Single-Cell cDNA Amplification via Magnetic Bead Segmentation
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Solution Overview
Problem
Current methods for analyzing gene expression in single cells face challenges in efficiently converting trace amounts of mRNA into cDNA and amplifying it to sufficient quantities for large-scale DNA sequencing without bias, leading to sample loss and amplification biases.
Innovation Solution
A method involving immobilizing mRNA on magnetic beads using a first DNA probe with a poly T sequence, adding a polynucleotide sequence, hybridizing a second DNA probe, and performing PCR amplification to generate a sufficient amount of cDNA with reduced bias, using a protocol that minimizes sample loss and artifact production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional methods are used to amplify cDNA from single cell mRNA, then amplification can be achieved, but amplification bias and sample loss occur
Solution Approach 1:
The patent divides the amplification process into multiple stages: first strand cDNA synthesis, removal of reagents, addition of polynucleotide sequence, second strand cDNA synthesis, and PCR amplification. This segmentation allows each step to be optimized independently, reducing cumulative bias and loss while achieving sufficient amplification.
Solution Approach 2:
The patent performs preliminary actions by immobilizing mRNA on magnetic beads before amplification, removing reagents after first strand synthesis, and adding polynucleotide sequences in advance. These preliminary steps prepare the sample in a controlled manner that minimizes bias during subsequent amplification.
2Measurement precision
If trace amount of mRNA is used for analysis, then single cell resolution is achieved, but conversion to cDNA becomes inefficient
Solution Approach 1:
The patent uses magnetic beads as an intermediary to immobilize mRNA during the conversion process. This intermediary allows efficient capture and retention of trace mRNA molecules, facilitating effective cDNA synthesis from minimal starting material while maintaining single cell resolution.
Solution Approach 2:
The patent maintains continuous useful action by keeping the cDNA synthesized on magnetic beads in the reaction vessel and performing subsequent steps (polynucleotide addition, second strand synthesis, amplification) without removing the cDNA- bead complex. This continuity prevents sample loss and maintains efficiency throughout the workflow.
3Adaptability or versatility
If comprehensive gene expression analysis is performed, then all genes can be analyzed, but quantitative performance deteriorates
Solution Approach 1:
The patent creates a universal amplification system that can simultaneously amplify all genes in the transcriptome while maintaining quantitative accuracy. The magnetic bead-based approach and standardized amplification protocol work universally across different genes and cell types, enabling comprehensive analysis without sacrificing quantitative performance.
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 enables accurate and unbiased amplification of cDNA from single cells, allowing for comprehensive gene expression analysis with reduced sample loss and artifact production, suitable for large-scale DNA sequencing.
Implementation Method 1
capturing mRNA derived from the cell by a first DNA probe containing a first tag sequence and a poly T sequence and being immobilized to a solid carrier
Implementation Method 2
synthesizing a first strand cDNA from the mRNA by a reverse transcription reaction
Implementation Method 3
hybridizing a second DNA probe containing a second tag sequence and a complementary sequence to the polynucleotide sequence with the cDNA to which the polynucleotide sequence is added
Implementation Method 4
performing a DNA amplification reaction using the second strand cDNA synthesized on the solid carrier as a template
Data Source
AI summary
The problem to be solved by the present invention is to provide a method for preparing a sample for comprehensively and accurately analyzing gene expression in a single cell or a few cells, for example, by a large-scale DNA sequencer. The present invention relates to a method for amplifying cDNA from mRNA in a cell, comprising: (1) capturing mRNA derived from the cell by a first DNA probe containing a first tag sequence and a poly T sequence and being immobilized to a solid carrier in a single reaction vessel, and synthesizing a first strand cDNA from the mRNA by a reverse transcription reaction; (2) removing a reaction reagent from the reaction vessel while keeping the first strand cDNA synthesized onto the solid carrier in the reaction vessel; (3) adding a polynucleotide sequence consisting of one type of nucleotides to 3′ terminal of the first strand cDNA on the solid carrier; (4) hybridizing a second DNA probe containing a second tag sequence and a complementary sequence to the polynucleotide sequence with the cDNA to which the polynucleotide sequence is added, and synthesizing a second strand cDNA; and (5) performing a DNA amplification reaction using the second strand cDNA synthesized on the solid carrier as a template.