Enzymatic DNA Cleavage for Methylated Nucleotide Mapping
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Solution Overview
Problem
Current methods for identifying methylated DNA in mammalian genomes are cumbersome and experimentally difficult to implement reproducibly, particularly due to the complexity of using restriction enzymes like HpaII and MspI, and the bisulfite modification method involves complicated chemical modifications and primer optimization.
Innovation Solution
Development of a set of double-stranded oligonucleotide fragments enzymatically cleaved from large DNA containing modified nucleotides, specifically methylated or hydroxymethylated cytosines, using enzymes that recognize these modifications and cleave at non-random distances, generating fragments of similar or varying sizes depending on DNA modification status.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If restriction enzymes like HpaII and MspI are used to identify methylated DNA, then methylation detection capability is improved, but experimental complexity and difficulty increase
Solution Approach 1:
The patent extracts the methylation detection function from complex enzymatic assays and chemical modification protocols, isolating it into a simple PCR amplification step. By designing primers that specifically amplify only methylated DNA sequences, the method extracts the essential detection capability while eliminating the complexity of enzyme sensitivity differences and chemical modification optimization.
Solution Approach 2:
The patent replaces the mechanical/enzymatic system of restriction enzyme digestion with a biochemical amplification system based on PCR. Instead of relying on enzyme-catalyzed cleavage patterns, the method uses DNA polymerase to amplify methylated sequences, substituting a more robust and easily controllable biochemical process that can be standardized and automated.
2Measurement precision
If bisulfite modification method is used for methylated DNA identification, then detection capability is improved, but procedural complexity increases
Solution Approach 1:
The patent extracts the methylation detection function from the multi-step bisulfite modification protocol, isolating it into a single PCR amplification step. By designing primers that specifically amplify only methylated DNA sequences, the method extracts the essential detection capability while eliminating the complexity of chemical modification and primer optimization.
Solution Approach 2:
The patent replaces the chemical modification system (bisulfite treatment) with a biochemical amplification system (PCR). This substitution eliminates the need for harsh chemical treatments and complex optimization, using instead a robust enzymatic amplification process that is easier to standardize and reproduce.
3Measurement precision
If complex chemical modification and amplification steps are used, then methylation detection accuracy is improved, but time consumption increases
Solution Approach 1:
The patent extracts the methylation detection function from lengthy chemical modification and amplification protocols, condensing it into a single PCR amplification step. By using primers that specifically target methylated sequences, the method achieves accurate detection while dramatically reducing the time required, eliminating multiple processing steps.
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
Enables efficient and reliable identification and mapping of modified nucleotides in DNA, simplifying the generation of a methylome and facilitating high-throughput sequencing for determining phenotypic properties and disease susceptibility.
Implementation Method 1
one or more enzymes that recognize a modified nucleotide in a DNA, wherein the modification is 5-methylation or 5-hydroxymethylation
Implementation Method 2
each enzyme is capable of cleaving the DNA at a site that is a non-random distance from the modified nucleotide
Data Source
Figure 1A~1B
Figure 1C
Figure 1D
AI summary
Compositions, methods and related uses are provided relating to cleaving modified DNA. For example, a set of DNA fragments obtainable by enzymatic cleavage of a large DNA is described where at least 50% are similarly sized and have a centrally positioned modified nucleotide. In addition, an enzyme preparation is provided that includes one or more enzymes that recognize a modified nucleotide in a DNA and cleave the DNA at a site that is at a non-random distance from the modified nucleotide. The one or more enzymes are further characterized by an N- terminal conserved domain with greater than 90% amino acid sequence homology to WXD(X)10YXGD. The related uses include creating a methylome, methods of purifying DNA fragments containing a modified nucleotide and diagnostic applications.