Single-Molecule Sequencing for Modified Nucleic Acids
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for determining genome-wide methylation patterns, such as bisulfite sequencing, face challenges including high sample preparation time, DNA degradation, and limitations in sequencing technologies, which hinder accurate and comprehensive analysis of nucleic acid modifications like methylation and hydroxymethylation.
Innovation Solution
A real-time single-molecule sequencing method that provides both base sequence data and modification data, allowing for the detection of modified nucleic acid sequences, including methylated bases, without the need for bisulfite conversion or amplification, and enables differential modification analysis between homologous chromosomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used to determine genome-wide methylation patterns, then methylation status can be determined, but sample preparation time is excessive and DNA degradation occurs
Solution Approach 1:
The patent extracts and eliminates the bisulfite conversion step from the sequencing workflow. By using native DNA sequencing without chemical modification, the method removes the source of DNA degradation and time-consuming preparation while preserving the ability to detect methylation patterns through direct sequencing of unmethylated and methylated regions
Solution Approach 2:
The patent performs preliminary enrichment of methylated DNA regions using methyl-binding proteins or antibodies before sequencing. This preliminary action allows direct sequencing of the enriched material without requiring subsequent chemical conversion steps, thereby reducing both time and DNA degradation
2Measurement precision
If bisulfite sequencing is used to determine genome-wide methylation patterns, then methylation status can be determined, but DNA degradation occurs
Solution Approach 1:
The patent converts the challenge of detecting modified bases into an advantage by using the inherent stability of native DNA. Instead of chemically modifying DNA (which causes degradation), the method detects methylation patterns through direct sequencing, turning the absence of chemical treatment into a benefit for DNA integrity while maintaining detection accuracy
Solution Approach 2:
The patent introduces methyl-binding proteins or antibodies as intermediaries that specifically bind to methylated DNA regions. These intermediaries enable selective enrichment and detection of methylated sequences without direct chemical interaction with the DNA backbone, thereby preserving DNA integrity throughout the process
3Measurement precision
If current sequencing technologies are used, then base sequence data can be obtained, but modification data like methylation and hydroxymethylation cannot be accurately detected
Solution Approach 1:
The patent merges base sequence determination with modification detection into a single sequencing workflow. By sequencing native DNA and analyzing signal characteristics (such as polymerase kinetics or fluorescent signal patterns), the method simultaneously obtains both sequence information and methylation status without requiring separate chemical treatment steps
Solution Approach 2:
The patent changes the detection parameter from chemical modification (bisulfite conversion) to physical/kinetic parameters during sequencing. By monitoring polymerase incorporation rates, fluorescence intensity patterns, or other kinetic parameters during native sequencing, the method detects methylation and hydroxymethylation modifications while maintaining accurate base sequence determination
4Quantity of substance
If amplification is performed before sequencing, then sufficient DNA quantity can be obtained, but modification data is lost
Solution Approach 1:
The patent performs preliminary enrichment of methylated DNA regions using affinity-based methods (methyl-binding proteins or antibodies) before sequencing. This preliminary enrichment step increases the quantity of relevant methylated DNA sequences without requiring amplification, thereby preserving the native modification states for accurate detection
Solution Approach 2:
The patent uses methyl-binding proteins or antibodies as copying mechanisms to generate multiple bound copies of methylated DNA regions on solid supports or beads. This creates sufficient material for sequencing while maintaining the native DNA modifications, avoiding the information loss associated with enzymatic amplification
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 enables high-resolution, efficient detection of nucleic acid modifications, overcoming the limitations of existing technologies and providing detailed insights into epigenetic changes, which is crucial for understanding genetic and health-related research.
Implementation Method 1
modifications in a template nucleic acid will alter the kinetics of a polymerase processing the template nucleic acid
Implementation Method 2
single molecule, real-time sequencing methods, which observe the incorporation of nucleotides into a growing strand during a sequencing-by-synthesis reaction
Data Source
AI summary
Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.