In Situ Genomic DNA Amplification via Combinatorial Barcoding
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for single-cell genomic DNA sequencing require physical separation of cells and specialized equipment, making them expensive, labor-intensive, and limited in scalability, whereas existing technologies for RNA sequencing do not need DNA amplification and are biased towards transcriptional profiles.
Innovation Solution
A method that amplifies and barcodes genomic DNA in situ within cells using isothermal polymerases and combinatorial barcoding, allowing for the sequencing of multiple cells without physical separation and using common laboratory equipment, enabling the generation of unique barcodes for each cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If physical separation of cells into separate containers is performed, then unique barcodes can be associated with each cell, but the process becomes labor-intensive and requires specialized equipment
Solution Approach 1:
The patent extracts the cell from the separation process entirely. Instead of separating cells into droplets or wells, the method performs amplification and barcoding directly within the intact cell, eliminating the need for physical separation while maintaining barcode assignment accuracy through in situ reactions
Solution Approach 2:
The cell itself serves as the reaction container for DNA amplification and barcoding. The cell membrane integrity is maintained throughout the process, allowing the cell to contain and facilitate the biochemical reactions needed for genomic DNA sequencing without external separation apparatus
2Measurement precision
If physical separation of cells is performed, then single-cell resolution is achieved, but the cost and equipment requirements increase
Solution Approach 1:
The patent removes the requirement for specialized microfluidic devices, flow cytometers, or droplet generation equipment by performing all single-cell operations within standard laboratory wells using common equipment, while maintaining single-cell resolution through well-based isolation
Solution Approach 2:
The cell serves multiple functions simultaneously: it contains the genomic DNA, provides the reaction environment for amplification, maintains barcode association through membrane integrity, and enables sequencing without requiring specialized single-cell equipment
3Productivity
If RNA sequencing methods are used, then transcriptional profiles are obtained, but genomic DNA sequencing is not achieved
Solution Approach 1:
The patent changes the biochemical parameters from RNA-based sequencing to DNA-based sequencing by using DNA polymerases, DNA-specific primers, and DNA amplification protocols within intact cells, enabling genomic DNA sequencing while maintaining high throughput through combinatorial barcoding
Solution Approach 2:
The method performs preliminary genomic DNA amplification and barcoding within intact cells before any separation or processing steps, ensuring that the genomic DNA is already prepared and uniquely identified before downstream sequencing operations
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 approach reduces the cost and complexity of single-cell genomic DNA sequencing, allows for the analysis of heterogeneous populations, and provides deeper insights into genetic diversity and evolutionary processes, while maintaining cellular membrane integrity and enabling the sequencing of a larger fraction of the genome.
Implementation Method 1
amplifying genomic DNA while it remains inside of each cell to create barcoded molecules under conditions that maintain cellular membrane integrity
Implementation Method 2
a targeting region comprising at least one of: (iii) random hexamers sequences; wherein the random hexamers sequences hybridize to complementary sequences on genomic DNA of the cells
Data Source
AI summary
Disclosed herein is an in situ, high throughput, single-cell whole-genome sequencing technology developed for sequencing genomes in large heterogeneous cell populations. More specifically, the invention disclosed herein does not require cell sorting or isolation because it uses the cell membrane to separate each genome.