Nucleic acid purification
Inventors
Assignees
Publication Number
US-10717976-B2
Publication Date
2020-07-21
Expiration Date
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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, including a method for nucleic acid purification from biological samples extracted with phenol-based denaturing solvents that does not require phase separation or nucleic acid precipitation. The invention further provides methods for binding a nucleic acid molecule to a substrate in the presence of phenol by contacting a sample comprising a nucleic acid and phenol with a silica substrate in the presence of a binding agent comprising a chaotropic salt, an alcohol or a combination thereof.
The methods enable direct binding of nucleic acids from an aqueous/organic suspension to a silica substrate without substantially separating organic and aqueous phases and without a centrifugation step prior to binding. The invention encompasses binding DNA, RNA or small RNAs (e.g., siRNAs or miRNAs), sequential purification of DNA and RNA from the same suspension, use of a variety of silica substrates (including magnetic beads), and formats amenable to automation and kit/reagent embodiments.
Claims Coverage
Independent claim 1 is the principal independent claim and its coverage can be summarized into six main inventive features.
Contact with a denaturing solvent
Contacting a sample comprising whole blood or blood cells with a denaturing solvent comprising a guanidinium thiocyanate and phenol [procedural detail omitted for safety], the denaturing solvent having an acidic pH [procedural detail omitted for safety].
Addition of a binding agent
Adding a binding agent to the sample, wherein the binding agent comprises a chaotropic salt, a lower alcohol or a mixture thereof.
Contacting with a silica substrate
Contacting the sample with a silica substrate in the presence of phenol [procedural detail omitted for safety], thereby binding the ribonucleic acid in the sample to the silica substrate.
Washing and elution steps
Washing the silica substrate and bound RNA with a wash solution and eluting the RNA from the silica substrate with an elution buffer, thereby providing purified RNA.
Retention of RNA in solution prior to binding
At least 90% of the RNA in the sample is unprecipitated prior to binding of the ribonucleic acid to the silica substrate [procedural detail omitted for safety].
Absence of 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 ribonucleic acid to the silica substrate [procedural detail omitted for safety].
Claim 1 covers methods that combine: (i) treatment of whole blood or blood cells with a phenol-containing guanidinium denaturing solvent; (ii) addition of a chaotropic salt and/or lower alcohol binding agent; (iii) direct binding of RNA to a silica substrate in the presence of phenol; (iv) wash and elution to yield purified RNA; and (v) preservation of RNA in solution without separate phases or precipitation prior to binding.
Stated Advantages
Does not require separation of organic and aqueous phases prior to nucleic acid binding.
Does not require nucleic acid precipitation prior to binding to silica.
Reduces opportunity for nuclease attack and reduces transfers between containers, lowering risk of exogenous nuclease introduction.
Amenable to automation and suitable for high-throughput processing (including 96- or 384-well formats).
Increases recovery of small RNAs compared to conventional phase separation methods (reported example of improved recovery for RNAs <40 nt).
Enables efficient isolation of both DNA and RNA, including from whole blood, and allows sequential purification of DNA and RNA from the same sample.
Documented Applications
Purification of RNA, including small RNAs such as siRNAs, miRNAs, piRNAs and synthetic small RNAs.
Purification of DNA (genomic or plasmid) and sequential purification of DNA and RNA from the same sample suspension.
Purification of nucleic acids directly from phenol-containing denaturing solvent lysates without phase separation or precipitation.
Isolation of nucleic acids from whole blood or blood cells and from various biological samples including animal, plant, cell line or tissue bank sources.
Automated nucleic acid purification using silica magnetic beads or column arrays (including 96- or 384-well formats) and robot-implemented workflows.
Kits and reagents comprising a denaturing solvent comprising phenol, binding agent(s) and a silica substrate (e.g., magnetic beads), and packaged components for workflows.
Selective purification/enrichment of small RNA molecules using elution strategies to separate small RNAs from larger RNA species.
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