Multiplex Nucleic Acid Variant Detection via Termination Control
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid detection methods struggle to efficiently identify and quantify low-abundance variants among high-abundance variants in a single reaction, often resulting in false negatives and reduced sensitivity due to interference from more abundant species.
Innovation Solution
A multiplex method involving the preparation of amplicons from target nucleic acid species, hybridization with oligonucleotides specific to both low-abundance and high-abundance variants, followed by single base extension reactions using terminating nucleotides with a capture agent, and subsequent capture and release steps to detect the presence or absence of variants, allowing for the quantification of low-abundance variants relative to high-abundance variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid detection methods are used, then the detection process is simple, but the sensitivity to detect low-abundance variants is reduced due to interference from high-abundance variants
Solution Approach 1:
The detection process is divided into distinct functional stages: amplicon preparation, hybridization with oligonucleotides, single base extension with terminating nucleotides, capture, and release. This segmentation allows each step to be optimized independently, with the extension step specifically designed to differentiate low-abundance variants through controlled incorporation of terminating nucleotides at low concentrations, thereby improving sensitivity without requiring complete redesign of the entire system
Solution Approach 2:
Terminating nucleotides serve as intermediary molecules that mediate the differentiation between low-abundance and high-abundance variants. These nucleotides incorporate selectively during the extension step and enable subsequent capture and detection, acting as a bridge between the hybridization step and final detection, allowing sensitive identification of low-abundance variants through their unique extension products
2Reliability
If high concentrations of terminating nucleotides are used, then the extension reaction is robust, but false negative results increase due to interference from high-abundance variants
Solution Approach 1:
The concentration of terminating nucleotides is precisely controlled and kept low during the extension reaction. This parameter change allows the extension to proceed robustly enough to detect variants while preventing excessive formation of extension products from high-abundance variants that would cause false negatives. The low concentration ensures that only low-abundance variants with their specific extension products are reliably detected
Solution Approach 2:
Different terminating nucleotides are used with different concentrations tailored to their specific detection requirements. The system applies local quality control by adjusting nucleotide concentrations according to the specific variant being detected, allowing optimized detection conditions for each variant type while maintaining overall system reliability
3Productivity
If multiple target nucleic acid species are detected in a single reaction, then the productivity increases, but the measurement precision for low-abundance variants decreases due to signal interference
Solution Approach 1:
The extension reaction system is designed with universal applicability to detect multiple target nucleic acid species simultaneously. The same extension composition with controlled terminating nucleotide concentrations can be applied across different variants, and the capture system can accommodate multiple species, enabling high productivity while maintaining precision through consistent low-concentration extension conditions that prevent interference
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
This approach enables the sensitive detection and quantification of low-abundance variants, providing a positive control for false negatives and improving the signal-to-noise ratio, thereby enhancing the accuracy and reliability of nucleic acid identification.
Implementation Method 1
In a single reaction hybridizing the amplicons to a plurality of oligonucleotide species
Implementation Method 2
Contacting the hybridized oligonucleotides with an extension composition comprising a terminating nucleotide specific for the low-abundance variants
Implementation Method 3
Contacting the extended oligonucleotides with a solid phase under conditions in which the capture agent interacts with the solid phase, thereby capturing the extended oligonucleotides onto the solid phase
Implementation Method 4
releasing the extended oligonucleotides in by contacting the solid phase at elevated temperature conditions with a competitor, wherein the competitor comprises the free form of the capture agent that interacts with the solid phase
Data Source
Figure 1
AI summary
Provided herein are multiplex methods for detecting the presence or absence and amount of variants of a plurality of target nucleic acid species having low-abundance variants and high-abundance variants.