Methyl-Binding Peptide Matrix for Mitochondrial DNA Enrichment
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Solution Overview
Problem
Current methods for enriching target DNA from environmental samples are inadequate, particularly when the target DNA is present in small amounts amidst a high background of host genomic material, and often require viable cells, which is not always feasible.
Innovation Solution
A composition and method using a matrix coated with methyl-binding domain peptides to selectively bind chromosomal DNA, allowing mitochondrial, chloroplast, or prokaryotic DNA to be enriched in a buffer, thereby separating and concentrating these DNA types from eukaryotic cell DNA, with the aid of a buffer containing salt and a non-ionic detergent.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If selective lysis of human-nucleated cells using chaotropic reagent is used, then human DNA contamination is reduced by 99.5%, but total bacterial DNA recovery is low at only 30%
Solution Approach 1:
The patent extracts and removes human chromosomal DNA from the sample using methyl-binding domain peptides that specifically bind to methylated cytosine residues in human DNA. This selective extraction eliminates the need for cell lysis and DNase treatment, thereby preserving bacterial DNA integrity and achieving both low human DNA contamination (99.5% reduction) and high bacterial DNA recovery (greater than 90%).
Solution Approach 2:
The patent introduces methyl-binding domain peptides as an intermediary substance that selectively binds to human DNA through recognition of methylated cytosine residues. This intermediary mediator enables specific separation of human DNA from bacterial DNA without directly contacting or damaging bacterial cells, thus achieving high purity enrichment while maintaining bacterial DNA recovery above 90%.
2Quantity of substance
If physical separation of bacterial cells from human blood cells based on soft inertial force-induced migration is used, then bacteria are enriched 300-fold, but this method can only reduce background contamination if the cells are viable
Solution Approach 1:
The patent replaces the mechanical soft inertial force-induced migration system with a chemical-biological recognition system using methyl-binding domain peptides. This substitution eliminates the requirement for cell viability and mechanical separation apparatus, enabling DNA-level separation that works regardless of cell integrity while achieving greater than 90% bacterial DNA recovery.
Solution Approach 2:
The patent changes the separation parameter from mechanical physical properties (density, size) to biochemical properties (methylation status of DNA). By targeting the epigenetic mark of methylated cytosine in human DNA, the method achieves specific separation without relying on cell viability or mechanical forces, thereby enabling enrichment of bacterial DNA from samples containing degraded or non-viable cells.
3Quantity of substance
If no enrichment method is applied, then target DNA remains in small amounts amidst high background of host genomic material, but applying conventional enrichment methods causes loss of target DNA
Solution Approach 1:
The patent converts the previously harmful effect of methylated cytosine residues (which are abundant in human DNA and caused non-specific binding in earlier methods) into a beneficial selective recognition feature. By designing methyl-binding domain peptides that specifically recognize methylated cytosine, the method transforms the chemical characteristic that caused problems into the basis for highly specific separation, achieving greater than 90% bacterial DNA recovery while reducing human DNA to less than 0.5%.
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 achieves significant enrichment of mitochondrial, chloroplast, or bacterial DNA, reducing genomic DNA contamination by up to 99.5% and facilitating sequencing while maintaining high recovery rates of target DNA, without the need for viable cells.
Implementation Method 1
a matrix coated with methyl binding domain peptide for selectively binding chromosomal DNA
Data Source
AI summary
Compositions and methods are provided for enriching mitochondrial DNA and optionally chloroplast DNA from eukaryotic cells in a simple rapid method that provides greater than 100 fold enrichment. Affinity protein-coated substrate in a buffer is used to efficiently bind chromosomal DNA and thereby remove it from the buffer. Mitochondrial sequencing reads reveal that non-biased sequence selection providing representation of a substantial proportion of mitochondrial DNA in the eukaryotic cells analyzed.