Methods for Nucleic Acid Extraction

Abstract

The present disclosure relates to particle-based methods and kits for extracting nucleic acids from a wide variety of biological samples using magnetic particles or magnetically responsive particles, etc. The disclosed methods and kits can be used to extract nucleic acids from simple to complex biological samples with high purity and recovery (compared to traditional methods such as using spin columns). The disclosed methods and kits can be used in high-throughput extractions or can be suitable for use in high-throughput applications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 63 / 432,862, filed December 15, 2022. No. 60 / 699,997, filed on Oct. 1, 2007, the entire contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to nucleic acid extraction, more particularly to methods for extracting nucleic acids from biological samples, and even more particularly to methods for extracting nucleic acids from a wide range of simple and complex biological samples. [Background technology]

[0003] Methods and / or kits for isolating nucleic acid are commercially available.However, many of these methods and kits cannot efficiently isolate nucleic acid from a wide range of biological samples, including simple and complex samples.Although the spin column-based approach of nucleic acid extraction may be preferred due to conventional practices, it does not scale up well, and tends to have low nucleic acid purity and recovery rate.The particle-based approach for extracting nucleic acid is not designed for a wide range of biological samples, and typically does not work well when extracting nucleic acid from more complex samples, such as whole blood, organoid and whole organ, which has the problem of low purity and recovery rate.

[0004] In the diagnostic and medical fields, there is a need for reliable and efficient kits and methods suitable for isolating nucleic acids with optimal purity and recovery from a wide range of biological samples, including simple and complex biological samples, as well as for the efficient extraction of nucleic acids from a large number of samples in a relatively short period of time. Nucleic acid extraction methods that are amenable to automation would also offer significant advantages in such settings.

[0005] Therefore, there is a particular need for improved kits and methods for extracting nucleic acids from a wide range of biological samples, including simple samples such as single cell suspensions and complex samples such as whole blood or tissue. Summary of the Invention

[0006] The present disclosure relates to kits and / or methods for extracting nucleic acids from biological samples.

[0007] In one embodiment of the present disclosure, a method for extracting nucleic acid from a biological sample is provided. The method of the present disclosure may include: i) treating a biological sample with a lysis buffer to prepare a sample lysate; ii) incubating the sample lysate at a temperature above room temperature and below 60°C; iii) adding particles to the sample lysate to bind nucleic acid; iv) separating the particles and the nucleic acid bound to the particles from the sample lysate; and v) contacting the particles and the nucleic acid bound to the particles with an elution buffer to release the nucleic acid from the particles.

[0008] In one embodiment, the particles are magnetic particles, and the magnetic particles are separated from the lysate in the presence of a magnetic field.

[0009] In one embodiment, the purity and / or recovery of the released nucleic acids is greater than the purity and / or recovery of the released nucleic acids when the biological sample in step i) above is incubated at a temperature above room temperature compared to when the biological sample is at room temperature.

[0010] In one embodiment, the pH of the lysis buffer is between 4 and 10. In one embodiment, the purity of the eluted nucleic acid is not compromised when the pH of the lysis buffer is in the range between 6 and 10.

[0011] In one embodiment, the disclosed method may further comprise preheating the elution buffer to a temperature above room temperature (RT) but less than 60° C. In one embodiment, the temperature of the elution buffer is between 30° C. and 60° C. In one embodiment, preheating the elution buffer improves the purity and / or recovery of the released nucleic acids compared to the purity and / or recovery of nucleic acids released in room temperature elution buffer.

[0012] In one embodiment, the particles are added to the sample lysate at a concentration greater than 1 mg / mL of sample lysate and less than 2.2 mg / mL of sample lysate, hi one embodiment, the concentration of the particles is between about 1.2 mg / mL of sample lysate and 2 mg / mL of sample lysate.

[0013] In one embodiment, the biological sample is whole blood, suspended cells, isolated cells, PBMCs, liver tissue, extracellular vesicles, leukapheresis products, single cell suspensions, organoids, plasma, and viruses.

[0014] In one embodiment, the biological sample is a complex sample.

[0015] In one embodiment, the biological sample is pre-processed. In one embodiment, the biological sample is not pre-processed.

[0016] In one embodiment, the biological sample is 1 ~10 9 Contains cells.

[0017] In one embodiment, the methods of the present disclosure may further comprise directly subjecting the released nucleic acid to one or more downstream applications.

[0018] In one embodiment, the method of the present disclosure may further comprise adding fresh particles to the released nucleic acid to recapture the released nucleic acid.

[0019] In one embodiment, the disclosed method may further comprise releasing the recaptured nucleic acid using an elution buffer of the disclosure.

[0020] In one embodiment, the method does not include washing the particles and the nucleic acids bound thereto before contacting them with an elution buffer.

[0021] In one embodiment, the method does not include washing the first and / or second particles and the nucleic acids bound thereto.

[0022] In one embodiment, the purity and / or recovery of the recaptured nucleic acid released from the fresh particles is greater than the purity and / or recovery of the nucleic acid released in step v) above.

[0023] In one embodiment, the method of the present disclosure may further comprise separating the particles and the supernatant containing the released nucleic acids by aspirating or pouring.

[0024] In one embodiment, the particles are silica-based.

[0025] In one embodiment, the method lasts for 30 to 45 minutes.

[0026] In another aspect of the present disclosure, a kit for extracting nucleic acid from a biological sample is provided.The kit of the present disclosure can include a lysis buffer (as described herein), one or more vials of particles (as described herein), a washing buffer (as described herein), and an elution buffer (as described herein).In one embodiment, the kit of the present disclosure further includes a priming buffer (as described herein).

[0027] Other features and advantages of the presently described subject matter will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating preferred embodiments of the described subject matter, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0028] For a better understanding of the various embodiments described herein, and to show more clearly how these may be practiced, reference will now be made, by way of example, to the accompanying drawings, which illustrate at least one exemplary embodiment, and which are not intended to limit the scope of the teachings described herein. [Brief explanation of the drawings]

[0029] [Figure 1] Bar graphs show the purity and recovery of nucleic acids extracted from LeukoPak PBMCs when the pH of the lysis buffer used in the disclosed method was varied from 4 to 10. The purity of the extracted DNA (salt and protein contaminants) was assessed by the 260 / 280 and 260 / 230 absorbance ratio values ​​obtained from a spectrophotometer (A). The amount of DNA and RNA (µg) extracted per 1 x 10 LeukoPak PBMCs (B) was measured using a Qubit fluorometer. Error bars represent the mean ± SD of two technical replicates. [Figure 2] Box plots show the purity and recovery of nucleic acids extracted from unwashed leukopack cells from three human donors using the disclosed method, varying only the concentration of magnetic particles in 90% isopropyl alcohol: 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, and 2.4 mg / mL sample lysate. The 260 / 230 absorbance ratio value assessed the purity of the extracted nucleic acids (free from salt and protein contaminants) (A). The amount of nucleic acid extracted (ng / μL) per 1 × 10 leukopack cells (B) was measured using a Qubit fluorometer. [Figure 3] The recovery rate of RNA extracted from liver organoid samples is compared using conventional spin columns ("Spin") or the exemplary particle-based workflow ("EasySep™") of the present disclosure. Liver organoid lysates are prepared using either a phenol-chloroform-based reagent (TRIzol) or the lysis buffer (IH-LB) of the present disclosure. Data points represent three technical replicates. [Figure 4] 1 shows UV spectrophotometric absorption spectra of nucleic acids extracted from a sample of whole blood using a standard workflow, a spin column workflow, and an improved workflow according to an exemplary embodiment of the present disclosure. [Figure 5]This figure compares the purity and recovery of nucleic acids extracted from whole blood samples using the disclosed two-step particle-based workflow ("improved workflow"). The control nucleic acid extraction workflow included a pre-lysed RBC sample and a spin-column-based method. Conditions 1-4 involved recapture of eluted nucleic acids onto the same particles as the "improved workflow," but varied the number of wash steps before elution and recapture as follows: no wash (condition 1), one wash (condition 2), two washes (condition 3), and three washes (condition 4). The purity of the recaptured nucleic acids (260 / 230) was determined by UV spectrophotometry (A), and the recovery of extracted nucleic acids (µg) per 1 x 106 input cells was measured using a NanoDrop2000 (B). n = 1 in duplicate. [Figure 6] 1 shows UV spectrophotometric absorption spectra of nucleic acids extracted from a sample of whole blood according to the two-step method of the present disclosure in the presence or absence of a priming buffer that primes the initial elution of nucleic acids before recapture by newly added particles. [Figure 7] Bar graphs comparing the purity and recovery of nucleic acids extracted from whole blood samples using the disclosed two-step method ("Improved"), a standard one-step particle-based workflow (e.g., single particle addition) ("Standard"), and conventional spin-column extraction. Purity (free from salt and protein contaminants) of extracted nucleic acids was assessed by the 260 / 230 absorbance ratio value obtained from a spectrophotometer (A). n=3. Error bars represent ±1 standard deviation. The amount of DNA (B) and RNA (C) extracted (μg) per 1×10 cells, as measured using a Qubit fluorometer, is shown. n=7. Error bars represent ±1 standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure relates to methods and / or kits for extracting nucleic acids from biological samples. More specifically, the present disclosure relates to extracting nucleic acids from a variety of biological samples, including samples ranging from simple to more complex samples, by performing the disclosed methods and / or using kits. Even more specifically, the present disclosure relates to techniques for high-throughput extraction of nucleic acids from a variety of biological samples, including samples ranging from simple to more complex samples.

[0031] As used in this disclosure, the term "nucleic acid" refers to linear, branched, or circular DNA and RNA. Examples of RNA can include mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA, ribozymes, viral RNA, or extracellular or cell-free RNA. Examples of DNA can include plasmid DNA, genomic DNA, mitochondrial DNA, viral DNA, and extracellular or cell-free DNA.

[0032] As used in this disclosure, the term "particle" refers to a particle that reversibly binds to nucleic acids such as DNA and / or RNA. In certain embodiments, the particle is a magnetic particle or a magnetically responsive particle, and may more specifically be referred to as a ferromagnetic particle, a ferrimagnetic particle, a paramagnetic particle, or a superparamagnetic particle. The magnetic particle or magnetically responsive particle may include a magnetically rich core encapsulated by a polymer shell. Any particle of the present disclosure, including the magnetic particle or the magnetically responsive particle, is commercially available and would be known to those skilled in the art. The particle may be a silica-coated particle, and the silica may be in the form of silica gel, silica particles, silica fibers, siliceous oxides, silicon dioxide, alkylsilica, aluminum silicate, borosilicate, solid silica, such as glass or diatomaceous earth, glass fiber, or a mixture of two or more of the above. Silica should be present at least partially on the surface of the bead. For efficient binding, it is preferable that silica be present on nearly all or substantially most (e.g., >50%, >60%, >70%, >80%, >90%, or >95%) of the particle surface. In one embodiment, the silicon oxide-coated (magnetic) particles also have an adsorption surface of hydrated silicon oxide. Target nucleic acids such as DNA or RNA bind to the adsorption surface of the particles, while other substances from the sample lysate, particularly harmful contaminants such as nucleases, can be limited from binding.

[0033] In one embodiment, the particles (e.g., magnetic particles) of the present disclosure function as a solid phase. The term solid phase encompasses suitable materials that are porous or non-porous, or permeable or impermeable. In one embodiment, the surface of the solid phase (e.g., silica solid phase) is not modified with a functional group or the like. In one embodiment, the surface of the solid phase (e.g., silica solid phase) is modified with a functional group or the like. In one embodiment, the solid phase may have an anion-exchange functional group capable of binding to the nucleic acid of interest.

[0034] As used in this disclosure, the term "chaotropic agent" refers to an agent that disrupts hydrogen-bonding networks in aqueous solutions and destabilizes the native state of macromolecules (e.g., proteins, nucleic acids) in solution. Chaotropic agents denature nucleic acid-associated proteins, thus weakening the hydrophobic interactions between such proteins and nucleic acids. Non-limiting examples of chaotropic agents include guanidinium thiocyanate, guanidine, urea, and thiourea. Preferably, the chaotropic agent is a guanidinium salt (e.g., guanidinium hydrochloride, guanidinium thiocyanate, and / or guanidinium isothiocyanate). In one embodiment, the concentration of the chaotropic agent in a buffer, such as a lysis buffer, can range from 0.1 M to 10 M, from 1 M to 8 M, from 3 M to 7 M, or from 4 M to 6 M.

[0035] As used in this disclosure, the terms "purity" or "purity ratio" refer to the purity of extracted nucleic acids, which can be assessed from the 260 / 280 and 260 / 230 absorbance ratios. Pure DNA has an A260 / A280 nm ratio (acceptable range: 1.7-2.0), and pure RNA has an A260 / A280 ratio (acceptable range: 1.9-2.2). A significantly low 260 / 280 ratio (≤1.6) may indicate the presence of proteins, phenols, or other contaminants that absorb at or near 280 nm. Expected A260 / A230 values ​​for DNA and RNA, respectively, are generally within the range of 1.8-2.2. A 260 / 230 ratio outside the aforementioned range may indicate the presence of contaminants that absorb at 230 nm (e.g., proteins, carbohydrates, lipids, salts, EDTA, or phenol). The 260 / 230 ratio is widely used as a secondary measure of nucleic acid purity.

[0036] As used in this disclosure, the term "recovery" refers to the amount or concentration (e.g., ng or μg) of extracted or isolated nucleic acid (e.g., DNA and / or RNA) and may be expressed relative to the input number of cells. Nucleic acid recovery is typically measured with a spectrophotometer, microspectrophotometer, or fluorometer.

[0037] method In one aspect of the present disclosure, a method for extracting nucleic acids from a biological sample is provided. In embodiments, the method for extracting nucleic acids from a biological sample can be performed on a wide range of biological samples, including simple and complex samples. In embodiments, the method for extracting nucleic acids from a biological sample can be performed on a wide range of biological samples, from simple to complex samples, without substantially compromising the purity and / or recovery rate of the nucleic acids.

[0038] The biological sample of the present disclosure is not limited to, provided that it contains nucleic acid. Thus, the biological sample may be of prokaryotic, viral, or eukaryotic origin. In certain embodiments, the biological sample is of mammalian origin. The biological sample may be fresh, frozen, dried, or cryopreserved. The method (and kit) of the present disclosure may be performed on a wide range of biological samples, from relatively simple samples to relatively more complex samples.

[0039] As used in this disclosure, the term "simple biological sample" can refer to any sample that is simple in composition with respect to the presence / concentration of proteins, carbohydrates, lipids, chemicals, or other contaminants, and / or requires minimal or no pre-treatment or special handling prior to nucleic acid extraction. Examples of simple biological samples include, but are not limited to, cells suspended (enriched) in a buffer or medium (e.g., a single cell suspension), plasma, leukapheresis products (e.g., PBMCs), etc.

[0040] As used herein, the term "complex biological sample" may refer to any sample whose composition is complex with respect to the presence / concentration of proteins, carbohydrates, lipids, chemicals, or other contaminants and / or that requires pretreatment or special handling before nucleic acid extraction. The concepts of simple and complex biological samples are relative terms, with the latter including more contaminants than the former, particularly contaminants that those skilled in the art will recognize as potentially affecting the purity and / or recovery rate of particle-based nucleic acid extraction. Complex samples may contain a few or many contaminants, collectively at relatively high concentrations. Examples of contaminants in simple and / or complex samples may include salts, proteins, cells, and endogenous and / or exogenous small organic molecules (e.g., lipids, amino acids). In particular, sources of contaminants may come from non-nucleic acid cellular material, such as proteins, lipids, carbohydrates, or other macromolecules, intact cells, or cell debris. Therefore, relatively high concentrations of such contaminants may reduce the efficiency of nucleic acid extraction, particularly by reducing purity and recovery rates. Examples of complex biological samples include, but are not limited to, blood (e.g., whole blood, crude whole blood lysate, serum, plasma, or menstrual blood), bloody fluids (e.g., wound exudate, mucus, bile, etc.), tissue samples (e.g., tissue biopsy, skin biopsy, muscle biopsy, or lymph node biopsy), whole organs (e.g., liver), homogenized tissue samples, urine, CSF, BAL, SF, semen, feces, swallowed (e.g., purulent or hemoptytic sputum), organoids, etc.

[0041] The number of cells in the biological sample from which nucleic acids are extracted using the methods (and kits) of the present disclosure can vary. In one embodiment, the number of cells in the biological sample is about 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 It could be.

[0042] In one embodiment, the biological sample is whole blood, which may be considered a complex sample because it contains RBCs, WBCs, platelets, plasma, and their respective cytosols, macromolecules, and small molecules. In one embodiment, the whole blood contains an anticoagulant.

[0043] In one embodiment, the biological sample is a sample of PBMCs or leukapheresis product. A PBMC sample may contain relatively low levels of contaminating RBCs, granulocytes, and / or platelets, or may be free of them. In one embodiment, the biological sample is plasma.

[0044] In one embodiment, the biological sample comprises viruses and / or prokaryotes. In one embodiment, the biological sample comprises soil material and / or plants.

[0045] In one embodiment, the biological sample is a suspension of cells in a suitable buffer or medium. The suspension of cells may be isolated or concentrated using an isolation or concentration method such as immunomagnetic separation, centrifugation (e.g., density gradient centrifugation), or filtration.

[0046] In one embodiment, the biological sample is a leukapheresis product, hi one embodiment, the leukapheresis product is adjusted with an appropriate buffer to obtain a desired cell concentration for nucleic acid extraction.

[0047] In one embodiment, biological sample is or comprises organoid.Organoid can be contained in or on extracellular matrix (for example, matrigel dome), and therefore organoid sample can comprise one or more extracellular matrix proteins.When organoid is contained in or on extracellular matrix, they can be decomposed into complex by incubation in lysis buffer (for example, phenol-chloroform solution or enzyme solution), and / or can be mechanically crushed using pipette.

[0048] In one embodiment, the biological sample is a whole organ or an organ fragment, which may be decomposed (e.g., dissociated or homogenized) through one or more of mincing, mechanical grinding, incubation in an enzyme solution, or the like.

[0049] Thus, the present disclosure relates to methods for extracting nucleic acids from any available biological sample, where the sample contains nucleic acids to be extracted and / or analyzed.

[0050] The method of the present disclosure may include preparing a sample lysate by treating a biological sample with a lysis buffer. Many lysis buffers are known in the art. The sample lysate may be prepared in a lysis buffer containing one or more of the following: a) Proteinase K, such as at a concentration of 10-100 μl per ml of sample, to cleave peptide bonds and digest proteins. b) One or more detergents (e.g., SDS, Tween-20, and Sarkosyl), such as at a concentration of 3% to 20%, to lyse cells by solubilizing membrane proteins and lipids (releasing soluble proteins). c) Hyperosmolarity or osmolarity (to create osmotic pressure on the cells, causing water to enter the cells, increasing their internal pressure and causing the cells to lyse). d) One or more buffering agents to maintain a desired pH (e.g., 6.0-10.0), such as Tris, HEPES, sodium bicarbonate, ACES, PIPES, MPSO, imidazole, MOPS, triethanolamine, pyrophosphate, sodium chloride, deoxycholate, or any combination. e) A binding inducer such as a chaotropic agent or a chaotropic salt, which may be provided alone or in combination with a binding additive such as an alcohol (eg, ethanol or isopropanol).

[0051] In one embodiment, the sample lysate is prepared in a lysis buffer that does not contain proteinase K.

[0052] The pH of the lysis buffer can range from 3 to 12, or from about 4 to 10. In one embodiment, the pH of the lysis buffer is about 6, about 7, about 8, about 9, or about 10. In one embodiment, the purity of the released nucleic acids is not compromised when the pH of the lysis buffer is in the range of 3 to 12, or more preferably about 4 to 10.

[0053] The sample lysate contains nucleic acids, among other cellular components. Examples of other cellular components can include fragments of cell and / or nuclear membranes, organelles, and macromolecules such as carbohydrates, proteins, and lipids. The nature of the components in the sample lysate environment can determine whether the biological sample is a complex or simple biological sample, as defined above.

[0054] The sample lysate may be prepared by heating the processed sample (or lysis buffer) to a temperature above room temperature and below 60°C. In one embodiment, the sample lysate is prepared by heating the processed sample (or lysis buffer) to a temperature above 30°C and below 60°C. In one embodiment, the sample lysate is prepared by heating the processed sample (or lysis buffer) to a temperature above 37°C and below 60°C. In one embodiment, the sample lysate is prepared by heating the processed sample (or lysis buffer) to a temperature preferably about 56°C ± 3°C. In one embodiment, the sample lysate is prepared by heating the processed sample (or lysis buffer) to a temperature of approximately room temperature ± 3°C.

[0055] The sample lysate can be prepared by incubating at the aforementioned temperature range or by incubating in a lysis buffer heated to the aforementioned temperature range. The sample lysate can be incubated for any amount of time as long as the nucleic acids do not begin to significantly denature. In one embodiment, the sample lysate is prepared by incubating at a given temperature (as described above) for about 3-30 minutes, about 5-20 minutes, or about 10-15 minutes. In one embodiment, the sample lysate is prepared by incubating at a given temperature (as described above) for about 5 minutes, about 10 minutes, or about 15 minutes.

[0056] The disclosed methods include adding particles, such as magnetic particles, to a sample lysate to bind nucleic acids. It may be necessary to incubate the sample lysate in the presence of the particles to allow the nucleic acids to complex or bind to the particles. In one embodiment, a volume of particles is added to the sample volume in approximately the same ratio (e.g., a 1:1 ratio). In one embodiment, a volume of particles is added to the sample volume in a different ratio (e.g., a 0.5:1 or 1.5:1, or 1:0.5 or 1:1.5 ratio). In one embodiment, the incubation is at room temperature. In one embodiment, the incubation is for about 5 minutes. In one embodiment, the incubation is for less than 5 minutes. In one embodiment, the incubation is for about 3 minutes. In one embodiment, the incubation is for about 1 minute.

[0057] In addition to binding to nucleic acids, particles can also nonspecifically bind to cellular components other than nucleic acids. In one embodiment, particles can nonspecifically bind to proteins or other macromolecules in a sample lysate (e.g., a cell lysate). In one embodiment, particles can nonspecifically bind to hemoglobin in the sample / cell lysate. The nature of the contaminants in the sample lysate, and therefore the nature of the substances that can nonspecifically bind to particles, can determine whether the biological sample is a complex biological sample or a simple biological sample.

[0058] The disclosed methods can include separating the particles and the nucleic acids bound thereto to concentrate the nucleic acids in the sample lysate from proteins and other debris in the sample lysate. Separation can be performed by any method that allows for the separation / isolation of the particles. As a non-limiting example, the particles can be centrifuged to form a pellet, or if the particles are magnetic particles, the particles can be exposed to a magnetic field and collected against a container surface closest to the magnetic field.

[0059] In certain embodiments where the particles are magnetic particles, the particles and their bound nucleic acids are separated from the sample lysate by applying a magnetic field to the sample lysate. It may be necessary to incubate the particles in the presence of the magnetic field for one minute or more to maximize co-localization of the particles with the magnetic field / wall of the container / tube closest to the magnetic field, etc. In one embodiment, the incubation is performed at room temperature. In one embodiment, the incubation is performed for about five minutes. In one embodiment, the incubation is performed for less than five minutes. In one embodiment, the incubation is performed for about three minutes. In one embodiment, the incubation is performed for about one minute.

[0060] Following collection of particles (e.g., magnetic particles) on or along the surface of the container (closest to the magnetic field), the supernatant can be removed. It may be important that the container be maintained near the magnetic field so that the particles can be fractionated from the remainder of the sample lysate. In one embodiment, the supernatant can be removed by pipetting. In one embodiment, the supernatant can be poured off.

[0061] After fractionating the particles from the supernatant, it may be important to wash the particles to remove contaminants entrained between the particles and / or contaminants that weakly interact with or are bound to the particles. In one embodiment, the pellet is washed with a wash buffer such as Buffer RW1 (Qiagen), AW1 (Qiagen), PBS, EasySep™ Buffer (STEMCELL Technologies), or any conventional wash buffer containing a high concentration of ethanol (or isopropanol). In one embodiment, the wash buffer contains an organic solvent, such as a lower alcohol (i.e., a C1-5 alcohol). In one embodiment, the wash buffer contains ethanol. In one embodiment, the wash buffer contains 40-80% ethanol or isopropanol.

[0062] Any number of washing cycles may be performed to increase purity. However, increasing the number of washes may result in a sacrifice of recovery. In one embodiment, the pellet is washed once with the wash buffer. In one embodiment, the pellet is washed twice with the wash buffer. In one embodiment, the pellet is washed three times with the wash buffer. In one embodiment, the pellet is washed more than three times, for example, five times or more, with the wash buffer.

[0063] The pH and solute composition and concentration of the wash buffer can be varied depending on the types of impurities expected to be present.

[0064] The disclosed method may include contacting the particles and the nucleic acid bound thereto with an elution buffer to release the nucleic acid from the particles. Elution buffers are known, and when eluting from a silica matrix such as may be contained on the particles of the present disclosure, elution efficiency may be affected by pH. The disclosed elution buffer may include one or more of the following: low salt, TE buffer, or about 10-100 mM Tris-HCl, pH about 8.0 or 8.5, and about 0.1-1 mM EDTA. Efficient elution of nucleic acid from particles may be achieved by incubating the particles in water or a buffer with low ionic strength. In one embodiment, particle-bound nucleic acid is eluted using distilled or deionized water or nuclease-free water.

[0065] The disclosed method may further comprise preheating the elution buffer prior to contacting the particles and the nucleic acid bound thereto with the elution buffer. In one embodiment, the elution buffer is preheated to a temperature above room temperature and below 60°C. In one embodiment, the elution buffer is preheated to a temperature above 30°C and below 60°C, or between 35°C and 55°C, or between 40°C and 50°C. In one embodiment, the elution buffer is preheated to 37°C ± 3°C. In one embodiment, the elution buffer is preheated to 56°C ± 3°C. In one embodiment, the disclosed method further comprises incubating the particles and the nucleic acid bound thereto at one of the aforementioned temperatures after they have been contacted with the elution buffer.

[0066] Preheating the elution buffer as described above (or incubating particles in the elution buffer at the aforementioned temperatures) may improve the purity and / or recovery of released nucleic acids. In one embodiment, preheating the elution buffer improves the purity of the released nucleic acids compared to the purity of nucleic acids released in elution buffer at room temperature. In one embodiment, preheating the elution buffer improves the recovery of released nucleic acids compared to the recovery of nucleic acids released in elution buffer at room temperature.

[0067] In one embodiment, the method can include preferentially releasing nucleic acids from the particles. Preferential elution can refer to conditions under which nucleic acids are substantially or completely released from the particles, while other cellular components may remain bound. In one embodiment, about 90-100% of the nucleic acids are released from the particles. In one embodiment, about 80-90% of the nucleic acids are released from the particles. In one embodiment, about 70-80% of the nucleic acids are released from the particles. In one embodiment, about 60-70% of the nucleic acids are released from the particles. In the same or a different embodiment, about 90-100% of the other components remain bound to the particles. In one embodiment, about 80-90% of the other components remain bound to the particles. In one embodiment, about 70-80% of the other components remain bound to the particles. In one embodiment, about 60-70% of the other components remain bound to the particles. In one embodiment, about 50-60% of the other components remain bound to the particles. In one embodiment, about 40-50% of the other components remain bound to the particles.

[0068] In one embodiment, the disclosed method does not include washing prior to the elution step. In one embodiment, the method does not include washing the particles and the nucleic acids bound thereto. In one embodiment, the method includes minimal washing of the particles and the nucleic acids bound thereto, for example, washing only once.

[0069] The method of the present disclosure may further include fractionating the particles and the supernatant containing the released nucleic acids. The particles and the released nucleic acids (in the supernatant) may be fractionated by aspirating or pouring them off. The particles and the supernatant can be fractionated by any method that allows the particles to colocalize, such as by pelleting the particles by centrifugation, or, if magnetic, by maintaining the particles in the presence of a magnetic field during fractionation. In one embodiment, fractionation is performed in the presence of a magnet or magnetic field, and the supernatant (e.g., the fraction containing nucleic acids) is transferred to another container or tube. Thus, the supernatant or eluate may be referred to as the first eluate.

[0070] The disclosed method may further include adding fresh particles to the released nucleic acids of the first eluate to rebind / recapture the nucleic acids in the first eluate. In one embodiment, the disclosed method includes adding fresh particles to the released and fractionated nucleic acids of the first eluate. The newly added particles may be referred to as second particles (e.g., second magnetic particles). The nucleic acids in the first eluate may be incubated in the presence of the second particles as described above, e.g., with respect to the relative volumes of the particles and the first eluate, incubation time, incubation temperature, etc. In one embodiment, the volume of fresh particles added to the first eluate contains alcohol, e.g., at a concentration of 40%, 50%, 60%, 70%, or more.

[0071] Prior to rebinding / recapture of nucleic acids in a first eluate to fresh / second particles, such as in a two-step extraction of the present disclosure, the nucleic acids may be primed for recapture. The priming buffer may alter the conditions of the first elution to facilitate recapture of the nucleic acids, such as by restoring salt concentration, pH, conformation, etc.

[0072] The nucleic acids in the first eluate may be contacted with a priming buffer, e.g., a buffer containing one or more of a chaotropic agent or salt, alcohol (e.g., at a high concentration), a cell lysis agent (as described above), and a binding additive such as alcohol. In one embodiment, priming involves supplementing the first eluate with a priming buffer, e.g., in a 1:1 or 1:1.5 ratio. In one embodiment, the alcohol is isopropanol. In one embodiment, the isopropanol used is 100%. In one embodiment, the priming buffer is the same as the lysis buffer described herein.

[0073] In one embodiment, the nucleic acids in the first eluate are primed (in a priming buffer) during contact with the second particles. Thus, a first eluate that has been treated or primed prior to rebinding the (eluted) nucleic acids to the second particles may be referred to as a treated or primed sample.

[0074] The disclosed method can further include separating the second particles and the nucleic acids bound thereto, essentially as described above. The second separation can further enrich the nucleic acids in the first eluate from proteins, debris, or other residues in the first eluate, such as those that may be carried into the first eluate from a complex biological sample, or the nucleic acids can be enriched regardless of whether the biological sample is simple or complex.

[0075] After separating the second particles and nucleic acids in the first eluate from the remainder of the first eluate, washing can be performed essentially as described above. In one embodiment, the disclosed method does not include washing before the second elution step.

[0076] As described above with respect to the first elution solution, the disclosed methods can further include contacting the second particles and the nucleic acids bound thereto with an elution buffer, wherein the nature (e.g., components, temperature, etc.) of the elution buffer and the duration of the contacting step are essentially as described above.

[0077] After releasing the recaptured nucleic acids from the fresh / secondary particles, the disclosed method may further include fractionating the second particles and the supernatant containing the nucleic acids, essentially as described above.

[0078] The particles of the present disclosure are not particularly limited, as many vendors have commercialized particles for nucleic acid extraction. Nucleic acids can be bound to the first (magnetic) particles and / or the second (magnetic) particles through various binding mechanisms. For example, the binding mechanism between the nucleic acid and the (magnetic) particles can be electrostatic / ionic interactions. Alternatively, in one embodiment, the binding mechanism between the nucleic acid and the (magnetic) particles can be hydrophobic interactions. Alternatively, the binding mechanism between the nucleic acid and the (magnetic) particles can be hydrogen bond / dipole bond interactions.

[0079] The particles (e.g., first and / or second particles), whether magnetic or not, can be added to the sample lysate at any desired concentration. The concentration of the first and / or second particles can be determined based on the volume of the sample lysate. In one embodiment, the concentration of the first and / or second particles ranges from 0.1 μg / mL to 1 μg / mL of sample lysate. In one embodiment, the concentration of the first and / or second particles ranges from about 1 μg / mL to 10 μg / mL of sample lysate, from about 1 μg / mL to 5 μg / mL of sample lysate, or from about 1 μg / mL to 3 μg / mL of sample lysate. In one embodiment, the first and / or second particles are Concentrated EasySep™ Total Nucleic Acid Rapidspheres™ (STEMCELL Technologies), and if concentrated, the particles may need to be diluted in the sample lysate or before being added to the sample lysate.

[0080] In one embodiment, the particles are added to the sample lysate at a concentration greater than 1 mg / mL of sample lysate and less than 2.2 mg / mL of sample lysate. In one embodiment, the particle concentration is between about 1.2 mg / mL of sample lysate and 2 mg / mL of sample lysate. In one embodiment, the particles are added at a concentration ranging from 0.5 to 3 mg per mL of sample lysate, or 0.75 to 2.75 mg per mL of sample lysate, or 1 to 2.5 mg per mL of sample lysate. In an embodiment, the particle concentration added to the sample lysate is about 1.2 mg / mL of sample lysate, or about 1.4 mg / mL of sample lysate, or about 1.6 mg / mL of sample lysate, or about 1.8 mg / mL of sample lysate, or about 2.0 mg / mL of sample lysate, or about 2.2 mg per mL of sample lysate.

[0081] In embodiments, the nucleic acids in the first eluate and / or the second eluate may be subjected to further treatment, such as with an RNase solution (to obtain purified or substantially purified DNA) or a DNase solution (to obtain purified or substantially purified RNA). In embodiments, the first eluate and / or the second eluate may be divided into separate containers, each treated with a DNAse and an RNAse, respectively. In one embodiment, the nucleic acids in the first eluate and / or the second eluate are not further treated.

[0082] The methods disclosed herein, whether using a one-step particle addition method or a two-step particle addition method, result in high nucleic acid purity (e.g., within an optimal A260 / 280 and / or A230 / 260 range). In one embodiment, the purity of nucleic acid released from the second particles is higher than the purity of nucleic acid released from the first particles. In one embodiment, the purity of nucleic acid released from the second particles and the purity of nucleic acid released from the first particles are both within an optimal A260 / 280 and / or A230 / 260 range. In one embodiment, the purity of nucleic acid released from the second particles is comparable to the purity of nucleic acid released from the first particles (e.g., within about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±12.5%, or ±15% of each other).

[0083] The methods disclosed herein, whether using a one-step particle loading method or a two-step particle loading method, can result in nucleic acid purity greater than that of nucleic acid eluted from a conventional spin column. In one embodiment, the purity of nucleic acid released from the first and / or second particles and the purity of nucleic acid eluted from a conventional spin column are both within the optimal A260 / 280 and / or A230 / 260 ranges. In one embodiment, the purity of nucleic acid released from the first and / or second particles is within the optimal A260 / 280 and / or A230 / 260 ranges, and the purity of nucleic acid eluted from a conventional spin column is not within the optimal A260 / 280 and / or A230 / 260 ranges. In one embodiment, the purity of the nucleic acid released from the first and / or second particles is comparable to the purity of the nucleic acid eluted from the spin column (e.g., within about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±12.5%, or ±15% of each other).

[0084] The disclosed two-step method can result in higher nucleic acid purity than that released from standard (one-step) particle-based workflows, whether commercially available or disclosed herein. In one embodiment, the purity of nucleic acid released from the second particles and the purity of nucleic acid released from the first particles are both within the optimal A260 / 280 and / or A230 / 260 ranges. In one embodiment, the purity of nucleic acid released from the second particles is within the optimal A260 / 280 and / or A230 / 260 ranges, and the purity of nucleic acid released from the first particles is not within the optimal A260 / 280 and / or A230 / 260 ranges. In one embodiment, the purity of the nucleic acid released from the second particle is comparable to the purity of the nucleic acid released from the first particle (e.g., purity within about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±12.5%, or ±15% of each other).

[0085] Improved nucleic acid purity may be applicable to extraction from a wide range of biological samples, from simple to complex samples. In one embodiment, improved nucleic acid purity using the nucleic acid extraction methods disclosed herein is particularly applicable to extraction from complex biological samples, such as samples containing high levels of contaminants, without the need for pretreatment to reduce the complexity of the sample.

[0086] The methods disclosed herein, whether one-step or two-step particle addition methods, provide high nucleic acid recovery (e.g., within 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of theoretical yield). In one embodiment, the recovery of nucleic acid released from the second particles is higher than the recovery of nucleic acid released from the first particles (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 2-fold higher). In one embodiment, the recovery of nucleic acid released from the second particles is equivalent to the recovery of nucleic acid released from the first particles (e.g., within about ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, ±40%, or ±50% of each other).

[0087] The methods disclosed herein, whether one-step or two-step particle addition methods, provide higher nucleic acid recovery rates (e.g., about 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 2-fold, or 3-fold, or 4-fold, or 5-fold higher) than elution from a conventional spin column. In one embodiment, the recovery of nucleic acid released from the first and / or second particles is comparable to the recovery of nucleic acid eluted from the spin column (e.g., within about ±1%, ±3%, ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, ±35%, ±40%, ±45%, or ±50% of each other).

[0088] The disclosed two-step method may result in higher nucleic acid recovery rates (e.g., about 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 2-fold higher) than those released from standard (one-step) particle-based workflows, whether commercially available or disclosed herein. In one embodiment, the recovery of nucleic acid released from the second particles is comparable to the recovery of nucleic acid released from the first particles (e.g., within about ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, ±35%, ±40%, ±45%, or ±50% of each other).

[0089] Improved nucleic acid recovery may be applicable to extraction from a wide range of biological samples, from simple to complex samples. In one embodiment, improved nucleic acid recovery using the nucleic acid extraction methods disclosed herein is particularly applicable to extraction from complex biological samples, such as samples containing high levels of contaminants, without the need for pretreatment to reduce the complexity of the sample.

[0090] In one embodiment, the nucleic acid extracted by the method of the present disclosure is directly used in downstream applications.In one embodiment, the extracted nucleic acid is directly used in downstream applications without the need for nucleic acid quantification.In one embodiment, the extracted nucleic acid is directly used in RT-qPCR without the need for DNAse treatment.

[0091] In one embodiment, the disclosed method does not include pre-treatment of the biological sample, such as to simplify or clarify the biological sample by, for example, filtration. In the example of whole blood, the sample is not pre-treated to reduce or remove red blood cells and / or other contaminants contained therein. In the example of a sample containing mucus, it is not treated with enzymes, sonication, etc. to liquefy the sample. Thus, in one embodiment, the only preparation of the biological sample may be to contact the sample with a lysis buffer.

[0092] The disclosed methods for extracting nucleic acids, whether using a one-step or two-step particle addition approach, may be suitable for high-throughput applications. Such methods may take 20 to 45 minutes. In embodiments for extracting nucleic acids from complex biological samples and using the two-step particle addition approach described above, the disclosed methods may take about 45 minutes or less, about 40 minutes or less, about 35 minutes or less, or about 30 minutes or less.

[0093] The disclosed method can be carried out in any suitable vessel, including a tube (e.g., a microcentrifuge tube) or a plate (e.g., a microplate). Depending on the vessel used, a suitable magnet is used in embodiments in which the particles are magnetic or respond to a magnetic field. In one embodiment, the magnet is an EasySep™ magnet that receives a tube therein. In one embodiment, the magnet is a plate magnet that receives a microplate thereon.

[0094] In one embodiment, the disclosed method provides various features and advantages over conventional nucleic acid extraction methods. In one embodiment, the method does not require pretreatment of the biological sample. In one embodiment, the first or second elution of nucleic acids can be used directly in downstream assays. In one embodiment, the disclosed method (whether one-step or two-step) extracts nucleic acids with optimal purity and recovery, even when the starting biological sample is complex.

[0095] The following non-limiting examples illustrate the present disclosure. [Example]

[0096] Example 1: Sample preparation The sample preparation method was changed depending on the type of sample.

[0097] Samples from the leukapheresis product were prepared by adjusting the cell concentration to the desired concentration. For example, the leukapheresis product was diluted to 5 × 10 in D-PBS. 6 May be diluted to cells / ml.

[0098] Samples of non-adherent cells were prepared by pelleting and resuspending in D-PBS to the desired concentration. Samples from 2D adherent cells were prepared in much the same way, except that they may need to be dissociated from the surface using a selected dissociation reagent and then quenched. Samples of cells isolated using the EasySep™ (STEMCELL Technologies) kit can be resuspended in an appropriate amount of D-PBS or EasySep™ buffer.

[0099] Whole blood samples were used directly for nucleic acid extraction. If nucleic acids are not extracted immediately from the blood sample, it may be necessary to treat the collected whole blood with an anticoagulant such as ACD-A, heparin, or K-EDTA.

[0100] Organoids maintained in dome culture (at least 4-6 confluent domes) were used for nucleic acid extraction. Briefly, organoids within the Matrigel domes were mechanically triturated with an electronic pipette to disintegrate the domes and fragment the organoids. The resulting cells were pelleted and resuspended in RLT buffer (Qiagen), a phenol-chloroform-based solution (e.g., TRIzol), or the lysis buffer disclosed herein. Alternatively, lysis buffer containing proteinase K was added directly to the domes, incubated, and then pipetted to lyse the domes.

[0101] Liver tissue samples (e.g., mouse) were prepared by cutting harvested mouse livers into small pieces in a dish containing cold DMEM / F-12. The liver pieces were allowed to settle by gravity on ice for 2 minutes, after which the supernatant was removed. 10 ml of RT Tissue Dissociation Cocktail (STEMCELL Technologies) was added to the liver pieces and incubated in a 37°C water bath for 20 minutes.

[0102] PBMC samples were prepared from unprocessed human whole blood. To avoid loss of monocytes, EDTA was added to the whole blood sample to a final concentration of 6 mM before labeling and isolation. PBMC samples were prepared directly from human umbilical cord blood and leukoreduced samples by immunomagnetic negative selection (STEMCELL Technologies).

[0103] Plasma samples were prepared by centrifuging whole blood at 2000 x g for 10 minutes, transferring the plasma layer to a tube, and centrifuging it for an additional 10 minutes at 2000 x g. The plasma supernatant was transferred to a new tube and centrifuged at 10,000 x g for 30 minutes to remove cellular debris and large vesicles. The resulting supernatant was transferred to the required tube and used to isolate nucleic acids. For nucleic acid extraction from pan-extracellular vesicles (EVs), EVs were isolated from plasma using the appropriate EasySep™ kit (STEMCELL Technologies).

[0104] The sample prepared as above was used in an amount / volume depending on whether a microcentrifuge tube or a 96-well plate was used. For extractions using microcentrifuge tubes, 25-300 μl of sample was used. For extractions using a 96-well plate, 10-50 μl of sample was used.

[0105] Example 2: Nucleic Acid Quantification and Analysis Nucleic acids extracted according to the disclosed method (see Example 3) and by other conventional methods were tested for purity and recovery. Average purity ratios, 260 / 280 and 260 / 230, were measured using a NanoDrop2000 (ThermoFisher). Recovery (μg) of nucleic acids (DNA and RNA) was measured using a Qubit™ Fluorometer (ThermoFisher).

[0106] Example 3: Total Nucleic Acid Extraction Samples prepared essentially as described in Example 1 were subjected to the following nucleic acid extraction method. To briefly summarize the above detailed description, samples, regardless of whether microcentrifuge tubes or 96-well plates were used, were treated with a 1:1 ratio of proteinase K-containing lysis buffer. The sample was incubated for approximately 10 minutes either at room temperature or in a 56°C heat block or water bath, and the sample lysate was contacted with a fixed amount of first magnetic particles (e.g., Diluted EasySep™ Nucleic Acid Rapidspheres™, STEMCELL Technologies), optionally in alcohol, at a ratio of 1.5:1 relative to the sample volume. The sample lysate and particles were incubated for approximately 2-5 minutes at room temperature to allow nucleic acid binding (e.g., first capture). The first capture product was concentrated under the influence of a magnetic field at room temperature for approximately 2-5 minutes. After removing the supernatant, whether by pipetting or pouring, the concentrated first capture product was resuspended in a suitable elution buffer, such as TE buffer or nuclease-free water, for approximately 5 minutes at room temperature. The eluted nucleic acids were fractionated from the particles either by centrifugation or by exposing the released particles to a magnetic field.

[0107] In an improved workflow, such as for complex samples, the nucleic acids in the eluate are primed or treated by exposure to a priming buffer containing a chaotropic agent or salt and, optionally, alcohol, and then contacted with a fixed amount of second magnetic particles (e.g., Diluted EasySep™ Nucleic Acid Rapidspheres™), optionally in alcohol, at a ratio of 1.5:1 to the elution volume for approximately 2-5 minutes at room temperature (e.g., second capture). The second capture product is concentrated under the influence of a magnetic field at room temperature for approximately 2-5 minutes. The second capture product is then washed approximately 1-3 times under the magnetic field with an appropriate wash buffer, such as PBS.

[0108] The eluted first and second captures can be quantified / analyzed as described in Example 2.

[0109] Example 4: Effect of Proteinase K Dissolution Temperature on Nucleic Acid Purity and Recovery Leukocyte-depleted samples prepared as described in Example 1 were treated with lysis buffer containing proteinase K at different temperatures (room temperature or 56°C) to investigate the effect on nucleic acid purity and recovery according to Example 3.

[0110] Samples treated with proteinase K and incubated at 56°C had better 260 / 280 and 260 / 230 ratios compared to samples incubated at room temperature. Recovery of nucleic acids (total nucleic acids and DNA) also improved between samples incubated at 56°C compared to room temperature (Tables 1A and 1B). [Table 1] [Table 2]

[0111] Example 5: Effect of lysis buffer pH on nucleic acid purity and recovery PBMC samples were prepared as described in Example 1 and treated with lysis buffer at different pH levels to investigate the effect on nucleic acid purity and recovery according to Example 3.

[0112] Regardless of the pH of the lysis buffer (e.g., 4.0, 6.0, 8.0, and 10.0), comparable 260 / 280 and 260 / 230 ratios of extracted nucleic acids were obtained (Figure 1A). DNA and RNA recoveries from samples lysed at pH 4.0, 6.0, 8.0, and 10.0 were comparable (Figure 1B).

[0113] Therefore, the lysis buffer of the present disclosure, which has a pH of 4.0 to 10.0, did not impair the purity and recovery rate of the extracted nucleic acid.

[0114] Example 6: Effect of particle concentration on nucleic acid purity and recovery Unwashed cells from leukapheresis samples from three donors were prepared as described in Examples 1 and 3 and contacted with different concentrations of particles (1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, and 2.4 mg of particles per mL of sample lysate in 90% IPA). Nucleic acid extraction was then performed as described in Example 3.

[0115] Recovery generally correlated with particle concentration, whereas purity correlated inversely with particle concentration. Purity was best at particle concentrations of 1.6 mg / mL or higher in sample lysates, but purity was acceptable even at sample lysate concentrations of 2.2 mg / mL or lower (Figure 2A). Furthermore, particle concentrations of 1.2 mg / mL or higher in sample lysates were more or less equivalent (Figure 2B).

[0116] Overall, a particle concentration range of 1-2.2 mg / mL of sample lysate provides a good balance between maximizing purity and recovery, more specifically 1.2-2 mg / mL of sample lysate.

[0117] Example 7: Effect of elution temperature on nucleic acid purity and recovery Whole blood samples were prepared as described in Example 1 and bound to particles as described in Example 3. The effect of elution buffer temperature (e.g., room temperature, 37°C, or 56°C) on nucleic acid purity and recovery was investigated in a spin column protocol compared to RT elution.

[0118] Recovery generally correlated with elution buffer temperature, exceeding that of the spin column at 56°C, while 260 / 280 purity correlated inversely with elution buffer temperature. [Table 3]

[0119] Example 8: Extraction of Nucleic Acids from EasySep-Isolated EVs Pan-EVs isolated from plasma using the EasySep™ kit (STEMCELL Technologies) were prepared as described in Example 1 and conjugated to particles as described in Example 3. We investigated whether the presence of particles used in EasySep™-mediated EV enrichment was an impediment to downstream particle-mediated nucleic acid extraction, as described in Example 3.

[0120] Table 3 below shows that the recovery of DNA compared to particle-free EVs was not affected by the presence of particles in the sample prior to nucleic acid extraction. [Table 4]

[0121] Example 9: Extraction of Nucleic Acids from Organoids Hepatic organoid (formed using the kit commercialized by STEMCELL Technologies) sample was prepared as described in Example 1 and bound to particles as described in Example 3. RNA was extracted from the sample using either a conventional spin column (RNeasy, Qiagen) or the one-step particle-based EasySep™ method of the present disclosure as described in Example 3.

[0122] Additionally, different combinations of lysis buffers (phenol-chloroform-based TRIzol and the lysis buffer of the present disclosure (IH-LB)) and extraction methods (Spin vs. EasySep™) were tested.

[0123] Regardless of sample lysis conditions, the particle-mediated EasySep™ RNA extraction of the present disclosure resulted in the highest recovery compared to traditional spin column approaches (Figure 3, Table 4), and the 260 / 280 purity ratio was consistent across experiments (Table 4). [Table 5]

[0124] Example 10: Extraction of nucleic acids from mouse liver tissue. Mouse liver samples were prepared as described in Example 1 and bound to particles as described in Example 3. RNA was extracted from the samples using either a conventional spin column (RNeasy, Qiagen) ("Spin") or the one-step particle-based EasySep™ method of the present disclosure as described in Example 3. The same lysis buffer and platform combination was tested as described in Example 9.

[0125] Regardless of sample lysis conditions, the particle-mediated EasySep™ RNA extraction of the present disclosure resulted in the highest recovery compared to the traditional spin column approach (Table 5), and the 260 / 280 purity ratio was consistent across experiments (Table 5). [Table 6]

[0126] Example 11: Suitability of extracted nucleic acids for downstream applications Nucleic acids extracted according to the disclosed method were tested by downstream RT-qPCR. Extracted RNA was either treated with DNase prior to reverse transcription or untreated, and the mean Ct values ​​of target genes were similar in both conditions, regardless of the amount of reverse-transcribed input RNA (Table 6).

[0127] Therefore, DNase treatment conditions of RNA extracted using the particle-based EasySep™ nucleic acid extraction platform prior to analysis by RT-qPCR do not adversely affect analysis by RT-qPCR. [Table 7]

[0128] Example 12: Extraction of Nucleic Acids from Whole Blood Using the Improved Method of the Present Disclosure As described in Example 3, nucleic acids were extracted from whole blood using either a one-step particle-based method ("standard workflow") or a two-step particle-based method ("improved workflow") and compared to a conventional spin-column approach.

[0129] Spectrophotometric absorption spectra showed that nucleic acids extracted from complex samples according to the improved workflow were improved in both recovery and purity compared to both the spin-column and standard workflows (Table 7). Furthermore, nucleic acids from both the spin-column and improved workflows were less contaminated compared to the standard workflow (Figure 4).

[0130] Thus, the results demonstrate that the improved two-step particle-based method of the present disclosure provides optimal purity ratios and highest recoveries for nucleic acids extracted from complex biological samples. [Table 8]

[0131] Example 13: Changing the washing cycle in the two-step nucleic acid extraction method Whole blood samples were prepared essentially as described in Example 1. Recapture of nucleic acids eluted from a first particle with a second particle and recapture of eluted nucleic acids with the same particles were evaluated for purity and recovery essentially as described in Example 3.

[0132] When nucleic acids were recaptured onto fresh particles ("improved workflow"), the highest 260 / 230 purity (Figure 5A) and comparable recovery (Figure 5B) were obtained compared to recapture onto the same particles (conditions 1-4) or the spin column method. The addition of fresh particles for the second capture resulted in purity and recovery comparable to the RBC lysis method of nucleic acid extraction from whole blood (Figure 5).

[0133] The effect of the number and timing of washes on nucleic acid purity and recovery was also examined to determine whether a modified two-step approach (in which nucleic acids are recaptured by the original particles) could improve purity. The no-wash condition (condition 1) showed the highest recovery, but it also resulted in the lowest purity (Figure 5). Performing one, two, or three washes during the first capture cycle (conditions 2, 3, and 4, respectively) and three washes during the second cycle of recapture on the same particles only slightly improved purity (Figure 5A).

[0134] Thus, all particle-based approaches outperformed spin-column nucleic acid extraction from complex biological samples (e.g., whole blood) in terms of recovery, with only the no-wash control (condition 1) yielding lower purity than the spin-column extract (Figure 5B). Thus, according to the examples disclosed herein, particle-based extraction from whole blood is superior to spin columns for nucleic acid extraction from complex samples. Best results were obtained using fresh particles in each step of the disclosed two-step capture and release EasySep™ protocol.

[0135] Example 14: Priming buffer required to recapture nucleic acids onto secondary particles Nucleic acid recovery was tested in the methods described in Examples 12 and 13 in the presence or absence of a priming buffer (prepared to match the lysis buffer of the present disclosure) during the first elution (before the second capture).

[0136] Addition of priming buffer to the first eluate significantly increased nucleic acid recovery compared to when the second capture was performed in the absence of priming buffer (Figure 6).

[0137] Example 15: Purity and Recovery in Spin Column and Two-Step Nucleic Acid Extraction Whole blood samples were prepared as described in Example 1 and subjected to column-based nucleic acid extraction (RNEasy) and one-step or two-step particle-based EasySep™ approaches ("standard" or "improved" workflows) as described in Examples 3, and 12-14.

[0138] The purity of nucleic acids (260 / 230 ratio) was highest using the improved workflow and the column-based workflow (Figure 7A), with the improved workflow being slightly more pure. The normalized recovery of nucleic acids (µg / 10 6 cells) were higher for both DNA (Figure 7B) and RNA (Figure 7C) compared to the recoveries using the spin column approach.

Claims

1. 1. A method for extracting nucleic acids from a biological sample, comprising: i) preparing a sample lysate by treating the biological sample with a lysis buffer; ii) incubating the sample lysate at a temperature equal to or greater than room temperature (RT) and less than 60°C; iii) adding particles to the sample lysate to bind the nucleic acids; iv) separating the particles and the nucleic acids bound thereto from the sample lysate; and v) contacting the particles and the nucleic acid bound thereto with a lysis buffer to release the nucleic acid from the particles.

2. 2. The method of claim 1, wherein the purity and / or recovery of the released nucleic acids is higher when the biological sample of step i) is incubated at a temperature above room temperature compared to when the biological sample is at room temperature.

3. 3. The method of claim 1, wherein the pH of the lysis buffer is about 4 to 10.

4. 4. The method of any one of claims 1 to 3, further comprising preheating the lysis buffer to a temperature above room temperature but below 60°C.

5. 5. The method of claim 4, wherein the temperature of the lysis buffer is between 30°C and 60°C.

6. 6. The method of claim 4 or 5, wherein pre-heating the elution buffer improves the purity and / or recovery of the released nucleic acids compared to the purity and / or recovery of the nucleic acids released in room temperature elution buffer.

7. 7. The method of any one of claims 1 to 6, wherein the particles are added to the sample lysate at a concentration greater than 1 mg / mL of the sample lysate and less than 2.2 mg / mL of the sample lysate.

8. 8. The method of claim 7, wherein the concentration of the particles is from about 1.2 mg / mL of the sample lysate to 2 mg / mL of the sample lysate.

9. The method of any one of claims 1 to 8, wherein the biological sample is whole blood, suspended cells, isolated cells, PBMCs, liver tissue, extracellular vesicles, leukapheresis products, single cell suspensions, organoids, plasma, and viruses.

10. The method according to any one of claims 1 to 9, wherein the biological sample is a complex sample.

11. The method according to any one of claims 1 to 10, wherein the biological sample is pre-treated or not pre-treated.

12. The biological sample is 10 １ ~10 ９ The method of any one of claims 1 to 11, comprising cells.

13. 13. The method of any one of claims 1 to 12, further comprising directly subjecting the released nucleic acid to one or more downstream applications.

14. 14. The method of any one of claims 1 to 13, further comprising adding fresh particles to the released nucleic acids to recapture the released nucleic acids.

15. 15. The method of claim 14, further comprising releasing the recaptured nucleic acid.

16. 16. The method of claim 14 or 15, wherein the method does not include washing the particles and the nucleic acids bound thereto before contacting them with the elution buffer.

17. 17. The method of any one of claims 14 to 16, wherein the purity and / or recovery of the recaptured nucleic acid released from the fresh particles is higher than the purity and / or recovery of the nucleic acid released in step v).

18. 18. The method of any one of claims 1 to 17, further comprising separating the particles from a supernatant containing the released nucleic acids by aspirating or pouring.

19. The method of any one of claims 1 to 18, wherein the magnetic particles are silica-based.

20. The method of any one of claims 1 to 19, wherein the method lasts for 30 to 45 minutes.

21. 21. The method of any one of claims 1 to 20, wherein the particles are magnetic particles, and the magnetic particles and the nucleic acids bound thereto are separated from the sample lysate by applying a magnetic field to the sample lysate.

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