DNA Methylation Detection via Enzymatic Ligation and Restriction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting DNA methylation, such as methylation-sensitive restriction endonuclease assays and sodium bisulfite-based approaches, face challenges like false positives, false negatives, and insensitivity, especially when detecting low levels of methylated DNA in the presence of a majority of unmethylated DNA, which is crucial for early cancer detection.
Innovation Solution
A method involving methylation-sensitive restriction enzyme digestion, oligonucleotide probe sets, and ligation reactions to specifically identify target nucleic acid molecules with methylated residues, using 5' nuclease activity and thermostable ligases to form ligation products that are then distinguished based on methylation status.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If methylation-sensitive restriction endonuclease assays are used to detect methylated DNA, then the method can identify methylated sequences, but false positives occur due to incomplete digestion of unmethylated DNA
Solution Approach 1:
The patent combines restriction enzyme digestion with oligonucleotide ligation reactions to create a multi-step detection system. The restriction enzyme cleaves unmethylated DNA at recognition sites, and subsequent ligation of oligonucleotide probes to the cleaved fragments provides confirmatory evidence of methylation status, reducing false positives from incomplete digestion
Solution Approach 2:
The patent performs restriction enzyme digestion as a preliminary step before ligation and amplification. By pre-cleaving unmethylated DNA fragments, the method prepares the sample in advance to enable selective ligation of probes only to methylated templates, improving detection accuracy
2Measurement precision
If sodium bisulfite conversion is used to detect DNA methylation, then methylation status can be determined through sequence analysis, but the method produces false negatives and has reduced sensitivity
Solution Approach 1:
The patent uses oligonucleotide probes as intermediaries that hybridize to methylated DNA sequences after restriction digestion. These probes serve as mediators between the methylated template and the detection system, providing specific and sensitive detection without the artifacts of bisulfite conversion
Solution Approach 2:
The patent replaces the chemical conversion mechanism of bisulfite treatment with an enzymatic approach using restriction enzymes and ligases. This substitution maintains detection capability while avoiding the DNA degradation and false negatives associated with harsh chemical conversion
3Ease of operation
If single marker detection is used for early cancer detection, then the method is simple to implement, but false-positive and false-negative results are unacceptably high
Solution Approach 1:
The patent divides the detection system into multiple independent components: multiple oligonucleotide probes targeting different methylation markers, each capable of being detected separately. This segmentation allows simultaneous monitoring of multiple cancer-related genes (e.g., CDKN2A, RASSF1A, APC) while maintaining operational simplicity through a unified assay format
Solution Approach 2:
The patent creates a universal detection platform using common oligonucleotide probes and restriction enzymes that can detect multiple different methylation markers. The same basic assay components serve multiple functions across different cancer detection applications, maintaining simplicity while improving diagnostic reliability through multiplexing
4Measurement precision
If detection methods are designed to detect very low levels of methylated DNA in predominantly unmethylated samples, then early cancer detection sensitivity improves, but the complexity of the detection system increases
Solution Approach 1:
The patent performs restriction enzyme digestion as a preliminary enrichment step that selectively cleaves unmethylated DNA fragments before the ligation and amplification steps. This pre-processing action removes the overwhelming background of unmethylated DNA, enabling sensitive detection of low-abundance methylated targets without requiring overly complex assay designs
Solution Approach 2:
The patent uses oligonucleotide probes that copy or replicate the methylation-specific sequence information through ligation and subsequent amplification. This copying mechanism amplifies the signal from rare methylated molecules, enhancing detection sensitivity while using standard, relatively simple molecular biology techniques
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 enhances sensitivity and specificity for detecting low-abundance methylated DNA, reducing false positives and negatives, and allows for accurate identification of methylation status even in samples with predominantly unmethylated DNA.
Implementation Method 1
The sample is subjected to a methylation sensitive restriction enzyme digest to cleave target nucleic acid molecules in the sample having non-methylated residues within the at least one methylation sensitive restriction enzyme sequence
Implementation Method 2
contacting the sample and the one or more oligonucleotide probe sets under conditions effective for first and second oligonucleotide probes of a probe set to hybridize at adjacent positions in a base specific manner to their corresponding target nucleic acid molecule
Implementation Method 3
The overlapping identical nucleotide of the second oligonucleotide probe is cleaved with an enzyme having 5' nuclease activity, thereby liberating a 5' phosphate on the second oligonucleotide probe
Implementation Method 4
The first and second oligonucleotide probes of the one or more oligonucleotide probe sets are ligated together at the junction to form a ligation product hybridized to its complementary target nucleic acid molecule
Data Source
Figure 1(a)~1(d)
Figure 2A~2I
Figure 3A~3I
AI summary
The present invention is directed to methods for identifying the presence of one or more methylated or unmethylated target nucleotide sequences in a sample that involve coupled methylation sensitive restriction enzyme digestion-ligation and/or extension processes. In some embodiments, the ligation and primary extension products formed in the reaction processes of the present invention are subsequently amplified using a polymerase chain reaction. The ligation products or primary extension products are detected, and the presence of one or more methylated or unmethylated target nucleotide sequences in the sample is identified based on the detection.