Preparation of templates for methylation analysis
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Solution Overview
Problem
Existing methods for genome-wide methylation analysis require prior knowledge of template sequences for primer design, are costly, and introduce artifacts, limiting their applicability to complex mixtures of templates.
Innovation Solution
A method using methylated universal adaptors to ligate with fragmented nucleic acids, followed by bisulfite treatment to convert unmethylated cytosines to uracil, allowing methylation status analysis without prior sequence knowledge, and enabling solid-phase amplification and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If prior methods for methylation analysis are used, then sequence-specific information can be obtained, but prior knowledge of template sequences is required for primer design
Solution Approach 1:
The patent employs universal primers that can bind to common sequences flanking the region of interest, allowing the same primer set to amplify multiple different target sequences. This universal approach enables methylation analysis of unknown sequences while maintaining sequence-specific detection capability through the bisulfite conversion process.
Solution Approach 2:
The patent introduces a bisulfite treatment step as an intermediary process that converts unmethylated cytosines to uracils while leaving methylated cytosines unchanged. This chemical transformation serves as a mediator that preserves methylation information without requiring prior sequence knowledge for primer design, as the universal primers bind to sequences that remain unchanged after bisulfite treatment.
2Productivity
If high throughput methylation analysis is implemented, then productivity increases, but cost increases
Solution Approach 1:
The patent combines multiple functions into a single workflow: bisulfite treatment for methylation detection, universal primer amplification for sequence enrichment, and solid-phase sequencing for high-throughput analysis. This integrated approach achieves high throughput methylation profiling while reducing per-sample costs by eliminating the need for separate experiments for different targets.
3Adaptability or versatility
If conventional amplification methods are used, then unknown sequences can be amplified, but artifacts are introduced
Solution Approach 1:
The patent performs bisulfite treatment before amplification, converting unmethylated cytosines to uracils in advance. This preliminary action preserves the methylation status of the original DNA and prevents artifacts that would otherwise be introduced during subsequent PCR amplification, as the template for amplification already contains the methylation information in a stable form.
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
Facilitates genome-wide methylation analysis by preserving methylation status and allowing amplification of unknown sequences, reducing costs and artifacts, and enabling high-throughput methylation profiling.
Implementation Method 1
treating the adaptor-ligated double stranded nucleic acid target fragments with a reagent that converts the non-methylated cytosine bases to uracil
Implementation Method 2
ligating universal adaptors to the fragmented double stranded nucleic acid target fragments to produce adaptor-ligated double stranded nucleic acid target fragments
Data Source
AI summary
The invention relates to a method of preparing and using a library of template polynucleotides suitable for use as templates in solid-phase nucleic acid amplification and sequencing reactions to determine the methylation status of the cytosine bases in the library. In particular, the invention relates to a method of preparing and analysing a library of template polynucleotides suitable for methylation analysis.