Methylation Detection via Cytosine Conversion Sequencing
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Solution Overview
Problem
Current methods for detecting modified cytosines, such as 5-methylcytosine, in nucleic acid sequences are inefficient and lack accuracy, particularly in minimizing sample loss and maintaining sequence quality and complexity.
Innovation Solution
A method involving the synthesis of double-stranded nucleic acid templates where a conversion reagent converts modified cytosines to thymine or uracil, allowing for their detection by sequencing, using boron-based reducing agents or cytidine deaminases, and subsequent selective processing and amplification techniques to enhance signal intensity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used for detecting modified cytosines, then detection can be performed, but accuracy and resolution are insufficient
Solution Approach 1:
The patent applies preliminary action by treating the target polynucleotide with a conversion reagent before sequencing. This reagent converts modified cytosines (5mC, 5hmC, 5fc, 5cc) to thymine or uracil, creating a permanent chemical signature that enables accurate detection during sequencing. This pre-treatment step is crucial for maintaining detection accuracy and reliability throughout the sequencing process.
Solution Approach 2:
The patent utilizes parameter changes by altering the chemical state of cytosine bases through conversion to thymine or uracil. This chemical transformation creates distinguishable signals during sequencing, allowing precise identification of modified cytosine positions. The parameter change from cytosine to thymine/uracil provides a clear signal for detection.
2Quantity of substance
If conventional sequencing methods are used, then sequence data can be obtained, but sample loss occurs and quality deteriorates
Solution Approach 1:
The conversion reagent treatment is performed as a preliminary action on the target polynucleotide before sequencing. This pre-treatment converts modified cytosines to thymine or uracil, creating stable chemical signatures that persist through sequencing. By performing this conversion before sequencing rather than during, the method prevents sample loss and maintains sequence quality throughout the analysis.
3Productivity
If rapid detection methods are implemented, then detection speed increases, but accuracy may compromise
Solution Approach 1:
The patent achieves rapid detection by performing the conversion of modified cytosines to thymine or uracil as a preliminary step before sequencing. This pre-treatment creates permanent chemical signatures that enable fast and accurate detection during sequencing. The conversion reagent treatment prepares the sample in advance, allowing rapid and accurate simultaneous detection of multiple modification types.
Solution Approach 2:
The chemical parameter change from modified cytosine to thymine or uracil creates distinct, easily detectable signals during sequencing. This parameter transformation enables rapid detection while maintaining high accuracy, as the converted bases produce unambiguous signals that can be quickly and reliably identified.
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 quick and accurate detection of modified cytosines with minimal sample loss, improving the resolution and reliability of epigenetic modification analysis.
Implementation Method 1
the conversion reagent is configured to convert a modified cytosine to thymine or a nucleobase which is read as thymine/uracil
Implementation Method 2
the enzyme comprises a cytidine deaminase
Implementation Method 3
the chemical agent comprises a boron-based reducing agent
Data Source
AI summary
The invention relates to methods of detecting modified cytosines in nucleic acid sequences.