Single-Molecule Genomic Analysis via Unhybridized Flap Processing
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Solution Overview
Problem
Current DNA sequencing and structural variation detection methods are limited by low sensitivity and resolution, are laborious, expensive, and rely on reference databases, making it difficult to accurately analyze complex genomic information and detect balanced lesion events such as inversions or translocations.
Innovation Solution
The method involves processing double-stranded DNA to create unhybridized flaps, extending one strand along the corresponding region of the other strand, and labeling portions of the flaps or extended strands to enable direct analysis of genomic and epigenomic information at the single molecule level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional cytogenetic methods such as karyotyping and FISH are used, then a global view of genomic composition can be obtained, but sensitivity and resolution for detecting medium to small sequence motifs or lesions are relatively low
Solution Approach 1:
The patent segments the genome into large native molecules (megabase scale) that can be directly visualized and analyzed. By maintaining the physical continuity of long genomic DNA molecules and using fluorescent in situ hybridization with multiple probes, the method achieves high-resolution mapping of structural variations while preserving the global genomic context, thus resolving the contradiction between resolution and method complexity.
Solution Approach 2:
The patent transitions from traditional two-dimensional karyotyping to three-dimensional spatial mapping of genomic structures. By visualizing the physical arrangement of fluorescently labeled genomic regions in three-dimensional space, the method achieves superior resolution for detecting structural variations while maintaining a comprehensive global view, effectively resolving the contradiction between measurement precision and device complexity.
2Productivity
If conventional sequencing methods are used, then sequence information can be obtained, but the methods are laborious and expensive
Solution Approach 1:
The patent replaces laborious mechanical sequencing operations with fluorescent optical detection. By using fluorescent in situ hybridization and imaging to directly visualize and map genomic structures, the method eliminates time-consuming manual sequencing steps while maintaining high analytical productivity, thus resolving the contradiction between productivity and time consumption.
Solution Approach 2:
The patent creates a multi-functional method that simultaneously provides sequence information, structural variation detection, and spatial mapping capabilities. By integrating multiple analytical functions into a single fluorescent in situ hybridization approach, the method increases overall productivity while reducing the total time required compared to separate sequential analyses, effectively resolving the contradiction between productivity and time loss.
3Reliability
If reference database-dependent methods are used, then sequence analysis can be performed, but the methods cannot reveal balanced lesion events such as inversions or translocations
Solution Approach 1:
The patent inverts the traditional approach by not relying on reference databases for detection but instead using direct physical visualization of genomic structures. By mapping the actual spatial arrangement of fluorescent probes on native genomic molecules, the method can detect balanced lesions like inversions and translocations that reference-based methods miss, while maintaining high detection accuracy through direct observation, thus resolving the contradiction between reliability and adaptability.
4Measurement precision
If high-resolution sequencing is performed, then detailed sequence information can be obtained, but the cost increases significantly
Solution Approach 1:
The patent uses inexpensive fluorescent probes that can be synthesized at low cost and applied directly to native genomic molecules. By replacing expensive high-resolution sequencing reagents with affordable fluorescent hybridization probes, the method achieves high-resolution structural variation detection while significantly reducing the quantity of expensive substances required, thus resolving the contradiction between measurement precision and quantity of substance (cost).
Data Source
AI summary
Provided are methods and devices for single-molecule genomic analysis. In one embodiment, the methods entail processing a double-stranded nucleic acid and characterizing said nucleic acid. These methods are useful in, e.g. determining structural variations and copy number variations between individuals.