Nucleic acid purification
Inventors
Assignees
Publication Number
US-9051563-B2
Publication Date
2015-06-09
Expiration Date
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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 that allow binding of nucleic acids directly from a phenol-containing sample suspension to a silica substrate, and that do not require phase separation or nucleic acid precipitation. The methods include contacting a nucleic acid containing sample extracted with a denaturing solvent comprising phenol with a binding agent comprising a chaotropic salt, an alcohol or a combination thereof and contacting the sample with a silica substrate to bind the nucleic acid, and may be used for differential isolation of RNA and DNA.
The background problem addressed is that phenol-based reagents are widely used to solubilize proteins and lipids but must later be removed because phenol is toxic and interferes with downstream processes such as sequencing or hybridization, and that conventional protocols require time-consuming phase separation and nucleic acid precipitation which can lead to loss of nucleic acid and increased exposure to nucleases. The invention thus aims to avoid organic/aqueous phase separation and nucleic acid precipitation and to reduce processing steps and transfers.
Claims Coverage
The independent claim recites a multi-step method for purification of RNA with five main inventive features.
Denaturing solvent comprising chaotropic salt and phenol
Contacting a nucleic acid containing sample with a denaturing solvent comprising a chaotropic salt and [procedural detail omitted for safety] phenol.
Binding agent comprising a lower alcohol or a chaotropic salt
Adding a binding agent to the sample wherein the binding agent comprises a lower alcohol, or a chaotropic salt or a mixture thereof.
Binding RNA to a silica substrate in presence of phenol
Contacting the sample with a silica substrate, in the presence of [procedural detail omitted for safety] phenol, thereby binding RNA to the silica substrate.
Washing and eluting bound RNA from silica substrate
Washing the silica substrate with bound RNA with a wash solution and eluting the RNA from the silica substrate with an elution buffer to provide purified RNA.
Binding without separate aqueous and organic phases
A limitation that, after addition of the binding agent, the sample does not comprise separate aqueous and organic phases prior to binding the RNA to the silica substrate.
The independent claim centers on combining a phenol-containing denaturing solvent with a specified binding agent to enable direct binding of RNA to a silica substrate in the presence of phenol, followed by washing and elution, and includes the limitation that binding can occur without prior separation of aqueous and organic phases.
Stated Advantages
No need to separate organic and aqueous phases, avoiding time-consuming centrifugation and laborious phase removal.
No need to precipitate nucleic acid, reducing sample loss especially for low-abundance nucleic acids.
Reduced opportunity for nuclease attack and reduced chance of introducing exogenous nuclease due to fewer processing steps and container transfers.
Suitability for modern high throughput protocols due to reduced labor input.
Documented Applications
Purification of RNA, DNA or a combination of RNA and DNA from biological samples.
Differential or sequential isolation of DNA and RNA from the same sample suspension by use of different binding agents.
Kits and reagents for nucleic acid purification comprising a denaturing solvent comprising phenol, one or more binding agents and a silica substrate.
Purification of nucleic acid from multiple samples and automation of one or more steps of the method by a robot or microfluidic device for higher throughput processing.
Application to a variety of sample sources, including animal-derived samples such as blood, urine, fecal samples, tissue samples (e.g., biopsies), saliva samples, or hair samples.
Use of purified nucleic acids directly in downstream analyses such as sequencing or hybridization.
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