Spatial Sequencing of Modified Sequences with In Situ Amplification
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Solution Overview
Problem
Existing single-cell technologies struggle to provide precise sequencing information for spatial gene and protein co-expression patterns, which are crucial for understanding tissue development and disease progression, particularly in cancers like breast cancer.
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 nucleic acids, then gene and protein expression can be quantified, but precise sequencing information for spatial patterns cannot be obtained
Solution Approach 1:
The method segments the sequencing process into distinct spatial steps: (1) in situ hybridization of oligonucleotide primers to target nucleic acids within intact tissue sections, (2) circularization of primers to generate padlock probes that remain anchored at their original spatial locations, and (3) rolling circle amplification to generate readable sequences while preserving spatial coordinates. This segmentation allows simultaneous获得 of sequencing precision and spatial pattern information.
Solution Approach 2:
The patent introduces oligonucleotide padlock probes as intermediary molecules that bridge the gap between in situ spatial preservation and sequencing information extraction. These probes hybridize to target nucleic acids in their native spatial context, then undergo circularization and amplification to generate readable sequences while maintaining their original spatial positions as physical intermediaries between the tissue architecture and sequencing data.
2Loss of information
If nucleic acids are extracted for sequencing, then sequencing information can be obtained, but spatial location information is lost
Solution Approach 1:
The method performs preliminary in situ hybridization and circularization of padlock probes to target nucleic acids within intact tissue sections before any extraction or amplification steps. This preliminary action anchors the sequencing primers at their exact spatial locations, ensuring that subsequent rolling circle amplification and sequencing operations preserve the original spatial coordinates while generating high-quality sequencing data.
Solution Approach 2:
The patent adds a spatial dimension to traditional sequencing by performing all amplification and sequencing operations in situ within the tissue architecture rather than in extracted nucleic acid solutions. The rolling circle amplification occurs while the padlock probes remain anchored to their target sequences within the intact tissue section, thereby preserving spatial information in the x-y-z dimensions while generating sequencing reads.
3Quantity of substance
If traditional amplification methods are used, then nucleic acid copies can be generated, but spatial sequencing information is compromised
Solution Approach 1:
The method employs rolling circle amplification, a self-service amplification mechanism where a single circularized padlock probe serves as its own template for continuous synthesis. The strand-displacing polymerase automatically displaces newly synthesized strands and uses them as templates for further synthesis, generating multiple copies of the target sequence while the entire process occurs in situ at the original spatial location, thereby maintaining both copy number and spatial 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 of nucleic acids in situ, allowing for identification of agent-mediated sequences and cell responses, thereby providing valuable 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 sample comprising a cell.