Target-Specific Universal Primers for Multiplex Nucleic Acid Amplification
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Solution Overview
Problem
Current nucleic acid amplification methods face challenges in efficiently amplifying multiple targets in a single reaction due to primer interactions, leading to decreased assay sensitivity and the need for extensive primer design and testing in multiplex assays.
Innovation Solution
The use of target-specific universal (TSU) primers, which include both target-specific and universal sequences, allows for initial target capture and amplification, followed by universal primer amplification in isothermal conditions, minimizing primer interactions and increasing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple primers are used to amplify multiple targets in a single reaction, then the ability to detect multiple targets is improved, but primer interactions occur leading to decreased assay sensitivity
Solution Approach 1:
The primer design is segmented into two functional parts: a universal sequence region and a target-specific sequence region. This segmentation allows the primer to serve dual purposes - the universal region enables amplification of multiple different targets while the target-specific region ensures specificity, thereby reducing unwanted primer interactions while maintaining the ability to detect multiple targets
Solution Approach 2:
The invention employs universal primers that contain a universal sequence region that can bind to a common sequence present in multiple different target nucleic acids. This multi-functionality allows a single primer design to amplify multiple different targets (such as different viral genomes) in a single reaction without requiring separate primer pairs for each target, thereby reducing primer-primer interactions while maintaining assay sensitivity
2Adaptability or versatility
If multiple primers are used for multiplex amplification, then multiple targets can be amplified, but the complexity of primer design and testing increases
Solution Approach 1:
The invention employs universal primers that contain a universal sequence region that can bind to a common sequence present in multiple different target nucleic acids. This multi-functionality allows a single primer design to amplify multiple different targets (such as different viral genomes) in a single reaction without requiring separate primer pairs for each target, thereby reducing primer-primer interactions while maintaining assay sensitivity
3Productivity
If conventional amplification methods are used, then amplification can be achieved, but the need for extensive primer design and testing in multiplex assays increases
Solution Approach 1:
The invention employs universal primers that contain a universal sequence region that can bind to a common sequence present in multiple different target nucleic acids. This multi-functionality allows a single primer design to amplify multiple different targets (such as different viral genomes) in a single reaction without requiring separate primer pairs for each target, thereby reducing primer-primer interactions while maintaining assay sensitivity
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 approach enhances amplification efficiency, simplifies multiplex assay design, and reduces the need for multiple primers, enabling sensitive and rapid detection of multiple targets in a single reaction, suitable for high-throughput and automated systems.
Implementation Method 1
The TSU primer is specific for a target nucleic acid sequence and hybridizes thereto
Implementation Method 2
compositions and methods that are useful for amplifying target nucleic acid sequences in vitro
Implementation Method 3
enzymatic in vitro nucleic acid synthesis by an RNA polymerase to transcribe RNA transcripts from the dsDNA
Data Source
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AI summary
There is described a composition comprising: a TSU promoter oligonucleotide that includes a 5' promoter sequence, an internal first universal sequence (U1), and a 3' first target specific sequence (TS1) that binds specifically to a target sequence contained in a target nucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoter primer that has a 3' terminus that is capable of being extended by a polymerase, or is a TSU promoter provider oligonucleotide that has a blocked 3' terminus that is incapable of being extended by a polymerase, a TSU non-promoter primer oligonucleotide made up of a 5' second universal sequence (U2) and a 3' second target specific sequence (TS2) which is different from the TS1, and a means for directly or indirectly joining the TSU promoter oligonucleotide to the TSU non-promoter primer oligonucleotide, thereby forming a target specific universal (TSU) primer complex.