Sequential Probe Decoding for Overlapping Transcript Detection
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Solution Overview
Problem
Existing methods for detecting analytes in biological samples are limited by the diffraction limit of optical microscopes, leading to difficulties in differentiating spatially overlapping targets and providing inaccurate, complex, and expensive results, especially in cases where transcripts are close to each other or have high expression levels.
Innovation Solution
A method involving two sets of analyte-specific probes with unique identifier sequences, combined with decoding oligonucleotides, allows for temporal separation of signal generation to detect analytes beyond the diffraction limit, maintaining sample integrity and enabling high-throughput detection without optical crowding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional optical microscopy methods are used to detect analytes, then the detection process is simple, but the resolution is limited by the diffraction limit and spatially overlapping targets cannot be differentiated
Solution Approach 1:
The detection process is segmented into multiple sequential rounds, with each round detecting a specific subset of analytes. Probe sets are divided into multiple groups that are applied in sequence, allowing resolution of spatially overlapping targets by temporal separation of detection events.
Solution Approach 2:
Probe sets are pre-designed with unique identifier sequences that enable subsequent decoding. The probes are prepared and applied to the sample before detection, establishing the basis for resolving overlapping signals through sequential rounds of hybridization and detection.
2Measurement precision
If multiple analytes are detected simultaneously using conventional methods, then throughput is high, but spatial overlap of targets leads to inaccurate differentiation
Solution Approach 1:
The detection process uses periodic action by performing multiple rounds of hybridization and detection in sequence. Each round detects a specific subset of analytes with unique identifier sequences, allowing accurate differentiation through temporal separation while maintaining high throughput via automated processing.
Solution Approach 2:
The system changes detection parameters by using different identifier sequences for different analytes and performing detections at different time points. This parameter variation enables accurate differentiation of spatially overlapping targets while maintaining high throughput through efficient sequential processing.
3Device complexity
If directly labeled probe sets are used for detection, then the detection process is straightforward, but the probes must be denatured after every detection round increasing complexity
Solution Approach 1:
The identifier sequence information is extracted from the probe structure and used for decoding, while the probes themselves remain bound to targets. This separation eliminates the need for denaturation and probe removal after each detection round, reducing complexity and time loss.
Solution Approach 2:
An intermediary decoding step is introduced that reads the identifier sequences from probes already bound to targets. This intermediary process allows multiple detection rounds without requiring probe denaturation or removal, simplifying the overall workflow and reducing time investment.
4Adaptability or versatility
If high multiplexing is achieved using conventional methods, then many analytes can be detected, but the cost and complexity increase significantly
Solution Approach 1:
The probe sets use universal identifier sequences that can be decoded across multiple detection rounds. This universality allows the same decoding mechanism to handle many different analytes, achieving high multiplexing capability without proportionally increasing complexity or cost.
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 multiplexing capability, allows detection of spatially overlapping transcripts, reduces complexity and cost, and improves accuracy by decoupling analyte-specific and signal oligonucleotides, facilitating the detection of a substantial portion of the transcriptome or proteome.
Implementation Method 1
hybridization of the target probes to different analytes in the sample
Implementation Method 2
hybridization of a first set of decoding oligonucleotides to the identifier sequences
Implementation Method 3
hybridization of a set of signal oligonucleotides to translator sequences
Data Source
AI summary
The technology provided herein relates to high resolution multiplex methods and kits for detecting different analytes in a sample, such as by sequential signal-encoding of said analytes. The methods allows a differentiation of targets which distance is below the diffraction limit of optical microscopes, that is, targets with spatial optical overlap. The disclosed methods also include in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.