Enhancer Oligonucleotides for Probe Hybridization Artifacts
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Solution Overview
Problem
Double-stranded DNA probes used in target enrichment assays suffer from self-annealing and cross-annealing artifacts, leading to decreased assay sensitivity and performance due to shared primer-binding sites, which are necessary for cost-effective manufacturing but impair hybridization efficiency.
Innovation Solution
Incorporating enhancer oligonucleotides that bind to universal primer-binding sites on the probes, preventing undesirable interactions and minimizing probe concatenation and re-annealing, thereby improving capture efficiency and uniformity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If double-stranded DNA probes with universal primer-binding sites are used for cost-effective manufacturing, then manufacturing cost is reduced and manufacturing ease is improved, but self-annealing and cross-annealing artifacts occur leading to decreased assay sensitivity and hybridization efficiency
Solution Approach 1:
The patent introduces a third component (blocker oligonucleotide) that acts as an intermediary to bind to the universal primer-binding sites on the probes. This blocker prevents the probes from self-annealing or cross-annealing through these sites, thereby eliminating the harmful artifacts while preserving the cost-effective manufacturing approach using universal primer-binding sites
2Measurement precision
If double-stranded DNA probes are used to capture both positive and negative strands, then data quality is improved by minimizing DNA strand capture bias, but self-annealing and cross-annealing occur resulting in decreased assay performance
Solution Approach 1:
The blocker oligonucleotide serves as a mediator that specifically binds to the universal primer-binding sites on double-stranded probes. This prevents the probes from forming self-annealing or cross-annealing artifacts that would otherwise reduce assay performance, while allowing the probes to maintain their ability to capture both DNA strands effectively
3Productivity
If universal primer-binding sites are included on probes, then probe manufacturing becomes more efficient and cost-effective, but hybridization efficiency is impaired due to probe concatenation and re-annealing
Solution Approach 1:
The blocker oligonucleotide acts as a protective intermediary that binds to universal primer-binding sites during the hybridization process. This prevents probe concatenation and re-annealing events that would reduce hybridization efficiency, while allowing the probes to retain their universal primer-binding sites for cost-effective manufacturing
Solution Approach 2:
The blocker oligonucleotide is introduced in advance to preemptively bind to the universal primer-binding sites on the probes before the probes can undergo unwanted self-annealing or cross-annealing reactions. This preliminary protective action prevents the formation of artifacts that would impair hybridization efficiency
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
The use of enhancer oligonucleotides enhances target enrichment performance by reducing duplicate reads and improving capture uniformity in a dose-dependent manner, leading to better hybridization outcomes and increased sensitivity in nucleic acid sequencing.
Implementation Method 1
one or more enhancer oligonucleotides hybridizing to the second primer binding region
Implementation Method 2
Incorporating enhancer oligonucleotides that bind to universal primer-binding sites on the probes, preventing undesirable interactions
Data Source
AI summary
The invention includes improved methods and compositions for nucleic acid hybridization wherein the improvement comprises the use of enhancer oligonucleotides. Target enrichment is performed using probe oligonucleotides, wherein each probe oligonucleotide comprising a target-binding region, and a first and a second primer-binding region, and one or more enhancer oligonucleotides capable of hybridizing to at least one of the primer binding regions. The forward and reverse primer binding sites can be universal primer binding sites.