Back to Search Start Over

Simple and Multiplexed Enrichment of Rare DNA Variants via Sequence-Selective and Temperature-Robust Amplification

Authors :
David Zhang
Abhijit A. Patel
Lucia R. Wu
Sherry X. Chen
Yalei Wu
Publication Year :
2017
Publisher :
Cold Spring Harbor Laboratory, 2017.

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°C to 64°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.

Details

Database :
OpenAIRE
Accession number :
edsair.doi.dedup.....f150eb92bc5fdd56a2a6445ebf7ff118