Cas12a Nucleic Acid Detection via Ternary Complex Cleavage
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Solution Overview
Problem
Current nucleic acid detection methods, particularly for pathogens and SNPs, often require complex procedures, are time-consuming, and involve expensive equipment, with RNA detection being challenging due to instability and the need for additional amplification steps.
Innovation Solution
A method utilizing Cas12a protein with a guide RNA and a fluorescently labeled nucleic acid probe for direct detection of nucleic acid molecules, which forms a ternary complex to cleave and indicate the presence of target DNA through fluorescence, allowing for rapid and sensitive detection without the need for extensive amplification or expensive equipment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional nucleic acid detection methods (PCR, Southern/Northern hybridization) are used, then detection accuracy and reliability are improved, but detection time and operational complexity increase significantly
Solution Approach 1:
The patent combines the specific recognition function of guide RNA with the cleavage function of Cas12a protein into a single detection system. The guide RNA directs Cas12a to specifically bind and cleave target nucleic acid sequences, integrating target recognition and detection into one step, thereby maintaining high detection accuracy while significantly reducing detection time compared to traditional multi-step methods
Solution Approach 2:
The patent introduces a fluorescent probe as an intermediary that reports the cleavage activity of Cas12a. When Cas12a cleaves the target nucleic acid, it also cleaves the fluorescent probe, generating a detectable fluorescence signal. This intermediary mechanism enables rapid and sensitive detection without requiring complex equipment or multiple processing steps
2Measurement precision
If expensive detection equipment (next-generation sequencing, Oxford Nanopore sequencing) is used, then detection precision and comprehensiveness are improved, but cost and device complexity increase
Solution Approach 1:
The patent employs disposable fluorescent probes and guide RNAs that can be synthesized at low cost. These single-use reagents eliminate the need for expensive, complex sequencing equipment while maintaining sufficient detection precision for nucleic acid identification. The low-cost reagent approach makes high-precision detection accessible without requiring sophisticated instruments
3Adaptability or versatility
If RNA molecules are detected using traditional methods, then detection capability is achieved, but operational difficulty increases due to RNA instability and degradation
Solution Approach 1:
The patent performs preliminary protection of RNA molecules by conducting the entire detection reaction at isothermal conditions (37°C) without thermal cycling. This preliminary action prevents RNA degradation that would occur during PCR thermal cycling, maintaining RNA stability throughout the detection process and greatly simplifying operational requirements while preserving detection capability
4Measurement precision
If nucleic acid amplification is performed before detection, then detection sensitivity is improved, but detection time and process complexity increase
Solution Approach 1:
The patent merges the amplification step (RPA or LAMP) with the detection step (Cas12a cleavage) into a one-pot reaction system. The amplification primers and Cas12a detection reagents are combined in the same reaction mixture, allowing amplification and detection to occur simultaneously without separate processing steps. This integration maintains high detection sensitivity while significantly reducing process complexity and detection time
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 fast, cost-effective, and sensitive detection of nucleic acid molecules, including pathogens and SNPs, with the ability to detect as low as 10 aM concentrations, and is applicable to both DNA and RNA, simplifying the detection process and reducing operational complexity.
Implementation Method 1
a fluorescently labeled nucleic acid probe for direct detection of nucleic acid molecules, which forms a ternary complex to cleave and indicate the presence of target DNA through fluorescence
Data Source
AI summary
The present invention provides a use of a Cas protein, and a method and a kit for detecting target nucleic acid molecules. The method for detecting target nucleic acid molecules comprises adding a guide RNA, a Cas12a, and a nucleic acid probe into a reaction system containing target nucleic acid molecules to be detected, and detecting it after the reaction is completed.