Digital Biomolecule Detection via Isothermal Amplification
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Solution Overview
Problem
Current methods for detecting biomolecules such as DNA, RNA, and proteins face challenges in achieving accurate and sensitive quantification due to issues like nonspecific amplification, reverse transcription bias, and the need for thermocycling, which limits their application in digital formats.
Innovation Solution
A digital method involving isothermal amplification using a mixture of enzymes, oligonucleotides, and partitioning agents that eliminates background amplification, allowing for absolute quantification and sensitive detection of biomolecules without continuous monitoring or calibration, suitable for various biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR or RT-qPCR is used for sensitive detection of nucleic acids, then detection sensitivity is improved, but the technique requires thermocycling, reverse transcription steps, and sophisticated primer design that increase device complexity and introduce quantification bias
Solution Approach 1:
The patent changes the temperature parameter from cyclic (PCR) to constant isothermal conditions, eliminating the need for complex thermocycling protocols while maintaining amplification capability. The reaction is performed at a single temperature (e.g., 65°C) using enzymes like Bst polymerase that function optimally at this constant temperature, thereby simplifying the device requirements while preserving detection sensitivity.
Solution Approach 2:
The patent extracts and eliminates the reverse transcription step from the detection workflow by using DNA-based amplification of RNA targets through a different mechanism, or by performing RT and amplification in a simplified coupled manner without separate thermocycling protocols. This removes a major source of quantification bias and simplifies the overall procedure.
2Measurement precision
If digital PCR or digital RT-qPCR is used for absolute quantification without calibration, then measurement precision is improved, but the requirement for single-molecule detection in small compartments increases device complexity and reduces ease of operation
Solution Approach 1:
The patent divides the sample into many small compartments (droplets or wells) where each compartment independently amplifies target molecules. This segmentation enables digital quantification by counting positive compartments, achieving absolute quantification without calibration curves while maintaining operational simplicity through high-throughput parallel processing.
Solution Approach 2:
The patent uses amplification of target molecules to generate detectable copies in each compartment. The exponential amplification creates sufficient signal copies from single-molecule inputs, enabling detection and quantification without requiring direct single-molecule detection capabilities, thereby simplifying the instrumentation.
3Device complexity
If isothermal amplification methods like EXPAR or LAMP are used, then device complexity is reduced by eliminating thermocycling, but nonspecific amplification increases and reduces measurement precision
Solution Approach 1:
The patent performs preliminary optimization of the isothermal amplification conditions, including enzyme selection (e.g., Bst polymerase), primer design, and buffer composition, to maximize specificity before the actual amplification. This preliminary preparation ensures that the simplified isothermal protocol produces specific amplification products without the need for complex real-time monitoring or correction during the reaction.
Solution Approach 2:
The patent incorporates feedback mechanisms through the design of the amplification system, where the reaction conditions and reagent compositions are optimized based on prior knowledge of specific amplification behavior. The system uses carefully designed primer sets and enzyme formulations that inherently provide feedback control to prevent nonspecific amplification, ensuring measurement precision without complex real-time intervention.
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 rapid, selective, and absolute quantification of biomolecules, improving sensitivity and specificity, and can be used for medical and agro-diagnosis, including diseases like cancer and infectious diseases, without the need for complex calibration or real-time monitoring.
Implementation Method 1
The present invention relates to a digital method for detecting and/or quantifying biomolecules such as DNA, RNA and proteins in a sample, based on isothermal amplification having specific molecular design
Implementation Method 2
mixing said sample with a mixture comprising a buffer, enzymes, a first oligonucleotide, a second oligonucleotide and a third oligonucleotide
Implementation Method 3
partitioning the mixture obtained in step a) into several compartments so that a fraction of the compartments does not contain the target biomolecule
Data Source
AI summary
The present invention relates to a digital method for detecting and/or quantifying at least one target biomolecules in a sample, said biomolecules being selected from DNA, RNA, and proteins based on isothermal amplification. The present invention further relates to different applications of the digital method and to a kit.