Universal Probe dPCR Multiplexing with Radial Fluorescence Clusters
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Solution Overview
Problem
Current methods for detecting multiple nucleic acid targets, such as in cancer diagnostics or pathogen detection, are time-consuming and costly due to the need for multiple assays and specific probe design for each target, especially in diverse samples like wastewater or circulating tumor DNA.
Innovation Solution
The use of universal oligonucleotide probes with universal tails in digital PCR, combined with target-specific primers, allows for multiplexed detection of multiple targets using a limited set of fluorescent probes, enabling detection in instruments with fewer channels through radial or intensity multiplexing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple specific probes are designed and synthesized for each target, then detection accuracy is improved, but cost and time consumption increase
Solution Approach 1:
The patent employs universal probes that can bind to multiple different target sequences through degenerate base pairing. Instead of designing a unique probe for each target, a single set of universal probes with degenerate bases can detect multiple targets simultaneously, eliminating the need for multiple custom probe syntheses and reducing both time and cost while maintaining detection accuracy
Solution Approach 2:
The universal probes are pre-designed with degenerate bases that anticipate multiple target sequences. This preliminary design allows the same probe set to be used for detecting multiple targets without requiring subsequent customization, thereby reducing the time and resources needed for each new detection assay
2Reliability
If multiple assays are run to detect different variants, then detection completeness is improved, but productivity decreases
Solution Approach 1:
The universal probes with degenerate bases can detect multiple target variants in a single assay run. This multi-functional capability allows comprehensive detection of all target variants simultaneously, eliminating the need to run multiple separate assays and thereby significantly improving productivity while maintaining detection completeness
Solution Approach 2:
The patent merges the detection of multiple target variants into a single assay by using universal probes that can bind to different targets. This combining of multiple detection functions into one assay increases throughput and productivity while ensuring all variants are detected
3Measurement precision
If custom probe synthesis is performed for each target, then detection specificity is improved, but device complexity increases
Solution Approach 1:
The universal probes with degenerate bases provide detection specificity for multiple targets without requiring custom synthesis for each target. This universal approach simplifies the assay design and reduces complexity by using a standardized probe set that maintains specificity through degenerate base pairing
Solution Approach 2:
The patent uses degenerate bases in the probe sequences to maintain detection specificity across multiple targets. By changing the base composition parameters to include degenerate positions, the probes can specifically bind to multiple different target sequences without requiring custom design, thereby reducing assay complexity
4Reliability
If more detection channels are used to detect more targets, then detection capacity is improved, but instrument cost increases
Solution Approach 1:
The universal probes enable a single detection channel to detect multiple targets simultaneously through degenerate base pairing. This multi-functional capability allows the same instrument channel to perform what would otherwise require multiple channels, reducing instrument cost while maintaining high detection capacity
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 simplifies and speeds up the detection process by reducing the need for custom probe synthesis, lowers costs, and enables the detection of multiple targets in a single assay using a universal probe set, even in instruments with fewer channels.
Implementation Method 1
The reagents also include universal probes-probes that are not designed to be complementary to genomic sequences of any organism of interest but instead designed to be complementary to the universal tails provided on the primers or their reverse complements
Implementation Method 2
the probes are preferably fluorescently-labeled oligos that may include fluorescent quenchers
Implementation Method 3
The synthesis of the reverse complements of the universal primer tails may occur during the amplification reaction in the partitions
Data Source
AI summary
The invention provides methods for the detection of molecular targets by digital PCR (dPCR) using a set of universal probes and target-specific tailed primers. Each target is amplified by a unique mixture of primers. The tailed amplicons anneal to a universal set of probes to detect the associated targets. Some targets are amplified using more than one tailed primer. Some targets are amplified using the same multiple tailed primers. In these embodiments, the primers are concentrated to produce a different number of amplicons for each tailed primer, resulting in a different probe-amplicon balance for each target. Two colors of fluorescence intensity are read and plotted as a 2D plot. In the plot, different targets contribute well-resolved clusters. Each cluster in the plot essentially lies a long its own radius allowing for radial multiplexing. The use of a universal set of probes in multiple assays provides for greater flexibility and throughput.