Phylogenetic-Based Cell Separation for Cleaner Eukaryotic DNA Sequencing
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Solution Overview
Problem
Current methods struggle to effectively separate and isolate eukaryotic (Animalia) DNA from plant and bacterial DNA in mixed biological samples, leading to complications in downstream analyses such as forensic and medical research.
Innovation Solution
A method utilizing size exclusion-based separation and differential enzymatic treatments, combined with an optional oligonucleotide 'pull-down' method, to differentially isolate eukaryotic DNA from plant and bacterial DNA, maintaining cell viability for downstream applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional DNA extraction methods are used on mixed biological samples, then all DNA (target and non-target) is extracted together, but the complexity of downstream analyses increases and target DNA interpretation becomes difficult
Solution Approach 1:
The patent segments the mixed biological sample into separate fractions based on cell size and type using differential centrifugation and filtration. Eukaryotic cells (animal and plant) are separated from prokaryotic cells (bacteria), and further separated into nuclear and cytoplasmic fractions. This segmentation allows targeted extraction of specific DNA types while maintaining total DNA yield from all sources.
Solution Approach 2:
The patent selectively extracts DNA from specific cell fractions rather than extracting all DNA simultaneously. By taking out eukaryotic nuclear DNA from the mixed sample, the method enables focused analysis on target organisms while preserving non-target DNA for separate analysis, thereby reducing downstream analysis complexity.
2Loss of information
If non-target organism DNA is present in the sample, then valuable information about the sample can be captured, but the reliability of target DNA analysis decreases due to over representation of non-target DNAs
Solution Approach 1:
The patent segments DNA extraction by cell type and compartment, separating eukaryotic nuclear DNA from prokaryotic DNA and from cytoplasmic DNA. This segmentation ensures that target eukaryotic nuclear DNA is extracted with high reliability while non-target prokaryotic DNA is separated into distinct fractions, preventing over representation in target analysis.
Solution Approach 2:
The patent uses cell size differences and differential centrifugation as intermediary mechanisms to separate DNA sources. The centrifugation process acts as an intermediary that exploits physical differences between cell types to achieve selective DNA extraction, maintaining both target DNA reliability and sample information capture.
3Manufacturing precision
If differential cell separation methods are implemented, then target DNA can be isolated with high purity, but the time and complexity of the separation process increases
Solution Approach 1:
The patent performs preliminary cell separation by size and type before DNA extraction. By pre-separating eukaryotic and prokaryotic cells through differential centrifugation and filtration, the method enables rapid subsequent DNA extraction from purified fractions, reducing the overall time required compared to extracting and then separating DNA.
Solution Approach 2:
The patent replaces complex mechanical separation systems with simpler differential centrifugation and filtration approaches. By using centrifugal force and filter membranes based on cell size differences, the method achieves high DNA isolation purity without requiring complex mechanical separation equipment or prolonged processing times.
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 enhances the interpretability of DNA samples by reducing complexity in sequencing applications, allowing for the accurate identification and characterization of microbial, plant, and animal DNA components.
Implementation Method 1
filtering the mixed plant, animal and bacterial solution a first time using a first wetted filter into a first centrifuge tube to separate a first residue from a first filtrate
Implementation Method 2
centrifuging the first wetted filter, the second wetted filter, the first residue, and the second residue in a second centrifuge tube to form a first pellet
Implementation Method 3
forming a first suspension of the first pellet in a first lysis solution, forming a second suspension of the second pellet in a second lysis solution
Data Source
AI summary
An approach for differentially isolating eukaryotic (plant and animal) DNA from bacterial DNA prior to sequencing using a combination of size exclusion-based separation and differential cell lysis. The method of the present invention exploits the differences of the cellular size and components of each type of organism to be separated. The composition and nature of the cell wall of plant cells, enzymatic sensitivity of bacterial and animal cells and overall size difference of bacterial and plant/animal cells allows one portion of a mixed sample to be lysed while retaining the integrity of the remaining organisms. Separation of one phylogenetic component then permits the remaining components to be extracted with minimal contribution from the preceding component. The separation of DNAs from differing contributing kingdoms in an unknown sample increases interpretability through decreasing complexity in subsequent sequencing applications.