Biopolymer Labeling for High-Resolution Genomic Mapping
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Solution Overview
Problem
Current nucleic acid sequencing and molecular imaging techniques face limitations in resolving structural features on small samples, particularly failing to achieve accurate genomic information at the level of single molecules or physically small samples, with existing methods limited to around 1000 bp accuracy.
Innovation Solution
The methods involve labeling specific locations on biopolymers with fluorescent labels, using sequence-specific nicking endonucleases, and incorporating dye-labeled nucleotides to determine the presence or relative positions of exons and structural features, enabling improved resolution and accuracy through linearization and imaging techniques such as SHRIMP and SHREC.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing techniques are used, then standard genomic information can be obtained, but resolution and accuracy are limited to around 1000 bp
Solution Approach 1:
The patent segments the DNA molecule into manageable portions by linearizing it and introducing nicks at specific intervals using nicking endonucleases. This allows the long DNA molecule to be analyzed in sections while maintaining its continuous structure, enabling high-resolution mapping without requiring analysis of the entire genome at once.
Solution Approach 2:
The patent transitions from sequence-based analysis to spatial dimension analysis by measuring the physical distances between fluorescent labels on linearized DNA. This dimensional shift allows resolution beyond the conventional 1000 bp limit by directly measuring nanometer-scale distances between labeled sites.
2Loss of information
If sample volume is reduced to single molecules or small samples, then population heterogeneity is eliminated, but ability to resolve structural features deteriorates
Solution Approach 1:
The patent uses fluorescent labels with distinct emission wavelengths (colors) attached to specific DNA sequences. By detecting the colors and their spatial positions, the system can identify and characterize structural features on single DNA molecules, eliminating the need for population averaging while maintaining or improving resolution.
Solution Approach 2:
The patent replaces conventional mechanical or chemical sequencing methods with optical detection of fluorescent labels. This substitution enables detection of structural features on single molecules by measuring the positions and colors of fluorophores, achieving both single-molecule sensitivity and high structural resolution.
3Measurement precision
If linearization and high-resolution imaging techniques are implemented, then resolution and accuracy are improved, but method complexity increases
Solution Approach 1:
The patent performs preliminary linearization of DNA molecules and introduces nicks with fluorescent labels before the actual measurement step. This preliminary preparation simplifies the subsequent imaging and analysis by ensuring that DNA is in the optimal configuration for high-resolution measurement, reducing the complexity of real-time manipulation during detection.
Solution Approach 2:
The patent uses fluorescent labels as intermediaries between the DNA structure and the detection system. These labels serve as mediators that convert nanometer-scale structural information into optically detectable signals, enabling high-resolution measurement without requiring direct mechanical manipulation or complex imaging of the DNA itself.
4Productivity
If conventional methods are used, then standard throughput is achieved, but ability to detect structural variations and alternative splicing deteriorates
Solution Approach 1:
The patent merges multiple functions into a single experimental workflow: linearization, nicking with fluorescent labels, and high-resolution imaging are combined in one process. This integration allows simultaneous detection of structural variations, alternative splicing events, and sequence information, achieving both high throughput and high precision without requiring separate assays.
Solution Approach 2:
The patent creates a universal platform that can detect multiple types of genomic features (structural variations, alternative splicing, sequence variations) using the same basic approach of fluorescent label positioning. This multi-functional system achieves high throughput by applying the same methodology across different detection targets without requiring separate optimized protocols for each feature type.
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
These methods enhance the resolution and accuracy of genomic information, allowing for the detection of structural variations and alternative splicing events at a much finer scale than conventional methods, improving the ability to identify and quantify RNA splicing variants and providing high-throughput sequencing capabilities.
Implementation Method 1
labeling first and second locations on a biopolymer with, respectively, a first and a second label... linearizing and illuminating the nucleic acid biopolymer so as to determine the presence or relative positions
Data Source
AI summary
The present invention provides methods of obtaining structural information about a biopolymer sample. The methods include labeling portions of a biopolymer, such as DNA or RNA, linearizing the biopolymer in some cases, and determining the distance between the labels. The user can then compare different samples' between-label distances to qualitatively compare different samples and to assay a given sample for additions or deletions of nucleotides in the regions flanked by the labels. The methods also permit sequencing of biopolymers.