Multiplexed Minor Allele Detection With Skewed Chain Terminators
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Solution Overview
Problem
Existing nucleic acid detection methods struggle to accurately identify and quantify low-frequency nucleic acid variants due to high detection limits and interference from predominant wild-type species, leading to unreliable detection and quantitation of minor alleles and polymorphisms.
Innovation Solution
A multiplexed method involving simultaneous amplification and extension reactions using specific chain terminating reagents, where minor nucleic acid species are differentiated by unique chain terminators, allowing for the generation of chain-terminated extension products that are analyzed to identify and quantify the minor species relative to major species.
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 detection sensitivity is insufficient and cannot accurately identify low-frequency variants below 10-15% frequency
Solution Approach 1:
The detection method is divided into two distinct phases: (1) a pre-enrichment phase that selectively amplifies minor alleles using allele-specific oligonucleotides and selective priming, and (2) a detection phase that analyzes the enriched material. This segmentation allows the system to achieve high detection sensitivity for low-frequency variants while keeping each individual phase manageable in complexity
Solution Approach 2:
Before the actual detection of minor alleles, a preliminary enrichment step is performed using selective amplification conditions that favor the amplification of minor alleles over major alleles. This preliminary action increases the relative concentration of minor alleles in the sample, enabling their detection at frequencies below the conventional 10-15% threshold
2Measurement precision
If the wild type signal is removed to improve variant detection, then the detection sensitivity for low-frequency variants improves, but the ability to quantitate the relative amount of variant is lost
Solution Approach 1:
An intermediary enrichment step is introduced between the sample and the detection system. This intermediary process selectively amplifies minor alleles while preserving the ability to quantify them, serving as a bridge that enhances detection sensitivity without sacrificing quantitation capability. The selective priming and amplification conditions act as mediators that enrich the minor allele signal while maintaining proportionality for accurate measurement
3Productivity
If multiplexed detection of multiple nucleic acids is performed, then the screening efficiency increases, but the detection limit for low-frequency variants worsens due to signal overshadowing
Solution Approach 1:
The multiplexed detection system is segmented into multiple independent detection channels, each optimized for detecting specific minor alleles within a frequency range of 1-10%. By dividing the complex multiplexed signal into separable channels with selective amplification, the system maintains high screening efficiency while achieving detection limits for low-frequency variants that would be obscured in conventional multiplexed approaches
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 method achieves high detection sensitivity and accuracy in identifying and quantifying low-frequency nucleic acid variants, even at frequencies as low as 1-10% relative to the major species, with improved reliability and precision.
Implementation Method 1
simultaneously amplifying target regions of the mixture with amplification primer pairs under amplification conditions that include dNTPs
Implementation Method 2
contacting the amplified mixture with extension primers under extension conditions that include chain terminating reagents
Implementation Method 3
each of the one or more minor nucleic acid species has a chain terminating reagent that is specific for the minor nucleic acid species
Data Source
AI summary
Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.