Mutant DNA Enrichment via Mismatch Intercalation
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Solution Overview
Problem
Current methods face challenges in detecting and enriching low-abundance mutant DNA sequences due to their presence in vast excess of wild-type DNA, making it difficult to accurately identify and amplify rare mutations in biological samples.
Innovation Solution
The method involves using oligonucleotides with mismatches to selectively bind mismatch intercalating compounds with affinity labels, which are then captured by an affinity matrix, allowing for the enrichment and subsequent amplification of low-abundance mutant DNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional amplification methods are used to detect mutant DNA, then amplification can occur, but the detection precision is poor due to the vast excess of wild-type DNA overwhelming the signal from rare mutations
Solution Approach 1:
The patent extracts and enriches mutant DNA from the complex mixture by using mismatch-specific binding agents that selectively capture mutant sequences. The affinity purification step separates mutant DNA from the overwhelming excess of wild-type DNA, concentrating the rare target molecules for subsequent detection
Solution Approach 2:
The patent introduces mismatch-specific oligonucleotides and affinity purification reagents as intermediary agents. These mediators selectively bind to mutant DNA through mismatch recognition, enabling specific capture and enrichment of mutant sequences without being affected by the presence of wild-type DNA
2Measurement precision
If enrichment methods are applied to increase mutant DNA signal, then detection sensitivity improves, but the process complexity increases due to additional purification steps
Solution Approach 1:
The patent changes the binding parameters of oligonucleotides to create intentional mismatches that specifically recognize mutant sequences. By adjusting hybridization conditions and using mismatch-specific affinity reagents, the method achieves selective enrichment while maintaining a relatively simple workflow
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 effectively enriches and amplifies low-abundance mutant DNA, enabling accurate detection even in samples where wild-type DNA vastly outnumber mutant DNA, improving the sensitivity and specificity of mutation detection.
Implementation Method 1
contacting the duplex polynucleotides with a mismatch intercalating compound that is attached with an affinity label to generate a reaction mixture, wherein said mismatch intercalating compound is capable of binding to the duplex polynucleotides that contain a mismatch
Implementation Method 2
subjecting the reaction mixture to an affinity matrix that recognizes and binds to the affinity label on the mismatch intercalating compound
Implementation Method 3
providing conditions suitable for hybridization of the oligonucleotide to the target nucleic acid to generate duplex polynucleotides consisting of the oligonucleotide and one strand of either variant of the target nucleic acid sequence
Data Source
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AI summary
The detection of the presence of rare somatic mutations from a biological sample is often challenging due to the simultaneous presence of a vast excess of wild-type DNA. The present invention describes novel compounds and methods that would allow the enrichment of mutant DNA by depleting amplifiable wild-type DNA.