Hybrid DNA Probe Spatial Localization and NGS Sequencing
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Solution Overview
Problem
Current methods fail to simultaneously detect all expressed genes on a subcellular level and obtain their sequence and spatial information with high resolution.
Innovation Solution
A method using a hybrid circular/linear DNA probe with Unique Molecular Identifier (UMI) for spatial localization of mRNA, followed by reverse transcription, PCR amplification, and Rolling Circle Amplification (RCA) to sequence the target sequence, allowing for the retrieval and sequencing of UMI-containing cDNA strands from tissue sections.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used to detect expressed genes, then gene detection is possible, but simultaneous acquisition of spatial location and sequence information with high resolution is not achieved
Solution Approach 1:
The method segments the detection process into distinct functional components: spatial localization using fluorescently labeled probes, sequence information capture through reverse transcription and PCR, and molecular identity tagging using UMIs. This segmentation allows each component to be optimized independently while achieving high-resolution simultaneous measurement of both spatial and sequence data.
Solution Approach 2:
The methodology embeds multiple layers of information within nested molecular structures: UMIs are incorporated within cDNA molecules, which themselves contain sequence information and spatial localization markers. This nested arrangement allows compact storage of multiple data types (spatial coordinates, sequence, molecular identity) within a single molecular entity that can be simultaneously analyzed.
2Adaptability or versatility
If comprehensive gene detection is performed, then all expressed genes can be detected, but obtaining sequence and spatial information simultaneously remains challenging
Solution Approach 1:
The method employs multi-functional probes and reagents that simultaneously perform multiple functions: fluorescent probes provide both spatial localization and serve as templates for reverse transcription; the same cDNA molecules carry both sequence information and UMI tags for molecular identity; the assay can detect all expressed genes while maintaining high spatial resolution through the fluorescent signaling system.
3Measurement precision
If high resolution spatial and sequence information is obtained, then detection accuracy improves, but the complexity of the methodology increases
Solution Approach 1:
The methodology uses multiple copying steps to amplify and preserve information: reverse transcription copies RNA to cDNA, PCR amplifies specific regions of cDNA, and RCA amplifies the entire cDNA molecule. These copying steps create multiple identical copies of the molecular information, enabling high-accuracy detection while allowing the original fragile molecular structures to be replaced with more stable amplified copies for analysis.
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 the simultaneous acquisition of spatial and sequence information of target mRNA strands with higher resolution than existing technologies, facilitating the analysis of mutations or nucleotide variants on a tissue sample.
Implementation Method 1
hybridizing the locator probe with the RNA anchor region to the m-RNA strand
Implementation Method 2
complementing the RNA anchor region of the locator probe using the m-RNA strand as template thereby obtaining a reversed transcribed c-DNA strand
Implementation Method 3
complementing the circular locator starting from the first primer region using the circular locator as template
Implementation Method 4
ligating the resulting oligomer with the locator anchor region of the linear locator thereby obtaining an extended reversed transcribed c-DNA strand
Implementation Method 5
multiplying the circular locator by RCA starting from the second primer region on the tissue sample creating at least one first rolony
Implementation Method 6
spatially resolution sequencing the at least one first rolony thereby obtaining the spatial and sequence information
Implementation Method 7
amplification of the primed single stranded oligomer by PCR
Data Source
Figure 1(A)~1(B)
Figure 2a
Figure 2b
AI summary
The invention is directed to a method to simultaneously obtain both the spatial location and sequence information of a target sequence with a higher resolution than the known technologies. The method comprises steps to spatially localize the mRNA expressed on a tissue by the use of a hybrid circular/linear DNA probe with an UMI and - after several amplification steps, the obtaining sequence information by NGS.