Nucleic Acid Methylation Detection via Transient Probe Binding
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Solution Overview
Problem
Current methods for detecting epigenetic modifications in nucleic acids, such as methylation and hydroxymethylation, are cumbersome, require large sample amounts, and suffer from DNA degradation and limitations of sequencing technologies like bisulfite sequencing.
Innovation Solution
A method involving transient binding of short oligonucleotides to nucleic acids, monitoring binding kinetics, and using distinct signatures like dwell time and repeat binding events to classify modifications, allowing direct detection without DNA amplification or harsh chemical treatments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used to detect methylation, then methylation status can be determined, but DNA degradation occurs and large starting amounts of sample are required
Solution Approach 1:
The patent changes the chemical parameters of the detection method by using formic acid treatment instead of bisulfite conversion. This alternative chemical approach achieves methylation detection without the harsh conditions that cause DNA degradation, thereby maintaining measurement precision while eliminating the harmful effect of DNA damage.
Solution Approach 2:
The patent extracts only the essential function of bisulfite sequencing (methylation detection) while removing the harmful aspect (DNA degradation). By using a different chemical method that achieves the same detection goal without the damaging conversion process, the invention separates the useful function from the harmful effect.
2Measurement precision
If bisulfite sequencing is used to detect methylation, then methylation status can be determined, but large starting amounts of sample are required
Solution Approach 1:
The patent changes the methodological parameters from bisulfite conversion requiring amplification to formic acid treatment allowing direct sequencing. This parameter change eliminates the need for DNA amplification steps, enabling accurate methylation detection with much smaller starting sample amounts.
3Measurement precision
If transient binding of short oligonucleotides is used, then detection resolution is improved and sample requirements are reduced, but binding kinetics monitoring complexity increases
Solution Approach 1:
The patent replaces complex mechanical binding kinetics monitoring with a simpler chemical approach using formic acid treatment followed by direct sequencing. This substitution eliminates the need for real-time binding monitoring while achieving high-resolution detection through the chemical sensitivity of the sequencing method to modified bases.
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 high-resolution, efficient detection of epigenetic modifications with reduced sample requirements and minimal DNA degradation, providing accurate classification of methylation and hydroxymethylation states.
Implementation Method 1
a first set of one or more positions on the fixed single stranded nucleic acid that are complementary to the respective oligonucleotide probe species
Data Source
AI summary
Systems, methods, and compositions for detecting epigenetic modifications in nucleic acids are provided. The invention comprises methods, compositions, and systems for determining the modification status of a nucleic acid molecule by using probes to detect a difference in signal when the nucleic acid is modified compared to when it is not. The modification may comprise a covalent modification such as methylation on a nucleobase.