Optical Mapping DNA Repeat Analysis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current Next Generation Sequencing (NGS) methods are limited in analyzing repetitive DNA sequences due to random fragmentation and short read lengths, making it difficult to accurately quantify copy number and methylation status of repeat arrays, particularly for diseases like facioscapulohumeral muscular dystrophy (FSHD) associated with D4Z4 macrosatellite arrays.
Innovation Solution
A method combining optical mapping with fluorescence-based methylation detection, using sequence-specific nicking enzymes and methyltransferases to label and visualize DNA repeats, allowing for simultaneous copy number quantification and methylation status analysis at the single-molecule level without fragmentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Next Generation Sequencing (NGS) methods are used for analyzing DNA, then high throughput and speed are achieved, but repetitive DNA sequences cannot be accurately analyzed due to random fragmentation and short read lengths
Solution Approach 1:
The patent segments the DNA analysis process into two distinct labeling steps: first labeling repetitive sequences with one fluorescent marker, then labeling CpG sites with another marker. This segmentation allows each labeling reaction to target specific features independently, enabling accurate analysis of repetitive regions that would be indistinguishable in traditional NGS assembly approaches.
Solution Approach 2:
The patent transitions from one-dimensional sequence reading (NGS reads) to two-dimensional spatial mapping by visualizing the physical positions of fluorescent labels along stretched DNA molecules. This dimensional change allows direct observation of repeat copy numbers and methylation patterns without relying on computational assembly of short reads.
2Productivity
If Next Generation Sequencing (NGS) methods are used for analyzing DNA, then high throughput is achieved, but methylation status of repeat arrays cannot be detected
Solution Approach 1:
The patent merges copy number analysis and methylation status detection into a single optical mapping experiment by performing sequential fluorescent labeling. First, repetitive sequences are labeled to determine copy number, then CpG sites are labeled to determine methylation status, both on the same DNA molecule, achieving both measurements simultaneously in one high-throughput assay.
Solution Approach 2:
The patent uses different fluorescent colors to distinguish between repetitive sequence labels and CpG methylation labels. This color-based differentiation allows simultaneous detection of both copy number and methylation status through multi-color fluorescence imaging, making methylation detection as straightforward as sequence labeling.
3Length of moving object
If current NGS methods are used, then short reads are obtained, but assembly into long contiguous sequences is fundamentally limited
Solution Approach 1:
The patent creates a physical copy of the long DNA molecule's structural information through fluorescent labeling, bypassing the need for computational assembly. By directly visualizing the positions of labels along the stretched DNA, the method captures long-range structural information that would be lost in short-read NGS assembly, achieving both long effective read length and high reliability.
4Quantity of substance
If bulk measurements are performed, then averaged information is obtained, but intercellular variability is masked
Solution Approach 1:
The patent allows individual DNA molecules to serve themselves as separate analytical units. Each stretched DNA molecule is independently labeled and imaged, preserving the unique characteristics of each molecule. This self-service approach enables analysis of intercellular variability while maintaining high throughput, as each molecule's information is captured independently without being averaged with others.
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 precise and high-throughput analysis of DNA repeat arrays, providing detailed information on repeat number and methylation status, which is inaccessible with current NGS methods, and can distinguish between healthy and FSHD-affected individuals, potentially leading to personalized treatment approaches.
Implementation Method 1
sequence-specific nicking enzymes and methyltransferases to label and visualize DNA repeats
Implementation Method 2
CpG methylation is a major epigenetic modification responsible for genetic regulation
Implementation Method 3
methyltransferases to label and visualize DNA repeats
Implementation Method 4
fluorescence-based methylation detection, using sequence-specific nicking enzymes and methyltransferases to label and visualize DNA repeats
Data Source
AI summary
A method of diagnosing a disease associated with a DNA repeat sequence is disclosed. The method comprises:(a) determining the number of repeats of a DNA sequence in DNA molecules of a sample of the subject; and(b) determining the CpG methylation status of the DNA molecules, wherein the number of repeats of the DNA sequence and the CpG methylation status of the DNA molecules is indicative of the disease.