Genomic DNA Methylation Analysis via Bisulfite-Resistant Complementary Copies
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Solution Overview
Problem
Current methods for determining DNA methylation status are limited by the complexity reduction after bisulfite conversion, making it difficult to align converted fragments with the genome, especially with short sequences, and there is a need for methods that preserve the genomic context and facilitate identification of methylation patterns in multiplex formats.
Innovation Solution
The method involves generating complementary copies of DNA using bisulfite-resistant cytosine analogs, subjecting them to bisulfite treatment, and comparing the sequences of converted and non-converted strands to determine the methylation status, while preserving the complexity of the nucleic acid by keeping track of paired strands, allowing for the identification of methylation patterns in a multiplex format without the need for separating sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite conversion is used to determine DNA methylation status, then methylation detection capability is improved, but genomic complexity is reduced making alignment difficult
Solution Approach 1:
The patent generates complementary copies of the template nucleic acid using bisulfite-resistant cytosine analogs during complementary base pairing. These copies preserve the original genomic complexity and sequence information while the template undergoes bisulfite conversion. By comparing the converted template with the unconverted complementary copy, the method maintains alignment capability while enabling methylation detection.
Solution Approach 2:
The complementary copy acts as an intermediary that preserves genomic context. It serves as a reference that has not undergone bisulfite conversion, allowing researchers to align and compare sequences while the template provides the converted data needed for methylation status determination.
2Measurement precision
If bisulfite conversion is applied to DNA, then methylation status can be determined, but sequence alignment with genome becomes difficult especially with short sequences
Solution Approach 1:
A complementary copy is generated that retains the original sequence information without bisulfite conversion effects. This copy serves as an alignable reference that preserves genomic context, making it easier to map short converted sequences back to the reference genome by comparing against the unconverted complementary copy.
3Productivity
If traditional methylation analysis methods are used, then methylation patterns can be identified, but multiplex format analysis requires separating sequences which increases complexity
Solution Approach 1:
The patent combines the template nucleic acid and its complementary copy into a single nucleic acid pair that can be analyzed together in multiplex formats. By maintaining the physical association between converted and unconverted strands through tagging, multiple samples can be processed simultaneously without requiring separate handling or purification steps for each sequence.
Solution Approach 2:
The complementary copy serves as an internal reference that provides feedback for accurate methylation determination. By having both converted and unconverted sequences available in the same reaction, the system can self-correct and accurately identify methylation patterns without requiring complex external controls or separation procedures.
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 effectively preserves the complexity of the nucleic acid sequences, enabling accurate determination of methylation status and patterns, particularly in multiplex formats, by comparing the sequences of bisulfite-converted and non-converted strands, thus overcoming the limitations of reduced genomic complexity post-bisulfite conversion.
Implementation Method 1
subjecting the template nucleic acid and the complementary copy to conversion treatment to convert cytosine residues in the template nucleic acid into residues comprising the other base
Data Source
AI summary
The present invention relates to compositions, methods and systems for analyzing the methylation state of nucleic acids. Some embodiments relate to a compositions, methods and systems for analyzing the methylation state of DNA with a gene array.