Circularized Nucleic Acid Detection for Long Target Sequences
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Solution Overview
Problem
Current real-time polymerase chain reaction (RT-PCR) methods are less precise and sensitive when detecting and quantifying longer target nucleic acids due to the need for longer DNA elongation times, which complicates the detection and quantification process.
Innovation Solution
A method involving a first polynucleotide that hybridizes with a target nucleic acid, followed by extension with a nucleic acid polymerase, ligation to form a cyclic structure, and subsequent amplification using a primer set to produce an amplification product, allowing for the detection of longer target nucleic acids with high sensitivity and precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional RT-PCR methods are used to detect longer target nucleic acids, then the detection process can be performed, but the precision and sensitivity of detection decrease due to longer elongation times required
Solution Approach 1:
The invention divides the long target nucleic acid into two separate regions, each targeted by a distinct polynucleotide (first and second polynucleotides). Each polynucleotide binds to a specific region and extends for a shorter, optimized distance, avoiding the need to elongate the entire long sequence in one step. This segmentation allows precise detection while reducing elongation time requirements.
Solution Approach 2:
The invention introduces polynucleotides as intermediary elements that bind to the target nucleic acid and serve as templates for extension. These polynucleotides act as mediators between the detection system and the long target sequence, enabling indirect detection through their shorter extension products rather than direct amplification of the entire long target.
2Adaptability or versatility
If the target nucleic acid region is extended to cover longer sequences, then more comprehensive detection is achieved, but the detection precision and sensitivity are reduced
Solution Approach 1:
The detection system segments the long target nucleic acid into multiple detectable regions by using separate polynucleotides targeting different segments. Each segment is detected with high precision through its specific polynucleotide, while collectively providing comprehensive coverage of the entire long target sequence.
Solution Approach 2:
Instead of attempting to detect the entire long target sequence in a single amplification reaction, the invention uses multiple polynucleotides that each perform partial detection of specific regions. The combined results provide comprehensive detection capability while maintaining high precision for each individual region.
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 the precise and sensitive detection and quantification of longer target nucleic acids, improving upon the limitations of conventional RT-PCR by enhancing the amplification and detection process.
Implementation Method 1
the target nucleic acid and the first polynucleotide hybridize to form a hybridization product
Implementation Method 2
incubating the hybridization product in the presence of a first nucleic acid polymerase to extend the first polynucleotide
Implementation Method 3
incubating the extended product in the presence of a nucleic acid ligase to prepare a second polynucleotide having a cyclic structure
Implementation Method 4
incubating the second polynucleotide having a cyclic structure in the presence of a primer set and a second nucleic acid polymerase to amplify the second polynucleotide
Data Source
AI summary
A method of detecting a target nucleic acid, and a polynucleotide and a composition for detecting a target nucleic acid.