Methods for nucleic acid capture
Inventors
KEMP, Ryan • Claypool, Jonathan A. • Van Eden, Marc E. • JIA, Xi-Yu
Assignees
Publication Number
US-11236325-B2
Publication Date
2022-02-01
Expiration Date
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Abstract
Solutions, reagents, and methods for nucleic acid purification. In certain aspects, cationic surfactant and, optionally, an anionic surfactant solutions are provided which can be used for phase separation and capture of nucleic acids, such as plasmid or genomic DNA, to a solid phase carrier, such as a mineral matrix.
Core Innovation
The invention provides solutions, reagents, and methods for nucleic acid purification that use a cationic surfactant and, optionally, an anionic surfactant for phase separation and capture of nucleic acids to a solid phase carrier such as a mineral matrix. The methods are described for capture of nucleic acids, including plasmid and genomic DNA, to mineral matrices such as silica-based matrices including borosilicate glass fiber, with subsequent washing and elution to isolate nucleic acid.
In certain embodiments the methods are based on an alkaline lysis procedure and employ a phase separation reagent comprising a cationic surfactant of Formula I, with domiphen bromide (DB) disclosed as a preferred phase separation reagent. The disclosure describes that DB-based phase separation provides selectivity and tunability for preferential capture of large nucleic acids, a treatment of captured nucleic acid with a salt solution that increases retention, and improved capture capacity of silica relative to chaotropic salt-driven binding.
The background identifies a need for improved nucleic acid purification methods that are easier, faster, and provide increased yield and reliability for high-level purification of plasmids and other nucleic acids. The specification further describes kits, prepared sample compositions, and application of the phase separation reagents and mineral matrices to isolate nucleic acids from a variety of source materials.
Claims Coverage
One independent claim was identified. The main inventive features concern a method of isolating plasmid DNA by alkaline lysis that combines phase separation with domiphen bromide and capture to a mineral matrix followed by treatment and washes to enhance retention and isolation.
Method of isolating plasmid DNA by alkaline lysis
A multi-step method comprising: resuspending cells comprising plasmid DNA in a first aqueous solution [procedural detail omitted for safety]; lysing the cells with a second solution to generate a lysate [procedural detail omitted for safety]; neutralizing the lysate with a third solution [procedural detail omitted for safety]; capturing the plasmid DNA to a mineral matrix with a phase separation solution comprising domiphen bromide and in the presence of LiCl [procedural detail omitted for safety]; removing the phase separation solution from the mineral matrix and captured plasmid DNA; washing the mineral matrix and captured plasmid DNA with a salt solution to enhance retention of captured plasmid DNA [procedural detail omitted for safety]; washing the mineral matrix and captured plasmid DNA with an organic wash solution; and eluting the plasmid DNA from the mineral matrix, thereby isolating plasmid DNA.
The independent claim describes a workflow that integrates alkaline lysis with a domiphen bromide based phase separation capture onto a mineral (silica-based) matrix, followed by a salt-based retention enhancement wash, organic washing, and elution to isolate plasmid DNA.
Stated Advantages
Fast, reliable, and efficient method for isolation of substantially purified genomic or plasmid DNA.
Increased quality and yield of DNA in a small elution volume.
High selectivity and tunability for preferential capture of large nucleic acids (e.g., plasmid or genomic DNA) over smaller nucleic acids such as degraded RNA.
Increased capture capacity of silica (noted as nearly 17-fold compared to chaotropic salt-driven binding).
Treatment of captured nucleic acid with a salt solution enhances retention and boosts recovery.
Isolated nucleic acid suitable for sensitive molecular biological applications and described as essentially free of RNA, endotoxin, and/or PCR inhibitors.
Resulting nucleic acids have stability sufficient for prolonged storage at room temperature (as stated).
Documented Applications
Purification and isolation of nucleic acids, including plasmid DNA and genomic DNA, using phase separation reagents and mineral matrices.
Isolation of plasmid DNA from bacterial lysates following alkaline lysis workflows.
Selective isolation of genomic DNA or plasmid DNA by tuning phase separation conditions and salts.
Use of mineral matrices such as silica-based borosilicate glass fiber, including in spin columns and magnetic-silica particles, as solid phase carriers for captured nucleic acids.
Applications in downstream molecular biology techniques including PCR, restriction digests, ligation, sequencing, transcription, reverse transcription, cloning, and transfection (explicitly stated as suitable applications).
Isolation of nucleic acids from a variety of sources explicitly listed: cultured cells, bacteria, yeast, blood, solid tissues, plant tissues, sputum, lymph fluid, cerebrospinal fluid, urine, serum, sweat, various aspirates, and adhesive/resin sources such as water-soluble tape.
Provision of kits for nucleic acid purification comprising a phase separation reagent comprising a cationic surfactant of Formula I, mineral matrix, solutions and components for capture, wash, and elution, and instructions.
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