Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

  • Nature Biomedical Engineering 1714723 (2017)
  • doi:10.1038/s41551-017-0126-5
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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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The authors thank J. H. Bae and J. S. Wang for experimental advice; C. Lee and G. Bao for advice and for use of their ddPCR instrument; D. Khodakov for initial testing of BDA on the MiniPCR platform; P. Song for assistance with gel electrophoresis; and A. Narayan for testing BDA in A.A.P.'s lab at the Yale School of Medicine. This work was funded by CPRIT grant RP140132 and NIH grant R01CA203964 to D.Y.Z., and NIH grant R01CA197486 to A.A.P.

Author information


  1. Department of Bioengineering, Rice University, Houston, TX, 77030, USA

    • Lucia R. Wu
    • , Sherry X. Chen
    •  & David Yu Zhang
  2. Thermo Fisher, San Francisco, CA, 94080, USA

    • Yalei Wu
  3. Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, 06510, USA

    • Abhijit A. Patel


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L.R.W. conceived the project, designed and conducted the experiments, analysed the data, and wrote the paper. S.X.C. designed the experiments, wrote automated design software, and analysed the data. Y.W. conducted the experiments and analysed the data. A.A.P. provided clinical samples and analysed the data. D.Y.Z. conceived the project, guided experimental design, analysed the data, and wrote the paper.

Competing interests

There is a patent pending on the BDA system described in this work. D.Y.Z. is a co-founder and significant equity holder of Nuprobe Global, a startup commercializing BDA technology

Corresponding author

Correspondence to David Yu Zhang.

Electronic supplementary material

  1. Supplementary Information

    Supplementary discussion, figures, methods, data and code.