Modified Cytosine Phasing on Long DNA Without Fragmentation
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Solution Overview
Problem
Conventional methods for phasing modified nucleotides, such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are limited by DNA fragmentation and sequence bias, making it impossible to determine whether these nucleotides are linked on the same DNA molecule over long distances and introducing errors in sequencing results.
Innovation Solution
The use of glucosylation and oxidation reactions, facilitated by enzymes like β-glucosyltransferase (BGT) and methylcytosine dioxygenase, to protect modified nucleotides from deamination, followed by cytidine deaminase treatment, allows for the differentiation between unmodified and modified cytosines without DNA fragmentation, enabling accurate phasing at a single molecule level.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite sequencing is used to analyze modified nucleotides, then single-nucleotide resolution information about methylation status is obtained, but DNA fragmentation occurs making it impossible to determine whether modified nucleotides are linked on the same DNA molecule
Solution Approach 1:
The patent changes the chemical parameters of the treatment process by using enzymes (TET dioxygenase for oxidation, β-glucosyltransferase for glucosylation, cytidine deaminase for deamination) instead of harsh chemical reagents (sodium bisulfite). This enzymatic approach maintains DNA integrity while achieving the desired nucleotide modification detection, resolving the contradiction between measurement precision and DNA stability.
2Measurement precision
If conventional bisulfite sequencing methods are used, then methylation status can be determined, but sequence bias toward cytosine adjacent to certain nucleotides introduces errors
Solution Approach 1:
The patent replaces the mechanical/chemical conversion process of bisulfite sequencing with an enzymatic cascade system. The enzymes (TET dioxygenase, β-glucosyltransferase, cytidine deaminase) provide sequence-independent recognition and modification of cytosine residues, eliminating the sequence bias inherent in bisulfite chemistry while maintaining reliable methylation status detection.
3Loss of information
If long-distance phasing of modified nucleotides is attempted using conventional methods, then information about linked nucleotides is needed, but DNA fragmentation prevents determination of linkage on the same molecule
Solution Approach 1:
The patent applies preliminary enzymatic treatments (oxidation by TET dioxygenase, glucosylation by β-glucosyltransferase) to protect modified nucleotides before deamination by cytidine deaminase. This preliminary action preserves the structural integrity of DNA molecules throughout the process, enabling long-distance phasing by maintaining continuous DNA templates that span multiple modified nucleotide sites.
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 provides accurate and efficient phasing of modified nucleotides, reducing sequencing errors and preserving long DNA molecules, thereby enhancing the resolution and reliability of epigenetic studies.
Implementation Method 1
treating an aliquot of a nucleic acid sample with methylcytosine dioxygenase and glucosyltransferase in the same reaction mix to produce a reaction product in which substantially all modified cytosines (Cs) are oxidized and 5hmCs are glucosylated
Implementation Method 2
treating an aliquot of a nucleic acid sample with methylcytosine dioxygenase and glucosyltransferase in the same reaction mix to produce a reaction product in which substantially all modified cytosines (Cs) are oxidized
Implementation Method 3
treating the reaction product with cytidine deaminase to produce a second reaction product in which substantially all unmodified Cs are converted to U
Data Source
AI summary
Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.