Nucleotide Tag Encoding for Multiplexed Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting specific nucleic acid sequences in high-throughput assays are limited in their ability to analyze multiple targets and samples simultaneously across a broad range of concentrations, necessitating the development of more efficient techniques for increased throughput and cost-effectiveness.
Innovation Solution
The method involves encoding reactions that produce tagged target nucleotide sequences with sample-specific and target-specific tags, allowing for the mixing of samples and subsequent amplification using unique primer pairs to detect multiple targets in batches, thereby increasing the number of samples and targets that can be analyzed in a single assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple samples and targets are analyzed simultaneously in a single assay, then throughput and cost-effectiveness are improved, but the complexity of the assay system increases
Solution Approach 1:
The patent segments the assay system into modular components: sample-specific nucleotide tags, target-specific nucleotide tags, and unique primer pairs. Each sample is assigned a unique combination of tags, allowing systematic identification and analysis of multiple targets across multiple samples through structured segmentation of the detection system
Solution Approach 2:
The patent employs universal amplification primers that can amplify multiple different target sequences, and universal detection methods that can detect multiple different amplification products. This multi-functionality allows a single assay system to analyze numerous targets across numerous samples without requiring separate specialized reagents for each target-sample combination
2Productivity
If the number of targets and samples analyzed in a single assay is increased, then the number of samples and targets that can be analyzed simultaneously is improved, but the assay complexity and resource requirements increase
Solution Approach 1:
The patent adds a dimensional layer of sample-specific nucleotide tags to the traditional target-detection approach. Instead of only detecting targets, the system now detects combinations of sample tags and target tags, effectively adding a sample-identification dimension that enables multiplexing of both samples and targets in the same assay space
Solution Approach 2:
The patent introduces nucleotide tags as intermediary elements that bridge samples and targets. These tags serve as mediators that allow the amplification and detection systems to distinguish between different samples and targets through unique tag combinations, enabling high-throughput analysis without direct complex sample-target pairing
3Productivity
If samples are analyzed in batches with encoding reactions and tagged sequences, then throughput is improved, but the difficulty of detecting and measuring specific targets increases
Solution Approach 1:
The patent applies local quality by making each amplification primer pair unique and specific to a particular target sequence, while the sample-specific tags provide local identification of the sample source. This localized specificity at the primer and tag level ensures that even in complex batched assays with multiple samples and targets, each target can be detected with high specificity through its unique primer pair and tag combination
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 efficient detection of multiple target nucleic acids in multiple samples, enhancing throughput and reducing costs by allowing for the analysis of up to 9216 targets in a single assay, while maintaining specificity and accuracy.
Implementation Method 1
a forward or a reverse amplification primer that anneals to a target nucleotide sequence; and a reverse or a forward amplification primer, respectively, that anneals to a sample-specific nucleotide tag
Data Source
AI summary
The present invention provides assay methods that increase the number of samples and/or target nucleic acids that can be analyzed in a single assay. In certain embodiments, an assay method entails separately subjecting S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence including a sample-specific nucleotide tag and a target nucleotide sequence. In some embodiments, an assay method entails separately subjecting S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence including a first nucleotide tag linked to a target nucleotide sequence, which is linked to a second nucleotide tag. In either case, the tagged target nucleotide sequences from the S samples can be mixed to form an assay mixture and subsequently assayed.