RNA Detection via Sensor DNA Hybridization and PCR Amplification
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Solution Overview
Problem
Current methods for detecting RNA, especially those with short base sequences, lack sensitivity and accuracy, particularly in diagnosing diseases such as infectious diseases and cancer at early stages.
Innovation Solution
A method involving sensor DNA that hybridizes with target RNA, followed by polymerization, amplicon generation, and analysis, which includes designating a unique barcode region in the target RNA and ligating amplicons to enable quantitative detection of RNA with high sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional fluorescence intensity detection is used to detect double-stranded DNA, then the detection process is simple, but the sensitivity and measurement precision are insufficient for detecting RNA at very low concentrations
Solution Approach 1:
The detection method is segmented into multiple distinct steps: RNA extraction, reverse transcription to cDNA, PCR amplification to generate amplicons, and sequencing. This segmentation allows each step to be optimized for its specific function, achieving high sensitivity through amplification while maintaining manageable complexity through standardized protocols.
Solution Approach 2:
The method performs preliminary actions including RNA extraction and reverse transcription before the actual detection step. By converting RNA to cDNA and amplifying it to generate sufficient amplicons before sequencing, the method ensures that even trace amounts of RNA can be detected with high precision.
2Adaptability or versatility
If RNA with short base sequences is targeted for detection, then the detection scope is expanded to include more RNA types, but the detection accuracy and sensitivity deteriorate
Solution Approach 1:
The method changes the concentration parameter by amplifying the target RNA through PCR to generate numerous amplicons. This parameter change ensures that even short RNA sequences are present in sufficient quantities for accurate sequencing detection, maintaining high precision across diverse RNA types including those with short base sequences.
3Reliability
If quantitative detection of RNA is performed with high sensitivity, then the diagnostic capability for early disease detection is improved, but the detection method complexity increases
Solution Approach 1:
The detection method uses universal components including standard PCR primers, common sequencing adapters, and standardized library preparation protocols. These universal elements can detect various RNA targets (viral, bacterial, cancer-related) using the same workflow, achieving high diagnostic reliability without proportionally increasing complexity.
Solution Approach 2:
The method introduces cDNA as an intermediary between RNA and the detection system. By converting RNA to stable cDNA and amplifying it to generate amplicons, the method creates a robust intermediate form that enhances detection sensitivity and reliability while using well-established molecular biology techniques.
4Measurement precision
If the detection limit is reduced to femtomole and attomole levels, then the sensitivity is dramatically improved, but the difficulty of detecting and measuring increases
Solution Approach 1:
The method performs preliminary amplification of the target RNA through PCR to generate numerous amplicons before the actual measurement step. This preliminary action ensures that even femtomole and attomole levels of original RNA are amplified to detectable quantities, dramatically improving sensitivity while using standard PCR technology to manage measurement difficulty.
Solution Approach 2:
The method creates multiple copies of the target RNA by generating amplicons through PCR amplification. Each original RNA molecule is copied thousands to millions of times, transforming trace amounts at femtomole/attomole levels into sufficient quantities for accurate sequencing detection, thereby improving sensitivity without requiring specialized measurement equipment.
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 achieves very low detection limitations at femtomole and attomole levels, providing remarkable sensitivity and accuracy in detecting RNA, including specific viral genes like COVID-19 ORF7, enabling early diagnosis and infection level assessment.
Implementation Method 1
hybridizing a sensor DNA as a primer comprising a complementary sequence of a target RNA to be detected with a target RNA
Implementation Method 2
polymerizing with a polymerase using the target RNA as a template and the sensor DNA as a primer
Implementation Method 3
generating an amplicon by amplifying using a primer corresponding to the strand polymerized in step b)
Implementation Method 4
when a plurality of amplicons are produced in step c), a step d') ligating the produced amplicons
Data Source
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AI summary
The present invention relates to a method of analyzing and detecting RNA. In particular, the present invention is capable of analyzing RNA with short base sequences while quantitatively detecting RNA with high sensitivity and accuracy, and thus can be widely used for diagnosis of various diseases such as infectious diseases and cancer.