Isothermal Nucleic Acid Detection via Sequence Conversion and Cascade Amplification
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Solution Overview
Problem
Current nucleic acid detection methods, such as PCR, face challenges in maintaining accurate temperatures and suffer from non-specific amplification, high background signals, and reduced selectivity and sensitivity, especially at low concentrations and under isothermal conditions.
Innovation Solution
The use of sequence conversion DNA (SC DNA) and cascade signal amplifier DNA (cSA DNA) interactions to generate unique signal DNAs through endonuclease recognition sites and polymerase activity, allowing for selective and sensitive detection of target nucleic acids under low temperature, isothermal conditions, with the ability to amplify signals and simplify sample preparation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR is used for nucleic acid amplification, then amplification efficiency is improved, but temperature control complexity and time loss increase
Solution Approach 1:
The patent changes the temperature parameter from variable (PCR) to constant (isothermal), using enzymes that function optimally at a single temperature (37°C) to eliminate the need for temperature cycling while maintaining amplification capability
Solution Approach 2:
The patent replaces the mechanical thermal cycling system with a biochemical system using temperature-stable enzymes (polymerase and endonuclease) that operate at constant temperature, substituting mechanical control with enzymatic function
2Device complexity
If isothermal amplification is used to simplify temperature control, then temperature control complexity is reduced, but non-specific amplification and high background signals increase
Solution Approach 1:
The patent divides the amplification process into distinct functional modules: sequence conversion DNA for specific target recognition, cascade signal amplifier DNA for controlled signal generation, and endonuclease for regulated cleavage. This segmentation allows each component to perform its function with high specificity under isothermal conditions
Solution Approach 2:
The patent introduces sequence conversion DNA as an intermediary that first binds to the target nucleic acid with high specificity, then triggers the cascade amplification. This intermediary step ensures that amplification only occurs when the correct target is present, reducing non-specific amplification even under simple isothermal conditions
3Quantity of substance
If conventional detection methods are used, then detection capability is maintained, but sensitivity and selectivity at low concentrations are reduced
Solution Approach 1:
The patent implements a continuous cascade amplification system where sequence conversion DNA binds to target, triggers polymerase extension, which creates substrates for endonuclease cleavage, which releases products that trigger further amplification. This continuous chain reaction amplifies even trace amounts of target nucleic acid to detectable levels while maintaining specificity through multiple recognition steps
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 the detection of target nucleic acids at low concentrations with improved selectivity and sensitivity, allowing for the detection of nucleic acids in samples within a shorter time frame and reducing the complexity of sample preparation processes.
Implementation Method 1
a second oligonucleotide (cascade signal amplifier DNA 1 or cSA DNA 1) comprising, in the 5′ to 3′ direction, a second unique signal DNA generation sequence (D), an endonuclease recognition site (E)
Implementation Method 2
a polymerase
Data Source
AI summary
Disclosed are methods for detecting a target nucleic acid in a sample. The methods include contacting the sample, in the presence of a polymerase and an endonuclease, with a first oligonucleotide comprising, in the 5′ to 3′ direction, a first signal DNA generation sequence, an endonuclease recognition site, and a sequence complementary to the 3′ end of a target nucleic acid; a second oligonucleotide comprising, in the 5′ to 3′ direction, a second signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the first signal DNA generation sequence of the first oligonucleotide; a third oligonucleotide comprising, in the 5′ to 3′ direction, a third signal DNA generation sequence, an endonuclease recognition site, and a sequence that is homologous to the second signal DNA generation sequence of the second oligonucleotide.