Methylation Pattern Retention in Nucleic Acid Amplification
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Solution Overview
Problem
Existing sequencing technologies fail to retain the complexity of methylation patterns, which is crucial for accurately determining the origin of cancer at an early stage.
Innovation Solution
The method involves generating an immobilized methylated complement template polynucleotide by hybridizing a methylated template polynucleotide with a primer attached to a solid support, extending the primer with a polymerase, and then contacting the resulting non-methylated complement template with a DNA methyltransferase reagent to introduce methylation patterns.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing sequencing technologies are used to analyze nucleic acids, then sequencing can be performed, but the complexity of methylation patterns is lost
Solution Approach 1:
The patent applies preliminary action by performing methylation preservation steps before sequencing. Specifically, the method involves hybridizing methylated nucleic acid to a template, extending with polymerase to create a complement strand that preserves methylation information, and then using this preserved template for subsequent sequencing operations. This preliminary preparation ensures methylation patterns are maintained before the actual sequencing measurement occurs.
Solution Approach 2:
The patent uses an intermediary approach by introducing a complement template polynucleotide as a mediator between the original methylated nucleic acid and the sequencing process. The complement template serves as an intermediate carrier that preserves methylation information (through methylated cytosine residues) while being suitable for sequencing, thus transferring the methylation complexity information through the sequencing process without loss.
2Productivity
If standard nucleic acid amplification is performed, then amplification efficiency is high, but methylation status is not retained in amplification products
Solution Approach 1:
The method performs preliminary methylation preservation by creating a complement template strand that is extended from a methylated template using polymerase. This complement strand is then used as the template for amplification, ensuring that the methylation information is preserved in the amplification products while maintaining high amplification efficiency through standard polymerase-based methods.
Solution Approach 2:
The patent uses copying by creating a complement template polynucleotide that copies the methylation pattern from the original template. The polymerase extends the primer using the methylated template, incorporating nucleotides that preserve the methylation information. This copied template with retained methylation status then serves as the basis for further amplification, ensuring methylation information is propagated through the amplification process.
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 maintains the methylation status and patterns of the template nucleic acid within the amplification products, improving sequencing accuracy by distinguishing between methylated and non-methylated cytosine nucleobases.
Implementation Method 1
extending the first immobilized primer with a polymerase to generate an immobilized non-methylated complement template polynucleotide hybridized to the methylated template polynucleotide
Implementation Method 2
contacting the immobilized non-methylated complement template polynucleotide with a DNA methyltransferase reagent to generate an immobilized methylated complement template polynucleotide
Implementation Method 3
hybridizing a methylated template polynucleotide to a first immobilized primer at a first temperature
Data Source
AI summary
Disclosed herein, inter alia, are compositions, methods, and kits useful for detecting nucleobase modifications on one or both strands of a double-stranded nucleic acid fragment.