Micro RNA Detection via Single-Stranded DNA Probe Hybridization
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Solution Overview
Problem
Current methods for detecting microRNAs (miRNAs) are complex, costly, and require specialized equipment, making them inconvenient and burdensome for patients and testers, and there is a need for a simple, rapid, and highly specific method that does not rely on such equipment.
Innovation Solution
A nucleic acid detection method involving reverse transcription to extend the length of small RNAs, followed by PCR amplification to create a double-stranded DNA fragment with a single-stranded region, which can be bound to an oligonucleotide probe immobilized on a solid phase for visual detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional qRT-PCR or microarray assay methods are used to detect miRNAs, then detection accuracy can be maintained, but the device complexity and testing costs increase significantly
Solution Approach 1:
The invention extracts only the essential detection function from complex qRT-PCR systems by using a simplified isothermal amplification approach with a single primer containing both recognition and extension sites, eliminating the need for sophisticated thermal cycling equipment while maintaining detection capability
Solution Approach 2:
The patent creates a simplified copy of the detection function using a single-stranded DNA probe with embedded recognition sequence that can bind directly to target miRNA, replacing the need for complex reverse transcription and amplification machinery while preserving detection accuracy
2Measurement precision
If conventional multi-step qRT-PCR protocols are used, then detection sensitivity can be achieved, but the time required for testing increases
Solution Approach 1:
The single primer is pre-designed with the target recognition sequence embedded within it, allowing direct binding and amplification without preliminary reverse transcription or separate hybridization steps, thus reducing testing time while maintaining sensitivity
Solution Approach 2:
The invention merges the recognition function and extension function into a single primer molecule, eliminating multiple sequential steps (reverse transcription, amplification, detection) into one isothermal reaction, thereby reducing testing duration while preserving detection sensitivity
3Ease of operation
If simple detection methods are used, then ease of operation improves, but measurement precision decreases
Solution Approach 1:
The single-stranded DNA probe acts as an intermediary that specifically binds to the target miRNA sequence, enabling simple isothermal amplification while maintaining high detection accuracy through sequence-specific recognition, thus bridging simplicity and precision
4Stability of the object's composition
If small RNA length is kept natural (18-25 nucleotides), then biological fidelity is maintained, but amplification efficiency decreases
Solution Approach 1:
The invention extends the detection into a different dimensional space by adding a single-stranded DNA tail to the amplified product, allowing the short miRNA to be amplified efficiently while maintaining its original sequence integrity, thus resolving the conflict between natural composition and amplification efficiency
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 allows for accurate and rapid detection of miRNAs without specialized equipment, reducing testing costs and burdens, and enables visual analysis of amplified DNA products with higher sensitivity compared to single-stranded DNA detection.
Implementation Method 1
carrying out a reverse transcription reaction using a target RNA as a template and a reverse transcription primer having on its 5′-end side a sequence non-complementary to the target RNA
Implementation Method 2
hybridizing the single-stranded region of the amplified double-stranded DNA fragment to an oligonucleotide probe immobilized on a solid phase
Data Source
AI summary
Provided is a method for detecting small RNAs in a simple and highly accurate manner. Provided is a nucleic acid detection method including the following steps (a) to (c): (a) carrying out a reverse transcription reaction using a target RNA as a template and a reverse transcription primer having on its 5′-end side a sequence non-complementary to the target RNA to produce a reverse transcription product longer than the target RNA; (b) carrying out a nucleic acid amplification reaction using the reverse transcription product as a template and two primers to produce an amplified double-stranded DNA fragment having a single-stranded region at least at one end; and (c) hybridizing the single-stranded region of the amplified double-stranded DNA fragment to an oligonucleotide probe immobilized on a solid phase.