Tag-Sequence Two-Dimensional cDNA Library for Single-Cell Gene Expression
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Solution Overview
Problem
Current methods for gene expression analysis in individual cells face limitations such as low accuracy in quantitative analysis, limited frequency of measurement operations, high costs, and the need for large and expensive apparatuses due to the requirement for cell isolation and nucleic acid amplification, which restrict the number of genes and cells that can be analyzed simultaneously while maintaining positional information.
Innovation Solution
A method involving the hybridization of test nucleic acids to a nucleic acid probe with a cell recognition tag sequence immobilized on a support, followed by cDNA library preparation and nucleic acid amplification, allowing for the analysis of gene expression in multiple cells while preserving positional information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR amplification and DNA chip methods are used for gene expression analysis, then the analysis can be performed on a group of cells, but the accuracy of quantitative analysis is low
Solution Approach 1:
The invention segments the analysis by performing quantitative PCR on individual cells rather than on a group of cells. Each cell's mRNA is separately converted to cDNA and analyzed, eliminating the averaging effect that reduces accuracy in group analyses while maintaining high throughput through automated processing of multiple cells
Solution Approach 2:
The invention creates multiple copies of cDNA from the original mRNA through reverse transcription and PCR amplification. This copying process enables sufficient material for accurate quantitative analysis while preserving the ability to analyze multiple genes from a single cell's original mRNA population
2Measurement precision
If quantitative PCR is performed on a single cell, then the quantitative analysis accuracy is improved, but the number of genes that can be measured is limited due to sample division requirements
Solution Approach 1:
The invention performs preliminary reverse transcription to convert all mRNA from a single cell into cDNA before dividing the sample for analysis of multiple genes. This preliminary action ensures that the total mRNA population is preserved and can be distributed across multiple PCR reactions without losing low-expression genes
Solution Approach 2:
The cDNA library prepared from a single cell serves multiple functions: it can be used for analyzing multiple different genes through different PCR primers, and the same cDNA pool can be repeatedly analyzed for different gene targets without requiring additional sample division that would compromise low-expression gene detection
3Productivity
If cells are inserted one by one into multiple reaction tanks with tag sequences for cell identification, then the number of cells that can be analyzed increases, but the cost and complexity of apparatus increase
Solution Approach 1:
The invention merges multiple cells into a single reaction tank rather than analyzing each cell in separate tanks. By using high-throughput sequencing technology, the system can identify and differentiate individual cells within the pooled sample, thereby reducing the number of reaction tanks and associated apparatus complexity while maintaining the ability to analyze many cells
Solution Approach 2:
The invention uses sequencing tags that are copied and attached to cDNA from individual cells, allowing cell identification through sequence data rather than through physical separation into multiple tanks. This copying of identification information into the nucleic acid sequence itself eliminates the need for complex physical indexing systems
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 accurate and high-throughput gene expression analysis across multiple cells, providing detailed information on gene flow and tissue responses, and reducing the cost and complexity of equipment needed.
Implementation Method 1
a step of hybridizing a test nucleic acid to serve as a target to a nucleic acid probe in a support in which the nucleic acid probe having a test nucleic acid capture sequence and a known sequence
Implementation Method 2
a step of synthesizing a complementary DNA strand to the test nucleic acid, thereby preparing a cDNA library constituted by the DNA complementary strand containing the tag sequence
Implementation Method 3
a step of performing nucleic acid amplification of the whole or part of the cDNA library
Data Source
AI summary
The present invention relates to a method, a device, and an apparatus for analyzing the expression of a gene in single cells. Specifically, the present invention relates to: a device for gene expression analysis, characterized by including a support, in which a nucleic acid probe having a test nucleic acid capture sequence and a known sequence, and further containing a cell recognition tag sequence which differs depending on the difference in position on the surface of the support or in the vicinity of the surface thereof, and a common primer sequence having a known sequence is two-dimensionally distributed and immobilized on the surface of the support or in the vicinity of the surface thereof; and a method and an apparatus using the device for gene expression analysis.