Cas12a Nucleic Acid Detection via Fluorescence
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Solution Overview
Problem
Current nucleic acid detection methods are often complex, costly, and time-consuming, particularly for rapid pathogen detection and SNP analysis, as they require multiple steps and expensive equipment, and RNA detection is challenging due to instability.
Innovation Solution
A method using Cas12a protein with a guide RNA and a fluorescently labeled nucleic acid probe for direct detection of target 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
1Measurement precision
If conventional nucleic acid detection methods (PCR, sequencing) are used, then detection accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The patent replaces complex mechanical detection systems (PCR thermocyclers, sequencers) with a biochemical system based on Cas12a protein and guide RNA that performs detection through fluorescence signal generation, eliminating the need for expensive thermal cycling equipment and complex instrumentation
Solution Approach 2:
The patent introduces fluorescently labeled nucleic acid probes as intermediary molecules that mediate between the target nucleic acid and the detection system, enabling signal amplification and detection through fluorescence rather than requiring complex direct detection methods
2Measurement precision
If multi-step detection protocols are used, then detection precision is improved, but loss of time increases
Solution Approach 1:
The patent merges the amplification step and detection step into a single integrated reaction system where Cas12a simultaneously performs target recognition and fluorescence signal generation, eliminating the need for separate amplification and detection protocols
Solution Approach 2:
The patent employs preliminary isothermal amplification of the target nucleic acid before detection, which prepares sufficient target material in advance to enable rapid subsequent detection without requiring time-consuming thermal cycling during the detection phase
3Ease of operation
If isothermal amplification methods are used, then ease of operation is improved, but measurement precision deteriorates
Solution Approach 1:
The patent uses fluorescently labeled nucleic acid probes as intermediary molecules that provide signal amplification and enhancement, enabling highly sensitive detection even when starting with low-abundance targets amplified through simple isothermal methods
Solution Approach 2:
The patent optimizes reaction parameters including probe concentration, Cas12a protein concentration, and incubation time to maximize detection sensitivity, achieving precise quantification of target nucleic acids despite using operationally simple isothermal amplification
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 method enables fast, low-cost, and efficient detection of nucleic acids, including pathogens and SNPs, with high sensitivity, capable of detecting as low as 10 aM concentrations, and can handle both DNA and RNA samples with minimal equipment, addressing the limitations of existing techniques.
Implementation Method 1
a fluorescently labeled nucleic acid probe for direct detection of target 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.