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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-9206469-B2
Publication Date
2015-12-08
Expiration Date
Abstract
Methods and composition for nucleic acid isolation are provided. In one embodiment, a method is provided for nucleic acid purification from biological samples, such as whole blood 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 of small RNAs (e.g., siRNAs or miRNAs) purification and are amenable to automation.
Core Innovation
The invention provides methods and compositions for nucleic acid isolation that permit binding a nucleic acid molecule to a silica substrate in the presence of phenol, including from samples extracted with phenol-based denaturing solvents, without requiring phase separation or nucleic acid precipitation. Methods according to the invention may be used for purification of RNA (including small RNAs such as siRNAs or miRNAs), DNA, or combinations thereof, and are amenable to automation.
The invention addresses the problem that phenol-based reagents, used to solubilize proteins and lipids, must later be removed because phenol is toxic and interferes with downstream analyses, and that conventional protocols require time-consuming phase separation, centrifugation and nucleic acid precipitation which can result in sample loss and increased exposure to nucleases. The invention provides direct binding of nucleic acids from an aqueous/organic suspension to a silica substrate in the presence of a binding agent comprising a chaotropic salt, an alcohol or a combination thereof, thereby avoiding separate aqueous/organic phase separation and precipitation steps and reducing opportunities for nuclease exposure. [procedural detail omitted for safety]
Claims Coverage
Overview: The independent claim recites eight main inventive features.
Denaturing solvent comprising chaotropic salt and at least 20% phenol
Contacting a nucleic acid containing sample with a denaturing solvent comprising a chaotropic salt and at least 20% phenol.
Binding agent comprising a chaotropic salt or lower alcohol
Adding a binding agent to the sample, wherein the binding agent comprises a chaotropic salt, or a lower alcohol or a mixture thereof.
Binding RNA to a silica substrate in presence of at least 10% phenol
Contacting the sample with a silica substrate, in the presence of at least 10% phenol, thereby binding RNA to the silica substrate.
Washing and eluting bound RNA
Washing the silica substrate and bound RNA with a wash solution and eluting the RNA from the silica substrate with an elution buffer to provide purified RNA.
No separate aqueous and organic phases prior to binding
After the addition of the binding agent, the sample does not comprise separate aqueous and organic phases prior to binding the RNA to the silica substrate.
Application to whole blood or blood cells
The method is performed wherein the sample comprises whole blood or blood cells.
RNA molecules less than 200 nucleotides
The method is performed wherein the RNA comprises RNA molecules less than 200 nucleotides in length.
Magnetic silica beads and automation
The method is performed wherein the silica substrate comprises magnetic silica beads and wherein one or more of steps (a)-(c) is automated.
Conclusion: The independent claim combines (i) use of a phenol-containing chaotropic denaturing solvent, (ii) addition of a binding agent comprising a chaotropic salt and/or lower alcohol, (iii) direct binding of RNA to a silica substrate in the presence of phenol without separate aqueous/organic phase formation, (iv) washing and elution steps, and (v) optional provisions for whole blood samples, small RNAs (<200 nt), magnetic beads and automation.
Stated Advantages
Eliminates the need to separate aqueous and organic phases, avoiding time-consuming centrifugation and laborious removal of phases.
Removes the need to precipitate nucleic acid, reducing labor and avoiding loss of nucleic acid, particularly when present in small amounts.
Reduces the opportunity for nuclease attack by minimizing additional precipitation, centrifugation steps and transfers between containers.
Amenable to automation and high-throughput protocols, reducing labor input.
Increases recovery of small RNAs (for example, ~4× higher recovery of RNAs <40 nt compared to conventional phase separation methods).
Documented Applications
Purification of RNA, including small RNAs such as siRNAs, miRNAs, piRNAs and synthetic RNA oligonucleotides.
Purification of DNA, including genomic or plasmid DNA, and combined or sequential purification of DNA and RNA from the same sample.
Purification of nucleic acids from whole blood or blood cells and other sample sources such as animal or plant material, cell lines, tissue, urine, feces, saliva and hair.
Automated nucleic acid purification using magnetic silica beads, columns or microfluidic/robotic devices and processing in 96- or 384-well formats.
Selective enrichment or selective purification of small RNA molecules (<200 nt) using selective elution and re-binding steps.
Kits and reagents for nucleic acid purification comprising a denaturing solvent comprising phenol, one or more binding agents, and a silica substrate (e.g., magnetic beads), optionally including wash agents and elution buffers.
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