Digital Drop-Off Assays Using Universal Probes for Multiplex Detection
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Solution Overview
Problem
Existing nucleic acid detection methods require target-specific probe design, limiting flexibility and efficiency in multiplexed detection, and often rely on costly optimization for each target sequence.
Innovation Solution
The use of tailed primers and molecular inversion probe-like oligonucleotides with universal probe binding regions allows for multiplexed detection by generating distinct signals through specific target 'barcoding', enabling the use of universal probes that do not need sequence alteration based on the target.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If target-specific probe design is used for each analyte, then detection specificity is improved, but device complexity and cost increase
Solution Approach 1:
The patent employs universal probes that can bind to multiple different analytes through a common binding motif. Instead of designing unique probes for each target, a single universal probe design recognizes multiple analyte-specific barcodes, thereby reducing probe design complexity while maintaining detection specificity through the barcode-probe pairing system
Solution Approach 2:
The patent introduces barcode sequences as intermediaries between the analyte and the universal probe. The barcode acts as a mediator that translates analyte-specific information into a format recognizable by universal probes, allowing specific detection without requiring target-specific probe design
2Measurement precision
If target-specific probe design is used for each analyte, then detection accuracy is improved, but manufacturing cost increases
Solution Approach 1:
The universal probe design allows a single probe sequence to detect multiple analytes, significantly reducing the number of probes that need to be manufactured. This multi-functionality maintains detection accuracy through specific barcode recognition while lowering manufacturing costs by eliminating the need for multiple target-specific probe synthesis processes
3Productivity
If multiple analytes are detected simultaneously, then productivity is improved, but signal detection complexity increases
Solution Approach 1:
The patent segments the detection system into analyte-specific barcode regions and universal probe binding regions. This segmentation allows multiple analytes to be detected simultaneously through their unique barcodes while using a common universal probe, thereby improving productivity without proportionally increasing signal detection complexity
Solution Approach 2:
The patent employs fluorophore-labeled universal probes that generate distinct fluorescent signals for different analyte-barcode-probe combinations. The use of fluorescent color changes enables simultaneous detection of multiple analytes through wavelength discrimination, improving productivity while managing signal detection complexity through optical differentiation
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
Enhances detection sensitivity and flexibility by allowing simultaneous detection of multiple analytes with high sensitivity, up to 99%, using universal probes that can identify different targets with distinct signal patterns.
Implementation Method 1
a primer oligonucleotide comprising a first region, wherein the first region hybridizes to said analyte
Implementation Method 2
b) subjecting said primer oligonucleotide to an extension reaction, thereby generating a probe binding nucleic acid
Implementation Method 3
a probe of said one or more probes comprises fluorophore
Data Source
AI summary
The present disclosure provides methods, systems, and compositions for the multiplexed detection and quantification of multiple analytes from a sample. Analytes may be nucleic acid analytes. Detection of analytes may comprise contacting one or more samples with primers and/or hybridization probes to generate cumulative signal measurements. The methods may comprise digital PCR or may comprise partitioning a sample into multiple partitions.