Multiplexed Blocker Displacement Amplification for Low-VAF Alleles
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Solution Overview
Problem
Existing methods struggle to simultaneously profile many sequence variations at low variant allele frequencies (VAF) due to limitations in sensitivity and cost-effectiveness of microarrays, digital PCR, and ultradeep NGS.
Innovation Solution
The use of blocker displacement amplification (BDA) oligo sets, comprising BDA forward primers, blockers, and reverse primers, to enrich low VAF sequence variations by 100-fold or more, allowing profiling via low-depth NGS or microarrays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If microarrays are used for profiling sequence variations, then cost is reduced, but sensitivity for low VAF variants deteriorates
Solution Approach 1:
The BDA method performs preliminary enrichment of variant alleles before the actual profiling step. By pre-amplifying variant-containing DNA using blockers that selectively inhibit wild-type amplification, the method prepares the sample in advance to achieve both cost-effectiveness and sensitivity.
Solution Approach 2:
The BDA oligos act as intermediaries between the original DNA sample and the final profiling step. These oligos selectively bind to and amplify variant-containing sequences, serving as a bridge that enables sensitive detection using cost-effective downstream methods.
2Measurement precision
If digital PCR is used for detecting low VAF variants, then sensitivity is improved, but cost increases
Solution Approach 1:
The BDA method creates multiple copies of variant-containing DNA sequences through selective amplification. By generating abundant copies of rare variants, the method enables sensitive detection using lower-cost methods that would otherwise be insufficient for detecting low VAF variants.
3Measurement precision
If ultradeep NGS is used for profiling sequence variations, then sensitivity for low VAF variants is improved, but cost and complexity increase
Solution Approach 1:
The BDA method extracts and enriches only the variant-containing sequences from the complex genomic DNA mixture. By isolating and amplifying only the relevant variant alleles, the method simplifies the subsequent profiling step and reduces the need for complex ultradeep sequencing.
4Ease of operation
If standard PCR is used for amplification, then simplicity is maintained, but ability to enrich low VAF variants deteriorates
Solution Approach 1:
The BDA method introduces local quality differentiation through blocker oligos that specifically target wild-type sequences. This creates a localized inhibition effect at the binding site, allowing standard PCR machinery to be used while achieving selective enrichment of variant sequences.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables simultaneous amplification and profiling of up to 1,000,000 genetic loci with low VAF variations, enhancing detection of rare mutations and cell line contamination through improved sensitivity and cost-effectiveness.
Implementation Method 1
each BDA blocker contains a 3' sequence or modification that prevents extension by DNA polymerase
Implementation Method 2
mixing a sample comprising DNA with a DNA polymerase and a blocker displacement amplification (BDA) oligo set
Data Source
AI summary
Provided herein are reagents and methods for simultaneously enriching many potential rare genetic variants at different genetic loci. The rare variants enriched can include single nucleotide polymorphisms (SNPs), single nucleotide variants, or small insertions and deletions. Embodiments of the invention include procedures for integration with downstream next generation sequencing (NGS) analysis. Embodiments of the invention include analysis of nonpathogenic SNPs for the determination of cell identity and detection of cell contamination using qPCR or NGS.