Method 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, such as the use of magnetically or magnetically responsive particles. The methods and kits of the present disclosure can be used to extract nucleic acids from simple to complex biological samples with high purity and recovery compared to conventional methods such as using centrifugal columns. The methods and kits of the present disclosure may be used in high throughput extraction or may be suitable for use in high throughput applications.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 432,862, filed on December 15, 2022, the entire content of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to nucleic acid extraction, and 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 Art

[0004] Methods and / or kits for separating nucleic acids are commercially available. However, many such methods and kits fail to effectively separate nucleic acids from a wide range of biological samples, including simple and complex samples. Spin - column - based nucleic acid extraction methods, while perhaps being preferred by convention, often do not scale well and result in low nucleic acid purity and recovery. Particle - based methods for extracting nucleic acids are not designed for a wide range of biological samples and perform poorly when extracting nucleic acids from more complex samples such as whole blood, organoids, and whole organs, typically having low purity and recovery.

[0005] In the diagnostic and medical fields, there is a need for reliable and effective kits and methods to separate nucleic acids with optimal purity and recovery from a wide range of biological samples, such as from simple and complex biological samples, and the kits and methods are suitable for effectively extracting nucleic acids from a large number of samples in a relatively short period of time. Nucleic acid extraction methods suitable for automation would also provide great benefits in such environments.

[0006] Accordingly, there is a particular need for improved kits and methods for extracting nucleic acids from a wide range of biological samples, such as simple samples like single - cell suspensions and complex samples like whole blood or tissues. Summary of the Invention

[0007] The present disclosure relates to kits and / or methods for extracting nucleic acids from biological samples.

[0008] In one aspect of the present disclosure, a method for extracting nucleic acids from a biological sample is provided. The method of the present disclosure may include: i) preparing a sample lysate by treating the biological sample with a lysis buffer; ii) incubating the sample lysate at a temperature at or above room temperature and below 60 °C; iii) adding particles to the sample lysate to bind nucleic acids; iv) separating the particles and the nucleic acids bound thereto from the sample lysate; and v) contacting the particles and the nucleic acids bound thereto with an elution buffer to release the nucleic acids from the particles.

[0009] In one embodiment, the particles are magnetic particles and the magnetic particles are separated from the lysate in the presence of a magnetic field.

[0010] In one embodiment, when the biological sample of step i) is incubated at a temperature higher than room temperature, the purity and / or recovery rate of the released nucleic acid is higher than that of the released nucleic acid when incubated at room temperature.

[0011] In one embodiment, the pH of the lysis buffer is between 4 and 10. In one embodiment, when the pH of the lysis buffer is in the range between 6 and 10, the purity of the eluted nucleic acids is not compromised.

[0012] In one embodiment, the methods of the present disclosure may further comprise preheating the elution buffer to a temperature above room temperature (RT) and below 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 increases the purity and / or recovery of the released nucleic acids compared to the purity and / or recovery of nucleic acids released in the room temperature elution buffer.

[0013] In one embodiment, the particles are added to the sample lysate at a concentration greater than 1 mg / mL sample lysate and less than 2.2 mg / mL sample lysate. In one embodiment, the concentration of the particles is between about 1.2 mg / mL sample lysate and 2 mg / mL sample lysate.

[0014] 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.

[0015] In one embodiment, the biological sample is a complex sample.

[0016] In one embodiment, the biological sample is pre-treated. In one embodiment, the biological sample is not pre-treated.

[0017] In one embodiment, the biological sample comprises 10 1 to 10 9 cells.

[0018] In one embodiment, the methods of the present disclosure may further comprise subjecting the released nucleic acids directly to one or more downstream applications.

[0019] In one embodiment, the method of the present disclosure may further comprise adding fresh particles to the released nucleic acids to recapture the released nucleic acids.

[0020] In one embodiment, the method of the present disclosure may further include releasing the recaptured nucleic acid using the elution buffer of the present disclosure.

[0021] In one embodiment, the method does not involve washing the particles and the nucleic acid bound thereto before contacting the particles and the nucleic acid bound thereto with the elution buffer.

[0022] In one embodiment, the method does not involve washing the first particle and / or the second particle and the nucleic acid bound thereto.

[0023] In one embodiment, the purity and / or recovery rate of the recaptured nucleic acid released from the fresh particles is higher than the purity and / or recovery rate of the nucleic acid released in step v) above.

[0024] In one embodiment, the method of the present disclosure may further include fractionating the particles and the supernatant containing the released nucleic acid by suction or pouring.

[0025] In one embodiment, the particles are silica-based.

[0026] In one embodiment, the method takes between 30 and 45 minutes.

[0027] In another aspect of the present disclosure, there is provided a kit for extracting nucleic acid from a biological sample. The kit of the present disclosure may include a lysis buffer (as described herein), one or more vials containing particles (as described herein), a wash 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).

[0028] Based on the following detailed description, other features and advantages of the presently described subject matter will become apparent. However, it should be understood that although the preferred embodiments of the described subject matter are indicated, the detailed description and the specific examples 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To better understand the various embodiments described herein and to more clearly show how these various embodiments may be implemented, reference will be made, by way of example, to the accompanying drawings, which show at least one exemplary embodiment and are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0030] Figure 1 shows a bar graph of the purity and recovery of nucleic acids extracted from leukopak PBMCs when the pH of the lysis buffer used in the method of the present disclosure varies between 4 and 10. The purity of the extracted DNA (salt and protein contaminants) was evaluated from the 260 / 280 and 260 / 230 absorbance ratios obtained from a spectrophotometric instrument (A). The amount of DNA and RNA (μg) extracted per 1 x 10 6 leukopak PBMCs as measured using a Qubit fluorometer (B). Error bars represent the mean ± S.D. of 2 technical replicates.

[0031] Figure 2 shows box plots of the purity and recovery of nucleic acids extracted from unwashed leukopak cells from three human donors when only the concentration of magnetic particles in 90% isopropanol varies in the method of the present disclosure: 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, and 2.4 mg / mL sample lysate. The 260 / 230 absorbance ratio evaluated the purity of the extracted nucleic acids (salt and protein contaminants) (A). The amount of nucleic acids extracted per 1 x 10 6 leukopak cells (in ng / μL) as measured using a Qubit fluorometer (B).

[0032] Figure 3 Compare the recovery of RNA extracted from samples of liver organoids using a conventional spin column (“Spin”) or the exemplary particle-based workflow of the present disclosure (“EasySep TM ”). Liver organoid lysates were prepared using a phenol-chloroform-based reagent (TRIzol) or the lysis buffer of the present disclosure (IH-LB). Data points represent three technical replicates.

[0033] Figure 4 Shows the UV spectrophotometric absorption spectra of nucleic acids extracted from whole blood samples using a standard workflow, a spin column workflow, and an improved workflow according to an exemplary embodiment of the present disclosure.

[0034] Figure 5 compares the purity and recovery of nucleic acids extracted from whole blood samples using the two-step particle-based workflow of the present disclosure (“improved workflow”). The control nucleic acid extraction workflow included pre-lysed RBC samples and a spin column-based method. Conditions 1-4 included re-capturing the eluted nucleic acids onto the same particles as in the “improved workflow”, but with different numbers of wash steps before elution and re-capture, as follows: no wash (Condition 1), 1 wash (Condition 2), 2 washes (Condition 3), and 3 washes (Condition 4). The purity of the re-captured nucleic acids (260 / 230) was determined by UV spectrophotometry (A), and the amount per 1 x 10 6Recovery amount (μg) of nucleic acid extracted from individual input cells. n = 1, in duplicate.

[0035] Figure 6 Shows the UV spectrophotometric absorption spectra of nucleic acids extracted from whole blood samples according to the two-step method of the present disclosure, in the presence or absence of primer buffer, for the first eluate of nucleic acids to prime before recapturing the nucleic acids by freshly added particles.

[0036] Figure 7 shows a bar graph comparing the purity and recovery of nucleic acids extracted from whole blood samples using the two-step method of the present disclosure (“improved”), a standard one-step bead-based workflow (e.g., single bead addition) (“standard”), and conventional spin column extraction. The purity (salt and protein contaminants) of the extracted nucleic acids was evaluated by the 260 / 230 absorbance ratio obtained from a spectrophotometric instrument (A). n = 3. Error bars represent ±1 standard deviation. Amounts (μg) of DNA (B) and RNA (C) extracted per 1 x 10 6 cells as measured using a Qubit fluorometer. n = 7. Error bars represent ±1 standard deviation. Detailed Description

[0037] 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 range of biological samples (such as samples ranging from simple to more complex samples) by practicing the disclosed methods and / or by using the kits. Still more specifically, the present disclosure relates to high-throughput extraction of nucleic acids from a range of biological samples (such as samples ranging from simple to more complex samples).

[0038] As used in the present disclosure, the term “nucleic acid” refers to linear, branched, or circular DNA and RNA. Examples of RNA may include mRNA, siRNA, miRNA, snRNA, tRNA, hnRNA, ribozymes, viral RNA, or extracellular or cell-free RNA. Examples of DNA may include plasmid DNA, genomic DNA, mitochondrial DNA, viral DNA, and extracellular or cell-free DNA.

[0039] As used in the present disclosure, the term "particle" refers to a particle that reversibly binds nucleic acids such as DNA and / or RNA. In a specific embodiment, the particle is a magnetic particle or a magnetoresponsive particle, and may more specifically be referred to as a ferromagnetic, ferrimagnetic, paramagnetic, or superparamagnetic particle. The magnetic or magnetoresponsive particle may comprise a magnetic-rich core encapsulated by a polymer shell. Any particle in the present disclosure, including magnetic particles or magnetoresponsive particles, is commercially available and will be well 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, alkyl silica, aluminosilicate, borosilicate, solid silica such as glass or diatomaceous earth, glass fibers, or a mixture of two or more of the foregoing. The silica should be present at least partially on the surface of the bead. For effective binding, it is preferred that the silica is present on all or substantially most (e.g., >50%, >60%, >70%, >80%, >90%, or >95%) of the particle surface. In one embodiment, the siliceous oxide-coated (magnetic) particle also has an adsorbed surface of hydrated siliceous oxide. The target nucleic acid such as DNA or RNA adheres to the adsorbed surface of the particle, while the binding of other materials from the sample lysate, especially harmful contaminants such as nucleases, may be restricted.

[0040] In one embodiment, the particles of the present disclosure (e.g., magnetic particles) act 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, such as not modified with functional groups. In one embodiment, the surface of the solid phase (e.g., silica solid phase) is modified, such as modified with functional groups. In one embodiment, the solid phase may carry an anion exchange functional group capable of binding the nucleic acid of interest.

[0041] As used in the present disclosure, the term "chaotropic agent" refers to an agent that disrupts the hydrogen-bonding network in an aqueous solution, which can destabilize the native state of macromolecules (e.g., proteins, nucleic acids) in the solution. The chaotropic agent denatures nucleic acid-related proteins, thereby weakening the hydrophobic interaction between such proteins and nucleic acids. Non-limiting examples of chaotropic agents include guanidine thiocyanate, guanidine, urea, and thiourea. Preferably, the chaotropic agent is a guanidine salt (e.g., guanidine hydrochloride, guanidine thiocyanate, and / or guanidine isothiocyanate). In one embodiment, the concentration of the chaotropic agent in a buffer (such as a lysis buffer) may be in the range of ≥0.1 M to ≤10 M, ≥1 M to ≤8 M, ≥3 M to ≤7 M, or ≥4 M to ≤6 M.

[0042] As used in the present disclosure, the term "purity" or "purity ratio" refers to the purity of the extracted nucleic acid as can be evaluated from the 260 / 280 and 260 / 230 absorbance ratios. The acceptable range for the A260 / A280nm ratio of pure DNA is 1.7 - 2.0, while the acceptable range for the A260 / 280 ratio of pure RNA is 1.9 - 2.2. If the 260 / 280 ratio is significantly lower (≤1.6), it may indicate the presence of proteins, phenols, or other contaminants that absorb at or near 280 nm. For each of DNA and RNA, the A260 / A230 value is expected to generally be in the range of 1.8 - 2.2. If the 260 / 230 ratio exceeds the foregoing range, it 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.

[0043] As used in the present disclosure, the term "recovery rate" refers to the amount or concentration (e.g., in ng or in μg) of the extracted or separated nucleic acid (such as DNA and / or RNA), and can be expressed relative to the number of input cells. The nucleic acid recovery rate is typically measured using a spectrophotometer, a microspectrophotometer, or a fluorometer.

[0044] Method

[0045] In one aspect of the present disclosure, a method for extracting nucleic acid from a biological sample is provided. In an embodiment, the method for extracting nucleic acid from a biological sample can be carried out on a wide range of biological samples (such as simple and complex samples). In an embodiment, the method for extracting nucleic acid from a biological sample can be carried out on a wide range of biological samples (such as simple and complex samples) with substantially no sacrifice of nucleic acid purity and / or recovery rate.

[0046] The biological samples of the present disclosure are not particularly limited, provided that they contain nucleic acid. Thus, the biological samples can be of prokaryotic, viral, or eukaryotic origin. In a specific embodiment, the biological sample is of mammalian origin. The biological sample can be fresh, frozen, dried, or cryopreserved. The methods (and kits) of the present disclosure can be carried out on a wide range of biological samples, ranging from relatively simpler samples to relatively more complex samples.

[0047] As used in the present disclosure, the term "simple biological sample" can refer to any sample that is simple in its composition in terms of the presence / concentration of proteins, carbohydrates, lipids, chemicals, or other contaminants and / or requires minimal or no pre-treatment or special treatment before nucleic acid extraction. Examples of simple biological samples include, but are not limited to, (enriched) cells suspended in a buffer or medium (such as a single cell suspension), plasma, leukapheresis products (such as PBMC), etc.

[0048] As used herein, the term "complex biological sample" may refer to any sample that is complex in its composition in terms of the presence / concentration of proteins, carbohydrates, lipids, chemicals, or other contaminants and / or requires pretreatment or special handling prior to nucleic acid extraction. It can be seen that the concepts of simple and complex biological samples are relative terms, with the latter containing more contaminants than the former, particularly those that may affect purity and / or recovery in particle-based nucleic acid extraction as would be understood by one of ordinary skill in the art. Complex samples may contain few or many contaminants, together at relatively high concentrations. Examples of contaminants in simple and / or complex samples can include salts, proteins, cells, and endogenous and / or exogenous small organic molecules (such as lipids, amino acids). In particular, the source of contaminants may come from non-nucleic acid cellular material, such as proteins, lipids, carbohydrates, or other macromolecules, intact cells, or cell debris. Thus, relatively high concentrations of such contaminants may reduce the efficiency of nucleic acid extraction, particularly by reducing purity and recovery. 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, sputum, bile, etc.), tissue samples (e.g., tissue biopsies, skin biopsies, muscle biopsies, or lymph node biopsies), whole organs (e.g., liver), homogenized tissue samples, urine, CSF, BAL, SF, semen, feces, sputum (e.g., purulent sputum or bloody sputum), organoids, and the like.

[0049] 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 can be on the order of 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 or 10 9 。

[0050] In one embodiment, the biological sample is whole blood, which can be considered a complex sample as it contains RBCs, WBCs, platelets, plasma, and their respective cytosol, macromolecules, and small molecules. In one embodiment, the whole blood contains an anticoagulant.

[0051] In one embodiment, the biological sample is a sample of PBMCs or a leukapheresis product. The PBMC sample may contain relatively low levels or no contaminating RBCs, granulocytes, and / or platelets. In one embodiment, the biological sample is plasma.

[0052] In one embodiment, the biological sample comprises a virus and / or a prokaryote. In one embodiment, the biological sample comprises soil and / or plant material.

[0053] In one embodiment, the biological sample is a cell suspension in a suitable buffer or medium. Separation or enrichment methods such as immunomagnetic separation, centrifugation (e.g., density gradient centrifugation), or filtration can be used to separate or enrich the cell suspension.

[0054] In one embodiment, the biological sample is a leukapheresis product. In one embodiment, the leukapheresis product is adjusted with a suitable buffer to obtain the desired cell concentration for nucleic acid extraction.

[0055] In one embodiment, the biological sample is an organoid or contains organoids. The organoids may be contained in or on an extracellular matrix (e.g., a Matrigel dome), so the organoid sample may contain one or more extracellular matrix proteins. If the organoids are contained in or on the extracellular matrix, their complexity can be disrupted by incubation in a lysis buffer (e.g., a phenol-chloroform solution or an enzyme solution) and / or mechanical grinding using a pipette.

[0056] In one embodiment, the biological sample is an entire organ or a fragment of an organ. The complexity of the organ or fragment can be disrupted (e.g., dissociated or homogenized) by one or more of chopping, mechanical grinding, incubation in an enzyme solution, or other means.

[0057] Accordingly, the present disclosure relates to a method for extracting nucleic acids from any available biological sample, provided that the sample contains nucleic acids to be extracted and / or analyzed.

[0058] The method of the present disclosure may include preparing a sample lysate by treating the biological sample with a lysis buffer. Many lysis buffers are known in the art. The sample lysate can be prepared in a lysis buffer comprising one or more of the following:

[0059] a) Proteinase K, such as at a concentration between 10 - 100 μl / ml of the sample, to cleave peptide bonds and digest proteins.

[0060] b) One or more detergents (e.g., SDS, Tween-20, and Sarkosyl), such as at a concentration between 3% and 20%, to lyse cells (release soluble proteins) by solubilizing membrane proteins and lipids.

[0061] c) High osmolarity or osmolality to impart an osmotic pressure to the cells and cause water to enter the cells, thereby increasing their internal pressure and causing cell lysis.

[0062] d) One or more buffering agents to maintain the 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 thereof.

[0063] e) A binding inducer, such as a chaotropic agent or chaotropic salt, which can be provided alone or in combination with a binding additive (such as an alcohol (e.g., ethanol or isopropanol)).

[0064] In one embodiment, the sample lysate is prepared in a lysis buffer that does not contain proteinase K.

[0065] The pH of the lysis buffer can be in the range between 3 and 12 or between about 4 and 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, when the pH of the lysis buffer falls within the range between 3 and 12, or more preferably between about 4 and 10, the purity of the released nucleic acid is not impaired.

[0066] The sample lysate will contain nucleic acids as well as other cellular components. Examples of other cellular components can include cell debris 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 biological sample or a simple biological sample, as defined above.

[0067] The sample lysate can be prepared by heating the treated sample (or lysis buffer) to a temperature that is at or above room temperature and below 60°C. In one embodiment, the sample lysate is prepared by heating the treated 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 treated 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 treated sample (or lysis buffer) to a temperature preferably about 56°C ± 3°C. In one embodiment, the sample lysate is prepared by heating the treated sample (or lysis buffer) to a temperature about room temperature ± 3°C.

[0068] A sample lysate can be prepared by incubating at the aforementioned temperature range or in a lysis buffer heated to the aforementioned temperature range. The sample lysate can be incubated for any amount of time, provided that the nucleic acid does not begin to significantly degenerate. In one embodiment, the sample lysate is prepared by incubating at a given temperature (as described above) for between about 3 - 30 minutes, between about 5 - 20 minutes, or between 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.

[0069] The methods of the present disclosure include adding particles (such as magnetic particles) to the 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 become complexed or bound to the particles. In one embodiment, a volume of particles is added in a ratio approximately the same as the sample volume (e.g., at a 1:1 ratio). In one embodiment, a volume of particles is added in a ratio different from the sample volume (e.g., 0.5:1 or 1.5:1, or 1:0.5 or 1:1.5). In one embodiment, the incubation is carried out at room temperature. In one embodiment, the incubation is carried out for about 5 minutes. In one embodiment, the incubation is less than 5 minutes. In one embodiment, the incubation is about 3 minutes. In one embodiment, the incubation is about 1 minute.

[0070] In addition to binding nucleic acids, the particles can also non - specifically bind cell components other than nucleic acids. In one embodiment, the particles can non - specifically bind proteins or other macromolecules in the sample lysate (such as a cell lysate). In one embodiment, the particles can non - specifically bind hemoglobin in the sample / cell lysate. The nature of the contaminants in the sample lysate and thus the substances that can non - specifically bind to the particles can determine whether the biological sample is a complex biological sample or a simple biological sample.

[0071] The methods of the present disclosure can include separating the particles and the nucleic acids bound thereto to enrich the nucleic acids in the sample lysate from the proteins and other debris in the sample lysate. The separation can occur in any manner capable of separating / isolating the particles. As a non - limiting example, the particles can be centrifuged to form a pellet, or if the particles are magnetic particles, they can be exposed to a magnetic field to collect on the surface of the container closest to the magnetic field.

[0072] In a specific embodiment where the particles are magnetic particles, the particles and the nucleic acid bound thereto 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 a magnetic field for one minute or longer to maximize the co-localization of the particles, such as co-localization against the wall of the container / tube closest to the magnet / magnetic field. In one embodiment, the incubation is carried out at room temperature. In one embodiment, the incubation is carried out for about 5 minutes. In one embodiment, the incubation is less than 5 minutes. In one embodiment, the incubation is about 3 minutes. In one embodiment, the incubation is about 1 minute.

[0073] After collecting the particles (e.g., magnetic particles) on or along the surface of the container (closest to the magnetic field), the supernatant can be removed. Importantly, the container is kept 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 pipette. In one embodiment, the supernatant can be poured off.

[0074] Once the particles have been fractionated from the supernatant, it may be important to wash the particles to remove contaminants entrapped in the particles and / or contaminants that interact weakly or bind to the particles. In one embodiment, the pellets are washed with a wash buffer such as Buffer RW1 (Qiagen), AW1 (Qiagen), PBS, EasySep TM 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., C1-5 alcohol). In one embodiment, the wash buffer contains ethanol. In one embodiment, the wash buffer contains 40%-80% ethanol or isopropanol.

[0075] Any number of wash cycles can be performed to increase purity; however, increasing the number of washes may come at the expense of recovery. In one embodiment, the pellets are washed once with the wash buffer. In one embodiment, the pellets are washed twice with the wash buffer. In one embodiment, the pellets are washed three times with the wash buffer. In one embodiment, the pellets are washed more than three times with the wash buffer, such as five or more times.

[0076] The pH, solute composition, and concentration of the wash buffer solution can vary depending on the type of impurities expected to be present.

[0077] The methods of the present disclosure may include contacting the particles and the nucleic acid bound thereto with an elution buffer to release the nucleic acid from the particles. The elution buffer is known and the elution efficiency may be affected by pH when eluting from a silica matrix (such as may be included on the particles of the present disclosure). The elution buffer of the present disclosure may comprise one or more of the following: a low salt concentration; TE buffer or about 10 - 100 mM Tris-HCl; a pH of about 8.0 or 8.5; and about 0.1 - 1 mM EDTA. Effective elution of the nucleic acid from the particles can be achieved by incubating the particles in water or a buffer with low ionic strength. In one embodiment, distilled water or deionized water or nuclease-free water is used to elute the nucleic acid bound to the particles.

[0078] The methods of the present disclosure may further include preheating the elution buffer before contacting the particles and the nucleic acid bound thereto with the elution buffer. In one embodiment, the elution buffer is preheated to a temperature at or 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 methods of the present disclosure further include incubating the particles and the nucleic acid bound thereto at any of the foregoing temperatures after contacting the particles and the nucleic acid bound thereto with the elution buffer.

[0079] Preheating the elution buffer as described above (or incubating the particles in the elution buffer at the foregoing temperatures) can improve the purity and / or recovery of the released nucleic acid. In one embodiment, preheating the elution buffer improves the purity of the released nucleic acid compared to the purity of the nucleic acid released in an elution buffer at room temperature. In one embodiment, preheating the elution buffer improves the recovery of the released nucleic acid compared to the recovery of the nucleic acid released in an elution buffer at room temperature.

[0080] In one embodiment, the method may include preferentially releasing nucleic acids from the particles. Preferential elution may refer to those conditions under which nucleic acids are substantially or completely released from the particles while other cellular components 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 different embodiments, 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.

[0081] In one embodiment, the methods of the present disclosure do not involve washing prior to the elution step. In one embodiment, the method does not involve washing the particles and the nucleic acids bound thereto. In one embodiment, the method involves minimal washing of the particles and the nucleic acids bound thereto, such as only a single wash.

[0082] The methods of the present disclosure may further include separating the particles and the supernatant containing the released nucleic acids. The particles and the released nucleic acids (in the supernatant) may be separated by aspiration or decanting. The particles and the supernatant may be separated in any manner that enables the particles to be co-localized, such as by centrifuging to precipitate the particles, or by maintaining the particles in the presence of a magnetic field (if magnetic) during the separation process. In one embodiment, the separation occurs in the presence of a magnet or magnetic field, and the supernatant (e.g., the fraction containing the nucleic acids) is transferred to a separate container or tube. Thus, the supernatant or eluate may be referred to as the first eluate.

[0083] The methods of the present disclosure 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 methods of the present disclosure include adding fresh particles to the released and separated nucleic acids of the first eluate. The freshly 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 (in terms of, for example, the relative volumes of the particles and the first eluate, the incubation time, the incubation temperature, etc.). In one embodiment, the volume of the fresh particles added to the first eluate includes an alcohol at a concentration such as 40%, 50%, 60%, 70% or higher.

[0084] Prior to re-binding / re-capturing the nucleic acids in the first eluate to fresh / second particles, such as in the two-step extraction of the present disclosure, the nucleic acids can be primed for re-capture. The priming buffer can alter the conditions of the first eluate to facilitate re-capture of the nucleic acids, for example, by restoring salt concentration, pH, conformation, etc.

[0085] The nucleic acids in the first eluate can be contacted with a priming buffer, such as a buffer comprising one or more of the following: a chaotropic agent or salt; an alcohol (e.g., at a high concentration); a cell lysing agent (as described above); and a binding additive, such as an alcohol. In one embodiment, priming includes supplementing the first eluate with the priming buffer, for example, at a ratio of 1:1 or 1:1.5. 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.

[0086] In one embodiment, the nucleic acids in the first eluate are primed (in the priming buffer) during contact with the second particles. The first eluate so treated or primed prior to re-binding (eluting) the nucleic acids to the second particles can be referred to as a treated or primed sample.

[0087] The method of the present disclosure can also include separating the second particles and the nucleic acids bound thereto, substantially 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 may be carried over from a complex biological sample into the first eluate; or concentrate the nucleic acids, regardless of whether the biological sample is simple or complex.

[0088] After the second particles and the nucleic acids of the first eluate have been separated from the remainder of the first eluate, washing can be performed, substantially as described above. In one embodiment, the method of the present disclosure does not involve washing prior to the second elution step.

[0089] As described above with respect to the first eluate, the method of the present disclosure can also include contacting the second particles and the nucleic acids bound thereto with an elution buffer. The nature of the elution buffer (e.g., components, temperature, etc.) and the duration of the contacting step are substantially as described above.

[0090] After releasing the re-captured nucleic acids from the fresh / second particles, the method of the present disclosure can also include fractionating the second particles and the supernatant containing the nucleic acids, substantially as described above.

[0091] The particles of the present disclosure are not particularly limited as many suppliers have commercialized particles for nucleic acid extraction. Nucleic acids can bind to the first (magnetic) particle and / or the second (magnetic) particle through various binding mechanisms. For example, the binding mechanism between the nucleic acid and the (magnetic) particle can be through electrostatic / ionic interactions. Alternatively, in one embodiment, the binding mechanism between the nucleic acid and the (magnetic) particle can be through hydrophobic interactions. Alternatively, the binding mechanism between the nucleic acid and the (magnetic) particle can be through hydrogen bond / dipole bond interactions.

[0092] The particles (e.g., the first particle and / or the second particle), whether magnetic or otherwise, can be added to the sample lysate at any desired concentration. The concentration of the first particle and / or the second particle can be determined based on the volume of the sample lysate. In one embodiment, the concentration of the first particle and / or the second particle ranges between 0.1 μg / mL and 1 μg / mL of the sample lysate. In one embodiment, the concentration of the first particle and / or the second particle ranges between about 1 μg / mL and 10 μg / mL of the sample lysate, between about 1 μg / mL and 5 μg / mL of the sample lysate, or between about 1 μg / mL and 3 μg / mL of the sample lysate. In one embodiment, the first particle and / or the second particle is concentrated EasySep TM TotalNucleic Acid Rapidspheres TM (STEMCELL Technologies), and if concentrated, the particles may need to be diluted in the sample lysate or before adding them to the sample lysate.

[0093] In one embodiment, the particles are added to the sample lysate at a concentration higher than 1 mg / mL of the sample lysate and lower than 2.2 mg / mL of the sample lysate. In one embodiment, the concentration of the particles ranges between about 1.2 mg / mL and 2 mg / mL of the sample lysate. In one embodiment, the particles are added at a concentration ranging between 0.5 and 3 mg per mL of the sample lysate, or between 0.75 and 2.75 mg, or between 1 and 2.5 mg. In embodiments, the concentration of the particles added to the sample lysate is about 1.2 mg / mL of the sample lysate, or about 1.4 mg / mL of the sample lysate, or about 1.6 mg / mL of the sample lysate, or about 1.8 mg / mL of the sample lysate, or about 2.0 mg / mL of the sample lysate, or about 2.2 mg / mL of the sample lysate.

[0094] In embodiments, the nucleic acids of the first eluate and / or the second eluate may undergo further processing, such as treatment with an RNase solution (to produce purified or substantially purified DNA) or treatment with a DNase (to produce purified or substantially purified RNA). In embodiments, the first eluate and / or the second eluate may be aliquoted into separate containers, each treated with DNase and RNase, respectively. In one embodiment, no further processing is performed on the nucleic acids in the first eluate and / or the second eluate.

[0095] The methods disclosed herein, whether one-step particle addition or two-step particle addition, result in high nucleic acid purity (e.g., within an optimal A260 / 280 and / or A230 / 260 range). In one embodiment, the purity of the nucleic acids released from the second particle is higher than the purity of the nucleic acids released from the first particle. In one embodiment, the purity of the nucleic acids released from the second particle and the purity of the nucleic acids released from the first particle are both within the optimal A260 / 280 and / or A230 / 260 range. In one embodiment, the purity of the nucleic acids released from the second particle is comparable to the purity of the nucleic acids released from the first particle (e.g., purities within about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±12.5% or ±15% of each other).

[0096] The methods disclosed herein, whether one-step particle addition or two-step particle addition, can result in higher nucleic acid purity than nucleic acids eluted from conventional spin columns. In one embodiment, the purity of the nucleic acids released from the first particle and / or the second particle and the purity of the nucleic acids eluted from the conventional spin column are both within the optimal A260 / 280 and / or A230 / 260 range. In one embodiment, the purity of the nucleic acids released from the first particle and / or the second particle is within the optimal A260 / 280 and / or A230 / 260 range, and the purity of the nucleic acids eluted from the conventional spin column is not within the optimal A260 / 280 and / or A230 / 260 range. In one embodiment, the purity of the nucleic acids released from the first particle and / or the second particle is comparable to the purity of the nucleic acids eluted from the spin column (e.g., purities within about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±12.5% or ±15% of each other).

[0097] The two-step method of the present disclosure can produce nucleic acids with higher purity than those released from standard (one-step) particle-based workflows, whether commercially available or as disclosed herein. In one embodiment, the purity of the nucleic acids released from the second particle and the purity of the nucleic acids released from the first particle are both within the optimal A260 / 280 and / or A230 / 260 ranges. In one embodiment, the purity of the nucleic acids released from the second particle is within the optimal A260 / 280 and / or A230 / 260 ranges, and the purity of the nucleic acids released from the first particle is not within the optimal A260 / 280 and / or A230 / 260 ranges. In one embodiment, the purity of the nucleic acids released from the second particle is comparable to the purity of the nucleic acids released from the first particle (e.g., purities within about ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±12.5% or ±15% of each other).

[0098] The improvement in nucleic acid purity can be applicable to the extraction from a wide range of biological samples, from simple to complex samples. In one embodiment, using the nucleic acid extraction method disclosed herein to improve nucleic acid purity is particularly applicable to the extraction from complex biological samples (such as those containing high levels of contaminants) without pretreatment to reduce sample complexity.

[0099] The methods disclosed herein, whether one-step particle addition method or two-step particle addition method, produce high nucleic acid recovery rates (e.g., within 98%, or 95%, or 90%, or 85%, or 80%, or 75%, or 70%, or 65%, or 60%, or 55% or 50% of the theoretical yield). In one embodiment, the recovery rate of the nucleic acids released from the second particle is higher than the recovery rate of the nucleic acids released from the first particle (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 times higher). In one embodiment, the recovery rate of the nucleic acids released from the second particle is comparable to the recovery rate of the nucleic acids released from the first particle (e.g., within about ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, ±40% or ±50% of each other).

[0100] The methods disclosed herein, whether one-step particle addition or two-step particle addition, can produce higher nucleic acid recovery rates than elution from conventional spin columns (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). In one embodiment, the recovery rate of nucleic acids released from the first particle and / or the second particle is comparable to the recovery rate of nucleic acids eluted from a spin column (e.g., within about ±1%, ±3%, ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, ±35%, ±40%, ±45%, or ±50% of each other).

[0101] The two-step method disclosed herein can produce higher nucleic acid recovery rates than the recovery rates of nucleic acids released from standard (one-step) particle-based workflows, whether commercially available or as disclosed herein (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). In one embodiment, the recovery rate of nucleic acids released from the second particle is comparable to the recovery rate of nucleic acids released from the first particle (e.g., within about ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, ±35%, ±40%, ±45%, or ±50% of each other).

[0102] The increased nucleic acid recovery rate can be applicable to extraction from a wide range of biological samples (from simple to complex samples). In one embodiment, using the nucleic acid extraction method disclosed herein to increase nucleic acid recovery rate is particularly applicable to extraction from complex biological samples (such as those containing high levels of contaminants) without pretreatment to reduce sample complexity.

[0103] In one embodiment, the nucleic acids extracted by the methods disclosed herein are directly used for downstream applications. In one embodiment, the extracted nucleic acids are directly used for downstream applications without nucleic acid quantification. In one embodiment, the extracted nucleic acids are directly used for RT-qPCR without DNase treatment.

[0104] In one embodiment, the methods disclosed herein do not involve pretreatment of biological samples, such as to simplify or clarify biological samples, for example, by filtration. In the example of whole blood, the sample is not pretreated to reduce or remove the red blood cells and / or other contaminants contained therein. In the example of a sample containing mucus, no enzymatic, ultrasonic, etc. treatment is performed to liquefy the sample. Thus, in one embodiment, the only preparation of the biological sample can be to contact the sample with a lysis buffer.

[0105] The methods for nucleic acid extraction of the present disclosure can be high-throughput or suitable for high-throughput applications, whether in a one-step or two-step particle addition method. Such methods can take between 20 and 45 minutes. In embodiments where nucleic acids are extracted from complex biological samples and the two-step particle addition method as described above is used, the methods of the present disclosure take about 45 minutes or less, about 40 minutes or less, about 35 minutes or less, or about 30 minutes or less.

[0106] The methods of the present disclosure can be carried out in any suitable container, including tubes (e.g., microcentrifuge tubes) or plates (e.g., microtiter plates). Depending on the container used, a suitable magnet is used in embodiments where the particles are magnetic or responsive to a magnetic field. In one embodiment, the magnet is an EasySep TM magnet in which the tube is received. In one embodiment, the magnet is a plate magnet on which the microtiter plate is received.

[0107] In one embodiment, compared to conventional nucleic acid extraction methods, the methods of the present disclosure offer various features and advantages. In one embodiment, the method does not require pretreatment of the biological sample. In one embodiment, the first or second eluate of the nucleic acid can be directly used in downstream assays. In one embodiment, the methods of the present disclosure (whether one-step or two-step) extract nucleic acids with optimal purity and recovery, even if the starting biological sample is a complex sample.

[0108] The following non-limiting examples illustrate the present disclosure.

[0109] Example

[0110] Example 1: Sample Preparation

[0111] The sample preparation method varies depending on the sample type.

[0112] Samples from leukapheresis products are prepared by adjusting to the desired cell concentration. For example, leukapheresis products can be diluted to 5x10 6 cells / ml in D-PBS.

[0113] Samples of non-adherent cells are prepared by precipitation in D-PBS and resuspension to the desired concentration. Samples from 2D adherent cells are prepared in substantially the same manner, except that the cells are dissociated from the surface using a selected dissociation reagent that may need to be quenched subsequently. Samples of cells isolated using the EasySep TM (STEMCELL Technologies) kit can be resuspended in an appropriate volume of D-PBS or EasySep TM buffer.

[0114] Whole blood samples are used directly for nucleic acid extraction. If nucleic acids are not immediately extracted from the blood samples, the collected whole blood may need to be treated with an anticoagulant (such as ACD-A, heparin, or K-EDTA).

[0115] Organoids maintained in dome cultures (at least 4 - 6 confluent domes) are used for nucleic acid extraction. Briefly, the organoids in Matrigel domes are mechanically ground using an electronic pipette to disrupt the domes and break up the organoids. The resulting cell pellet is resuspended in RLT buffer (Qiagen), a phenol-chloroform-based solution (such as TRIzol), or the lysis buffer of the present disclosure. Alternatively, a lysis buffer containing proteinase K is added directly to the dome, incubated, and pipetted up and down to dissolve the dome.

[0116] Samples of liver tissue (e.g., mouse) are prepared by chopping the harvested mouse liver into small pieces in a Petri dish containing cold DMEM / F-12. After the liver fragments are allowed to sediment by gravity on ice for 2 minutes, the supernatant is removed. 10 ml of RT tissue dissociation mixture (STEM CELL Technologies) is added to the liver fragments and incubated in a 37°C water bath for 20 minutes.

[0117] Samples of PBMCs are prepared from untreated human whole blood. To avoid loss of monocytes, EDTA is added to the whole blood sample to a final concentration of 6 mM before labeling and isolation. Samples of PBMCs are prepared directly from human cord blood and leukapheresis samples by immunomagnetic negative selection (STEMCELL Technologies).

[0118] Plasma samples are prepared by centrifuging whole blood at 2000 x g for 10 minutes and transferring the plasma layer to a tube, then centrifuging at 2000 x g for another 10 minutes. The plasma supernatant is transferred to a new tube and centrifuged at 10,000 x g for 30 minutes to remove cell debris and large vesicles. The resulting supernatant is transferred to the desired tube and used for nucleic acid isolation. To extract nucleic acids from pan-cellular extracellular vesicles (EVs), an appropriate EasySep TM kit (STEMCELL Technologies) is used to isolate EVs from the plasma.

[0119] The samples prepared as above are used in amounts / volumes depending on whether microcentrifuge tubes or 96-well plates are used. For extractions using microcentrifuge tubes, 25 - 300 μl of sample is used. For extractions using 96-well plates, 10 - 50 μl of sample is used.

[0120] Example 2: Quantification and analysis of nucleic acids

[0121] The purity and recovery rate of nucleic acids obtained according to the method of the present disclosure (see Example 3) and by other conventional methods were tested. The average purity ratios 260 / 280 and 260 / 230 were measured using a NanoDrop 2000 (Thermo Fisher). The recovery rate (μg) of nucleic acids (DNA and RNA) was measured using a Qubit TM fluorometer (Thermo Fisher).

[0122] Example 3: Total nucleic acid extraction

[0123] The samples prepared substantially as described in Example 1 were subjected to the following nucleic acid extraction method. Briefly summarizing the above detailed description, the samples were treated with a lysis buffer containing proteinase K at a 1:1 ratio, whether using microcentrifuge tubes or 96-well plates. The samples were incubated at room temperature or in a 56 °C heating block or water bath for approximately 10 minutes, and the sample lysates were contacted with a certain volume of first magnetic particles (such as diluted EasySep TM Nucleic Acid Rapidspheres TM , STEMCELL Technologies) optionally in alcohol at a ratio of 1.5:1 to the sample volume. The sample lysates and particles were incubated at room temperature for about 2 - 5 minutes to achieve nucleic acid binding (such as the first capture). The first capture was enriched under the influence of a magnetic field at room temperature for about 2 - 5 minutes. After removing the supernatant by pipetting or pouring, the enriched first capture was resuspended in a suitable elution buffer (such as TE buffer or nuclease-free water) at room temperature for about 5 minutes. The eluted nucleic acids were fractionated from the particles by centrifugation or by exposing the released particles to a magnetic field.

[0124] In an improved workflow, such as for complex samples, the nucleic acids in the eluate were primed or treated by exposure to a priming buffer containing a chaotropic agent or chaotropic salt and optionally alcohol, and contacted with a certain volume of second magnetic particles (such as diluted EasySep TM Nucleic Acid Rapidspheres TM ) optionally in alcohol at a ratio of 1.5:1 to the elution volume at room temperature for about 2 - 5 minutes (such as the second capture). The second capture was enriched under the influence of a magnetic field at room temperature for about 2 - 5 minutes. The second capture was washed about 1 - 3 times with a suitable wash buffer (such as PBS, etc.) under the magnetic field.

[0125] The eluted first capture and second capture can be quantified / analyzed as described in Example 2.

[0126] Example 4: Effect of proteinase K lysis temperature on the purity and recovery rate of nucleic acids

[0127] According to Example 3, the leukapheresis samples prepared as described in Example 1 were treated with a lysis buffer containing proteinase K at different temperatures (room temperature or 56 °C) to study the effect on the purity and recovery of nucleic acids.

[0128] Samples treated with proteinase K and incubated at 56 °C produced better 260 / 280 and 260 / 230 ratios compared to samples incubated at room temperature. The recovery of nucleic acids (total nucleic acids and DNA) was also increased in samples incubated at 56 °C compared to room temperature (Tables 1A and 1B)

[0129]

[0130] Table 1A. Leukopak samples were lysed with proteinase K at 56 °C for 10 minutes.

[0131]

[0132] Table 1B. Leukopak samples were lysed with proteinase K at room temperature for 3 minutes.

[0133] Example 5: Effect of lysis buffer pH on the purity and recovery of nucleic acids

[0134] According to Example 3, PBMC samples prepared as described in Example 1 were treated with lysis buffers having different pH levels to study the effect on the purity and recovery of nucleic acids.

[0135] Comparable 260 / 280 and 260 / 230 ratios of the extracted nucleic acids were obtained regardless of the lysis buffer pH (e.g., 4.0, 6.0, 8.0, and 10.0) Figure 1A ). The DNA and RNA recoveries from samples lysed at pH 4.0, 6.0, 8.0, and 10.0 were comparable Figure 1B ).

[0136] Thus, the lysis buffers of the present disclosure having a pH of 4.0 to 10.0 do not compromise the purity and recovery of the extracted nucleic acids.

[0137] Example 6: Effect of particle concentration on the purity and recovery of nucleic acids

[0138] Unwashed cells of leukapheresis samples from 3 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 (in 90% IPA) per mL of sample lysis buffer) prior to nucleic acid extraction as described in Example 3.

[0139] Although the recovery rate is generally related to the particle concentration, the purity is negatively correlated with the particle concentration. The purity is optimal at a particle concentration of 1.6 mg / mL sample lysate or higher, while a concentration of 2.2 mg / mL sample lysate or lower yields a purity within an acceptable range ( Figure 2A ). Additionally, the particle concentrations of 1.2 mg / mL sample lysate and higher are roughly equivalent ( Figure 2B ).

[0140] Overall, a particle concentration range between 1 and 2.2 mg / mL sample lysate, inclusive, strikes a balance between maximizing purity and recovery rate, and more specifically, a particle concentration range between 1 and 2.2 mg / mL sample lysate.

[0141] Example 7: Effect of Elution Temperature on the Purity and Recovery Rate of Nucleic Acids

[0142] A whole blood sample was 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 the purity and recovery rate of nucleic acids was investigated relative to room temperature elution in a spin column protocol.

[0143] Although the recovery rate is generally related to the elution buffer temperature and exceeds the spin column recovery rate at 56 °C, the 260 / 280 purity is negatively correlated with the elution buffer temperature.

[0144]

[0145] Table 2. Effect of Elution Temperature on the Purity and Recovery Rate of DNA from Whole Blood.

[0146] Example 8: Extraction of Nucleic Acids from EVs Isolated by EasySep

[0147] Pan-EVs isolated from plasma using the EasySepTM kit (STEMCELL Technologies) were prepared as described in Example 1 and bound to particles as described in Example 3. It was investigated whether the presence of particles used in EasySepTM-mediated EV enrichment would hinder downstream particle-mediated nucleic acid extraction, as described in Example 3.

[0148] The following Table 3 shows that the recovery rate of DNA is not affected by the presence of particles in the sample prior to nucleic acid extraction, relative to particle-free EVs.

[0149] Qubit (N = 3) With particle purification Without particle purification Average DNA / isolation (ng / ul) 0.055 0.051 Average ng DNA / isolation 1.80 1.64

[0150] Table 3. Recovery Rates (ng / μl and ng / extraction) of DNA from Pan-EVs With or Without Particle Purification. n = 3

[0151] Example 9: Extraction of nucleic acids from organoids

[0152] Prepare a sample of liver organoids (formed using a kit commercialized by STEMCELL Technologies) as described in Example 1 and bind it to particles as described in Example 3. As described in Example 3, use a conventional centrifugal column (RNeasy, Qiagen) or the one-step particle-based EasySep of the present disclosure TM method to extract RNA from the sample.

[0153] In addition, 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 TM ) were tested.

[0154] Regardless of the sample lysis conditions, compared with the conventional centrifugal column method, the particle-mediated EasySep of the present disclosure TM RNA extraction produced the highest recovery rate ( Figure 3 , Table 4), while the 260 / 280 purity ratio was consistent in the experiments (Table 4).

[0155]

[0156] Table 4. Purity and recovery rate of RNA extracted using different combinations of lysis conditions and separation platforms. The shown recovery rate values are the average of 3 technical replicates.

[0157] Example 10: Extraction of nucleic acids from mouse liver tissue

[0158] Prepare a mouse liver sample as described in Example 1 and bind it to particles as described in Example 3. As described in Example 3, use a conventional centrifugal column (RNeasy, Qiagen) (“Spin”) or the one-step particle-based EasySep of the present disclosure TM method to extract RNA from the sample. Test the same combination of lysis buffer and platform as described in Example 9.

[0159] Regardless of the sample lysis conditions, compared with the conventional centrifugal column method, the particle-mediated EasySep of the present disclosure TM RNA extraction produced the highest recovery rate (Table 5), while the 260 / 280 purity ratio was consistent in the experiments (Table 5).

[0160]

[0161] Table 5. Purity and recovery rate of RNA extracted from whole mouse liver tissue by the method of the present disclosure.

[0162] Example 11: Suitability of Extracted Nucleic Acids for Downstream Applications

[0163] The nucleic acids extracted according to the methods of the present disclosure were tested in downstream RT-qPCR. The extracted RNA was treated with DNase or not treated with DNase before reverse transcription, and the average Ct values of the target genes were comparable under both conditions, independent of the amount of input RNA for reverse transcription (Table 6).

[0164] Thus, the DNase treatment status of RNA extracted using the bead-based EasySep TM nucleic acid extraction platform does not negatively impact the analysis by RT-qPCR.

[0165]

[0166] Table 6. Ct values of RNA extracted from day 7 organoids are comparable regardless of DNase treatment status (n = 1).

[0167] Example 12: Extraction of Nucleic Acids from Whole Blood by the Improved Method of the Present Disclosure

[0168] Nucleic acids were extracted from whole blood using the one-step bead-based method (“standard workflow”) or the two-step bead-based method (“improved workflow”) described in Example 3 and compared to a conventional spin column method.

[0169] Spectrophotometric absorption spectra showed that nucleic acids extracted from complex samples according to the improved workflow had improved recovery and purity relative to the spin column workflow and the standard workflow (Table 7). Additionally, nucleic acids from the spin column workflow and the improved workflow had less contamination compared to the standard workflow ( Figure 4 ).

[0170] Thus, the results indicate that the improved two-step bead-based method of the present disclosure yields the best purity ratio and highest recovery for nucleic acids extracted from complex biological samples.

[0171] Standard workflow Improved workflow Spin column workflow Avg 260 / 280 1.74+0.07 1.87+0.02 1.87+0.05 Avg 260 / 230 0.52+0.10 2.28+0.04 1.98+0.44 Avg μg DNA 2.53+0.88 2.78+0.47 1.97+0.30 Avg μg RNA 0.28+0.16 0.30+0.10 0.21+0.06

[0172] Table 7. Comparison of nucleic acid purity and DNA and RNA recovery using the one-step bead-based workflow (standard workflow), the improved two-step bead-based workflow (improved workflow), and the conventional spin column method (spin column workflow). Values are the average of 2 technical replicates of 3 biological replicate samples.

[0173] Example 13: Different Washing Cycles in the Two-Step Nucleic Acid Extraction Method

[0174] A whole blood sample was prepared essentially as described in Example 1. The re-capture of nucleic acids eluted from the first particles by the second particles was evaluated in terms of purity and recovery essentially as described in Example 3, and compared to the re-capture of eluted nucleic acids by the same particles.

[0175] When nucleic acids were re-captured onto fresh particles (“improved workflow”), the highest 260 / 230 purity ( Figure 5A ) and comparable recovery ( Figure 5B ) were obtained compared to both re-capture onto the same particles (conditions 1 - 4) or the spin column method. Adding fresh particles for the second capture resulted in purity and recovery comparable to the RBC lysis method for nucleic acid extraction from whole blood (Figure 5).

[0176] The effect of the number and duration of washes on nucleic acid purity and recovery was also explored to see if purity could be improved in the modified two-step method where nucleic acids were re-captured by the original particles. While the no-wash condition (condition 1) showed the highest recovery, 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 re-capture cycle only slightly improved purity ( Figure 5A ).

[0177] Thus, in terms of recovery, all particle-based methods were superior to spin column nucleic acid extraction from complex biological samples such as whole blood, and only the no-wash control (condition 1) produced a purity lower than spin column extraction ( Figure 5B ). Therefore, particle-based extraction from whole blood according to the embodiments disclosed herein is better for nucleic acid extraction from complex samples. Using fresh particles at each step of the two-step capture and release EasySep TM protocol gave the best results.

[0178] Example 14: Primer Buffer Required for Re-capture of Nucleic Acids onto the Second Particles

[0179] In the methods as described in Examples 12 and 13, nucleic acid recovery was studied in the first eluate (before the second capture) in the presence or absence of primer buffer (formulated consistent with the lysis buffer of the present disclosure).

[0180] Adding primer buffer to the first eluate resulted in a significant increase in nucleic acid recovery ( Figure 6 ) compared to performing the second capture in the absence of primer buffer.

[0181] Example 15: Purity and Recovery of Spin Column and Two-step Nucleic Acid Extraction

[0182] Prepare whole blood samples as described in Example 1 and perform column-based nucleic acid extraction (RNEasy) and one- or two-step bead-based EasySep TM methods (“standard” or “modified” workflows) as described in Examples 3 and 12-14.

[0183] Using the modified workflow and the column-based workflow, the nucleic acid purity (260 / 230 ratio) was highest ( Figure 7A ), with slightly higher purity for the modified workflow. Compared to the recovery using the spin column method, the normalized nucleic acid recoveries of DNA ( Figure 7B ) and RNA ( Figure 7C ) were higher using the modified workflow (μg / 10 6 cells).

Claims

1. A method for extracting nucleic acids from a biological sample, the method comprising: i) preparing a sample lysate by treating the biological sample with a lysis buffer; ii) incubating the sample lysate at a temperature at or above room temperature (RT) and below 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 acids bound thereto with an elution buffer to release the nucleic acids from the particles.

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

3. The method according to claim 1 or 2, wherein the pH of the lysis buffer is between about 4 and 10.

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

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

6. The method according to claim 4 or 5, wherein preheating the elution buffer increases the purity and / or recovery rate of the released nucleic acids compared to the purity and / or recovery rate of the nucleic acids released in a room temperature elution buffer.

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

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

9. The method according to 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 pretreated or not pretreated.

12. The method according to any one of claims 1 to 11, wherein the biological sample comprises 10 1 to 10 9 cells.

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

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

15. The method according to claim 14, further comprising releasing the recaptured nucleic acids.

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

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

18. The method according to any one of claims 1 to 17, further comprising fractionating the particles and the supernatant containing the released nucleic acid by suction or pouring.

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

20. The method according to any one of claims 1-19, wherein the method takes between 30 and 45 minutes.

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

Images