Nucleic Acid Detection via Restriction Digestion Enrichment
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Solution Overview
Problem
Current nucleic acid detection methods are inadequate for sensitive and efficient detection of rare genetic variants and mutations, particularly due to limitations in sensitivity, specificity, and workflow complexity, especially when dealing with DNA or RNA samples where most sequences are wild-type, hindering the analysis of rare genetic variants and mutations.
Innovation Solution
The method involves using proteins with nucleic acid cleaving activity, such as restriction endonucleases, Cas enzymes, Ago enzymes, or their complexes, to digest non-target nucleic acids before or during amplification, allowing for the enrichment and detection of target nucleic acids with specific alterations, using techniques like recombinase polymerase amplification (RPA) and CRISPR-based detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR-based detection is used for rare genetic variants, then detection sensitivity is improved, but the method becomes less effective when majority sequences are wild-type
Solution Approach 1:
The patent applies preliminary action by performing restriction digestion of wild-type sequences before amplification. The method uses restriction enzymes to selectively digest wild-type DNA sequences, thereby enriching the target sequences with mutations before amplification. This preliminary enrichment step resolves the contradiction by preparing the sample in advance to overcome the low frequency of target sequences in wild-type majority samples.
Solution Approach 2:
The patent extracts the harmful wild-type sequences from the sample through restriction digestion. By using restriction enzymes that recognize and cut wild-type sequences but leave mutant sequences intact, the method removes the interfering wild-type background before amplification. This extraction of harmful elements enables sensitive detection of rare mutations without being overwhelmed by the majority wild-type sequences.
2Measurement precision
If restriction digestion is used to enrich target sequences, then detection sensitivity is enhanced, but workflow complexity increases
Solution Approach 1:
The patent merges the restriction digestion step with the amplification step into a single reaction system. The restriction enzymes, amplification primers, and nucleic acid templates are combined in one tube, allowing simultaneous restriction digestion and amplification. This merging eliminates the need for separate workflow steps, thereby reducing procedural complexity while maintaining the sensitivity enhancement from wild-type sequence enrichment.
Solution Approach 2:
The patent creates a multi-functional reaction system where a single reaction mixture performs multiple functions: restriction digestion of wild-type sequences, amplification of target sequences, and detection of mutations. The reaction system integrates enzymes and reagents that simultaneously accomplish enrichment and amplification, reducing the number of separate operations needed and simplifying the overall workflow.
3Adaptability or versatility
If next-generation sequencing is used for mutation detection, then comprehensive profiling is achieved, but time consumption and cost increase
Solution Approach 1:
The patent extracts only the relevant information needed for mutation detection by using restriction digestion to enrich target sequences before amplification. Instead of sequencing entire genomes or exomes as required by NGS, the method selectively enriches and amplifies only the regions containing mutations of interest. This extraction of relevant sequences dramatically reduces the amount of data that needs to be processed, thereby reducing time and cost while maintaining profiling capability for the target mutations.
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 highly sensitive and specific detection of nucleic acid alterations down to 0.001% sensitivity, significantly improving the detection of rare mutations, such as FLT3-D835 mutations, and can be completed within an hour, making it suitable for point-of-care cancer diagnosis and precision medicine.
Implementation Method 1
proteins having an activity of cleaving nucleic acid that recognizes the base(s) at the specified site
Implementation Method 2
restriction endonucleases (BstUI/HhaI) coupling with an RPA-assisted CRISPR/Cas 13a system
Implementation Method 3
recombinase polymerase amplification (RPA)
Implementation Method 4
Cas13a-based detection
Implementation Method 5
Cas13a-based detection has been applied to SARS-COV-2 diagnosis
Data Source
AI summary
The present disclosure provides methods, kits, and materials for amplifying, enriching or detecting a target nucleic acid, especially in a small amount, in a sample by digesting or degrading non-target nucleic acids in the sample before or during amplification and may detect the target nucleic acids. Interference of the non-target nucleic acids before or during amplification or detection will be minimized and the sensitivity, accuracy and efficiency of the amplification or detection will be greatly increased.