比野生型序列扩增1000倍。这可以很容易地检测和定量数百个潜在的变异，最初≤0.1%的等位基因频率。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，在56°C至64°C的退火温度范围内实现相当的富集性能。为了证明BDA序列的普遍性，我们证明了156个snv的富集和对个位数拷贝的可靠检测。我们还表明，从肺癌患者血浆中提取的无细胞DNA样本中，BDA检测罕见驱动突变与使用分子谱系标签的深度测序高度一致，接收器算子特征准确率为95%。
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%.
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影响因子：
--
作者：
Kidess E;Heirich K;Wiggin M;Vysotskaia V;Visser BC;Marziali A;Wiedenmann B;Norton JA;Lee M;Jeffrey SS;Poultsides GA
通讯作者：
Poultsides GA
影响因子：
7
作者：
Hiatt JB;Pritchard CC;Salipante SJ;O'Roak BJ;Shendure J
通讯作者：
Shendure J
影响因子：
3.7
作者：
Morlan J;Baker J;Sinicropi D
通讯作者：
Sinicropi D
影响因子：
14.9
作者：
Forbes SA;Bindal N;Bamford S;Cole C;Kok CY;Beare D;Jia M;Shepherd R;Leung K;Menzies A;Teague JW;Campbell PJ;Stratton MR;Futreal PA
通讯作者：
Futreal PA
影响因子：
48
作者：
Narayan, Azeet;Bommakanti, Ananth;Patel, Abhijit A.
通讯作者：
Patel, Abhijit A.