Simple And Multiplexed Enrichment Of Rare DNA Variants Via Sequence-Selective And Temperature-Robust Amplification

Abstract

Rare DNA sequence variants hold important clinical and biological information, but are chal-lenging for existing methods (e.g. PCR, NGS) to profile in an inexpensive, multiplexed, simple-to-implement, and sequence-general way. Here, we present Blocker Displacement Amplification (BDA), a temperature-robust PCR method that selectively amplifies all sequence variants within a roughly 20 nt window by 1000-fold over wildtype sequences, allowing easy detection and quantitation of hundreds of potentials variants originally at [≤]0.1% allele frequency. BDA employs a rationally designed competitive hybridization reaction to achieve similar enrichment performance across anneal temperatures ranging from 56{degrees}C to 64{degrees}C. This temperature robustness facilitates multiplexed enrichment of many different variants across the genome, and furthermore enables the use of in-expensive and portable thermocycling instruments for rare DNA variant detection. To show the sequence generality of BDA, we demonstrated enrichment on 156 single-nucleotide variants (SNVs). BDA has been validated on multiple different PCR platforms, DNA polymerases, and sample types including clinical cell-free DNA samples collected from the blood plasma of lung cancer patients. BDA quantitation of mutation allele fraction is generally consistent with deep sequencing results.