Amplification Oligomer Complexes for Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current multiplex nucleic acid amplification methods face challenges such as preferential amplification, complex primer selection, cross-reactivity, and reduced sensitivity due to competition for reagents, limiting the number of targets that can be analyzed simultaneously.
Innovation Solution
The use of amplification oligomer complexes, including primer and promoter oligomers with universal tag sequences, allows for the formation of stable complexes that can be captured and amplified independently, reducing interference and enabling efficient amplification of multiple targets in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional multiplex PCR amplification is used to amplify multiple target sequences simultaneously, then the number of targets that can be detected increases, but preferential amplification occurs where certain targets amplify more efficiently than others, reducing measurement precision
Solution Approach 1:
The invention divides the amplification process into two distinct phases: a first amplification phase using target-specific primer pairs for each individual target sequence, and a second amplification phase using universal primer pairs that amplify all targets simultaneously. This segmentation prevents preferential amplification by isolating target-specific amplification events from the multiplex amplification event, ensuring uniform amplification efficiency across all targets while enabling detection of multiple targets.
2Adaptability or versatility
If multiple primer pairs are used in multiplex amplification to target different sequences, then the versatility of the assay increases, but the complexity of primer selection and optimization increases significantly
Solution Approach 1:
The invention introduces universal primer pairs that can amplify multiple different target sequences simultaneously. The first amplification phase uses target-specific primer pairs to generate initial amplification products, and the second amplification phase uses universal primer pairs that recognize common structural features across all targets. This universality allows a single set of universal primers to amplify multiple targets, dramatically reducing the complexity of primer design and selection while maintaining high multiplexing capability.
3Productivity
If multiple amplification reactions are performed simultaneously in a single reaction mixture, then productivity increases, but competition for reagents occurs which reduces the sensitivity of detection
Solution Approach 1:
The invention segments the amplification process into two sequential phases performed in separate reaction mixtures. The first amplification phase uses target-specific primer pairs in individual reactions to generate sufficient initial amplification products for each target. The second amplification phase then uses universal primer pairs in a multiplex reaction to simultaneously amplify all targets. This segmentation eliminates reagent competition during the critical target-specific amplification step, ensuring high sensitivity while maintaining high productivity through the subsequent multiplex amplification.
4Adaptability or versatility
If multiple primer pairs are combined in a single reaction to amplify different targets, then the number of targets analyzed simultaneously increases, but cross-reactivity between primers occurs leading to false results
Solution Approach 1:
The invention separates target-specific primer binding events from the multiplex amplification event by using two distinct phases. In the first phase, target-specific primer pairs bind to their respective targets in individual reactions, ensuring high specificity without cross-reactivity. In the second phase, universal primer pairs amplify the generated products in a multiplex reaction. This segmentation ensures that each target-specific primer pair operates in isolation, eliminating cross-reactivity while maintaining the ability to analyze multiple targets simultaneously.
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 enables the simultaneous amplification of multiple nucleic acid sequences with improved sensitivity and reduced interference, allowing for the detection of a larger number of gene targets from a small initial quantity of nucleic acid without extensive optimization, thereby overcoming the limitations of existing multiplex amplification techniques.
Implementation Method 1
hybridizing the at least two amplification oligomer complexes to different target nucleic acid sequences
Data Source
AI summary
Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products.