Phylogenetic-based differential cell separation and DNA extraction
Inventors
Marciano, Michael • Dunegan, Molly
Assignees
Publication Number
US-12344834-B2
Publication Date
2025-07-01
Expiration Date
2040-11-02
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Abstract
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.
Core Innovation
The invention provides a method to differentially isolate eukaryotic (animal and plant) DNA from bacterial DNA prior to sequencing by exploiting differences in cellular size, cell wall composition, and enzymatic sensitivities of each organism type. This method combines size exclusion filtration and differential enzymatic lysis to separate the components of a mixed biological sample into distinct organismal fractions before DNA extraction and sequencing. By using specific pore-sized filters and targeted lysis buffers, the method retains the integrity of some cells while lysing others, allowing for a stepwise extraction of DNA from bacteria, plants, and animals with minimal cross-contamination.
The problem addressed by the invention arises from the challenge of analyzing DNA from mixed biological samples containing bacterial, plant, and animal cells. In typical sequencing, especially with shotgun next-generation sequencing, the abundance and smaller genome size of microbial DNA can dominate sequencing results, obscuring the target organism's DNA and increasing interpretability difficulties. Prior art methods do not effectively separate these mixed kingdoms, causing complications in fields like forensic analysis, medical microbiome studies, and environmental DNA research. There is a need for a simple, cost-effective process that enhances the accuracy and interpretability of DNA sequencing results by reducing complexity through prior physical and chemical separation of sample components.
The method involves initially forming a suspension of bacterial, plant, and animal cells in phosphate-buffered saline, followed by sequential filtration through multiple wetted filters of approximately 5 μm pore size to separate cell types based on size. The residues and filtrates from filters are washed and centrifuged to form cell pellets enriched in specific cell types. These pellets are then subjected to differential lysis using buffers tailored to the cell wall properties of the target organisms, such as lysozyme-containing buffers for bacteria and SDS detergent for animal cells, with subsequent incubation steps to ensure selective lysis. A final filtration step post-lysis further purifies the desired cell fractions. This systematic approach allows separation of DNA from different phylogenetic kingdoms, thereby facilitating downstream DNA sequencing with reduced complexity.
Claims Coverage
The claims include one independent claim describing a multi-step method for differentially processing a mixture of bacterial, plant, and animal cells prior to sequencing. The inventive features relate to the specific sequential filtration, washing, centrifugation, suspension in tailored lysis solutions, and further filtration steps to effect the separation and isolation of distinct cell types from a mixed sample.
Sequential size exclusion filtration with wetted filters
The method uses first and second wetted filters to filter a mixed solution of bacterial, plant, and animal cells prior to any lysing step, thus separating the mixture into residues and filtrates based on size.
Washing filter residues into a collection tube
The residues retained on the first and second wetted filters are washed with defined amounts of phosphate-buffered saline into a first collection tube to recover retained cells for further processing.
Centrifugation to separate distinct cell pellets
Centrifuging the first and second wetted filters along with their respective residues in a second centrifuge tube to form a first pellet containing a first cell type, and separately centrifuging the first centrifuge tube to form a second pellet containing a second, different cell type.
Differential suspension of cell pellets in specific lysis solutions
Forming suspensions of the first and second pellets in first and second lysis solutions respectively, where the lysis solutions differ to target selective cell lysis according to cell type.
Third filtration and washing after lysis
Filtering the first suspension through a third wetted filter to separate a third residue from a filtrate, with washing of the third residue into a second collection tube, wherein the third residue contains a third distinct cell type different from the first two.
The claims encompass a stepwise physical and chemical separation method of a mixed biological sample using sequential filtrations, washings, centrifugations, and differential lysis suspensions, culminating in filtrations post-lysis to isolate distinct cell types from bacterial, plant, and animal sources prior to DNA sequencing.
Stated Advantages
Provides higher information content from biological samples by enabling identification of microbial, plant, and animal DNA signatures separately.
Improves human microbiome characterization by effectively removing non-target cells and DNAs from bacterial fractions.
Allows deconvolution of complex samples with multiple organisms, enhancing individual organism characterization in metagenomics and environmental DNA analyses.
Offers a cost-effective, simple, and time-efficient method that maintains cell viability and DNA integrity for downstream applications.
Facilitates more accurate and interpretable sequencing outcomes by reducing sample complexity before sequencing.
Documented Applications
Forensic analyses to separate human DNA from microbial and plant DNA in crime scene samples for improved target DNA identification.
Medical research involving human microbiome studies where separation of human and bacterial cells improves the analysis of bacterial populations.
Metagenomic and environmental DNA analyses to differentiate DNA contributions from bacteria, plants, and animals in mixed environmental samples.
General scientific research requiring differential extraction and sequencing of DNA from multiple phylogenetic kingdoms to improve interpretability and accuracy.
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