Enzymatic Detection of 5hmC and 5mC Modifications
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Solution Overview
Problem
Current methods for analyzing DNA methylation patterns, such as sodium bisulfite sequencing, struggle to distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) and require high DNA amounts and sequencing depth, while long-read amplicon sequencing is limited to specific genomic regions, hindering the study of the whole methylome and hydroxymethylome.
Innovation Solution
A method involving enzymatic conversion and whole genome sequencing library preparation, where DNA is fragmented, protected, denatured, and deaminated to convert cytosines into uracils, allowing for the detection and discrimination of 5mC and 5hmC without the need for expensive oligos, enabling analysis of the whole genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sodium bisulfite sequencing is used to analyze DNA methylation, then methylation patterns can be detected, but the method cannot distinguish between 5mC and 5hmC and requires high DNA amounts and sequencing depth
Solution Approach 1:
The patent segments the detection process into separate enzymatic reactions: one reaction specifically converts 5mC to uracil while leaving 5hmC unchanged, and another reaction converts both 5mC and 5hmC to uracil. By comparing the results of these segmented reactions, the method can distinguish between the two modifications without requiring high DNA amounts, as each reaction processes the sample independently and efficiently.
Solution Approach 2:
The patent introduces enzymatic intermediaries (specifically uracil DNA glycosylase and APOBEC1) that mediate the conversion of methylated cytosines to uracils. These enzymes act as selective intermediaries that can differentiate between 5mC and 5hmC based on their chemical properties, enabling precise detection without direct sequencing of the modified bases.
2Measurement precision
If long-read amplicon sequencing is used to detect 5mC and 5hmC, then single-base resolution can be achieved, but the method is restricted to analysis of a few genomic regions
Solution Approach 1:
The patent creates a universal enzymatic conversion system that can process the entire genome. The enzymes (uracil DNA glycosylase and APOBEC1) act throughout the whole genome simultaneously, converting all 5mC and 5hmC sites to uracils regardless of their location. This universal approach allows subsequent sequencing to cover the entire methylome and hydroxymethylome at single-base resolution, eliminating the need for region-specific amplicon sequencing.
3Ease of manufacture
If conventional library preparation methods are used, then sequencing libraries can be prepared, but expensive DNA oligos are required
Solution Approach 1:
The patent employs inexpensive, readily available enzymes (uracil DNA glycosylase and APOBEC1) as disposable reagents that can be easily replaced. These enzymes perform the critical function of converting methylated cytosines to uracils without requiring expensive synthetic oligos. The enzymatic reactions can be performed with standard laboratory reagents, significantly reducing the cost of library preparation while maintaining high detection accuracy.
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 method effectively detects and discriminates 5mC and 5hmC across the whole genome, reducing costs and improving analysis efficiency, particularly suitable for low-input samples like circulating cell-free DNA and single-cell analysis.
Implementation Method 1
performing enzymatic deamination of the ssNA product obtained in step d1) to convert cytosines (C) and methylated cytosines (5mC) into uracils (U)
Implementation Method 2
denaturing the product of step b) to obtain a modified single stranded nucleic acid sample (ssNA)
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3C
AI summary
The present invention discloses a method to detect 5-hydroxymethylcytosine (5hmC) and discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) from a nucleic acid that comprises enzymatic treatment and construction of nucleic acid library and a kit to perform said method.