Overview

The NuProbe team has pioneered the development of a unique and novel PCR-based enrichment method called Blocker Displacement Amplification (BDA). The BDA technology enables the selective amplification of low abundant sequence variants (SNV and indels) in a background of wildtype DNA.

Systematic experimental validation of BDA enrichment on hundreds of different SNVs and on a variety of samples ranging from synthetic templates to cell-line genomic DNA to clinical blood samples have demonstrated the robustness of BDA as a rare variant enrichment and detection method.

 

Key Features

  • Ultrasensitive and quantitative: BDA allows for easy detection and quantitation of hundreds of potential variants down to less than 0.1% allele frequency. We have shown that BDA yields a median enrichment greater than 1,000 fold across 200+ SNPs.
  • High multiplexing capabilities: BDA has a broad temperature range spanning a 8 °C window (56 – 64 °C) over which it effectively enriches variants. This temperature robustness is a key feature that provides practical advantages for nucleic acid testing by facilitating the highly-multiplexed enrichment of many SNVs.
  • Simple method: BDA requires little to no empirical protocol optimization, employs unmodified and broadly available DNA oligonucleotides, is broadly compatible with many enzymes, is uniquely robust to buffer conditions, and is tolerant to instrument temperature inaccuracies and non-uniformity.
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Mechanism

BDA Mechanism

  • BDA Mechanism

  • The BDA blocker is designed to be perfectly complementary to a known template sequence and to contain an overlap region with the forward primer to enable molecular competition between the primer and the blocker. When the BDA blocker is bound to a variant template, the resulting mismatch bubble between the blocker and the template increases the favorability of the primer to displace the blocker. This results in the differential amplification of the variant and the wildtype templates, yielding to 1,000-fold enrichment of the variant template when compounded over multiple cycles.
Multiplex Setting

  • Multiplex Setting

  • BDA can be used in a multiplex setting to simultaneously enrich DNA sequence variants in multiple. Because all wildtype template amplification is suppressed, the majority of PCR products will correspond to amplicons from initially low VAF DNA sequence variants.
qBDA workflow

  • qBDA

  • By integrating molecular barcoding technology with NuProbe’s Blocker Displacement Amplification (BDA) allele enrichment, Quantitative Blocker Displacement Amplification (QBDA) enables calibration-free accurate VAF quantitation with low-depth sequencing. QBDA integrates sequence-selective variant enrichment and unique molecular identifiers (UMIs) for quantitation of mutations below 0.01% VAF.
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Performance

Molecular Diagnostics

  • qPCR amplification results on BDA enrichment

  • Reference samples with various VAF values were prepared by mixing gBlock and WT genomic DNA at certain ratios. BDA reactions with 100 ng of DNA input were performed on a Bio-Rad CFX96 qPCR instrument to assess BDA enrichment.

Rare Mutations

  • BDA variant enrichment performance

  • Effective enrichment at 1% VAF level was observed in 100% (39/39) mutations and at 0.1% VAF level in 36/39 (92.3%) mutations.
qBDA for MRD

  • qBDA MRD detection of ≤ 0.01% VAF

  • With quantitative Blocker Displacement Amplification (qBDA) technology, we can detect minimal residual disease (MRD) at ≤ 0.01% VAF. Sample shown was prepared with FLT3/DNMT3A/IDH1/IDH2 mutations spiked into 1μg human PBMC gDNA.
BDA enrichment is tolerant to the DNA input

  • BDA enrichment is tolerant to the DNA input

  • Multiplex reactions with different VAF samples and linearly correlated log(VAF) values to ∆Ct. Correlation constant R2 values of greater than 0.99 and linear range between 0.1% and 100% demonstrate that quantitative VAF values can be inferred for comparison with NGS results. The linear relationship between ∆Ct and log(VAF) is preserved across a wide range of DNA input quantities, from 20 ng to 300 ng.
Sequence verification by Sanger

  • Variant verification of BDA product by Sanger sequencing

  • To confirm the identity of quantities detected and quantitated by qPCR, Sanger sequencing was performed on the qPCR amplicons. Standard Sanger sequencing protocols will be applied. Because BDA enrichment enriches the VAF of mutations by over 200-fold, Sanger’s limit of detection of 20% VAF is effectively improved to 0.1% VAF

Publication References:

Calibration-free NGS quantitation of mutations below 0.01% VAF

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Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing

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

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