CpG/TpG Primer Composition for Low-Level DNA Methylation Detection

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Solution Overview

Problem

Conventional methods for determining DNA methylation, such as qMSP, MethyLight, and MS-HRM, suffer from limitations including biased amplification, high type 1 and type 2 errors, and difficulty in measuring low methylation ratios, especially in samples like blood, due to non-specific binding and probe interference.

Innovation Solution

A method using a primer set comprising a methylation primer with a CpG recognition site and an unmethylation primer with a TpG recognition site, adjusted for binding opportunities through temperature and concentration, allowing for sensitive methylation detection without probes, even in trace amounts of target nucleic acids.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If qMSP or MethyLight methods are used to determine methylation level, then sensitivity is improved, but specificity deteriorates due to biased amplification and non-specific amplification

Engineering Contradiction:

ImprovesensitivityVSAvoidspecificity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent divides the primer into two distinct segments: a methylation-specific primer that binds only to methylated DNA sequences and an unmethylation-specific primer that binds only to unmethylated DNA sequences. This segmentation allows separate amplification of methylated and unmethylated forms, eliminating cross-interference and improving both sensitivity and specificity simultaneously

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces a probe as an intermediary element that specifically binds to the amplified DNA sequence. The probe contains a fluorescent reporter and quencher that enable detection through FRET mechanism, serving as a mediator between the amplified DNA and the detection system, thereby enhancing specificity without compromising sensitivity

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If MethyLight method adds a fluorescent probe to increase specificity, then specificity is improved, but device complexity and cost increase

Engineering Contradiction:

ImprovespecificityVSAvoidanalysis test design difficulty

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent designs a universal probe structure that can be applied across different methylation analysis targets. The probe contains universal fluorescent reporter and quencher components that work with various target sequences, making the system multi-functional and reducing the need for custom design for each application, thereby simplifying overall system complexity

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The patent optimizes probe parameters including fluorescent dye selection, quencher type, and probe length to achieve optimal binding characteristics. By carefully adjusting these parameters, the probe maintains high specificity while ensuring efficient amplification and detection, avoiding the need for complex additional components

Inventive Principle:

Principle #35Parameter changes

3Productivity

If MS-HRM method uses primers without CpG in binding region to avoid biased amplification, then amplification efficiency is improved, but measurement precision deteriorates due to unmethylation bias

Engineering Contradiction:

Improveamplification efficiencyVSAvoidmethylation ratio accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent segments the primer design into two specialized primers: one optimized for binding methylated sequences and another for unmethylated sequences. Each primer is designed with appropriate CpG content for its specific target, eliminating the need to compromise amplification efficiency while maintaining accurate methylation ratio measurement

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent applies local quality optimization by designing primers with CpG sequences specifically in the regions where they are needed for methylated DNA binding, while keeping other regions optimized for general amplification. This localized optimization allows each primer to perform its specific function effectively without interfering with the other

Inventive Principle:

Principle #3Local quality

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

The method achieves high sensitivity and specificity in detecting methylation levels, enabling accurate diagnosis of cancer through semi-quantification of DNA methylation in biological samples, particularly in low concentrations of methylated DNA.

Implementation Method 1

a methylation primer which binds to a CpG site of the methylated nucleic acid

Methodology Applied

Scientific EffectCpG recognition:

Implementation Method 2

an unmethylation primer which binds to a TpG site of the unmethylated nucleic acid

Methodology Applied

Scientific EffectTpG recognition:

Implementation Method 3

amplifying a target site by treating the biological sample with the composition

Methodology Applied

Scientific EffectPCR amplification:

Data Source

PatentUS20250270639A1Composition for determining whether nucleic acid is methylated and method for determining whether nucleic acid is methylated

Publication Date: 2025.08.28 LEPIDYNE CO LTD

AI summary

The present application relates a composition for determining whether a nucleic acid is methylated and a method for determining whether a nucleic acid is methylated.