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Assignees
MemberZymo ResearchZymo ResearchZymo Research is a global biotechnology company specializing in innovative products and services for sample collection, nucleic acid purification, microbiomics, epigenetics, and next-generation sequencing (NGS). With over 30 years of experience, Zymo Research is recognized for simplifying complex scientific workflows and providing reliable solutions for life science research, clinical diagnostics, and molecular biology applications. The company offers a comprehensive portfolio of kits, standards, and services designed to ensure accuracy, reproducibility, and efficiency in molecular analyses, supporting researchers worldwide.
Zymo Research is a global biotechnology company specializing in innovative products and services for sample collection, nucleic acid purification, microbiomics, epigenetics, and next-generation sequencing (NGS). With over 30 years of experience, Zymo Research is recognized for simplifying complex scientific workflows and providing reliable solutions for life science research, clinical diagnostics, and molecular biology applications. The company offers a comprehensive portfolio of kits, standards, and services designed to ensure accuracy, reproducibility, and efficiency in molecular analyses, supporting researchers worldwide.
Publication Number
US-10464961-B2
Publication Date
2019-11-05
Expiration Date
Abstract
Methods and composition for nucleic acid isolation are provided. In one embodiment, the invention provides a method for nucleic acid purification from biological samples extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for differential isolation of RNA and DNA.
Core Innovation
The invention provides methods and compositions for nucleic acid isolation. In one embodiment, the invention provides a method for nucleic acid purification from biological samples extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for differential isolation of RNA and DNA.
The background describes a need to separate nucleic acids from proteins and lipids after denaturation with organic solvents such as phenol, and states that phenol-based reagents must later be carefully removed because phenol is toxic and interferes with downstream processes such as sequencing or hybridization. The patent identifies phase separation, nucleic acid precipitation and centrifugation as time consuming steps that can cause loss of nucleic acid and increase exposure to nucleases and labor in existing protocols.
The invention addresses this problem by binding nucleic acids directly to a silica substrate from a sample containing phenol by adding a binding agent comprising a chaotropic salt, an alcohol or a combination thereof, thereby allowing nucleic acid binding from an aqueous/organic suspension without substantially separating organic and aqueous phases or precipitating nucleic acid. The methods can be used to purify RNA, DNA, RNA and DNA, or to sequentially purify DNA and RNA, and the invention further provides kits and reagents comprising a denaturing solvent, a binding agent and a silica substrate.
Claims Coverage
The patent contains two independent claims: one composition claim and one method claim. Ten inventive features are identified below, addressing compositional requirements and steps for binding RNA from a high-phenol monophasic solution to a silica substrate.
pH of 3.5 to 6.0
A composition at a pH of 3.5 to 6.0 is recited as part of the claimed composition.
Guanidinium thiocyanate concentration at least 0.68 M
The composition comprises guanidinium thiocyanate with a concentration of at least 0.68 M with respect to the composition.
Organic component comprising at least 10% phenol by volume
The composition comprises an organic component wherein the organic component comprises at least 10% phenol by volume.
Silica substrate with RNA molecules bound thereto
The composition comprises a silica substrate having RNA molecules bound thereto.
Monophasic composition lacking separate aqueous and organic phases
The composition and the monophasic solution of the method are defined as not comprising separate aqueous and organic phases (i.e., monophasic).
At least 90% free of precipitated RNA molecules
The composition is claimed to be at least 90% free of precipitated RNA molecules, and the method specifies that RNA molecules are at least 90% unprecipitated during contacting with silica.
Obtaining a high phenol monophasic solution at pH 3.5 to 6.0
The method claim recites obtaining a high phenol monophasic solution at pH 3.5 to 6.0 comprising an aqueous component, a cell sample including RNA molecules, guanidinium thiocyanate at least 0.68 M, and an organic component with at least 10% phenol by volume.
Contacting the monophasic solution with a silica substrate to bind RNA while unprecipitated
The method recites contacting the monophasic solution with a silica substrate thereby binding RNA molecules to the silica substrate, wherein during contacting the monophasic solution does not comprise separate aqueous and organic phases and the RNA molecules are at least 90% unprecipitated.
Washing the silica substrate with a wash buffer
The method recites washing the silica substrate comprising bound RNA molecules with a wash buffer as a claimed step.
Eluting the RNA molecules from the silica substrate
The method recites eluting the RNA molecules from the silica substrate as a claimed step.
The independent claims define a composition and a method that require a monophasic high-phenol environment containing guanidinium thiocyanate and a silica substrate with RNA bound, and recite method steps of obtaining such a monophasic solution, contacting it with silica while RNA remains largely unprecipitated, washing the silica, and eluting RNA.
Stated Advantages
Does not require phase separation prior to binding nucleic acid.
Does not require nucleic acid precipitation prior to binding.
Reduces opportunity for nuclease attack by reducing exposure time in aqueous buffers and reducing sample transfers.
Reduces labor input and is suited for high throughput protocols.
Eluted RNA molecules are claimed to be sufficiently purified for sequencing without additional extraction with organic compounds or RNA precipitation steps (claim 11).
Documented Applications
Purification of RNA, DNA, or a combination thereof.
Differential or sequential isolation of RNA and DNA from the same sample suspension.
Use of silica substrates including beads, fibers or porous silica matrices for nucleic acid binding and purification.
Kits for purification of nucleic acid comprising a denaturing solvent comprising phenol, at least a first binding agent, and a silica substrate.
Reagents comprising a denaturing solvent, a binding agent and a silica substrate packaged as a bottle, vial or column.
Purification from a variety of sample types including samples obtained from animal subjects, plants, cell lines or tissue banks, including blood, urine, fecal, tissue (e.g., biopsy), saliva, and hair samples.
Automation of one or more steps of the method by a robot or a microfluidic device and use in high-throughput protocols.
Downstream analysis of eluted nucleic acids such as sequencing or hybridization.
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