Methyl-Binding Affinity Matrix for Microbiome DNA Enrichment
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Solution Overview
Problem
Current methods for enriching target DNA from environmental samples with high background contamination of mammalian genomic DNA are inefficient, often requiring viable cells and resulting in low recovery of target DNA, especially in scenarios where the DNA of interest is present in small amounts amidst a high background of host genomic material.
Innovation Solution
A composition comprising a mixture of methylated and unmethylated polynucleotides, an affinity matrix coated with methyl-binding proteins, and a buffer with salt and non-ionic detergent, which selectively binds methylated polynucleotides, allowing for the enrichment of unmethylated polynucleotides by separating them from the mixture, thereby reducing mammalian genomic DNA contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If selective lysis of human-nucleated cells using chaotropic reagent is used to isolate bacterial DNA, then human DNA contamination is reduced by 99.5%, but bacterial DNA recovery is low at only 30%
Solution Approach 1:
The patent changes the chemical parameters of the binding buffer by incorporating specific concentrations of salt (10mM-800mM) and non-ionic detergent, which modifies the binding characteristics of the affinity matrix to selectively bind methylated DNA while allowing unmethylated bacterial DNA to remain in solution, thereby achieving both high removal of human DNA and high recovery of bacterial DNA
2Quantity of substance
If soft inertial force-induced migration in microfluidic device is used to separate bacterial cells from human blood cells, then bacteria are enriched 300-fold, but the method only works if cells are viable
Solution Approach 1:
The patent replaces the mechanical separation system (soft inertial force-induced migration in microfluidic devices) with a chemical/biochemical system based on differential DNA methylation patterns. This substitution allows separation to occur at the DNA level rather than requiring intact viable cells, enabling analysis of non-viable or environmental samples
3Object-affected harmful factors
If salt-resistant DNase is used to degrade human DNA from lyzed cells, then human DNA is removed, but total bacterial DNA recovery is low at only 30%
Solution Approach 1:
The patent introduces an intermediary substance (affinity matrix with methyl-binding proteins) that selectively binds to methylated human DNA in the presence of salt and non-ionic detergent. This intermediary allows specific capture of human DNA without affecting bacterial DNA, enabling both effective human DNA removal and high bacterial DNA recovery through subsequent separation of the bound complex
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 achieves rapid and efficient enrichment of unmethylated polynucleotides, with up to 97% removal of mammalian genomic DNA and 90% recovery of bacterial DNA, enhancing the analysis of microbiome DNA by minimizing host DNA interference without requiring viable cells.
Implementation Method 1
a matrix coated with a protein or a polypeptide domain to form an affinity matrix that is capable of selectively binding the methylated polynucleotides containing methylated cytosines but not the unmethylated polynucleotides
Data Source
AI summary
Compositions and methods are provided for enriching non-target polynucleotides from a mixture of non-target and target polynucleotides where differences between the target polynucleotides and the non-target polynucleotides include the extent of modified bases that are present in a greater density in the target polynucleotides than in the non-target polynucleotides. This permits the target polynucleotides to be selectively and rapidly bound to an affinity matrix such as affinity protein-coated magnetic beads providing enrichment of the non-target polynucleotides in the supernatant. One use of this enrichment is to remove human genomic DNA from a mixture of DNAs obtained from human tissue samples to enrich for polynucleotides in a microbiome so as to characterize the microbiome by DNA sequencing. The modified bases are preferably methylated cytosines that can be bound to an affinity matrix comprising a methyl-binding domain (MBD).