Rolling Circle Amplification with Restriction Enzyme for Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid-based cancer diagnostics face challenges in detecting genetic mutations, particularly point mutations, due to limitations in distinguishing between normal and mutated genes, high costs, and the inability to multiplex detection, which are not effectively addressed by existing methods like next-generation sequencing (NGS) and droplet digital PCR (ddPCR).
Innovation Solution
A method utilizing rolling circle amplification (RCA) with a linear template containing target nucleic acid binding regions, a primer binding region, a restriction enzyme binding region, and a barcode generation region, where the restriction enzyme is used to produce amplicons with predefined barcodes, allowing for simultaneous detection of multiple target nucleic acids without the need for expensive enzymes like CRISPR, using a surface measurement sensor for detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If next generation sequencing (NGS) is used to detect point mutations, then massively parallel identification of many regions is achieved, but difficulty in distinguishing errors from real data and high costs are incurred
Solution Approach 1:
The invention segments the detection process into two distinct stages: first, rolling circle amplification to generate abundant target nucleic acid copies, and second, digital PCR for precise quantification and error distinction. This segmentation allows NGS to focus on its strength (parallel identification) while digital PCR handles the precision requirement (distinguishing errors from real mutations).
Solution Approach 2:
The invention introduces rolling circle amplification as an intermediary step between sample preparation and NGS/digital PCR analysis. This intermediary amplification process generates sufficient target material to enable both massively parallel identification and precise error distinction without directly compromising the measurement precision of the downstream detection methods.
2Measurement precision
If droplet digital PCR (ddPCR) is used to detect point mutations, then precise detection of specific mutations is achieved, but only one mutation can be identified per reagent tube and high costs are incurred due to multiple reagents needed
Solution Approach 1:
The invention segments the detection workflow by using rolling circle amplification as a universal pre-amplification step that can handle multiple target sequences simultaneously, followed by digital PCR for precise detection. This segmentation allows multiplexed detection of multiple mutations in a single tube while maintaining the measurement precision of digital PCR.
Solution Approach 2:
The rolling circle amplification system is designed with universal primers and adapters that can amplify multiple different target nucleic acid sequences using the same reagents. This multi-functionality enables a single ddPCR tube to detect multiple mutations, eliminating the need for separate reagents for each mutation and reducing overall costs.
3Ease of operation
If general detection methods using DNA complementary binding are used, then detection of nucleic acids is achieved, but inability to distinguish between normal genes and mutation genes occurs
Solution Approach 1:
The invention applies local quality by designing the rolling circle amplification template with specific sequences that are complementary only to the mutant nucleic acid sequence at the critical binding region. This localized specificity ensures that amplification occurs only when the target mutation is present, while the rest of the template structure remains simple and easy to operate with.
Solution Approach 2:
The invention introduces rolling circle amplification with mutation-specific templates as an intermediary step between simple DNA binding and final detection. This intermediary step provides the discrimination power to distinguish mutant from normal genes by selectively amplifying only the target mutation sequences, while the final detection can use simple complementary binding methods.
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 high-sensitivity detection of multiple target nucleic acids simultaneously, reducing costs and overcoming the limitations of existing technologies by using existing nucleic acid detection systems, making it suitable for genetic mutation detection and molecular diagnostics.
Implementation Method 1
a linear template including i) target nucleic acid binding regions arranged at both ends to complementarily bind to a target nucleic acid sequence
Implementation Method 2
followed by ligation to form a circular template-target nucleic acid complex
Implementation Method 3
performing rolling circle amplification in the presence of a primer binding to the primer binding region and a cleavable nucleic acid having the same sequence as that of the restriction enzyme binding region to produce a single-stranded amplicon
Implementation Method 4
treating the single-stranded amplicon with a restriction enzyme such that regions where the single-stranded amplicon complementarily binds to the cleavable nucleic acid are cut to obtain first amplicons including barcodes
Implementation Method 5
wherein the barcodes are detected using a surface measurement sensor comprising a probe complementary to the barcodes, and wherein the surface measurement sensor operates based on a method selected from the group consisting of fluorescence, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), and cantilevers
Implementation Method 6
wherein the barcodes are detected using a surface measurement sensor comprising a probe complementary to the barcodes, and wherein the surface measurement sensor operates based on a method selected from the group consisting of fluorescence, surface plasmon resonance (SPR)
Data Source
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AI summary
The present invention relates to a method of detecting a target nucleic acid on the basis of rolling circle amplification (RCA), and more specifically, to a method of detecting a target nucleic acid, the method in which a target nucleic acid (a nucleic acid having a target nucleic acid sequence), when present, forms a circular template with a template for performing an amplification reaction, wherein during the amplification reaction, a restriction enzyme is added to further induce a new RCA reaction, thus increasing the reaction rate and sensitivity, and to an RCA composition for implementing the method. The method of detecting a target nucleic acid according to the present invention, by detecting a barcode sequence predefined according to the type of the target nucleic acid, enables multiple detections of the presence of the target nucleic acid without sequencing, is inexpensive for not using costly enzymes, such as CRISPR, can detect barcode sequences, and can utilize various existing nucleic acid detection systems, and thus, can be useful in the detection of gene mutations.