Methods for rapid separation and purification of DNA topological forms
Inventors
Shoura, Massa • Levene, Stephen • Girata, David
Assignees
University of Texas System • Leland Stanford Junior University
Publication Number
US-12351796-B2
Publication Date
2025-07-08
Expiration Date
2041-07-21
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Abstract
Methods are provided for the isolation and analysis of circular DNA from complex samples, based on the topology of the DNA molecule. A sample comprising DNA species is combined with a chaotropic dense salt solution. A fraction containing the circular DNA of interest is isolated and dialyzed to remove excess salt. In some embodiments salt gradients are generated by ultracentrifugation in the absence of intercalating dyes, e.g. ethidium bromide; and in the absence of protease digestion. The circular DNA thus isolated is substantially pure, e.g. greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95% of DNA in the isolated fraction is comprised of circular DNA.
Core Innovation
The invention provides methods for the isolation and analysis of circular DNA from complex samples based on the topology of the DNA molecule. A sample comprising DNA species is combined with a chaotropic dense salt solution. Ultracentrifugation is used to generate a gradient wherein different DNA topologies are stratified, allowing isolation of a fraction containing circular DNA of interest. Excess salt is subsequently removed by dialysis, and the circular DNA thus isolated is substantially pure, often more than 90% purity.
This invention addresses the technical challenge of efficiently purifying large circular DNA, such as extrachromosomal circular DNA (eccDNA, microDNA, double minutes, or ecDNA), which constitutes a tiny mass fraction within organisms’ DNA. Purification has been difficult due to the high likelihood of DNA breakage and degradation. The methods herein proceed without intercalating dyes like ethidium bromide and without protease digestion, preserving DNA topology and enabling isolation from various sample types including fixed chromatin, naked DNA, bodily fluids, tissues, and whole organisms.
Claims Coverage
The patent contains one independent claim focusing on a method for rapid and topology-dependent purification of circular DNA. The claim outlines several inventive features related to the use of chaotropic dense salt solutions, stratification by ultracentrifugation, and isolation of purified circular DNA fractions.
Topology-dependent DNA purification method
A method combining a sample containing DNA species with a chaotropic dense salt solution absent intercalating dye and protein-dependent digests, stratifying circular DNA topologies by ultracentrifugation, isolating fractions of specific topological states, and removing excess salt to yield purified circular DNA.
Characterization of purified circular DNA
Subsequent characterization of purified circular DNA using methods such as gel electrophoresis, capillary electrophoresis, single-molecule electrophoretic analysis, chromatography, high-resolution imaging, and next-generation sequencing (NGS).
Stratification into distinct DNA forms
Ultracentrifugation stratifies DNA into closed circular DNA, nicked circular DNA, and linear DNA for selective isolation based on topology.
Use with low DNA input amounts
Applicable to DNA samples comprising less than 10 ng of DNA, allowing processing of small quantities of DNA species.
Compatibility with extrachromosomal DNA types
Applicable to eukaryotic extrachromosomal circular DNA types, including eccDNA, double-minute elements, ecDNA, and microDNA, present as circular DNA.
Embedding in polymer matrices
Embedding the sample in a polymer matrix prior to combination with the chaotropic dense salt solution, using materials such as agarose, gelatin, polysaccharides, alginate, and pluronic polymers.
High purity of isolated circular DNA
Isolated DNA fractions achieve greater than about 90% purity for the circular DNA of specific topological states.
Maintenance of DNA topology and size
The method maintains the topology and size of the DNA during purification.
Process conditions at ambient temperature
Combination of sample and chaotropic salt solution performed at room temperature without intercalating dyes or protease digestion.
Applicable sample types
Samples include nuclear DNA, circulating DNA, cells, tissues (hard and soft), whole organisms, and bodily fluids containing these samples.
Preservation of chromatin and epigenetic marks
The method supports sample preparation with fixed chromatin, maintenance of chromatin structure and chromatin marks post purification, including immunoprecipitation or affinity isolation for epigenetic analysis.
Preservation of DNA methylation and histone modifications
Methylated DNA, histone modifications, and associated methylation and modification patterns are preserved during purification and can be assayed.
Use of defined chaotropic salts
Chaotropic dense salt solutions employed include Rb trichloroacetic acid (TCA), CsTCA, BaTCA, Rb thiocyanate (SCN), CsSCN, or combinations thereof, for generating the salt gradient.
The claim covers a comprehensive topology-dependent purification method that integrates chaotropic salt-mediated stratification without intercalating dyes or proteolytic digestion, supports diverse sample types and DNA forms, achieves high purity, preserves DNA and chromatin structures, and facilitates downstream DNA characterization.
Stated Advantages
Isolation of substantially pure circular DNA (>90% purity) from complex samples.
Efficient enrichment of low abundance circular DNA species, including ecDNA and eccDNA, from minimal DNA input (<10 ng).
Preservation of DNA topology and chromatin structure, enabling analysis of epigenetic modifications.
Avoids use of intercalating dyes and protease digestion, reducing DNA damage and degradation during purification.
Compatibility with a broad range of sample types including cells, tissues, bodily fluids, and whole organisms.
Method allows embedding samples in polymer matrices to prevent mechanical shearing of DNA.
Provision of a rapid, topology-dependent purification that facilitates downstream sequencing, imaging, and topological characterization of circular DNA.
Documented Applications
Isolation and analysis of circular DNA in eukaryotic cells including extrachromosomal circular DNA (eccDNA), double minutes (ecDNA), and microDNA.
Extraction of circular DNA from complex samples such as bodily fluids (e.g., plasma, synovial fluid, cerebrospinal fluid), tissues, cells in suspension, and whole organisms.
Molecular diagnostics and genomic research on cancer and other diseases involving DNA topological variants as biomarkers.
Analysis of chromatinized circular DNA including preservation and assay of chromatin marks and modifications.
Sequencing applications including next-generation sequencing (NGS) pipelines to determine sequence, copy number, epigenetic state, and topological characteristics of circular DNA.
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