UNIT-DNA Spatial Barcoding for High-Resolution Sequencing
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Solution Overview
Problem
Existing in situ sequencing methods face limitations in the number of measurable mRNA sequences and expression dynamics due to spatial constraints, such as the size of cells and the density of rolonies, which impairs signal discrimination during optical detection.
Innovation Solution
The method employs optical coding using universal template-directed DNA synthesis (UNIT-DNA) to insert barcodes into DNA sequences, allowing for precise spatial barcoding of nucleic acids with a resolution of up to one micron, enabling each cell to receive a unique code.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If rolling circle amplification is used to amplify mRNA signals in situ, then signal amplification is achieved, but the number of measurable mRNA sequences is limited by cell size and rolonie density
Solution Approach 1:
The patent extracts the amplification process from the cellular context by capturing mRNA molecules onto a surface outside the cell, then performing amplification ex situ. This removes the spatial constraint of cell size that previously limited the number of measurable mRNA sequences.
Solution Approach 2:
The patent transitions from two-dimensional in situ amplification within cells to a three-dimensional ex situ amplification process on a surface, allowing unlimited expansion of amplification products without being constrained by cell volume.
2Quantity of substance
If high density of rolonies is formed within cells, then signal amplification is enhanced, but discrimination of single mRNA signals during optical detection is impaired
Solution Approach 1:
The patent segments the detection process by first capturing individual mRNA molecules onto distinct surface locations, then performing amplification at each captured site. This creates spatially separated amplification products that can be individually resolved by optical detection, maintaining signal discrimination while enabling amplification.
Solution Approach 2:
The patent introduces a surface with barcoded primers as an intermediary between the mRNA molecules and the detection system. This intermediary allows amplification to occur while maintaining spatial information and enabling optical discrimination of individual signals.
3Loss of information
If in situ capturing is used with barcoded primers, then spatial information is preserved, but RNA capture efficiency is restricted and resolution is poor
Solution Approach 1:
The patent performs preliminary capture of mRNA molecules onto a surface with barcoded primers before amplification. This preliminary action preserves spatial information through the barcode while enabling subsequent high-resolution optical detection of the amplified products.
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 significantly enhances the resolution and variability of the barcode, overcoming the spatial limitations of existing methods, allowing for high-resolution, single-cell analysis and improved mRNA expression dynamics.
Implementation Method 1
the blocking groups are removed from the incorporated nucleotides by irradiation with light
Data Source
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AI summary
The invention is directed to a method to provide a polynucleotide molecule comprising a first and a second strand with a barcode nucleotide sequence characterized in that the first strand is provided at its 5´ end with an overhang of at least one universal base and the corresponding recessed 3´ end of the second strand of the polynucleotide with at least one nucleotide provided with a blocking group, wherein the blocking groups are removed from the incorporated nucleotides by irradiation with light.