Tumor-Infiltrating Cell Detection by In Situ Circularized Sequencing
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Solution Overview
Problem
Existing single-cell technologies struggle to accurately profile the genome, epigenome, and transcriptome for understanding cell co-localization and disease spread, particularly in cancer, due to limitations in quantifying gene and protein expression and sequencing precision.
Innovation Solution
A method involving hybridization of oligonucleotide primers to target nucleic acids, circularization, amplification using strand-displacing polymerase, and sequencing of extension products to obtain precise sequencing information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing single-cell technologies are used for profiling genome, epigenome, and transcriptome, then basic cellular composition information can be obtained, but measurement precision and sequencing accuracy are insufficient for understanding cell co-localization and disease spread
Solution Approach 1:
The patent segments the nucleic acid profiling process into distinct functional modules: in situ hybridization for spatial localization, circularization for template preparation, strand-displacing polymerase amplification for signal enhancement, and sequencing for data generation. This segmentation allows each module to be optimized independently, improving overall measurement precision while maintaining reliability through specialized function assignment.
Solution Approach 2:
The patent introduces circularized oligonucleotide templates as intermediaries between the target nucleic acids and the sequencing process. These circular templates serve as stable, amplifiable intermediates that bridge the gap between in situ hybridization and sequencing, enabling precise measurement while maintaining the spatial context of the original sample.
2Quantity of substance
If traditional amplification methods are used, then sufficient signal for detection can be obtained, but spatial information and in situ context are lost
Solution Approach 1:
The patent performs in situ hybridization and circularization before amplification, establishing the spatial context and template structure in place. This preliminary action ensures that subsequent amplification occurs at the original location, preserving spatial information while generating sufficient signal through the strand-displacing polymerase reaction.
Solution Approach 2:
The patent implements a nested structure where circularized oligonucleotides are amplified within the in situ context, creating multiple copies of the template that remain spatially anchored. This nesting allows signal amplification while maintaining the spatial relationship between different nucleic acid targets in the original sample.
3Manufacturing precision
If complex multi-step protocols are implemented to improve sequencing accuracy, then identification precision increases, but device complexity and ease of operation deteriorate
Solution Approach 1:
The patent employs universal reagents and conditions that can be applied across different nucleic acid targets and sample types. The in situ hybridization protocol, circularization reaction, and strand-displacing polymerase amplification can be used for genome, epigenome, and transcriptome profiling with the same basic methodology, reducing operational complexity while maintaining high identification precision.
Solution Approach 2:
The circularized oligonucleotide templates serve as self-amplifying structures that automatically generate sufficient signal through the strand-displacing polymerase reaction without requiring external intervention. This self-service mechanism simplifies the protocol by eliminating the need for complex manual manipulation steps while maintaining high identification precision.
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
Enables precise sequencing and identification of agent-mediated nucleic acid sequences, allowing for the detection of cells responding to genetically modifying agents and identifying agents, thereby providing insights into cellular processes and disease mechanisms.
Implementation Method 1
hybridizing an oligonucleotide primer to the target nucleic acid, wherein the oligonucleotide primer includes a first region at a 3′ end that hybridizes to a first complementary region of the target nucleic acid
Implementation Method 2
amplifying the circular oligonucleotide by extending an amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a tissue sample.