In Situ Isothermal Amplification for Spatially Resolved Sequencing
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Solution Overview
Problem
Current methods for sequencing nucleic acids within biological samples require the destruction of the sample and extraction of target sequences, making it difficult to achieve spatially resolved sequencing data.
Innovation Solution
A method involving isothermal amplification and in situ sequencing within the biological sample, using a set of primers to generate hairpin loops and a strand displacing polymerase to amplify and sequence the target sequence without destroying the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional sequencing methods are used, then sequencing can be performed, but the biological sample must be destroyed and target sequences extracted prior to sequencing
Solution Approach 1:
The method performs amplification and sequencing steps in situ within the biological sample before any extraction or destruction occurs. By carrying out these preparatory actions (amplification and sequencing) while the sample remains intact, the invention preserves the spatial information and eliminates the need for subsequent sample destruction and extraction that would otherwise be required.
Solution Approach 2:
The invention introduces an isothermal amplification system as an intermediary process that occurs within the intact biological sample. This amplification system enables sufficient target sequence copies to be generated in situ, allowing sequencing to proceed without extracting the targets from the sample matrix, thereby maintaining spatial resolution while avoiding sample destruction.
2Ease of operation
If isothermal amplification is used, then amplification can be performed at constant temperature, but the amplification efficiency must be sufficient for sequencing
Solution Approach 1:
The invention changes the temperature parameter from the variable thermal cycling required by conventional PCR to a constant isothermal temperature. This parameter change simplifies the operational complexity of temperature control while the patent ensures sufficient amplification efficiency through the use of isothermal amplification methods that are optimized to achieve robust copy number increases at the maintained constant temperature.
3Loss of information
If amplification and sequencing are carried out within the biological sample, then spatially resolved sequencing data can be generated, but the method complexity increases
Solution Approach 1:
The invention merges multiple previously separate steps (target extraction, amplification, and sequencing) into a unified in situ process. By combining these steps and performing them within the intact biological sample, the method preserves spatial information while the integrated nature of the approach actually reduces overall procedural complexity compared to performing each step separately with multiple transfers and extractions.
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
This method allows for fast, robust, and efficient amplification and sequencing of nucleic acid target sequences within the biological sample, enabling spatially resolved data generation and applications in proteomics, genomics, and cell therapy monitoring.
Implementation Method 1
amplifying within the biological sample the target sequence with an isothermal amplification method in order to generate at least one amplified target sequence
Implementation Method 2
the primers of the set of primers are configured to hybridise to the target sequence
Implementation Method 3
a strand displacing polymerase to amplify and sequence the target sequence
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
A method for sequencing within a biological sample is provided comprising the following steps: providing the biological sample comprising at least one target sequence; amplifying within the biological sample the target sequence with an isothermal amplification method in order to generate at least one amplified target sequence; sequencing within the biological sample the amplified target sequence.