Identifier-Sequence RCA Sequencing to Reduce Optical Crowding
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Solution Overview
Problem
Plex-scalability of in situ detection methods is limited by optical crowding, where signals from large or high-intensity features overlap with smaller or weaker signals, compromising detection quality and decoding, especially in multiplexed assays.
Innovation Solution
Base-by-base sequencing methods are employed using sequencing primers that hybridize to identifier sequences, generating signal code sequences through nucleotide incorporation or binding events, with staggered detection of ON and OFF signals to minimize optical crowding and decode analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If large size features (RCA products, nucleic acid probes, or nucleic acid complexes) and/or high intensity signals are used in multiplexed assays, then signal strength is improved, but optical crowding increases causing signal overlap and masking
Solution Approach 1:
The patent segments the detection process by using multiple fluorophores with different emission wavelengths (e.g., F1, F2, F3) to detect different analytes simultaneously. Each analyte is assigned a specific fluorophore, allowing spatial separation of signals through wavelength discrimination rather than physical separation, thus reducing optical crowding while maintaining high signal intensity.
Solution Approach 2:
The patent transitions from spatial dimension (physical separation of signals) to spectral dimension (wavelength-based separation). By using multiple fluorophores emitting at different wavelengths, the system can multiplex many analytes without increasing optical crowding, as signals are distinguished by their spectral properties rather than spatial proximity.
2Adaptability or versatility
If complex pools of oligonucleotide probes are used, then detection capability is improved, but cost and time for detection and decoding increase
Solution Approach 1:
The patent extracts and uses only the essential information needed for detection - fluorescent labels with specific emission wavelengths - rather than employing complex pools of oligonucleotide probes. This simplification maintains detection capability for multiple analytes while dramatically reducing the time and complexity required for detection and decoding.
Solution Approach 2:
The patent employs simple, stable fluorophore molecules that can be easily attached to probes and maintained throughout the assay, replacing complex oligonucleotide probe pools. These fluorophores are inexpensive, stable, and require minimal handling, reducing both cost and time while maintaining versatile detection capability.
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
The method effectively decodes identifier sequences in situ by minimizing optical crowding, allowing for accurate detection and identification of multiple analytes in a cell or tissue sample, reducing the need for complex probe pools and enhancing detection efficiency.
Implementation Method 1
a sequencing primer hybridizes to a priming site 3' to an identifier sequence in the sample
Implementation Method 2
the sequencing primer can be extended by a polymerase in a base-by-base fashion using a sequence of the identifier sequence as a template
Implementation Method 3
contacting the biological sample with detectably labeled nucleotides in a cyclic series of nucleotide incorporation or nucleotide binding steps and detecting signals associated with the incorporation or binding events
Data Source
AI summary
Methods and compositions for performing a rolling circle amplification (RCA) reaction using circularized template comprising identifier sequences are provided. Sequencing is performed on the RCA product using a polymerase to incorporate a plurality of cognate nucleotides into the sequencing primer or an extension product thereof to generate an extension product.