Oligonucleotide Mixture for Low Abundance Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting low abundance target polynucleotides, such as mutant or rare alleles, are either ineffective at low copy numbers, excessively costly, or time-consuming, failing to accurately identify genetic defects in cancerous cells due to heterogeneity and low abundance of circulating tumor DNA.
Innovation Solution
A mixture comprising specific oligonucleotides, including a target sequence-specific primer, a locus-specific primer, and a target site-specific probe, designed to hybridize and amplify low abundance target polynucleotides, with optimized concentrations of potassium chloride and ammonium sulfate to enhance discrimination and amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods are used to detect low abundance target polynucleotides, then detection capability is limited, but cost and time consumption become excessively high
Solution Approach 1:
The detection method is divided into distinct phases: an enrichment phase that selectively amplifies target sequences containing the variant nucleotide, followed by a detection phase. This segmentation allows the system to focus resources on detecting low abundance targets without the time penalty of analyzing entire genomes, resolving the contradiction between detection capability and time consumption.
Solution Approach 2:
The enrichment phase performs preliminary action by selectively amplifying target sequences before the actual detection occurs. This preliminary enrichment of low abundance target polynucleotides enables subsequent rapid detection, solving the contradiction by preparing the sample in advance to reduce detection time while maintaining high sensitivity.
2Measurement precision
If current methods are used to detect low abundance target polynucleotides, then detection accuracy is limited, but cost becomes excessively high
Solution Approach 1:
The method extracts and isolates only the relevant target sequences containing the variant nucleotide through selective enrichment, discarding the vast majority of non-target DNA. This extraction approach achieves high detection accuracy for low abundance targets without the excessive cost of analyzing entire genomes, as resources are focused only on the clinically relevant sequences.
Solution Approach 2:
The invention changes key parameters of the detection system by using variant-specific primers and probes with optimized nucleotide sequences that selectively bind to target sequences containing specific variant nucleotides. This parameter optimization enables high accuracy detection at lower costs by improving binding specificity and reducing off-target amplification.
3Reliability
If selective enrichment is performed to improve detection of low abundance targets, then specificity increases, but method complexity increases
Solution Approach 1:
The method segments the detection process into enrichment and detection phases, with each phase having specific objectives. The enrichment phase uses variant-specific primers to selectively amplify target sequences, while the detection phase identifies the enriched targets. This segmentation achieves high specificity without excessive complexity by dividing the workflow into manageable, purpose-driven steps.
Solution Approach 2:
The invention introduces variant-specific primers and probes as intermediary molecules that mediate between the complex genomic DNA and the detection system. These intermediaries selectively bind to target sequences containing variant nucleotides, enabling high specificity detection while keeping the overall method relatively simple through standardized PCR and probe-based detection protocols.
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 the detection and differentiation of low abundance target polynucleotides, including mutant alleles, with high specificity and efficiency, even at very low copy numbers, improving cancer diagnosis and treatment strategies.
Implementation Method 1
a first oligonucleotide configured to hybridize to a first sequence in a first target polynucleotide strand
Implementation Method 2
the first oligonucleotide further has a nucleotide residue at its 3'-end that is positioned to hybridize to the target variant nucleotide
Implementation Method 3
methods for amplification of nucleic acids
Data Source
AI summary
This disclosure relates to reagents, mixtures, kits and methods for use in detecting low-frequency target polynucleotides, such as rare allelic variants.