In Situ Single-Cell DNA/RNA Sequencing with Polymerase Quenching
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for single-cell genomic DNA and RNA sequencing require labor-intensive cell isolation, are costly, and lack the ability to simultaneously sequence both DNA and RNA without converting DNA to RNA, and existing technologies face interference between DNA and RNA sequencing reactions.
Innovation Solution
A method that amplifies and barcodes genomic DNA and RNA within the same cell using isothermal polymerases, quenching the polymerase to prevent interference, and uses unique barcodes for each cell to distinguish DNA and RNA sequences, followed by a template switch reaction to prepare mixed pools for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cell isolation and sorting are performed to enable single-cell sequencing, then measurement precision is improved, but loss of time and productivity deteriorate due to labor-intensive procedures
Solution Approach 1:
The cell serves as its own reaction vessel, eliminating the need for external isolation and sorting. The in situ amplification occurs directly within the intact cell, allowing the cell to 'service itself' for sequencing preparation without requiring labor-intensive manual handling or specialized equipment.
Solution Approach 2:
The patent extracts the amplification reaction from the cell and places it back into the cell environment. By performing in situ amplification, the method removes the need for cell lysis and external processing steps, thereby eliminating time-consuming isolation procedures while maintaining single-cell resolution.
2Manufacturing precision
If DNA is amplified using polymerase for single-cell genomic sequencing, then manufacturing precision is improved, but object-generated harmful factors worsen due to polymerase interference with RNA sequencing
Solution Approach 1:
The patent segments the amplification process into distinct phases: DNA amplification using polymerase followed by RNA amplification using transcriptase. By separating these reactions temporally and using different enzymes, the method eliminates polymerase interference with RNA sequencing while maintaining high precision for both DNA and RNA amplification.
Solution Approach 2:
The patent introduces an intermediary step where the polymerase reaction is completed and then the enzyme is inactivated or removed before initiating RNA amplification. This intermediary action prevents the polymerase from interfering with subsequent RNA sequencing while preserving the benefits of accurate DNA amplification.
3Manufacturing precision
If sequential reactions are performed for DNA and RNA sequencing, then manufacturing precision is improved, but device complexity and ease of operation worsen due to requiring two separate reaction protocols
Solution Approach 1:
The patent merges DNA amplification and RNA amplification into a single integrated protocol that occurs sequentially within the same cell. By combining these reactions in a unified workflow with automated enzyme addition and in situ processing, the method maintains high sequencing accuracy while reducing overall procedural complexity compared to separate handling of DNA and RNA.
Solution Approach 2:
The patent creates a universal in situ amplification platform that can handle both DNA and RNA sequencing within the same cell using the same basic workflow. The system is designed to accommodate different enzyme additions and reaction conditions, making it a multi-functional solution that simplifies operation while maintaining precision for both nucleic acid types.
4Measurement precision
If barcodes are added after cell sorting into separate containers, then measurement precision is improved, but ease of operation and productivity deteriorate due to difficulty in washing and quenching reactions
Solution Approach 1:
The cell serves as its own reaction container, eliminating the need for external washing and quenching steps. Barcodes are added in situ within the cell environment, and the cell's own structures facilitate the reaction completion and enzyme inactivation without requiring manual handling of tiny volumes in separate containers.
Solution Approach 2:
The patent extracts the barcode addition process from the context of sorted cell containers and performs it directly within the intact cell. This removes the operational difficulties associated with washing and quenching reactions in micro-scale external containers while preserving the precision of cell-specific barcode assignment.
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 high-throughput, cost-effective, and user-friendly simultaneous sequencing of single-cell genomes and transcriptomes without specialized equipment, allowing for multiplexing and accurate association of genetic changes with phenotypic differences.
Implementation Method 1
amplifies and barcodes genomic DNA and RNA within the same cell using isothermal polymerases
Implementation Method 2
reverse transcribing the RNA present in the cell to generate cDNA
Implementation Method 3
capturing the released amplicons by contacting the barcoded amplicons comprising the affinity moiety and the barcoded cDNA comprising the affinity moiety with an affinity capture reagent
Data Source
AI summary
Disclosed herein is an in situ, high throughput, single-cell region(s) of interest (ROI) or whole-genome sequencing technology developed for sequencing gene(s) or genomes in large heterogeneous cell populations combined with single-cell RNA sequencing. More specifically, the technology disclosed herein does not require cell sorting or isolation because it uses the cell membrane to separate each genome whereupon single genomes and transcriptomes are concurrently sequenced. While other in situ single-cell sequencing technologies are only able to work with RNA, and must first convert DNA to RNA, the method of this disclosure operates directly on DNA as well as on RNA. Thus, it simultaneously sequences each single cell's genome and transcriptome.