Isothermal Oligonucleotide Amplification with AEGIS Nucleotides
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Solution Overview
Problem
Isothermal amplification methods for nucleic acids, such as LAMP, face challenges in visualization and sequence specificity, often resulting in false positives and inefficient multiplexing due to primer-primer interactions and interference from natural nucleic acids in complex biological samples.
Innovation Solution
The use of reverse displaceable probes with non-standard nucleotides and self-avoiding molecular recognition systems, where the fluorescent species is attached to the displaced oligonucleotide and the quencher to the priming oligonucleotide, allowing for real-time monitoring and preventing primer interactions, and the employment of AEGIS nucleotides to prevent invasion by natural nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard nucleotides are used in LAMP amplification, then the amplification process can proceed, but false positives occur due to invasion by natural nucleic acids and primer-primer interactions
Solution Approach 1:
The probe is divided into two separate oligonucleotides: a priming oligonucleotide and a displaced oligonucleotide. This segmentation allows the system to distinguish between specific target binding and non-specific interactions, preventing false positives while maintaining amplification efficiency
Solution Approach 2:
A fluorescent reporter and quencher are introduced as intermediary molecules that mediate the detection process. The fluorescent species is attached to the displaced oligonucleotide and the quencher to the priming oligonucleotide, enabling real-time monitoring without direct interference from natural nucleic acids
2Measurement precision
If conventional visualization methods are used for LAMP amplification, then amplification can be detected, but sequence specificity is poor leading to false positives
Solution Approach 1:
The conventional mechanical/chemical visualization methods are replaced with a fluorescence-based optical system. The fluorescent reporter-quencher system provides real-time, sequence-specific detection with high precision, eliminating false positives associated with traditional methods
Solution Approach 2:
The system utilizes fluorescence emission (color change) as the detection mechanism. When the priming oligonucleotide binds to the target and displaces the displaced oligonucleotide, the fluorescent species transitions from a quenched state to an emissive state, providing real-time monitoring with high sequence specificity
3Productivity
If isothermal amplification is performed without temperature cycling, then amplification speed is improved, but primer-primer interactions cause inefficiency and false positives
Solution Approach 1:
The priming oligonucleotide is pre-designed with a quencher attached, and the displaced oligonucleotide is pre-loaded with a fluorescent species. This preliminary configuration ensures that fluorescence is only generated when specific target binding occurs, preventing false positives while maintaining rapid isothermal amplification
Solution Approach 2:
The fluorescence signal provides real-time feedback on the amplification process. As the priming oligonucleotide binds to the target and displaces the displaced oligonucleotide, the fluorescence intensity increases, allowing continuous monitoring and confirmation of specific amplification without temperature cycling
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 specific and efficient amplification with reduced false positives, allowing for continuous monitoring and improved multiplexing capabilities, facilitating rapid detection of pathogens in complex samples.
Implementation Method 1
the fluorescent species is attached to the displaced oligonucleotide and the quencher to the priming oligonucleotide, allowing for real-time monitoring
Data Source
AI summary
Processes are disclosed that create derivative DNA molecules in large numbers under isothermal conditions, where amplification of a looped structure derived from a target is achieved by a process that separately or in combination includes (a) a displaceable probe that (b) may be captured in a separate site, optionally containing (c) components of an artificially expanded genetic information system, where the primers optionally contain (d) components of a self-avoiding molecular recognition system, and optionally (e) involving direct transfer of DNA or RNA from a sample from a capture medium with immobilized quaternary ammonium salts.