Methods, Compositions, and Kits for Size-Selective Enrichment of Nucleic Acids

Abstract

[0003] Methods for isolating and concentrating nucleic acids of a selected target size (e.g., in increments of less than 1000 base pairs) from a biological liquid mixture are provided, comprising combining the biological liquid mixture with a first aqueous two-phase system (ATPS) formed from a first-phase polymer or surfactant component dissolved in a first-phase solution and a second-phase solution, whereby target nucleic acid fragments below the desired target size partition into the second-phase solution and contaminants partition into the first-phase solution; extracting and mixing the second-phase solution with a second ATPS formed from a second-phase polymer or surfactant component dissolved in a third-phase solution and a fourth-phase solution, whereby the target nucleic acid fragments partition into and are concentrated in the third-phase solution; extracting and mixing; and recovering the concentrated target nucleic acid fragments from the third-phase solution. Also provided are compositions and kits for isolating and concentrating nucleic acids of a selected target size as described above.

Description

Technical Field

[0001] The present invention relates to the isolation, concentration and / or purification of nucleic acid fragments in a continuous aqueous phase system. In particular, the present invention provides sample preparation methods, compositions and kit components for the isolation, concentration and / or purification of nucleic acid fragments from biological materials.

Background Art

[0002] Methods for the isolation and purification of nucleic acids (such as DNA and RNA) from complex matrices such as blood, tissue, urine and forensic samples and bacterial and mammalian cell cultures are important in gene research, nucleic acid probe diagnosis, forensic DNA testing and other areas that require amplification, processing or analysis of nucleic acids. The purified nucleic acids must be of high quality and in sufficient quantity so that they can be used in various downstream applications including detection, sequencing, clinical diagnosis and the like. Obtaining purified nucleic acids is a complex task due to the presence of large amounts of contaminating cellular materials (such as proteins and carbohydrates) present in the complex environment in which nucleic acids are identified, including urine, blood, plasma, serum, saliva and other biological fluids. Current methods for the extraction and purification of nucleic acids from biological samples are usually time-consuming, cumbersome, costly, involve the use of harmful organic solvents, and are often suitable for capturing only nucleic acids above a certain size, for example 1000 bp.

[0003] In the case of a target analyte present at very low concentrations in biological fluids such as urine and blood, it is required to obtain large volumes of biological fluids in order to obtain sufficient amounts of the target analyte for subsequent detection by molecular techniques. Detecting the presence of an analyte with extremely low concentrations has been a major challenge. The analyte can be a biomarker of a disease such as cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or protein present in samples such as a patient's saliva, blood, urine, and other body fluids. Many of the existing diagnostic or detection methods may falsely report the absence of the analyte when the analyte concentration is too low. For example, the gold standards of diagnosis such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) may produce false-negative results when the target analyte has extremely low amounts outside the detection range of the assay.

[0004] When biomarkers are present in small amounts, accurate detection depends on isolation methods that can concentrate nucleic acids from the background. Depending on the isolation method, this burden can be complicated by variations in nucleic acid fragment size and the effects of test inhibitors. Various methods are used for nucleic acid purification. These include precipitation, ultrafiltration (Hirasaki et al., J. Membr. Sci., 106:123-129 (1995)), and adsorption using anion exchange columns. Other test methods, including commercial methods, have been reported to have lower DNA extraction yields and be unable to extract small DNA fragments less than 200 bp in length (Fong et al, Clinical Chemistry 55(3), 587-589, 2009). Ribeiro et al. (Biotechnol. Bioeng. May 20, 2002; 78(4):376-84) described an aqueous two-phase system (ATPS) using polyethylene glycol (PEG) as the polymer component and dipotassium hydrogen phosphate (K2HPO4) as the salt component. The isolation of plasmid pCF1-CFTR from Escherichia coli (E. Coli) DH5a has been reported. In this case, the cells were first disrupted through alkaline lysis (lysis buffer containing NaOH and SDS), and then the lysate was neutralized with 3M sodium acetate. Subsequently, cell debris, proteins, and genomic DNA (gDNA) were removed by batch centrifugation. In the above aqueous two-phase system, the clarified lysate was utilized. However, this process has the drawback of being very expensive in terms of equipment and time. The above papers are hereby incorporated by reference in their entirety. Commercially available bench-top solid-phase kits, such as those provided by Qiagen® and Invitrogen®, enable relatively rapid extraction and purification of nucleic acids but require bench-top laboratory equipment, meaning that extraction and purification are limited mainly to laboratories with basic equipment and power supplies. Summary of the Invention Problems to be Solved by the Invention

[0005] Accordingly, there is a great desire for simplified methods and apparatuses to achieve higher concentrations and higher numbers of molecules of nucleic acids with small target fragment sizes in an inexpensive manner suitable for various industrial, clinical, and research applications, such as more accurate higher signal-to-noise for downstream applications, point-of-care testing, etc.

Means for Solving the Problems

[0006] To overcome the aforementioned limitations of existing technologies, novel methods, compositions, and kits are disclosed for size-selective concentration, isolation, and analysis of analytes in an inexpensive, rapid, and fully liquid process that utilizes continuous aqueous two-phase systems (ATPS) without the need for complex equipment. The methods and compositions can achieve the following multiple operations, including cell lysis, removal of non-targeted biomolecules, and / or concentration of targeted analytes. In some embodiments, the analyte is a nucleic acid comprising nucleic acid fragments of less than a selected size, such as less than 1000 base pairs (e.g., less than 10,000 bp, 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp). In some embodiments, the analyte is single-stranded nucleic acid, while in other embodiments, the analyte is double-stranded nucleic acid. The target size cut-off can be selected via appropriate selection and specific order of the ATPS phases forming and fractionating the components, as well as centrifugation, mixing, and incubation steps. In some embodiments, the nucleic acid fragments can be isolated and concentrated with a target size cut-off accuracy of less than 1000 bp and more preferably an accuracy of 25 bp in the most advantageous range for analysis.

[0007] The present invention provides an improved method for purifying nucleic acids derived from biological samples and subsequently removing components of ATPS derived from the nucleic acids that would otherwise interfere with downstream applications such as disease detection, amplification, and genotyping. Exemplary biological samples can include blood, plasma, saliva, urine, cells, exosomes, proteins, cfDNA, RNA, and circulating tumor cells. The inventors have found that the disclosed stable and repeatable process achieves nucleic acid purification with little or no loss of nucleic acid (i.e., very high recovery rate) and extracts a single phase of native ATPS and mixes it with a phase-forming component having chemical properties different from those of the phase-forming components of the first ATPS, such that the target nucleic acid can form a second ATPS in which it is partitioned into a phase opposite to the phase partitioned in the first ATPS.

[0008] Provided in some embodiments is a method disclosed for isolating and concentrating nucleic acids of a desired target size from a liquid biological mixture containing nucleic acids and contaminants. The biological mixture can be combined with a phase-forming polymer or surfactant component dissolved in a first phase solution and a first ATPS formed from a second phase solution, whereby target nucleic acid fragments below the target size are isolated by partitioning into the second phase solution while contaminants are partitioned into the first phase solution. Next, the second phase solution can be extracted and mixed with a phase-forming polymer or surfactant component dissolved in a third phase solution and a second ATPS formed from a fourth phase solution, whereby the target nucleic acid fragments are concentrated by partitioning into the third phase solution. Next, the concentrated target nucleic acid fragments can be recovered from the third phase solution by any number of methods.

[0009] In some embodiments, the polymer or surfactant component forming the first and second phases can include one or more polymers, one or more surfactants, and combinations thereof. Possible polymers that can be utilized include polyalkylene glycols (PEG) such as hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers such as hydrophobically modified poly(oxyalkylene) copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starches, alkoxylated celluloses, alkyl hydroxyalkyl celluloses, silicon-modified polyethers, and poly N-isopropylacrylamide and its copolymers, but are not limited thereto. In another embodiment, the polymer forming the first phase includes polyethylene glycol, polypropylene glycol, or dextran.Possible surfactants that can be used include anionic surfactants such as Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfonates, sulfates, sulfated natural oils and fats, etc., sulfated esters, sulfated alkanolamides, ethoxylated and sulfated nonionic surfactants such as alkylphenols, ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan anhydride esters, glycol esters of fatty acids, carboxylic acid amides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines having amide bonds, cationic surfactants such as polyoxyethylene alkyl and alicyclic amines, etc., n,n,n’,n’-tetrasubstituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline and amphoteric surfactants such as n-coco-3-aminopropionic acid / sodium salt, n-tallow-3-iminodipropionate, disodium salt, n-carboxymethyl-n-dimethyl-n-9-octadecenyl ammonium hydroxide, n-cocoamidoethyl-n-hydroxyethyl glycine and sodium salt, etc., but are not limited thereto.

[0010] In some embodiments, the first polymer or surfactant component has a higher molecular weight than the second polymer or surfactant component.

[0011] In some embodiments, the molar concentration of the first polymer or surfactant component in the first-phase solution of the NA isolation ATPS is higher than the molar concentration of at least one of the second polymer or surfactant component in the third-phase solution, the second polymer or surfactant component in the fourth-phase solution, and the second polymer or surfactant component in the NA-enriched ATPS.

[0012] In some embodiments, the mass concentration of the first polymer or surfactant component in the first-phase solution of the NA isolation ATPS is higher than at least one of the mass concentrations of the second polymer or surfactant component in the third-phase solution, the second polymer or surfactant component in the fourth-phase solution, and the second polymer or surfactant component in the NA-concentrated ATPS. In one embodiment, the concentration of the first polymer or surfactant component in the first-phase solution ranges from about 0.01 wt% to about 90 wt% (w / w) of the total weight of the aqueous solution. In various embodiments, the first-phase solution is selected from polymer or surfactant solutions that are about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, or about 1% w / w. In some embodiments, the first-phase solution is selected from polymer or surfactant solutions that are about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w, about 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, about 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w, and about 50% w / w.

[0013] In some embodiments, the second phase solution in the isolated ATPS contains the dissolved surfactant or polymer component as described above that forms at least one phase and / or dissolved salts such as dipotassium phosphate, monopotassium phosphate, and combinations thereof. In some embodiments, salts include, but are not limited to, inorganic salts containing cosmotrophic salts, chaotropic salts, cations such as linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and anions such as phosphate, sulfate, nitrate, chloride, and bicarbonate. In another embodiment, the salts can include NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, and combinations thereof. Other salts, such as ammonium acetate, can also be used. In another embodiment, the salts can be selected from magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, and aluminum salts. In some embodiments, the salts can be selected from bromide salts, iodide salts, fluoride salts, carbonates, sulfates, citrates, carboxylates, borates, and phosphates. In some embodiments, the salts include potassium phosphate. In some embodiments, the salts include ammonium sulfate. In one embodiment, the overall salt concentration ranges from about 0.01% to about 90%. One of ordinary skill in the art will understand that the amount of salt required to form an ATPS is affected by the molecular weight, concentration, and physical state of the polymer or surfactant. In various embodiments, the salt solution is from about 0.001% w / w to 90% w / w. In various embodiments, the salt solution is about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, or about 1% w / w.In some embodiments, the salt solution is about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w, about 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, about 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w and about 50% w / w. In one embodiment, the concentration of the salt is about 2% w / w to 40% w / w. In one embodiment, the concentration of the salt is about 3% w / w to 30% w / w. In one embodiment, the concentration of the salt is about 5% w / w to 20% w / w.

[0014] In some embodiments, the fourth phase solution in the concentrated ATPS contains at least one phase-forming dissolved salt, surfactant or polymer component as described above and combinations thereof.

[0015] In some embodiments, the second phase solution of the isolated ATPS exerts a weaker excluded volume interaction on the target nucleic acid fragment than the fourth phase solution of the concentrated ATPS. In some embodiments, the first phase solution of the isolated ATPS exerts a stronger excluded volume interaction on the target nucleic acid fragment than the third phase solution of the concentrated ATPS.

[0016] In some embodiments, the second-phase solution of the isolated ATPS exerts more favorable hydrophilic and hydrophobic interactions on the partitioning of the target nucleic acid fragment into the second phase than the fourth-phase solution of the concentrated ATPS. More specifically, there is a favorable change in free energy when the molecule of the target DNA moves from the first-phase solution to the second-phase solution than when the molecule of DNA moves from the third-phase solution to the fourth-phase solution. In some embodiments, the first-phase solution of the isolated ATPS exerts less favorable hydrophilic and hydrophobic interactions on the partitioning of the target nucleic acid fragment into the first-phase solution than the third-phase solution of the concentrated ATPS.

[0017] In some embodiments, the second-phase solution of the isolated ATPS exerts more favorable electrostatic interactions on the partitioning of the target nucleic acid fragment into the second phase than the fourth-phase solution of the concentrated ATPS. More specifically, there is a favorable change in free energy when the molecule of the target DNA moves from the first-phase solution to the second-phase solution than when the molecule of DNA moves from the third-phase solution to the fourth-phase solution. In some embodiments, the first-phase solution of the isolated ATPS exerts less favorable electrostatic interactions on the partitioning of the target nucleic acid fragment into the first-phase solution than the fourth-phase solution of the concentrated ATPS.

[0018] In one particular embodiment, the recovery of the concentrated target nucleic acid fragment in the second ATPS can include separating the third-phase solution from the fourth-phase solution and mixing the third-phase solution with at least one size-fractionating component selected from polymers, surfactants, salts, and combinations thereof to form a supernatant composed of the concentrated target nucleic acid and a precipitation pellet of nucleic acids above the target cut-off size. Next, the supernatant can be separated from the precipitation pellet of nucleic acids above the target cut-off size, and the target nucleic acid fragments below the selected cut-off size can be precipitated from the supernatant.

[0019] In some embodiments, the size-fractionating component can include a polymer or surfactant component having a higher molecular weight than the first polymer or surfactant component of the isolated ATPS, and the first polymer or surfactant component has a higher molecular weight than the second polymer or surfactant component of the concentrated ATPS.

[0020] In some embodiments, the molar concentration of at least one size-fractionated component in the supernatant can be less than the molar concentration of the first polymer or surfactant component in the first-phase solution, and the first polymer or surfactant component in the first-phase solution has a higher molar concentration than the molar concentration of the second polymer or surfactant component in the third-phase solution.

[0021] In some embodiments, the mass concentration of at least one size-fractionated component in the supernatant is less than the mass concentration of the first polymer or surfactant component in the first-phase solution, and the first polymer or surfactant component in the first-phase solution has a higher mass concentration than the mass concentration of the second polymer or surfactant component in the third-phase solution.

[0022] In some embodiments, at least one size-fractionated component can include one or more salts, polymers, and / or surfactants such as those described above, or combinations thereof.

[0023] In another main embodiment, a composition for isolating and concentrating nucleic acids of a selected small target size from a liquid (e.g., biological) mixture containing nucleic acids and contaminants is disclosed. The composition comprises a polymer or surfactant component forming a first phase dissolved in a first-phase solution and forming an NA isolation ATPS from a second-phase solution, such that when mixed with the liquid mixture, target nucleic acid fragments below the target size are partitioned into the second-phase solution, and contaminants are partitioned into the first-phase solution, components for forming, and a polymer or surfactant component forming a second phase dissolved in a third-phase solution and forming an NA concentration ATPS from a fourth-phase solution, such that when mixed with the second-phase solution, the target nucleic acid fragments are partitioned into and concentrated in the third-phase solution, components for forming, and the composition can also include materials for concentrating the target nucleic acid fragments from the third-phase solution.

[0024] In yet another embodiment, a kit for isolating and concentrating nucleic acids of a target size from a liquid mixture containing nucleic acids and contaminants is disclosed. The kit can include the composition components described in the composition embodiments, but can further include a container that can utilize a syringe or pipette for storage, filling, and / or reaction, and optionally equipment for manipulating aqueous solutions. Such containers and equipment can include columns, test tubes, capillary tubes, plastic test tubes, Falcon tubes, culture tubes, well plates, pipettes, and / or cuvettes.

[0025] These and other features and characteristics, as well as methods of operation and functions of the related components and economies of manufacture, will become more apparent upon consideration of the following detailed description in conjunction with the accompanying drawings and the appended claims, all of which form a part of this specification, and like reference numerals designate corresponding parts in the various figures. However, it should be clearly understood that the drawings are for illustrative and explanatory purposes only and are not intended to define the limitations of the claims. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Brief Description of the Drawings

[0026]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0027] Unless otherwise specified, the terms used in this specification, including technical and scientific terms, have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains, and detailed descriptions of well-known functions and configurations that may obscure the gist of the present invention are omitted.

[0028] "Aqueous", as used in this specification, refers to a characteristic property of a solvent / solute system in which the solvating substance mainly has hydrophilic properties. Examples of aqueous solvent / solute systems include those in which water or a composition containing water is the main solvent. The polymer and / or surfactant components described in the embodiments for use are "aqueous" in the sense that when combined with a solvent such as water, they form an aqueous phase. Further, as understood by those of ordinary skill in the art, in the context of this specification, the term "liquid'mixture'" simply refers to a combination of the components defined in this specification.

[0029] As used herein, an aqueous two-phase system (ATPS) means a liquid-liquid separation system capable of achieving isolation or concentration of an analyte by partitioning, and two phases, compartments, regions or components, etc., interact separately with at least one analyte to which they are exposed and optionally dissolved. An ATPS is formed when two immiscible phase-forming components such as a salt and a polymer having specific concentrations or two incompatible polymers (e.g., PEG and dextran) are mixed in an aqueous solution. The ATPS method is relatively inexpensive and scalable because it utilizes two-phase partitioning to separate analytes (e.g., nucleic acids) from contaminants.

[0030] The term "isolated", as used herein, refers to a nucleic acid removed from its original environment and thus altered from its original environment. Isolated nucleic acids are generally provided with a lesser amount of non-nucleic components (e.g., proteins, lipids) than the amount of components present in the original sample. A composition containing an isolated sample nucleic acid can be substantially isolated (e.g., free of non-nucleic components at about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99%).

[0031] As used herein, "concentrated" means that the mass ratio of the analyte of interest to the solution in which the analyte is suspended is higher than the mass ratio of said analyte in the pre-concentrated solution. For example, it may be slightly higher or more preferably at least 2-fold, 10-fold or 100-fold higher.

[0032] As used herein, "polymer" includes, but is not limited to, homopolymers, copolymers, terpolymers, random copolymers, and block copolymers. Block copolymers include, but are not limited to, block, graft, dendrimer, and star polymers. As used herein, copolymer refers to a polymer derived from two monomer species; similarly, terpolymer refers to a polymer derived from three monomer species. Polymers also include various forms including, but not limited to, linear polymers, branched polymers, random polymers, crosslinked polymers, and dendrimer-based polymers. By way of example, a polyacrylamide polymer refers to any polymer containing polyacrylamide, such as a homopolymer, copolymer, terpolymer, random copolymer, block copolymer, or terpolymer of polyacrylamide. Polyacrylamide can be a linear polymer, branched polymer, random polymer, crosslinked polymer, or dendrimer of polyacrylamide.

[0033] Preparation of Samples With respect to FIG. 1, an embodiment of an exemplary method may include an optional first step 10 of preparing a liquid mixture 12 by mixing a biological sample and a lysis buffer in a suitable container 14 and incubating for a sufficient time (e.g., preferably in the range of 1 minute to 60 minutes) at a suitable temperature (e.g., in the range of 15° C. to 40° C.) to release nucleic acid 16 from cells, exosomes, proteins, and / or other materials in the biological sample. Preferably, the lysis buffer may have a pH in the range of 4 to 11, preferably 7 to 10, and most preferably 8 to 9. The concentration of substances in the lysis buffer depends on the amount of biological material to be lysed and the mode of supply of said biological material. In principle, any separation method known to those skilled in the art may be suitable for releasing nucleic acid from a biological sample. Lysis methods that may be considered include, in particular, lysis by the action of heat, lysis by the action of mechanical force, lysis by an enzyme such as proteinase K, or lysis by contacting cells with a lysis buffer containing a surfactant or a chaotropic compound, or lysis by a hypotonic solution. Optionally, the aforementioned means may also be combined, for example, by mechanically disrupting cells in a lysis buffer containing a surfactant or a chaotropic compound, or by using a lysis buffer containing proteinase K together with a chaotropic compound.

[0034] Isolation of the analyte The isolation component 17 is added to the mixture 12 in an isolation step 18 to separate the target nucleic acid fragment 26 from the contaminants 20 and may form an aqueous two-phase system ATPS 19 with or without centrifugation. The isolation component 17 may include a polymer or surfactant component that forms a first phase dissolved in the first phase solution 22 and a second phase solution 24, whereby the target nucleic acid fragment 26 smaller than the selected target cut-off size is partitioned into the second phase solution 24 (e.g., the salt-rich lower phase), while proteins and other contaminants 20 are partitioned into the first phase solution 22 (e.g., the polymer-rich upper phase).

[0035] Several types of ATPS 19, including polymer-salt systems, polymer-polymer systems, polymer-surfactant systems, and micelle or reverse micelle systems, can be utilized. Phase separation partitioning can be affected by the appropriate selection of the isolated components 17 and factors such as specific order, pH, molecular weight, relative concentration, and centrifugation, mixing, and incubation steps.

[0036] The isolated component 17 may include a polymer or surfactant component that promotes the formation of the first-phase solution 22 and the second-phase solution 24. Suitable polymers may include polyalkylene glycols such as polyethylene glycol, polypropylene glycol, dextran, hydrophobically modified polyalkylene glycols; poly(oxyalkylene) polymers; poly(oxyalkylene) copolymers such as hydrophobically modified poly(oxyalkylene) copolymers; polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinylmethyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkylhydroxyalkyl cellulose, silicon-modified polyethers, and poly N-isopropylacrylamide and its copolymers. Suitable surfactants include Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfonates, sulfates, sulfated natural oils and fats and other anionic surfactants; sulfated esters, sulfated alkanolamides, alkylphenols, ethoxylated and sulfated nonionic surfactants such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan anhydride esters, glycol esters of fatty acids, carboxylic acid amides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines having amide bonds, polyoxyethylene alkyl and alicyclic amines and other cationic surfactants; n,n,n’,n’-tetrakis-substituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline and n-coco-3-aminopropionic acid / sodium salt, n-tallow-3-iminodipropionate, disodium salt, n-carboxymethyl-n-dimethyl-n-9-octadecenyl ammonium hydroxide, n-cocoamidoethyl-n-hydroxyethyl glycine and sodium salt and other amphoteric surfactants, but are not limited thereto.

[0037] In the polymer-salt embodiments of ATPS 19, the second phase solution 24 may include isolated components containing dissolved salts. Suitable salts include kosmotropic salts, chaotropic salts, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium cations, inorganic salts containing anions such as phosphate, sulfate, nitrate, chloride, and bicarbonate, NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, ammonium acetate, magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, aluminum salts, bromide salts, iodide salts, fluoride salts, carbonates, sulfates, citrates, carboxylates, borates, phosphates, potassium phosphate, ammonium sulfate, and combinations thereof, but are not limited thereto.

[0038] In some embodiments, the first phase solution 24 is formed by containing an isolated component 17 containing a polymer having an average molecular weight of 200 to 10,000 Da (e.g., 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 50,000, or 100,000 Da).

[0039] Concentration of the analyte It is desirable to reduce the process volume of the working solution containing nucleic acid fragment 26. In an ATPS, this can be achieved by reducing the volume of the upper phase. In some embodiments of the concentration step 30, the second phase solution 24 containing the target nucleic acid fragment 26 can be extracted from the container 14 and mixed with the concentrating component 31 in the second container 32 with or without centrifugation to form the second ATPS 34. In other embodiments, the first phase solution 22 with less nucleic acid can be extracted, and the container 14 can be used when performing the concentration step 30. The concentrating component 31 can include a polymer or surfactant component forming the second phase and a fourth phase solution 38 (e.g., the lower phase) dissolved in the third phase solution 36 (e.g., the upper phase). Thereby, the target nucleic acid fragment 26 is partitioned into and concentrated in the third phase solution 36 (e.g., the polymer-rich upper phase), while salts and other contaminants are partitioned into the fourth phase solution 38 (e.g., the salt-rich lower phase).

[0040] In some embodiments, the nucleic acid fragment 26 can be concentrated to an aqueous solution volume that is 1 / 10 of that of the pre-concentrated aqueous lower phase volume before being extracted from the container 14. In some embodiments, the analyte nucleic acid is concentrated at least 10-fold (e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000-fold or more) relative to its concentration before the formation of the ATPS 34. In some embodiments, a step that promotes the separation of the two phases can be utilized, such as by applying a force (e.g., gravity or centrifugation) to the ATPS 34.

[0041] In some embodiments, the third phase solution 36 and the fourth phase solution 38 have different volumes, and the nucleic acid fragment 26 is preferentially partitioned into the phase with the smaller volume. In some embodiments, the fourth phase solution 38 has a volume that is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or 100-fold or more of the third phase solution 36 of the ATPS 34.

[0042] In some embodiments, the molar concentration of the first polymer or surfactant component 17 in the first phase solution 22 may be higher than the molar concentration of at least one of the second polymer or surfactant component in the third phase solution 36, the second polymer or surfactant component in the fourth phase solution 38, and the second polymer or surfactant component 31 in the second ATPS 34.

[0043] In some embodiments, the mass concentration of the first polymer or surfactant component 17 in the first phase solution 22 is higher than the mass concentration of at least one of the second polymer or surfactant component 31 in the third phase solution 36, the fourth phase solution 38, or the overall second ATPS 34.

[0044] In some embodiments, the second polymer or surfactant component 31 including the fourth phase solution 38 includes one or more phase-forming dissolved salts, surfactants, or polymer components such as those described above and combinations thereof.

[0045] In some embodiments, the second phase solution 24 exerts a weaker excluded volume interaction on the target nucleic acid fragment 26 and a more favorable electrostatic interaction than the fourth phase solution 38.

[0046] In some embodiments, the first phase solution 22 exerts a stronger excluded volume interaction on the target nucleic acid fragment 26 and an electrostatic interaction that is not more favorable than the third phase solution 36.

[0047] In some embodiments, the second phase solution 22 exerts a more favorable hydrophilic / hydrophobic interaction on the partitioning of the target nucleic acid fragment 26 into the second phase 22 than that exerted by the fourth phase solution 38. In some embodiments, the first phase solution 22 exerts a hydrophilic / hydrophobic interaction that is not more favorable than that exerted by the third phase solution 36 on the partitioning of the target nucleic acid fragment 26 into the first phase solution 22. The relevance of the chemical potential of the target nucleic acid 26 in each phase immediately after mixing and immediately before reaching equilibrium is μ 1 DNA / μ2 DNA > μ 3 DNA / μ 4 DNA is.

[0048] Recovery of the analyte In the recovery step 40, the concentrated target nucleic acid fragment 26 can be recovered from the third-phase solution 36. FIG. 1 illustrates one optional method (steps 40a to 40c) for recovering the fragment 26, and the third-phase solution 36 can be combined with the size-fractionating component 42 with or without centrifugation in step 40a to precipitate unwanted nucleic acids (i.e., larger than the target fragment size). The supernatant 44 containing the target fragment 26 can be transferred to and combined with the precipitation component 46 with or without centrifugation in step 40b to isolate and desalt the target nucleic acid fragment 26 as a pellet 50 formed at the bottom of the container 48. In step 40c, the supernatant 44 can be removed, and the pellet 50 composed of the target nucleic acid fragment 26 can be resuspended.

[0049] In some embodiments, the size-fractionating component 42 can include one or more salts, polymers, and / or surfactants and combinations thereof. Suitable polymers can include, but are not limited to, polyalkylene glycols such as polyethylene glycol, polypropylene glycol, dextran, hydrophobically modified polyalkylene glycols; poly(oxyalkylene) polymers such as poly(oxyalkylene) copolymers, hydrophobically modified poly(oxyalkylene) copolymers; polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinylmethyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkylhydroxyalkylcellulose, silicon-modified polyethers, and poly N-isopropylacrylamide and its copolymers.

[0050] Suitable surfactants may include, but are not limited to, Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfonates, sulfates, sulfated natural oils and fats, etc. anionic surfactants, sulfated esters, sulfated alkanolamides, alkylphenols, ethoxylated and sulfated nonionic surfactants such as ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan anhydride esters, glycol esters of fatty acids, carboxylic acid amides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines having amide bonds, cationic surfactants such as polyoxyethylene alkyl and alicyclic amines, n,n,n’,n’-tetrakis-substituted ethylenediamine, 2-alkyl-1-hydroxyethyl-2-imidazoline and amphoteric surfactants such as n-coco-3-aminopropionic acid / sodium salt, n-tallow-3-iminodipropionate, disodium salt, n-carboxymethyl-n-dimethyl-n-9-octadecenyl ammonium hydroxide, n-cocoamidoethyl-n-hydroxyethyl glycine, etc.

[0051] Suitable salts include kosmotropic salts, chaotropic salts, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium cations, and inorganic salts containing anions such as phosphate, sulfate, nitrate, chloride, and bicarbonate, including but not limited to NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, ammonium acetate, magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, aluminum salts, bromide salts, iodide salts, fluoride salts, carbonate salts, sulfate salts, citrate salts, carboxylate salts, borate salts, phosphate salts, potassium phosphate, ammonium sulfate, and combinations thereof.

[0052] In some embodiments, the molar concentration of the size fraction component 42 in the supernatant is less than the molar concentration of the first polymer or surfactant component 17 in the first phase solution 22 and further has a higher molar concentration than the molar concentration of the second polymer or surfactant component 31 in the third phase solution 36.

[0053] In some embodiments, the mass concentration of at least one size fraction component 42 in the supernatant 44 is less than the mass concentration of the first polymer or surfactant component 17 in the first phase solution 22 and further has a higher mass concentration than the mass concentration of the second polymer or surfactant component 31 in the third phase solution 36.

[0054] Suitable precipitation components 46 can include those known in the art, such as isopropanol, polycationic polymer salts, compressants, and / or glycols.

Examples

[0055] The present invention will be described in more detail with reference to the following examples. Those skilled in the art will readily understand that the presented examples are for illustrative purposes only and are not intended to limit the scope of the present invention as defined by the following claims. All references shown below and elsewhere in this application are incorporated herein by reference.

[0056] Example 1 - Isolation of Nucleic Acids An experiment set was conducted to observe the effect of using combinations of polymers of different sizes and various concentrations on nucleic acid purification from contaminants by dividing between the upper and lower phases of the polymer-salt ATPS for isolation as described above. When biological samples are lysed, proteins, RNA, genomic DNA, cells, and cell debris are released. By centrifugation of the lysed samples, some of these contaminants can be removed, but many can be maintained. Intending a part of the isolation step experiment, 1 mL of 10% (w / v) BSA solution and preparations of different types of DNA ladders obtained from ThermoFisher Scientific or New England BioLabs were mixed in centrifuge tubes having components for isolating phase formation with pH varying from 7 to 11 at room temperature (15 - 30 °C) and centrifuged at various durations and speeds.

[0057] A glycol polymer obtained from Sigma Aldrich having a molecular weight of 400 - 1200 (in 30 wt% aqueous solution) was combined in a relative ratio with 67 μL of phase-forming salt prepared in 30 wt% aqueous solution within the range of 40 μL - 80 μL in water to form an isolating ATPS.

[0058] The upper and lower phases from the ATPS were qualitatively analyzed on an agarose gel (Figure 2). The agarose gel was run on a miniPCR blueGel electrophoresis system unit. A 0.01% (v / v) GelGreen nucleic acid stain (Biotium) with a 1% (w / v) agarose (Fisher BioReagents) gel was run at 48 V for 40 minutes. Next, the gel was analyzed and photographed under ultraviolet light. From the agarose gel electrophoresis, it can be observed that large-scale DNA fragments are highly partitioned into the salt-rich lower phase. The gel image and table illustrate the effect of changing the percentage of the isolated components of polymers of different sizes, which distributes nucleic acids into the polymer-rich upper phase while larger unwanted DNA fragments are distributed into the salt-rich lower phase.

[0059] Polymers, polypropylene glycols having a molecular weight in the range of 400 to 35000, polyethylene glycols having a molecular weight in the range of 200 to 35000, sodium poly(acrylate) having a molecular weight in the range of 8000 to 240000, dextran having a molecular weight in the range of 6000 to 65000, salts (phase-forming and non-phase-forming), 0.1 M to 5 M sodium chloride solution, sodium sulfate solution, ammonium sulfate solution, 30 to 65 wt% dipotassium phosphate, 20 to 50 wt% monopotassium phosphate and surfactants, Triton X-100, Triton X-114 and conditions (e.g., pH, temperature) were prepared in many combinations, vortex mixed, followed by centrifugation at various rotation times and speeds to screen their ability to separate into thermodynamically stable isolated ATPS when optimizing for yield, stability, partitioning, subsequent method steps and compatibility with downstream PCR. In a preferred embodiment, higher recovery of nucleic acids was achievable in ATPS consisting of polymers and salts, although other phase-forming isolation components were suitable. The molecular weight of the polymer was in the range of 200 to 240000, the concentration was 0.1 to 40, and the salt was 0.1 to 40. Generally, a pH of 7 to 8 and a temperature of 20 to 30 °C were found to result in more stable ATPS and relatively higher nucleic acid recovery rates. Furthermore, the process in the density of the phases was adjusted through the addition of cosolutes.

[0060] Example 2 - Selective isolation and concentration of short nucleic acid fragments using continuous ATPS A 500 ng GeneRuler Low Range DNA ladder manufactured by ThermoFisher Scientific was added to 500 μL of a 89 mg / mL BSA (Sigma Aldrich) solution. After equilibration at RT (24 °C), it was then mixed in a tube having an isolated component solution consisting of 22.5% (v / v) glycol polymer (200 molecular weight) and 18% (v / v) phase-forming salt at about pH 7 - 9. After thorough vortexing, the tube was rotated at 7000 rcf for 120 seconds to form the first isolated ATPS. With an upper phase:lower phase volume ratio of 5:1, contaminants were partitioned into the polymer-rich upper phase while most nucleic acids in a specific size range were partitioned into the salt-rich lower phase. In the test formulation, the percentage of nucleic acids recovered in the lower phase was measured and found to exceed 92% in the preferred embodiment (Run 2, Figure 3A).

[0061] To concentrate the nucleic acid fragments into a smaller volume, the nucleic acid-rich lower phase was extracted from the isolated ATPS container and mixed with various combinations of phase-forming concentrating components having a middle pH of about 7 - 9, including a 9.5% (v / v) glycol polymer 600MW (Sigma Aldrich) and a 25.8% (v / v) phase-forming salt at final concentrations. After thorough vortexing, the tube was rotated at 7000 rcf for 120 seconds to form the second concentrated ATPS. An increased concentration of the phase-forming salt results in a larger lower phase while the nucleic acids are partitioned into the smaller upper phase, which is freely adjustable and the concentration or partitioning of the nucleic acids is maximized mainly by changing the glycol polymer content.

[0062] The lower phase from the first ATPS was transferred by micropipette to a microcentrifuge tube having the second ATPS as described above. Contaminants were partitioned into the salt-rich lower phase while nucleic acids in a specific size range were partitioned into the polymer-rich upper phase and concentrated, which had a volume of 1 / 3 to 1 / 20 of the volume of the salt-rich lower phase. In different runs, the percentage of nucleic acids recovered in the upper phase was measured and found to exceed 90% in the preferred embodiment and more preferably be nearly 100% (Figure 3B).

[0063] Example 3 - Recovery Rate of Isolated and Concentrated Nucleic Acids A very large number of screening experiments were carried out to develop parameters suitable for recovering nucleic acids of a desired fragment size from solutions such as the isolated and concentrated upper phase of the concentrated ATPS as described above. In some embodiments, size fractionation and salting-out processes are utilized, where the Thermo GR1kb+ and GRL of MA size of 25 bp to 20 kbp, which are purified and concentrated nucleic acid solutions, are first mixed by vortexing, and then various concentrations of glycol polymers with a molecular weight of 15000 in the range of 1% to 12% and non-phase-forming salts in the concentration range of 0.1 M to 1.8 M are centrifuged at 10 krcf for 15 minutes at RT. Condensation of the polymer / salt yields nucleic acids exceeding the desired cut-off size, which can be freely adjusted beyond 100 bp, and precipitates them as pellets at the lower end of the microcentrifuge tube at the bottom of the mixing vessel, while a supernatant containing nucleic acid fragments smaller than the selected size is formed. Next, the supernatant having a final volume according to the formulation is transferred by a micropipette and mixed with a precipitation component containing polyethyleneimine, spermine, quatroquad, NaI, spermine HCl, trivalent spermidine, and salts, and the pH is neutralized to about 8 - 10. Next, the tube is rotated at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet and desalt it.

[0064] The cut-off size was found to be precisely adjustable by a very large number of factors including glycol polymers with different molecular weights from 2 k to 20 k at a final concentration of 1% to 12% (v / v), a non-phase-forming salt concentration of 0.1 M to 1.8 M, an incubation temperature of 0°C to 30°C, an incubation time of 5 to 20 minutes, and a centrifugation time and speed in the combination of 15 minutes at 10 krcf to 1 minute at 20 krcf. Testing different coprecipitants, several were found to be beneficial in removing large-scale DNA bands and stabilizing the selected cut-off.

[0065] In one embodiment, through several nucleic acid recovery experiments, Samples 1 to 9 were prepared, analyzed, and the effects of changing different parameters in the polymer / salt DNA precipitation system were evaluated. A sample solution of a nucleic acid mixture prepared by adding plasma (Thermo GR1kb+ and GRL, MA size of 25 bp to 20 kbp) was mixed with the first ATPS component in a tube and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the first isolated ATPS. Next, each of the first lower phases from the first ATPS was transferred to a new tube with the second ATPS component. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the second concentrated ATPS. Next, each of the second upper phases with the reaction volume (μL) from the second ATPS was transferred to a new tube respectively. A fraction component containing glycol polymer P10, salt S17, and additive EDTA was added to the second upper phase from the second ATPS to form a fraction mixture. After thorough vortexing, the tube was incubated at room temperature for 10 minutes (or 30 minutes for Sample 9) and rotated at about 16,000 rcf for 10 minutes. In each sample, a pellet "P" and a supernatant "S" were obtained. The supernatant "S" was transferred to a new tube, mixed with the precipitation component and salt as described above, and the pH was neutralized to about 8 - 10. Next, the tube was rotated at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in a buffer more suitable for analysis. The obtained pellet "P" was resuspended in a buffer for analysis. Tables 1 - 3 show the details of the first ATPS component, the second ATPS component, and the experimental conditions used in the nucleic acid recovery experiment in this embodiment example.

[0066]

Table 1

[0067]

Table 2

[0068]

Table 3

[0069] Figure 4 shows the gel image results of the pellets and supernatants in several nucleic acid recovery experiments. "L" represents the DNA ladder (Thermo GR1kb+ and GRL, MA size of 25 bp to 20 kbp) directly loaded into the reference gel, "P" represents the DNA recovered in the pellet, and "S" represents the DNA recovered in the supernatant obtained in the final step. These show how changes in several precipitation components (polymers, salts, coprecipitants) affect the size cutoff. According to the results, a decrease in the polymer concentration in the fractionation component from 26.0% to 14.3% would preferably change the size cutoff from 500 bp or less (Sample 1) to no cutoff, i.e., all sizes of DNA fragments are retained in the supernatant (Sample 2), while an increase in the polymer concentration in the fractionation component from 26.0% to 40.1% would preferably change the size cutoff from 500 bp or less (Sample 1) to 200 bp, i.e., only DNA fragments of approximately 200 bp or less are retained in the supernatant (Sample 3). According to the results, a decrease in the salt concentration in the fractionation component from 0.5% to 0.26% would cause a harmful effect on the DNA precipitation in the pellet, and it was shown that DNA fragments of all sizes are retained in the supernatant (Sample 4). Even an increase in the salt concentration in the fractionation component from 0.5% to 0.9% would cause a harmful effect on the DNA precipitation in the pellet (Sample 5), but DNA fragments larger than 500 bp are not retained in the supernatant. The results in Sample 6 (with decreased EDTA concentration) and Sample 7 (with increased EDTA concentration) show that fluctuations in the concentration of EDTA do not contribute to changes in the size cutoff. Furthermore, according to the results, a decrease in the total amount of added DNA from 500 ng to 100 ng (Sample 8) significantly decreases the final yield (i.e., the DNA band intensity is lighter), but it is shown that it does not affect the size cutoff. A longer incubation time (increase from 10 minutes to 30 minutes) does not increase the final yield (Sample 9).Generally, as shown in the gel image, by changing the concentration of the polymer used, the size cutoff changes from 500 bp to 200 bp or from 500 bp to no cutoff, while by changing the concentration of the salt used, the precipitation ability and the removal of large DNA fragments in response to the increase in salt concentration are affected. By attempting the double precipitation method, it was found that consistent results could be obtained, but the yield of nucleic acid fragments of the target size decreased. Furthermore, it was discovered that lower initial nucleic acid concentrations significantly decrease the yield and purity of the resulting nucleic acid products, but do not decrease the size cutoff. These observations can be resolved by adjusting the concentration of each component added (Figure 5).

[0070] In one embodiment, Samples 1 - 5 were prepared, analyzed, and the robustness of the recovery system experiment was evaluated. Tables 1 - 2 show the details of the first ATPS component and the second ATPS component, and Table 4 shows the experimental conditions used in the nucleic acid recovery experiment in this example of the embodiment.

[0071] [Table 4]

[0072] Figure 5 illustrates a gel image showing the robust ability of the recovery system embodiment to be relatively easily fine - tuned through the adjustment of design parameters at nucleic acid fragment size cutoffs within 50 bp increments from 100 bp to 300 bp. A total of 100 ng of NEB's 1 kb ladder and Thermo GRL were pre - added to the plasma sample, and the sample was run according to the present invention.

[0073] In the example of the embodiment, "cfDNA" represents a target DNA size example of 150 to 200 bp, for example, about 170 bp. In the results of Samples 1 to 2, most of the cfDNA precipitated into the pellet, while in the case of Samples 3 to 5, it was shown that the cfDNA was retained in the supernatant. By varying only the concentration of the components in the precipitation step of the polymer / non-phase-forming salt to be 1 to 12% (v / v) of the glycol polymer 15000 and a non-phase-forming salt concentration of 0.1 M to 1.8 M, the above-described desirable cut-off range can be achieved. This range was selected for illustrative purposes only and is related to the small sizes encountered in the analysis of cfDNA.

[0074] Example 4 - Comparison with the Qiagen QIAamp MinElute ccfDNA Mini Kit In this embodiment, the same DNA sample was used, and the ATPS system was compared with a commercially available kit. Thermo GR1kb+ and GRL with an MA size of 25 bp to 20 kbp were added to plasma to form a 1 kb ladder sample mixture for testing. The same amount of 1 kb ladder sample mixture was used for testing. For samples using the Qiagen QIAamp MinElute ccfDNA Mini kit (Qiagen kit), nucleic acids were prepared according to the manufacturer's instructions. For samples using the ATPS system, the prepared nucleic acid mixture solution was mixed with the first ATPS component in a tube and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the first isolated ATPS. Next, the first lower phase from the first ATPS was transferred to a new tube with the second ATPS component. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the second concentrated ATPS. Next, each of the second upper phases having the reaction volume (μL) from the second ATPS was transferred to a new tube. A fractionation component containing glycol polymer P10, salt S17, and additive EDTA was added to the second upper phase from the second ATPS to form a fractionation mixture. After thorough vortexing, the tube was incubated at room temperature for 10 minutes (or 30 minutes for sample 9) and rotated at about 16,000 rcf for 10 minutes. In each sample, a pellet "P" and a supernatant "S" were obtained. The supernatant "S" was transferred to a new tube and mixed with the precipitation component. Next, the tube was centrifuged at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in a buffer suitable for further analysis. The obtained pellet "P" was resuspended in a buffer for analysis. Tables 1-2 show the details of the first ATPS component and the second ATPS component, and Table 5 shows the experimental conditions used in the nucleic acid recovery experiment in this embodiment. The DNA samples recovered from the ATPS system and the commercially available kit for comparison were analyzed by qPCR (Roche).

[0075] [Table 5]

[0076] As shown in Figure 6, when using the present invention, almost 100% of the nucleic acid was analyzed by qPCR (Roche), and a 130-bp spike-in oligonucleotide was extracted from the plasma sample by the continuous ATPS method, while about 50% of such nucleic acid was extracted by the Qiagen kit. Therefore, the present invention showed improved performance compared to the QIAamp MinElute ccfDNA Mini kit.

[0077] Example 5 - Selective isolation and concentration of target nucleic acid fragments using glycol polymer / sodium poly(acrylate) and glycol polymer / Triton ATPS systems In this embodiment example, several nucleic acid recovery experiments were conducted to prepare, analyze, and evaluate examples of ATPS systems. A sample solution of a nucleic acid mixture prepared by adding plasma (Thermo GR1kb+ and GRL, MA size of 25 bp to 20 kbp) was mixed with the first ATPS component (glycol polymer / sodium poly(acrylate)) in a tube and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the first isolation ATPS. Next, each of the lower phases from the first ATPS was transferred to a new tube with the second ATPS component (glycol polymer / salt / Triton at concentrations of 0, 10, 20, and 30%). After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the second concentration ATPS. The upper (T) and lower (B) phases were transferred to a new tube and mixed with the precipitation component and salt. Next, the tube was centrifuged at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in a buffer more suitable for analysis. Tables 6 - 7 show the details of the first ATPS component and the second ATPS component in this embodiment example.

[0078] [Table 6]

[0079] [Table 7]

[0080] Figure 7 shows the gel image results of the upper (T) and lower (B) phases in the nucleic acid recovery experiment. These show how changes in several precipitation components (polymers, salts, coprecipitants) affect the size cut-off. According to the results, it was shown that most proteins and contaminants would be distributed in the first lower phase in the first ATPS system (glycol polymer / salt / sodium poly(acrylate)), and most of the nucleic acids would be retained in the first upper phase. Figure 7 shows that as the concentration of glycol polymer / salt / Triton in the second ATPS system increases, the nucleic acid size cut-off selection changes from no cut-off to about 300 bp, is retained in the second upper (T) phase, and for larger unwanted DNA, is distributed in the second lower (B) phase. Generally, as shown in the gel image, the desired nucleic acid size cut-off in the second upper phase will change due to the change in the concentration of glycol polymer / Triton used.

[0081] Example 6 - Selective isolation and concentration of target nucleic acid fragments using ATPS systems with different salt concentrations In this embodiment example, samples were prepared, analyzed, and various concentrations of sodium sulfate were evaluated through several DNA and protein recovery experiments. A sample solution (500 ng / μL) of a nucleic acid mixture with 89.3 mg / ml of protein was prepared, mixed with the first ATPS component with different concentrations of sodium sulfate in a tube, and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the first isolated ATPS. Next, each of the lower phases from the first ATPS was transferred to a new tube with the second ATPS component. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the second concentrated ATPS. The upper (T) and lower (B) phases were transferred to new tubes and mixed with the precipitate components and salts. Next, the tube was centrifuged at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in a buffer more suitable for analysis. The DNA recovery % and protein recovery % in the upper and lower phases were determined. Table 8 shows the experimental details in this embodiment example.

[0082] [Table 8]

[0083] Figure 8 shows the DNA and protein recovery results in the nucleic acid and protein recovery experiments. According to the results, it was shown that as the sodium sulfate salt concentration increased from 0.1% to 0.8%, higher DNA recovery of about 60% - 85% was brought about in the lower phase (Samples 2 and 3).

[0084] Example 7 - Selective Isolation and Concentration of Target Nucleic Acid Fragments Using Multiple Triton ATPS Systems In this embodiment, several DNA and protein recovery experiments were conducted to prepare, analyze samples, and evaluate sodium sulfate at various concentrations. A sample solution (1000 ng in total) of a nucleic acid mixture with 200 ul of plasma protein was prepared, mixed with the first ATPS component in a tube to form the first ATPS system (water / Triton ATPS system), and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the first isolated ATPS. Next, each of the lower phases from the first ATPS was transferred to a new tube with a precipitation component. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form a nucleic acid pellet in the salting-out system. The supernatant containing contaminants was removed. Next, the second ATPS component was added to the pellet. After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the second isolated ATPS (glycol polymer / salt ATPS system). The second upper phase containing smaller-sized nucleic acids was transferred to a new tube and mixed with the third ATPS component to form the third ATPS system (glycol polymer / Triton system). After thorough vortexing, the tube was centrifuged at 7000 rcf for about 1 minute to form the third ATPS (glycol polymer / Triton ATPS system) in the upper white suspension. Next, water was added to resuspend the white suspension to form a water / Triton system with an upper Triton-poor phase and a lower Triton-rich phase. The final extraction volumes used in Samples 1 and 2 were 25 ul and 20 ul, respectively. The upper Triton-poor phase is the final product containing clean target nucleic acids. The removed liquid, the final extract (i.e., the Triton-poor phase), and the Triton-rich phase were analyzed by gel electrophoresis. Figure 9 shows the gel image results of the removed liquid, the final extract, and the T-rich phase. "L" represents the DNA ladder (Thermo GR1kb+ and GRL, MA size of 25 bp to 20 kbp) directly loaded into the reference gel. The results showed that the targeted nucleic acids were present in all fractions and a partial decrease in the targeted nucleic acids was shown in a series of extraction steps. Furthermore, the results showed that the final extract contained a significant amount of the targeted nucleic acids.The 75 bp band of the target is retained in the final extract, removing the added proteins. Both the quality and quantity of the target nucleic acid in the final extract were sufficient for gel electrophoresis analysis. In short, multiple Triton ATPS systems are efficient when isolating, purifying, and concentrating target nucleic acids for downstream applications.

[0085] Example 8 - Recovery Rates of Isolated and Concentrated Nucleic Acids Using Polymers with Different Molecular Weights In this example embodiment, samples were prepared, analyzed, and the effect of varying the different molecular weights of the polymers in the second ATPS system on nucleic acid recovery was evaluated. A sample solution of approximately 50 ng of a nucleic acid mixture prepared by adding plasma was mixed in a tube with the same first ATPS components and incubated at approximately 37 °C for approximately 15 minutes. After thorough vortexing, the tube was centrifuged at 7000 rcf for approximately 1 minute to form the first isolation ATPS. Next, each of the first lower phases from the first ATPS was transferred to a new tube with a second ATPS component containing either a low molecular weight or medium molecular weight polymer. The polymers and salts in the second ATPS component in the total sample were maintained at the same concentrations of 7% and 35% respectively. After thorough vortexing, the tube was centrifuged at 7000 rcf for approximately 1 minute to form the second concentration ATPS. Next, each of the second upper phases with the reaction volume (uL) from the second ATPS was transferred to a new tube respectively. The supernatant was transferred to a new tube, mixed with the precipitate components and salts, and the pH was neutralized to approximately 8 - 10. Next, the tube was rotated at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in 15 ul of a buffer suitable for further analysis. The nucleic acid yield of the final product was determined by Qubit Fluorometric Quantification (Invitrogen), and the recovery rate was calculated from the yield. Table 9 shows the experimental details and the results of DNA yield and recovery rate in this example embodiment. In this example embodiment, the molecular weight difference from "low" to "medium" is within 100 - 1000 Da.

[0086]

Table 9

[0087] Table 9 shows that as the polymer molecular weight increases from "low" to "medium", it leads to a deterioration in the recovery rate, indicating that a polymer with a specific molecular weight may bring about remarkable performance in nucleic acid recovery rate.

[0088] Example 9 - Recovery Rate of Isolated and Concentrated Nucleic Acids with Different Upper Phase Volumes in the Second ATPS In this example embodiment, samples were prepared, analyzed, and the effect of changing the volume of the upper phase in the second ATPS system on the nucleic acid recovery rate was evaluated. One nucleic acid mixture was prepared, aliquoted into tubes with the same first ATPS components, and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tubes were centrifuged at 7000 rcf for about 1 minute to form the first isolation ATPS. Next, each of the first lower phases from the first ATPS was pooled together and aliquoted into new tubes with the second ATPS components having different volumes of polymer (PM2) and salt (SM2) according to Table 10. After thorough vortexing, the tubes were centrifuged at 7000 rcf for about 1 minute to form the second concentration ATPS. Next, the volume of each of the second upper phases from the second ATPS was measured. Each of the second upper phases was transferred to a new tube respectively. The supernatant was transferred to a new tube, mixed with the precipitate components and salts, and the pH was neutralized to about 8 - 10. Next, the tubes were rotated at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in a buffer more suitable for analysis. For each sample, the ability to form a clean pellet, the nucleic acid purity by the ratio of A260 / A280, the final product concentration, and the total yield were evaluated and determined. Table 10 shows the experimental details and results in this example embodiment.

[0089] [Table 10]

[0090] Having a larger upper phase volume is advantageous as it tends to minimize nucleic acid loss during transfer or extraction. However, Table 10 shows that as the upper phase volume increases, it either only produces acceptable DNA pellets (Samples 2 and 3) or even fails to produce DNA pellets (Samples 4, 6, 8 - 10). Samples with lower salt concentrations (Sample 2) produced insufficient and unstable pellets. Larger salt volumes (Samples 5 - 10) resulted in more stable pellets and reasonable yields. To achieve an increase in the volume of the upper phase, the polymer concentration needs to be increased accordingly. It was found that an upper phase volume exceeding 200 μL would be obtained when the polymer concentration was increased to exceed 8.7%. The data in Table 10 showed that an upper phase volume larger than 220 μL would lead to failure of the nucleic acid extraction process. Even if nucleic acid precipitation was successful, they produced unclean pellets (either unknown pellets or junk pellets), and the results were also reflected in the purity of the DNA (A260 / A280). In short, it has been discovered that when the polymer concentration and salt concentration in the second ATPS are less than 8.7% and 1% respectively, and the final volume of the upper phase is less than 200 μL, the yield and purity of the nucleic acid products obtained from the ATPS system are significantly improved.

[0091] Example 10 - Recovery Rates of Isolated and Concentrated Nucleic Acids by Polymers of Different Molecular Weights in the First and Second ATPS Systems In this exemplary embodiment, samples were prepared, analyzed, and the effects of using polymers with different molecular weights in the first and second ATPS systems on the nucleic acid recovery rate were evaluated. A sample solution of the nucleic acid mixture was prepared, aliquoted into tubes having the first ATPS component with different polymer molecular weights (according to Table 11), and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tubes were centrifuged at 7000 rcf for about 1 minute to form the first isolated ATPS. Next, each of the first lower phases from the first ATPS was transferred to a new tube having the second ATPS component with different polymer molecular weights (according to Table 11). After thorough vortexing, each of the tubes was centrifuged at 7000 rcf for about 1 minute to form the second concentrated ATPS. Each of the second upper phases was transferred to a new tube respectively. The supernatant was transferred to a new tube, mixed with the precipitate component and salts, and the pH was neutralized to about 8 - 10. Next, the tube was rotated at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in the same volume of buffer suitable for further analysis. For each sample, the nucleic acid purity and DNA concentration by the ratio of A260 / A280 were measured and determined by a spectrophotometer (Nanodrop). Table 11 shows the experimental details and results in this exemplary embodiment. In this exemplary embodiment, the molecular weight difference from "low" to "high" is within 200 - 2000 Da.

[0092]

Table 11

[0093] Table 11 showed that when a high molecular weight polymer was used instead of a lower molecular weight polymer in the second ATPS, there was a significant decrease in DNA concentration from about 24 ng / ul to about 17 ng / ul (Samples 1 and 2). It was also shown that when a low molecular weight polymer was used in the first ATPS, no further phase formation occurred in the first ATPS, and thus no nucleic acid was obtained. According to the results, it was shown that the selection of the molecular weight of the polymer in the first and second ATPS systems significantly affects the nucleic acid recovery rate in addition to the selection of the target size cut-off. If the first polymer component has a higher molecular weight than the second polymer component, the yield and purity of the nucleic acid product obtained from the ATPS system will be significantly improved.

[0094] Example 11 - Recovery Rates of Isolated and Concentrated Nucleic Acids by Different Molecular Weights and Concentrations of Polymers in Fractionation Components In this exemplary embodiment, samples were prepared, analyzed, and the effects of using different molecular weights and different concentrations of polymers in the fraction components on the nucleic acid recovery rate were evaluated. A sample mixture solution of a nucleic acid mixture of DNA ladder (Thermo GR1kb+ and GRL, MA size of 25 bp to 20 kbp) at about 200 ng / mL was prepared, aliquoted into tubes having the same first ATPS component, and incubated at about 37 °C for about 15 minutes. After thorough vortexing, the tubes were centrifuged at 7000 rcf for about 1 minute to form the first isolated ATPS. Each of the first lower phases from the first ATPS was transferred to a new tube having the second ATPS component with different polymer molecular weights (according to Table 11). After thorough vortexing, each of the tubes was centrifuged at 7000 rcf for about 1 minute to form the second concentrated ATPS. Each of the second upper phases was transferred to a new tube respectively. Next, each of the second upper phases from the second ATPS was transferred to a new tube respectively. Fraction components containing polymers of different molecular weights or different concentrations (see Table 12) were added to the second upper phases from the second ATPS to form fraction mixtures respectively. After thorough vortexing, each of the tubes was incubated at room temperature for 10 minutes (or 30 minutes for Sample 9) and rotated at about 16,000 rcf for 10 minutes. In each sample, a pellet "P" and a supernatant "S" were obtained. The supernatant "S" was transferred to a new tube, mixed with the precipitation component and salts, and the pH was neutralized to about 8 - 10. Next, the tube was rotated at 20 krcf for 2 minutes to isolate the target nucleic acid fragment as a pellet, desalted, and resuspended in 10 μL of a buffer (1× loading dye) suitable for further analysis. The obtained pellet "P" was resuspended in the same manner for analysis. Table 11 shows the experimental details and results in this exemplary embodiment. In this exemplary embodiment, the molecular weight difference from "low" to "high" is within 200 - 2000 Da.

[0095] Figure 10 shows the gel image results of the pellet "P" and the supernatant "S" in the nucleic acid recovery experiment in the embodiment example. The molecular weight difference from "low" to "medium" is within 100 - 1000 Da, while the molecular weight difference from "low" to "high" is within 100 - 1000 Da. "P" represents the DNA recovered in the pellet, and "S" represents the DNA recovered in the supernatant obtained in the final step. The dashed line indicates a molecular size of approximately 1500 bp. According to the results, when using a high molecular weight polymer in the fractionation component, it was shown that a cut-off size below approximately 1500 bp was achieved (Samples 1 and 2). When the molecular weight of the polymer changes from high molecular weight to medium molecular weight or even lower molecular weight, a cut-off size below approximately 1500 bp will not be achieved, and most of the nucleic acids with all fragment sizes tend to be retained in the supernatant (Samples 3 - 6). Furthermore, according to the results, when using a polymer at twice or four times the concentration, it was shown that most of the nucleic acids with all fragment sizes were retained in the pellet, so a satisfactory cut-off size was not achieved with a reasonable yield (Samples 7 - 10). In short, for a high molecular weight polymer at a specific concentration, satisfactory size cut-off and nucleic acid recovery rate results will be achieved.

[0096] Therefore, the exemplary embodiments of the present invention have been sufficiently described. Although the description refers to specific embodiments, it will be apparent to those skilled in the art that the present invention can be implemented with variations of these specific details. Therefore, the present invention should not be construed as being limited to the embodiments shown herein.

Claims

1. A method for isolating and concentrating target nucleic acid fragments smaller than a target size from a liquid mixture containing nucleic acids and contaminants, mixing the liquid mixture with a first aqueous two-phase system (ATPS) formed by a polymer dissolved in a first-phase solution and a salt dissolved in a second-phase solution, whereby the target nucleic acid fragments smaller than the target size are partitioned into the second-phase solution and the contaminants are partitioned into the first-phase solution; extracting the second-phase solution and mixing it with a second ATPS formed by a polymer forming the second phase dissolved in a third-phase solution and a salt dissolved in a fourth-phase solution, whereby the target nucleic acid fragments smaller than the target size are partitioned into and concentrated in the third-phase solution to obtain concentrated target nucleic acid fragments smaller than the target size; recovering the concentrated target nucleic acid fragments smaller than the target size from the third-phase solution The method comprising the above steps.

2. The method according to claim 1, wherein the polymer forming the first phase has a higher molecular weight than the polymer forming the second phase.

3. The method according to claim 1, wherein the molar concentration of the polymer forming the first phase in the first-phase solution is higher than the molar concentration of the polymer forming the second phase in the third-phase solution.

4. The method according to claim 1, wherein the mass concentration of the polymer forming the first phase in the first-phase solution is higher than the mass concentration of the polymer forming the second phase in the third-phase solution.

5. The salt dissolved in the second-phase solution is dipotassium phosphate, monopotassium phosphate, cosmotropic salts, chaotropic salts, inorganic salts containing cations selected from the group consisting of linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium and anions selected from the group consisting of phosphoric acid, sulfuric acid, nitric acid, chloride and bicarbonate, NaCl, Na 3 PO 4 and K 3 PO 4 and Na 2 SO 4 and potassium citrate, (NH 4 ) 2 SO 4 and sodium citrate, sodium acetate, ammonium acetate, magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, aluminum salts, bromide salts, iodide salts A fluoride salt, a carbonate, a sulfate, a citrate, a carboxylate, a borate, a phosphate, potassium phosphate, a combination thereof, The method according to claim 1, selected from the group consisting of.

6. The salt dissolved in the fourth-phase solution is dipotassium phosphate, monopotassium phosphate, a kosmotropic salt, a chaotropic salt, an inorganic salt containing a cation selected from the group consisting of linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, and an anion selected from the group consisting of phosphoric acid, sulfuric acid, nitric acid, chloride and bicarbonate, NaCl, Na 3 PO 4 and K 3 PO 4 and Na 2 SO 4 and potassium citrate, (NH 4 ) 2 SO 4 and sodium citrate, sodium acetate, ammonium acetate, a magnesium salt, a lithium salt, a sodium salt, a potassium salt, a cesium salt, a zinc salt, an aluminum salt, a bromide salt, an iodide salt, a fluoride salt, a carbonate, a sulfate, a citrate, a carboxylate, a borate, a phosphate, potassium phosphate, a combination thereof, The method according to claim 1, selected from the group consisting of.

7. The second-phase solution of the first ATPS exerts a weaker exclusion volume interaction on the target nucleic acid fragment smaller than the target size than the fourth-phase solution of the second ATPS, the method according to claim 1.

8. The first-phase solution of the first ATPS exerts a stronger exclusion volume interaction on the target nucleic acid fragment smaller than the target size than the third-phase solution of the second ATPS, the method according to claim 1.

9. The second-phase solution of the first ATPS exerts a more favorable hydrophilic / hydrophobic interaction on the partitioning of the target nucleic acid fragment smaller than the target size into the second phase than the fourth-phase solution of the second ATPS, the method according to claim 1.

10. The first-phase solution of the first ATPS exerts a less favorable hydrophilic / hydrophobic interaction on the partitioning of the target nucleic acid fragment smaller than the target size into the first-phase solution than the third-phase solution of the second ATPS, the method according to claim 1.

11. The method according to claim 1, wherein the second-phase solution of the first ATPS exerts a more favorable electrostatic interaction on the partitioning of the target nucleic acid fragments smaller than the target size into the second phase than the fourth-phase solution of the second ATPS.

12. The method according to claim 1, wherein the first-phase solution of the first ATPS exerts an electrostatic interaction that is not more favorable than the fourth-phase solution of the second ATPS on the partitioning of the target nucleic acid fragments smaller than the target size into the first-phase solution.

13. Recovering the concentrated target nucleic acid fragments smaller than the target size comprises separating the third-phase solution from the fourth-phase solution; mixing the third-phase solution with at least one size-fractionating component selected from polymers, surfactants, salts, and combinations thereof dissolved in an aqueous solution to form a supernatant composed of the concentrated target nucleic acid fragments smaller than the target size and a precipitation pellet of nucleic acids larger than the target size; separating the supernatant from the precipitation pellet of nucleic acids larger than the target size and precipitating the target nucleic acid fragments smaller than the target size from the supernatant The method according to claim 1.

14. The method according to claim 13, wherein the at least one size-fractionating component comprises a polymer having a higher molecular weight than the polymer forming the first phase, and the polymer forming the first phase has a higher molecular weight than the polymer forming the second phase.

15. The method according to claim 13, wherein the molar concentration of the at least one size-fractionating component in the supernatant is less than the molar concentration of the polymer forming the first phase in the first-phase solution, and the polymer forming the first phase in the first-phase solution has a higher molar concentration than the molar concentration of the polymer forming the second phase in the third-phase solution.

16. The method according to claim 13, wherein the mass concentration of the at least one size-fractionating component in the supernatant is less than the mass concentration of the polymer forming the first phase in the first-phase solution, and the polymer forming the first phase in the first-phase solution has a higher mass concentration than the mass concentration of the polymer forming the second phase in the third-phase solution.

17. The method according to claim 13, wherein the at least one size-fractionating component is selected from the group consisting of salts, polymers, surfactants, and combinations thereof.

18. The method according to claim 13, wherein the polymer forming the first phase and the polymer forming the second phase have a molecular weight of 200 to 10,000.

19. The method according to claim 1, wherein the liquid mixture comprises at least one of blood, plasma, cells, exosomes, proteins, cell-free DNA, RNA, and circulating tumor DNA.

20. The method according to claim 1, wherein the target size is less than 10,000 bp.

21. The method according to claim 1, wherein the target size is less than 1000 bp.

22. A kit for isolating and concentrating target nucleic acid fragments smaller than a target size from a liquid mixture containing nucleic acids and contaminants, having a composition that forms a first aqueous two-phase system (ATPS) comprising a polymer dissolved in a first-phase solution and a salt dissolved in a second-phase solution, such that when the composition forming the first aqueous two-phase system (ATPS) is mixed with the liquid mixture, the target nucleic acid fragments smaller than the target size are partitioned into the second-phase solution and the contaminants are partitioned into the first-phase solution, components for forming, wherein the polymer forming the first phase is polyalkylene glycol, poly(oxyalkylene) polymer, poly(oxyalkylene) copolymer, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, alkoxylated surfactant, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicon-modified polyether, and poly N-isopropylacrylamide and copolymers thereof, The salt dissolved in the second-phase solution is dipotassium phosphate, monopotassium phosphate, cosmotropic salts, chaotropic salts, inorganic salts containing a cation selected from the group consisting of linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium and an anion selected from the group consisting of phosphoric acid, sulfuric acid, nitric acid, chloride, and bicarbonate, NaCl, Na 3 PO 4 and K 3 PO 4 and Na 2 SO 4 and potassium citrate, (NH 4 ) 2 SO 4 and, sodium citrate, sodium acetate, ammonium acetate, magnesium salts, lithium salts, sodium salts, potassium salts A cesium salt, a zinc salt, an aluminum salt, a bromide salt, an iodide salt, a fluoride salt, a carbonate salt, a sulfate salt, a citrate salt, a carboxylate salt, a borate salt, a phosphate salt, potassium phosphate, combinations thereof, selected from the group consisting of; A composition for forming a second ATPS comprising a polymer forming a second phase dissolved in a third phase solution and a salt dissolved in a fourth phase solution, whereby when the composition for forming the second ATPS is mixed with the second phase solution, the target nucleic acid fragments smaller than the target size are partitioned into and concentrated in the third phase solution, components for forming; The polymer forming the second phase is polyalkylene glycol, poly(oxyalkylene) polymer, poly(oxyalkylene) copolymer, polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, alkoxylated surfactant, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicon-modified polyether and poly N-isopropylacrylamide and copolymers thereof, The salt dissolved in the fourth phase solution is dipotassium phosphate, monopotassium phosphate, a kosmotropic salt, a chaotropic salt, an inorganic salt containing a cation selected from the group consisting of linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium and an anion selected from the group consisting of phosphoric acid, sulfuric acid, nitric acid, chloride and bicarbonate, NaCl, Na 3 PO 4 and K 3 PO 4 and Na 2 SO 4 and potassium citrate, (NH 4 ) 2 SO 4 and sodium citrate, sodium acetate, ammonium acetate, a magnesium salt, a lithium salt, a sodium salt, a potassium salt, a cesium salt, a zinc salt, an aluminum salt, a bromide salt, an iodide salt, a fluoride salt, a carbonate salt, a sulfate salt, a citrate salt, a carboxylate salt, a borate salt, a phosphate salt, potassium phosphate, combinations thereof, selected from the group consisting of, A kit comprising materials for concentrating the target nucleic acid fragments from the third phase solution.

Images