Method and kit for isolating target nucleic acids smaller than the target size from a sample

Abstract

The present disclosure relates to methods, compositions, and kits for isolating target nucleic acids smaller than a target size from a sample containing nucleic acid components. In some embodiments, the methods involve one or more aqueous two-phase system (ATPS) compositions, at least one solid phase medium, and at least one buffer. Some embodiments provide kits including one or more ATPS compositions, at least one solid phase medium, and at least one buffer. Other embodiments provide methods for diagnosing a disease or condition using the methods described herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 381,933, filed November 2, 2022, the entire contents of which are incorporated herein by reference in their entirety for all purposes. [Technical Field]

[0002] The present application relates to a method and kit for isolating a target nucleic acid. More specifically, the present application relates to a method and kit for isolating a target nucleic acid of smaller than a target size from a sample. [Background technology]

[0003] Effectively concentrating and isolating target nucleic acids below a certain size from a sample is challenging, especially for isolating, purifying, and concentrating very rare and dilute nucleic acid fragments in a complex biological matrix background. Generally, the yield of relevant fragments is very low, and subsequent analysis may lack sufficient diagnostic sensitivity and specificity. For some applications, such as non-invasive prenatal testing (NIPT) and circulating tumor DNA enrichment, size difference is an important criterion for distinguishing target nucleic acids from non-target nucleic acids. Therefore, there is a need for an improved method that is simple, stable, robust, and effective for isolating target nucleic acids below a certain size from a sample. Summary of the Invention

[0004] Disclosed herein are novel methods and kits for isolating, concentrating, and / or purifying target analytes (eg, nucleic acids) below a target size using solid phase media (eg, beads or columns).

[0005] In some embodiments, a method for isolating a smaller-than-target-size target nucleic acid from a sample containing nucleic acid components is provided, the method comprising: (a) preparing a sample solution from the sample; (b) contacting a plurality of beads with the sample solution, wherein the nucleic acid components bind to the plurality of beads to form bead-analyte complexes; (c) mixing the bead-analyte complexes with a fractionation buffer containing at least one chaotropic agent to form a bulk fractionation solution, and releasing the smaller-than-target-size target nucleic acid from the bead-analyte complexes into the bulk fractionation solution; (d) immobilizing the bead-analyte complexes; and (e) separating the bulk fractionation solution containing the isolated smaller-than-target-size target nucleic acid from the immobilized bead-analyte complexes.

[0006] In some embodiments, a kit for isolating a target nucleic acid smaller than a target size from a sample containing nucleic acid components is provided, the kit including: (a) at least one ATPS component selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof; (b) a plurality of beads; (c) a fractionation buffer including at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide; and (d) a binding buffer including at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0007] In some embodiments, a method is provided for concentrating and purifying one or more target analytes from a sample solution, the method comprising: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the enriched one or more target analytes to obtain an enriched solution; (c) adding magnetic beads to the concentrated solution and allowing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; (d) recovering the one or more target analytes from the bead-analyte complexes to obtain a final solution containing concentrated and purified one or more target analytes.

[0008] In some embodiments, corresponding kits can be advantageously used in conjunction with and to practice the methods of each aspect of the present invention. In some embodiments, the kits can include the components described in the various embodiments, but can further include syringe- or pipette-accessible containers for storage, packaging, and / or reaction, and optional equipment for manipulating aqueous solutions. Such containers and equipment can include columns, test tubes, capillaries, plastic test tubes, falcon tubes, culture tubes, well plates, pipettes, cuvettes, and the like.

[0009] This specification has discussed other exemplary embodiments.

[0010] advantage

[0011] Various embodiments of the present disclosure have many advantages. For example, the methods and kits of the present disclosure uniquely and effectively concentrate and purify target analytes smaller than the target size from samples (e.g., clinical / biological samples). These methods and kits are particularly effective in purifying target analytes (e.g., free DNA) that are present in very small concentrations in biological samples. As shown in the examples, the methods of the present disclosure allow for precise size selection of recovered DNA molecules.

[0012] In some embodiments, the disclosed methods (referred to in some embodiments as "reverse fractionation") utilize complete binding of nucleic acid components in a sample, followed by selective debinding of target nucleic acids smaller than the target size from a solid phase medium (e.g., a magnetic bead-analyte complex or a solid phase extraction column). Compared to methods that utilize a first binding step to selectively bind and remove unwanted larger nucleic acids, the disclosed methods achieve significantly improved nucleic acid size fractionation, retaining target nucleic acids smaller than the target size in the supernatant for further purification by the solid phase medium.

[0013] In some embodiments, the disclosed methods are adaptable to varying sample volumes and can uniquely achieve stable and effective DNA size fractionation across different sample volumes, with stable DNA cutoffs and DNA recovery, particularly for small DNA fragments. In some embodiments, the disclosed methods are compatible with different sample types (e.g., plasma and urine) and have demonstrated stable and effective DNA size fractionation across different sample types. This allows for a wide range of applications and analyses, such as the diagnosis of diseases or conditions that require different types of clinical / biological samples.

[0014] The purified nucleic acids obtained by the disclosed methods can be used in a wide range of downstream applications, such as nucleic acid detection or analysis in forensic, diagnostic, or therapeutic applications, and laboratory procedures, such as sequencing, amplification, reverse transcription, labeling, digestion, blotting procedures, etc. The disclosed methods can improve the performance of downstream characterization or processing of nucleic acids.

[0015] In some embodiments, the disclosed methods and kits can be used in a variety of applications. For example, the disclosed methods and kits can be used for size-selective DNA fractionation during sequencing library production, i.e., isolating DNA molecules of a desired size or size range for subsequent sequencing applications, such as next-generation sequencing (NGS). In some embodiments, the disclosed methods and kits can be used to effectively isolate fetal nucleic acids from maternal nucleic acids, thereby enriching the fetal fraction in maternal samples for non-invasive prenatal detection (NIPT). In some embodiments, the disclosed methods and kits can be used to increase the ratio of circulating tumor DNA:free DNA and / or variant allele frequency (VAF) in clinical samples, which can be used for further analysis, such as cancer diagnostic assays.

[0016] These and other features and characteristics, and the method of operation and function of the associated components, will become more apparent from a consideration of the following detailed description and the appended claims, taken in conjunction with the drawings, all of which form a part of this specification, in which like reference numerals represent corresponding parts in the various views. It is to be expressly understood, however, that the drawings are used for purposes of illustration and description only and are not intended as a definition of the limits of the claims. [Brief explanation of the drawings]

[0017] [Figure 1A] Based on an exemplary embodiment, an exemplary operational flow of direct fractionation is shown. [Figure 1B] Based on an exemplary embodiment, an exemplary operational flow of reverse fractionation is shown. [Figure 2A] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma extracted from different volumes of the upper phase in a second ATPS using direct fractionation (expected DNA cutoff value is approximately 150 bp) are shown. [Figure 2B]According to an illustrative example, electropherograms of DNA oligonucleotide recovery from plasma extracted from different volumes of the upper phase in a second ATPS using reverse fractionation (expected DNA cutoff value of approximately 150 bp) are shown. [Figure 3A] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma extracted from different volumes of the upper phase in a second ATPS using direct fractionation (expected DNA cutoff value is approximately 300 bp) are shown. [Figure 3B] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma extracted from different volumes of the upper phase in a second ATPS using reverse fractionation (expected DNA cutoff value of approximately 300 bp) are shown. [Figure 4A] According to an illustrative example, electropherograms of DNA oligonucleotide recovery for different sample types using direct fractionation (expected DNA cutoff value is approximately 150 bp) are shown. [Figure 4B] According to an illustrative example, electropherograms of DNA oligonucleotide recovery for different sample types using reverse fractionation (expected DNA cutoff value is approximately 150 bp) are shown. [Figure 5A] According to an illustrative example, electropherograms of DNA oligonucleotide recovery for different sample types using direct fractionation (expected DNA cutoff value is approximately 300 bp) are shown. [Figure 5B] According to an illustrative example, electropherograms of DNA oligonucleotide recovery for different sample types using reverse fractionation (expected DNA cutoff value is approximately 300 bp) are shown. [Figure 6A] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6B]According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6C] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6D] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6E] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6F] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6G] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. [Figure 6H] According to an illustrative example, electropherograms of DNA oligonucleotide recovery of plasma samples using reverse fractionation with different reverse fractionation buffer formulations (Buffer R-015 to Buffer R-021, respectively) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless otherwise explained, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0019]

[0023] As used in this specification and the claims, the terms "comprise" (or any related form, e.g., "comprises" and "including"), "comprises" (or any related form, e.g., "comprises" or "comprising"), "containing" (or any related form, e.g., "contains" or "containing"), or "having" (or any related form, e.g., "has" or "having") mean the inclusion of the following elements, but do not exclude other elements. It is to be understood that for each example using the terms "comprises" (or any related form, e.g., "comprises" and "comprising"), "comprises" (or any related form, e.g., "comprises" or "comprising"), or "containing" (or any related form, e.g., "contains" or "containing"), the present disclosure / application further includes alternative examples in which the terms "comprises," "comprises," "contains," or "having" therein are replaced with "consisting essentially of" or "consisting of." These alternative embodiments using "consisting of" or "consisting essentially of" are understood to be smaller scope embodiments of the embodiments using "including," "comprising," or "containing."

[0020] For example, alternative embodiments of "a solution comprising A, B, and C" are "a solution consisting of A, B, and C" and "a solution consisting essentially of A, B, and C." The latter two embodiments are included in this disclosure / application even though they are not explicitly recited. It should be understood that the scope of the three embodiments listed above is different.

[0021] For clarity, the terms "comprise," "comprise," and "contain," and any related forms, are open terms, allowing for additional elements or features other than the required elements specified, and "consisting of" is closed terms, limited to the elements recited in the claim and not including any elements, steps, or ingredients not specified in the claim.

[0022] "Consisting essentially of" limits the claim to certain materials, elements, or steps ("essential elements") that do not have a substantial effect on the essential characteristic(s) of the invention for which protection is sought. In some embodiments, the essential characteristic(s) are one or more basic and novel characteristics of the invention for which protection is sought.

[0023] As used herein, the singular forms "a," "an," and "said" are intended to include the plural forms as well, unless the context clearly dictates otherwise. In some embodiments, the term "a" is interchangeable with terms such as "at least one" and "one or more."

[0024] When a range is referred to in the specification, it is understood that the range includes at least each discrete point within the range. For example, in some embodiments, 1 to 7 refers to 1, 2, 3, 4, 5, 6, and 7. Unless otherwise specified, a range is intended to include all values ​​within the range, including integers, fractions, parts, etc. For example, a range of 1 to 7 recited in the claims refers to ranges that include values ​​and subranges, such as 1, 1.5, 2-3, 6, and 7.

[0025] As used herein, the term "about" is understood to mean within the normal tolerance range of the art and not more than ±10% of the stated value. For example, about 50 means 45 to 55, including all values ​​therebetween. As used herein, the term "about" a particular value also includes the particular value, for example, about 50 includes 50.

[0026] As used herein, "aqueous" refers to a characteristic property of a solvent / solute system in which the solvate has predominantly hydrophilic properties. Examples of aqueous solvent / solute systems include those systems in which water or a water-containing composition is the primary solvent. The polymer and / or surfactant components (whose use is described in the Examples) are "aqueous" because they form an aqueous phase when combined with a solvent (e.g., water). As will be understood by those skilled in the art, the term liquid "mixture" as used herein refers only to the combination of components as defined herein.

[0027] As used herein, aqueous two-phase system (ATPS) refers to a liquid-liquid separation system that can achieve analyte isolation or concentration by partitioning, where two phases, moieties, regions, components, etc., interact differently with at least one analyte to which they are exposed and optionally dissolved. An ATPS forms when two immiscible phase-forming components with certain concentrations, such as a salt and a polymer, or two incompatible polymers (e.g., PEG and dextran), are mixed in an aqueous solution. ATPS methods are relatively inexpensive and scalable because they separate analytes (e.g., nucleic acids) and contaminants by two-phase partitioning.

[0028] As used herein, the term "isolated" refers to removing an analyte from its original environment, thereby changing it from its original environment. For example, the provided isolated nucleic acid typically has fewer non-nucleic acid components (e.g., proteins, lipids) than the amount of components present in the source sample. A composition containing an isolated analyte (e.g., sample nucleic acid) can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of non-analyte components (e.g., non-nucleic acid components)).

[0029] As used herein, "concentrated" refers to a mass ratio of the analyte under consideration to the solution in which it is suspended that is higher than the mass ratio of said analyte in its preconcentrated solution, e.g., it may be slightly higher, or more preferably at least 2-fold, 10-fold, 100-fold higher.

[0030] As used herein, a "biological sample" refers to any tangible substance obtained directly or indirectly from a living organism (e.g., a virus, bacteria, plant, animal, or human). Examples of biological samples include, but are not limited to, nucleic acids, proteins, cells, organelles, tissue extracts, tissues, organs, biological fluids (e.g., blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, cervical swabs, semen, breast milk, and other bodily fluids).

[0031] As used herein, a "clinical sample" refers to any sample obtained directly or indirectly from a subject (e.g., a human). In some embodiments, the subject is a human patient. Examples of clinical samples include, but are not limited to, blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swabs, vaginal swabs, cervical swabs, semen, breast milk, and other bodily fluids.

[0032] As used herein, "nucleic acid component" generally refers to nucleic acids extracted from a given sample, regardless of size. In some embodiments, the nucleic acid component comprises DNA, RNA, or a combination thereof. Examples of nucleic acid components include, but are not limited to, gDNA, cDNA, plasmid DNA, mitochondrial DNA, cfDNA, circulating tumor DNA (ctDNA), circulating fetal DNA, microbial free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or a combination thereof.

[0033] In some embodiments, a "target nucleic acid," "target analyte," or "small DNA fragment" refers to a nucleic acid fragment smaller than a selected size, e.g., a nucleic acid containing fewer than 1000 base pairs (e.g., less than 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 target nucleic acid / analyte is a single-stranded nucleic acid, while in other embodiments, the target nucleic acid / analyte is a double-stranded nucleic acid. In some embodiments, the target nucleic acid / analyte is DNA or RNA. Examples of target nucleic acids / analytes include, but are not limited to, gDNA, cDNA, plasmid DNA, mitochondrial DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microbial free DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA, long non-coding RNA (lncRNA), or combinations thereof.

[0034] As used herein, "free DNA" (cfDNA) is DNA that is present outside of cells, for example, DNA that is present in a sample obtained from a subject (e.g., blood, plasma, serum, or urine).

[0035] As used herein, the term "polymer" refers to any polymer containing at least one substituted or unsubstituted monomer. Examples of "polymer" include, but are 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.

[0036] As used herein, "copolymer" refers to a polymer derived from two monomer types; similarly, a terpolymer refers to a polymer derived from three monomer types. Polymers further include various morphologies, including, but not limited to, linear, branched, random, crosslinked, and dendrimer systems. In some embodiments, polymers further include chemically modified equivalents thereof, such as hydrophobically modified or silicone modified. For example, a polyacrylamide polymer refers to any polymer containing at least one substituted or unsubstituted acrylamide unit, such as a homopolymer, copolymer, terpolymer, random copolymer, block copolymer, or terpolymer of polyacrylamide. The polyacrylamide may be a linear, branched, random, crosslinked, or dendrimer of polyacrylamide, and the polyacrylamide may be a hydrophobically modified polyacrylamide or a silicone modified polyacrylamide.

[0037] In some embodiments, examples of polymers include, but are not limited to, polyethers, polyimides, polyalkylene glycols, alkoxylated surfactants, polysaccharides, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymers are hydrophobically or silicone-modified.

[0038] Examples of polyalkylene glycols ("PAG" or "poly(oxyalkylene)" or "poly(alkylene oxide)") include, but are not limited to, hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, hydrophobically modified poly(oxyalkylene) copolymers, dipropylene glycol, tripropylene glycol, polyethylene glycol (also referred to as "PEG"), and polypropylene glycol (also referred to as "PPG"). In some examples, examples of PAG copolymers include, but are not limited to, poly(ethylene glycol-propylene glycol) ("PEG-PPG" or "UCON") and poly(ethylene glycol-random-propylene glycol) ("PEG-random-PPG"). In some examples, PEG-PPG includes random copolymers, block copolymers, or combinations thereof. In some examples, PEG-PPG includes random copolymers and block copolymers. In some embodiments, PEG-PPG is PEG-random-PPG.

[0039] As used herein, "vinyl polymer" refers to a class of polymers derived from substituted vinyl (HC=CHR) monomers. Examples of vinyl polymers include, but are not limited to, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, and polyvinyl methyl ether.

[0040] Examples of polysaccharides include, but are not limited to, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, and maltodextrin. In some embodiments, the polysaccharide is an alkoxylated starch, alkoxylated cellulose, or alkyl hydroxyalkyl cellulose.

[0041] Examples of polyacrylamides include, but are not limited to, poly N-isopropylacrylamide.

[0042] Examples of polyimides include, but are not limited to, polyethyleneimine.

[0043] Examples of alkoxylated surfactants include, but are not limited to, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, sulfated alkylphenols, ethoxylated alkylphenols, sodium N-lauroyl sarcosinate (NLS), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxamides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

[0044] In some embodiments, the polymer has an average molecular weight of about 200-1,000 Da, 200-35,000 Da, 300-35,000 Da, 400-2,000 Da, or 400-35,000 Da. Examples include, but are not limited to, polyalkylene glycols (PAGs) having average molecular weights of about 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1,000 Da, 2,000 Da, 3,000 Da, 4,000 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, and 35,000 Da. In some embodiments, the PAG has an average molecular weight within either of these two molecular weight ranges.

[0045] Examples of PAGs include, but are not limited to, PEG 200, PEG 300, PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 2000, PEG 3000, PEG 4000, PEG 5000, PEG 6000, PEG 7000, PEG 8000, PEG 9000, PEG 10000, PEG 15000, PEG 20000, PEG 25000, PEG 30000, PEG 35000, PPG 425, PPG 725, PPG 900, PPG 1000, and PPG 2000. In some embodiments, the PEG has an average molecular weight within the range of either of the above two PEG molecular weights. In some embodiments, the PPG has an average molecular weight within the range of either of the above two PPG molecular weights.

[0046] In some embodiments, the polymer comprises ethylene oxide (EO) and propylene oxide (PO) units, and the ethylene oxide:propylene oxide (EO:PO) ratio is from 90:10 to 10:90. In some embodiments, the polymer has an EO:PO ratio of 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, or 90:10. In some embodiments, the polymer has an EO:PO ratio within either of these two ratios.

[0047] In some embodiments, the polymer is a PAG having an average molecular weight of about 980 to 12,000 Da and an EO:PO ratio of 50:50 to 75:25. Examples include, but are not limited to, PEG-PPG having average molecular weights of about 980 Da, 1,230 Da, 1,590 Da, 2,470 Da, 2,660 Da, 3,380 Da, 3,930 Da, 6,950 Da, and 12,000 Da. In some embodiments, the PEG-PPG has an average molecular weight within either of the above two PEG-PPG molecular weight ranges. In some embodiments, the PEG-PPG has an EO:PO ratio of 50:50 or 75:25. In some embodiments, the polymer is a PEG-random-PPG having an average molecular weight of about 2,500 or 12,000 Da and an EO:PO ratio of about 75:25.

[0048] In some embodiments, the polymer is a vinyl polymer having an average molecular weight of about 2,500 to 2,500,000 Da. Examples include, but are not limited to, polyvinylpyrrolidone having average molecular weights of about 2,500 Da, 10,000 Da, 40,000 Da, 100,000 Da, and 2,500,000 Da. In some embodiments, the vinyl polymer has an average molecular weight within either of these two molecular weight ranges.

[0049] In some embodiments, the polymer is a polysaccharide and has an average molecular weight of about 6,000 to 5,000,000 Da, examples of which include, but are not limited to, dextrans having average molecular weights of about 6,000 Da, 12,000 Da, 25,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 150,000 Da, 270,000 Da, 410,000 Da, 450,000 Da, 550,000 Da, 650,000 Da, 670,000 Da, 1,500,000 Da, 2,000,000 Da, 2,800,000 Da, 4,000,000 Da, and 5,000,000 Da. In some embodiments, the dextran has an average molecular weight within either of the above two molecular weight ranges.

[0050] In some embodiments, the polymer is a polyether and has an average molecular weight of about 200-35,000 Da, examples of which include, but are not limited to, silicone-modified polyethers (or "polyether-modified silicones") having an average molecular weight of about 200-35,000 Da.

[0051] In some embodiments, the polymer is polyacrylamide and has an average molecular weight of 1,000 to 5,000,000 Da. Examples include, but are not limited to, polyacrylamides or poly(N-isopropylacrylamide) having average molecular weights of 1,000 Da, 2,000 Da, 5,000 Da, 10,000 Da, 40,000 Da, 85,000 Da, and 5,000,000 Da. In some embodiments, the polyolefin has an average molecular weight within either of these two molecular weight ranges.

[0052] In some embodiments, the polymer is polyacrylic acid and has an average molecular weight of about 1,250 to 4,000,000 Da. Examples include, but are not limited to, polyacrylic acids having average molecular weights of 1,200 Da, 2,100 Da, 5,100 Da, 8,000 Da, 8,600 Da, 8,700 Da, 16,000 Da, and 83,000 Da. In some embodiments, the polyolefin has an average molecular weight within either of these two molecular weight ranges.

[0053] As used herein, the term "salt" refers to a substance containing a cation and an anion. Examples of salts include those whose cations are sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, or tetrabutylammonium, and / or those whose anions are phosphate, hydrogenphosphate, dihydrogenphosphate, sulfate, sulfide, sulfite, hydrogensulfate, carbonate, or carbonate. Examples of suitable salts include, but are not limited to, hydrogen carbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, or tris(hydroxymethyl)aminomethane. In some embodiments, the salt is a lyophilic salt, a discrete salt, or an inorganic salt.

[0054] In some embodiments, examples of "surfactants" include, but are not limited to, anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic surfactants, and amphoteric surfactants.

[0055] Examples of anionic surfactants include, but are not limited to, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols, and sulfated alkylphenols.

[0056] Examples of nonionic surfactants include, but are not limited to, ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid glycol esters, carboxamides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides.

[0057] Examples of cationic surfactants include, but are not limited to, quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and alicyclic amines, n,n,n',n' tetrasubstituted ethylenediamines, and 2-alkyl 1-hydroxyethyl 2-imidazolines. Examples of amphoteric surfactants include, but are not limited to, n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionic acid ester disodium salt, n-carboxymethyl n dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n hydroxyethylglycine sodium salt, and N-lauroyl sarcosinate sodium salt (NLS).

[0058] In some embodiments, the surfactant comprises a polymer, such as PAG. In some embodiments, the surfactant comprises EO. x -PO y -EO x where EO refers to an ethylene oxide unit, PO refers to a propylene oxide unit, and x and y are the numbers of monomers, respectively. In some embodiments, x is 2 to 136. In some embodiments, y is 16 to 62. In some embodiments, an example surfactant is (C2H4O) n C 14 H 22O (wherein n=4-10 (e.g., Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630)), Brij 58, Brij O10, Brij L23, EOx-POy-EOx (wherein x=2-136 and y=16-62 (e.g., Pluronic L-61, Pluronic F-127)), sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, N-lauroylsarcosine sodium salt (NLS), cetyltrimethylammonium bromide, or span 80.

[0059] As used herein, the term "chaotropic agent" refers to a substance that disrupts the hydrogen-bonding network between water molecules in a solution. For example, a chaotropic agent can include an anion selected from thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, or iodide. A chaotropic agent can include a cation selected from sodium, guanidine salt, lithium, or magnesium. Unless otherwise specified, chaotropic agents defined by a cation or anion include all compounds with the appropriate conjugated anion or cation, respectively. For example, 5M guanidine salt includes guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), etc. Examples of chaotropic agents include, but are not limited to, guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, urea, and the like.

[0060] DNA sizes and cutoff values ​​designated herein with reference to base pairs "bp" refer to the strand length of the DNA molecule and are therefore used to describe the length of single-stranded and double-stranded DNA molecules. Thus, when the DNA is a single-stranded DNA molecule, the above designations regarding size or length in "bp" refer to the size or length of the nucleotides in said single-stranded DNA molecule. Examples of the present invention

[0061] Example 1

[0062] In one aspect, a method is provided for concentrating and purifying one or more target analytes from a sample solution, the method comprising: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the enriched one or more target analytes to obtain an enriched solution; (c) adding magnetic beads to the concentrated solution and allowing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; (d) recovering the one or more target analytes from the bead-analyte complexes to obtain a final solution containing concentrated and purified one or more target analytes.

[0063] In some embodiments, step (b) comprises: (i) adding the isolated first phase containing the enriched target analyte(s) to a second ATPS to form a second mixture that separates into a third phase and a fourth phase, wherein the one or more target analytes are enriched in the third phase; (ii) isolating the third phase containing the enriched target analyte(s) in step (b) to form an enriched solution used in step (c).

[0064] In some embodiments, the concentrated solution of step (b) is mixed with a binding buffer, wherein the binding buffer contains at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea, to obtain the concentrated solution used in step (c).

[0065] In some embodiments, step (d) comprises: (i) mixing the bead-analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof, to form a fractionation solution, thereby releasing one or more target analytes smaller than a target size from the bead-analyte complex into the fractionation solution; (ii) immobilizing the bead-analyte complex using a magnetic stand; (iii) isolating the one or more target analytes smaller than the target size in a fractionation solution from the immobilized bead-analyte complexes.

[0066] In some embodiments, step (d) comprises: (iv) adding the isolated one or more target analytes smaller than the target size to a second binding buffer, wherein the second binding buffer comprises at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (v) adding magnetic beads to the mixture of the isolated one or more target analytes smaller than the target size and the second binding buffer, so that the magnetic beads bind to the one or more target analytes smaller than the target size to form second bead-analyte complexes; (vi) recovering the one or more target analytes from the second bead-analyte complex.

[0067] In some embodiments, the method further comprises: (e) performing a diagnostic assay on the final solution to detect and quantify the one or more target analytes.

[0068] In some embodiments, the one or more target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof.

[0069] In some embodiments, the one or more target analytes is DNA.

[0070] In some embodiments, the one or more target analytes is free DNA or circulating tumor DNA.

[0071] In some embodiments, the first ATPS comprises a first ATPS component capable of forming a first phase and a second phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0072] In some embodiments, the second ATPS comprises a second ATPS component capable of forming a third phase and a fourth phase when dissolved in an aqueous solution, wherein the second ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0073] In some embodiments, the polymer is dissolved in an aqueous solution at a concentration of 4% to 84% (w / w).

[0074] In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 1% to 80% (w / w). In some embodiments, the salt is dissolved in an aqueous solution at a concentration of 8% to 80% (w / w).

[0075] In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 10% (w / w). In some embodiments, the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 9.8% (w / w).

[0076] In some embodiments, step (a) comprises: (i) embedding components capable of forming a first ATPS into a porous material; (ii) contacting the sample solution with a porous material having components embedded therein, the components forming a first phase and a second phase as the sample solution passes through the porous material.

[0077] In one aspect, a method is provided for concentrating and purifying one or more target analytes from a sample solution, the method comprising: (a) adding a sample solution containing the one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the enriched target analyte(s); (c) adding the isolated first phase containing the enriched target analytes to a second ATPS to form a second mixture that separates into a third phase and a fourth phase, wherein the one or more target analytes are enriched in the third phase; (d) obtaining a concentrated solution by isolating a third phase containing the concentrated target analyte(s); (e) mixing the concentrated solution with a binding buffer, wherein the binding buffer comprises at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (f) adding magnetic beads to the mixture of the concentrated solution and binding buffer, and allowing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; (g) mixing the bead-analyte complex with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution, thereby releasing one or more target analytes smaller than a target size from the bead-analyte complex into the fractionation solution; (h) immobilizing the bead-analyte complex using a magnetic stand; (i) isolating the one or more target analytes smaller than the target size in a fractionation solution from the immobilized bead-analyte complex; (j) adding the isolated target analyte or analytes smaller than the target size to a second binding buffer, wherein the second binding buffer comprises at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (k) adding magnetic beads to a mixture of isolated target analytes or target analytes smaller than the target size and a second binding buffer, so that the magnetic beads bind to the isolated target analytes or target analytes smaller than the target size to form second bead-analyte complexes; (l) recovering one or more smaller target analytes from the second bead-analyte complexes to obtain a final solution containing one or more concentrated and purified smaller target analytes; (m) performing a diagnostic assay on the final solution to detect and quantify the one or more target analytes that are smaller than the target size.

[0078] Various ATPS systems that can be used in various embodiments of the present invention include, but are not limited to, polymer-polymer, polymer-salt, polymer-surfactant, salt-surfactant, surfactant, surfactant-surfactant, or polymer-salt-surfactant.

[0079] In one embodiment, the first and / or second ATPS comprises a polymer. In some embodiments, possible polymers that can be used include, but are not limited to, polyalkylene glycols (PAGs) (e.g., hydrophobically modified polyalkylene glycols), poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers (e.g., hydrophobically modified poly(oxyalkylene) copolymers), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. In some embodiments, the polymer is selected from the group consisting of polyethers, polyimides, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. In some embodiments, the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly N-isopropylacrylamide, and copolymers thereof.In some embodiments, the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly N-isopropylacrylamide. In some embodiments, the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid, and copolymers thereof. In some embodiments, the polymer is selected from the group consisting of dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch. In some embodiments, the polymer has an average molecular weight in the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 5,000,000 Da. In some embodiments, the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90.

[0080] In one embodiment, the polymer concentration of the first and / or second ATPS is in the range of about 4% to about 84% (w / w) based on the total weight of the aqueous solution. In various embodiments, the polymer solution may be about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% ​​w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w, about 17% w / w, about 17.5% w / w, about 18% w / w, about 18.5% w / w, about 19% w / w, about 19.5% w / w, about 20% w / w, about 20.5% w / w, about 21% w / w, about 21.5% w / w, about 22% w / w, about 22.5% w / w, about 23% w / w, about 23.5% w / w, about 24% w / w, about 24.5% w / w, about 35% w / w, about 35.5% w / w, about 36% w / w, about 36.5% w / w, about 37% w / w, about 37.5% w / w, about 38% w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, about 52% w / w, about 52.5% w / w, about 53% w / w, about 53.5% w / w, about 54% w / w, about 54.5% w / w, approximately 55% w / w, approximately 55.5% w / w, approximately 56% w / w, approximately 56.5% w / w, approximately 57% w / w, approximately 57.5% w / w, approximately 58% w / w, approximately 58.5% w / w, approximately 59% w / w, approximately 59.5% w / w, approximately 60% w / w, approximately 60.5% w / w, approximately 61% w / w, approximately 61.5% w / w, approximately 62% w / w, approximately 62.5% w / w, approximately 63% w / w, approximately 63.5% w / w, approximately 64% w / w, approximately 64.5% w / w, approximately 65% ​​w / w, approximately 65.5% w / w, approximately 66% w / w, approximately 66.5% w / w, approximately 67% w / w, approximately 67.5% w / w, approximately 68% w / w, approximately 68.5% w / w, approximately 69% w / w, approximately 69.5% w / w, approximately 70% w / w, approximately 70.5% w / w, approximately 71% w / w, approximately 71.5% w / w, approximately 72% w / w, approximately 72.5% w / w, approximately 73% The polymer solution is selected from about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w, about 80% w / w, about 80.5% w / w, about 81% w / w, about 81.5% w / w, about 82% w / w, about 82.5% w / w, about 83% w / w, about 83.5% w / w, and about 84% w / w.

[0081] In one embodiment, the first and / or second ATPS includes a salt, thereby forming a salt solution. In some embodiments, the salt includes, but is not limited to, a lyophilic salt, a discrete salt, or an inorganic salt containing a cation and an anion, such as, for example, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and the anion is, for example, phosphate, sulfate, nitrate, chloride, and bicarbonate. In another embodiment, the salt includes NaCl, Na3PO4, K3PO4, Na2SO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, and combinations thereof. Other salts, such as ammonium acetate, may also be used. In another embodiment, the salt is selected from magnesium salts, lithium salts, sodium salts, potassium salts, cesium salts, zinc salts, and aluminum salts. In some embodiments, the salt is selected from bromides, iodides, fluorides, carbonates, sulfates, citrates, carboxylates, borates, and phosphates. In some embodiments, the salt comprises potassium phosphate. In some embodiments, the salt comprises ammonium sulfate.

[0082] In one embodiment, the total salt concentration is in the range of about 0.01% to about 90%. One skilled in the art will appreciate that the amount of salt required to form an aqueous two-phase system is affected by the molecular weight, concentration, and physical state of the polymer.

[0083] In various embodiments, the salt concentration is between about 1% and 80% w / w. In various embodiments, the salt concentration is about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 3.5% w / w, about 4% w / w, about 4.5% w / w, about 5% w / w, about 5.5% w / w, about 6% w / w, about 6.5% w / w, about 7% w / w, about 7.5% w / w, about 8% w / w, about 8.5% w / w, about 9% w / w, about 9.5% w / w, about 10% w / w, about 10.5% w / w, about 11% w / w, about 11.5% w / w, about 12% w / w, about 12.5% ​​w / w, about 13% w / w, about 13.5% w / w, about 14% w / w, about 14.5% w / w, about 15% w / w, about 15.5% w / w, about 16% w / w, about 16.5% w / w, about 17% w / w, about 17.5% w / w, about 18% w / w, about 18.5% w / w, about 19% w / w, about 19.5% w / w, about 20% w / w, about 20.5% w / w, about 21% w / w, about 21.5% w / w, about 22% w / w, about 22.5% w / w, about 23% w / w, about 23.5% w / w, about 24% w / w, about 24.5% w / w, about 35% w / w, about 35.5% w / w, about 36% w / w, about 36.5% w / w, about 37% w / w, about 37.5% w / w, about 38% w / w, about 38.5% w / w, about 39% w / w, about 39.5% w / w, about 40% w / w, about 40.5% w / w, about 41% w / w, about 41.5% w / w, about 42% w / w, about 42.5% w / w, about 43% w / w, about 43.5% w / w, about 44% w / w, about 44.5% w / w, about 45% w / w, about 45.5% w / w, about 46% w / w, about 46.5% w / w, about 47% w / w, about 47.5% w / w, about 48% w / w, about 48.5% w / w, about 49% w / w, about 49.5% w / w, about 50% w / w, about 50.5% w / w, about 51% w / w, about 51.5% w / w, approximately 52% w / w, approximately 52.5% w / w, approximately 53% w / w, approximately 53.5% w / w, approximately 54% w / w, approximately 54.5% w / w, approximately 55% w / w, approximately 55.5% w / w, approximately 56% w / w, approximately 56.5% w / w, approximately 57% w / w, approximately 57.5% w / w, approximately 58% w / w, approximately 58.5% w / w, approximately 59% w / w, approximately 59.5% w / w, approximately 60% w / w, approximately 60.5% w / w, approximately 61% w / w, approximately 61.5% w / w, approximately 62% w / w, approximately 62.5% w / w, approximately 63% w / w, approximately 63.5% w / w, approximately 64% w / w, approximately 64.5% w / w, approximately 65% ​​w / w, approximately 65.5% w / w, approximately 66% w / w, approximately 66.5% w / w, approximately 67% w / w, approximately 67.5% w / w, approximately 68% w / w, approximately 68.5% w / w, approximately 69% w / w, approximately 69.5% w / w, approximately 70% w / w, approximately 70.5% w / w, approximately 71% w / w, approximately 71.5% w / w, approximately 72% w / w, about 72.5% w / w, about 73% w / w, about 73.5% w / w, about 74% w / w, about 74.5% w / w, about 75% w / w, about 75.5% w / w, about 76% w / w, about 76.5% w / w, about 77% w / w, about 77.5% w / w, about 78% w / w, about 78.5% w / w, about 79% w / w, about 79.5% w / w or about 80% w / w.

[0084] In one embodiment, the first and / or second ATPS comprises a surfactant. In some embodiments, possible surfactants that can be used include Triton-X, Triton-114, Igepal CA-630, and Nonidet P-40, anionic surfactants (e.g., carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural fats and oils, sulfated esters, sulfated alkanolamides, and alkylphenols), ethoxylated and sulfated nonionic surfactants (e.g., ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine condensates, and polyoxyethylene fatty acid amides), cationic surfactants (e.g., quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and alicyclic amines, n,n,n',n' tetrasubstituted ethylenediamines, and 2-alkyl 1-hydroxyethyl 2-imidazolines), and amphoteric surfactants (e.g., n-cocoyl 3-Aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate disodium salt, n-carboxymethyl n dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n hydroxyethylglycine and sodium salt).

[0085] In one embodiment, the concentration of the first and / or second ATPS surfactant is in the range of about 0.05% w / w to about 10% w / w. In various embodiments, the concentration of the surfactant is about 0.05% w / w, 0.1% w / w, about 0.2% w / w, about 0.3% w / w, about 0.4% w / w, about 0.5% w / w, about 0.6% w / w, about 0.7% w / w, about 0.8% w / w, about 0.9% w / w, about 1% w / w, 1.1% w / w, about 1.2% w / w, about 1.3% w / w, about 1.4% w / w, about 1.5% w / w, about 1.6% w / w, about 1.7% w / w, about 1.8% w / w, about 1.9% w / w, about 2% w / w, about 2.1% w / w, about 2.2% w / w, about 2.3% w / w, about 2.4% w / w, about 2.5% w / w, about 2.6% w / w, approx. 2.7% w / w, approx. 2.8% w / w, approx. 2.9% w / w, approx. 3% w / w, 3.1% w / w, approx. 3.2% w / w, approx. 3.3% w / w, approx. 3.4% w / w, approx. 3.5% w / w, approx. 3.6% w / w, approx. 3.7% w / w, approx. 3.8% w / w, approx. 3.9% w / w, approx. 4% w / w, approx. 4.1% w / w, approx. 4.2% w / w, approx. 4.3% w / w, approx. 4.4% w / w, approx. 4.5% w / w, approx. 4.6% w / w, approx. 4.7% w / w, approx. 4.8% w / w, approx. 4.9% w / w, approx. 5% w / w, approx. 5.1% w / w, approx. 5.2% w / w, approx. 5.3% w / w, approx. 5.4% w / w, about 5.5% w / w, about 5.6% w / w, about 5.7% w / w, about 5.8% w / w, about 5.9% w / w, about 6% w / w, 6.1% w / w, about 6.2% w / w, about 6.3% w / w, about 6.4% w / w, about 6.5% w / w, about 6.6% w / w, about 6.7% w / w, about 6.8% w / w, about 6.9% w / w, about 7% w / w, about 7.1% w / w, about 7.2% w / w, about 7.3% w / w, about 7.4% w / w, about 7.5% w / w, about 7.6% w / w, about 7.7% w / w, about 7.8% w / w, about 7.9% w / w, about 8% w / w, about 8.1% w / w, about 8.2% w / w, about 8.3% w / w, about 8.4% w / w, about 8.5% w / w, about 8.6% w / w, about 8.7% w / w, about 8.8% w / w, about 8.9% w / w, about 9% w / w, 9.1% w / w, about 9.2% w / w, about 9.3% w / w, about 9.4% w / w, about 9.5% w / w, about 9.6% w / w, about 9.7% w / w, about 9.8% w / w, about 9.9% w / w or about 10% w / w.

[0086] In one embodiment, the binding buffer (including the second binding buffer) comprises a chaotropic agent. In some embodiments, possible chaotropic agents that can be used include, but are not limited to, n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0087] In one embodiment, the concentration of the chaotropic agent in the binding buffer is in the range of about 0.1M to 8M. In various embodiments, the concentration of the chaotropic agent is about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, about 2 M, about 2.1 M, about 2.2 M, about 2.3 M, about 2.4 M, about 2.5 M, about 2.6 M, about 2.7 M, about 2.8 M, about 2.9 M, about 3 M, about 3.1 M, about 3.2 M, about 3.3 M, about 3.4 M, about 3.5 M, about 3.6 M, about 3.7 M, about 3.8 M, about 3.9 M, about 4 M, about 5 M, about 6 M, about 7 M, about 8 M, about 9 M, about 10 M, about 11 M, about 12 M, about 13 M, about 14 M, about 15 M, about 16 M, about 17 M, about 18 M, about 19 M, about 20 M, about 21 M, about 22 M, about 23 M, about 24 M, about 25 M, about 26 M, about 27 M, about 28 M, about 29 M, about 30 M, about 31 M, about 32 M, about 33 M, about 34 M, about 35 M, about 36 M, about 37 M, about 38 M, about 39 M, about 40 M, about 41 M, about 42 M, about M, about 4.1 M, about 4.2 M, about 4.3 M, about 4.4 M, about 4.5 M, about 4.6 M, about 4.7 M, about 4.8 M, about 4.9 M, about 5 M, about 5.1 M, about 5.2 M, about 5.3 M, about 5.4 M, about 5.5 M, about 5.6 M, about 5.7 M, about 5.8 M, about 5.9 M, about 6 M, about 6.1 M, about 6.2 M, about 6.3 M, about 6.4 M, about 6.5 M, about 6.6 M, about 6.7 M, about 6.8 M, about 6.9 M, about 7 M, about 7.1 M, about 7.2 M, about 7.3 M, about 7.4 M, about 7.5 M, about 7.6 M, about 7.7 M, about 7.8 M, about 7.9 M or about 8 M.

[0088] In some embodiments, the fractionation buffer comprises a polymer, a salt, a detergent, a chaotropic agent, or a combination thereof. In some embodiments, possible polymers, salts, detergents, and chaotropic agents that can be used include, but are not limited to, those listed above.

[0089] In some embodiments, possible magnetic beads that can be used include, but are not limited to, those listed in Table 1.1 below.

[0090] [Table 1.1]

[0091] Example 2

[0092] In some embodiments, a method for isolating a smaller-than-target-size target nucleic acid from a sample containing nucleic acid components is provided, the method comprising: (a) preparing a sample solution from the sample; (b) contacting a plurality of beads with the sample solution, wherein the nucleic acid components bind to the plurality of beads to form bead-analyte complexes; (c) mixing the bead-analyte complexes with a fractionation buffer containing at least one chaotropic agent to form a bulk fractionation solution, and releasing the smaller-than-target-size target nucleic acid from the bead-analyte complexes into the bulk fractionation solution; (d) immobilizing the bead-analyte complexes; and (e) separating the bulk fractionation solution containing the isolated smaller-than-target-size target nucleic acid from the immobilized bead-analyte complexes.

[0093] In some embodiments, step (a) further comprises: (a1) adding the sample to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first target-rich phase and a first target-poor phase, and the nucleic acid components are concentrated in the first target-rich phase; and (a2) isolating the first target-rich phase containing the concentrated nucleic acid components to obtain a sample solution.

[0094] In some embodiments, step (a) further comprises, after step (a2), (a3) ​​adding the sample solution in step (a2) to a second ATPS to form a second mixture separated into a second target-rich phase and a second target-poor phase, wherein the nucleic acid components are concentrated in the second target-rich phase; and (a4) isolating the second target-rich phase containing the concentrated nucleic acid components to form the sample solution in step (a).

[0095] In some embodiments, prior to step (b), the plurality of beads and the sample solution of step (a) are combined with a binding buffer, wherein the binding buffer comprises at least one chaotropic agent.

[0096] In some embodiments, step (e) further comprises: (e1) mixing the bulk fraction solution with a target binding buffer and a plurality of second beads, and allowing the plurality of second beads to bind to the target nucleic acids smaller than the target size to form second bead-analyte complexes, the target binding buffer comprising at least one chaotropic agent; and (e2) recovering the target nucleic acids smaller than the target size from the second bead-analyte complexes.

[0097] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

[0098] In some embodiments, the plurality of second beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

[0099] In some embodiments, the plurality of beads are magnetic beads, and step (b) further comprises: (b1) applying a magnetic field to immobilize the bead-analyte complexes and separate the bead-analyte complexes from a bulk supernatant; (b2) removing the bulk supernatant; and (b3) removing the magnetic field and proceeding to step (c).

[0100] In some embodiments, the plurality of second beads are magnetic beads, and the target nucleic acid recovery in step (e2) further comprises: (i) applying a first magnetic field to immobilize the second bead-analyte complexes and separating the second bead-analyte complexes from a first supernatant; (ii) removing the first supernatant; (iii) washing the immobilized second bead-analyte complexes with a wash buffer; (iv) discarding the wash buffer; (v) removing the first magnetic field; (vi) mixing the second bead-analyte complexes with an elution buffer to form a bulk elution solution, wherein the target nucleic acids smaller than the target size and the magnetic beads in the second bead-analyte complexes are separated and released into the bulk elution solution; (vii) applying a second magnetic field to immobilize the magnetic beads; and (viii) collecting the bulk elution solution containing the isolated target nucleic acids smaller than the target size.

[0101] In some embodiments, a method is provided that further comprises (f) performing a diagnostic assay on the isolated target nucleic acid for detection, quantitation, characterization, or a combination thereof, of the target nucleic acid.

[0102] In some embodiments, the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0103] In some embodiments, the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0104] In some embodiments, the at least one chaotropic agent has a concentration of about 1.5-8 M in the fractionation buffer.

[0105] In some embodiments, the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 to 3.9 M.

[0106] In some embodiments, the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8-3.0 M.

[0107] In some embodiments, the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylic acid ester.

[0108] In some embodiments, the at least one polymer is present in the fractionation buffer at a concentration of about 0.1% to 15% (w / w).

[0109] In some embodiments, the at least one polymer is present in the fractionation buffer at a concentration of about 1.0% to 5.0% (w / w).

[0110] In some embodiments, the at least one polymer has an average molecular weight ranging from 100 Da to 35,000 Da.

[0111] In some embodiments, the fractionation buffer further comprises one or more of a pH buffer, a metal chelator, or a combination thereof.

[0112] Examples of pH buffers include, but are not limited to, phosphate buffer, acetic acid-sodium acetate buffer, citrate-sodium citrate buffer, citrate-NaOH-HCl buffer, sodium borate buffer, carbonate buffer, HEPES buffer, MOPS buffer, TAE buffer, TBST buffer, Tris-HCl buffer, TE buffer, and TEN buffer.

[0113] Examples of metal chelators include, but are not limited to, 2,2'-bipyridine, dimercaptopropanol, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), the ion carrier nitrilotriacetic acid (NTA), salicylic acid, and triethanolamine (TEA).

[0114] In some embodiments, the nucleic acid component and / or the target nucleic acid is DNA, RNA, or a combination thereof.

[0115] In some embodiments, the nucleic acid component and / or target nucleic acid is cDNA, plasmid DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or a combination thereof.

[0116] In some embodiments, the first ATPS comprises a first ATPS component that is capable of forming the first target-rich phase and a first target-depleted phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0117] In some embodiments, the second ATPS comprises a second ATPS component that is capable of forming the second target-rich phase and a second target-depleted phase when dissolved in an aqueous solution, wherein the second ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof.

[0118] In some embodiments, the polymer is soluble in aqueous solution at a concentration of 0.5% to 80% (w / v).

[0119] In some embodiments, the polymer is selected from the group consisting of polyethers, polyimides, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starches, alkoxylated celluloses, alkylhydroxyalkyl celluloses, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof, hi some embodiments, the polymer is hydrophobically or silicone-modified.

[0120] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-ran-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly N-isopropylacrylamide, or copolymers thereof.

[0121] In some embodiments, the polymer is dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, or poly N-isopropylacrylamide.

[0122] In some embodiments, the polymer is polyacrylamide, polyacrylic acid, or a copolymer thereof.

[0123] In some embodiments, the polymer is polyacrylamide, polyacrylic acid, or a copolymer thereof. In some embodiments, the polymer is dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, or starch.

[0124] In some embodiments, the polymer has an average molecular weight ranging from 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 5,000,000 Da. In some embodiments, the polymer comprises ethylene oxide and propylene oxide units. In some embodiments, the polymer has an EO:PO ratio of 90:10 to 10:90.

[0125] In some embodiments, the polymer has an average molecular weight in the range of 980 to 12,000 Da or 3,400 to 5,000,000 Da.

[0126] In some embodiments, the polymer has an average molecular weight in the range of 100 to 10,000 Da.

[0127] In some embodiments, the salt is soluble in aqueous solution at a concentration of 0.1% to 80% (w / w).

[0128] In some embodiments, the salt is soluble in aqueous solution at a concentration of 0.1% to 50% (w / w).

[0129] In some embodiments, the salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, straight or branched chain trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

[0130] In some embodiments, the salt comprises an anion selected from the group consisting of phosphate, hydrogen phosphate, dihydrogen phosphate, sulfate, sulfide, sulfite, hydrogen sulfate, carbonate, bicarbonate, acetate, nitrate, nitrite, sulfite, chloride, fluoride, chlorate, perchlorate, chlorite, hypochlorite, bromide, bromate, hypobromite, iodide, iodate, cyanate, thiocyanate, isothiocyanate, oxalate, formate, chromate, dichromate, permanganate, hydroxide, hydrogen ion, citrate, borate, and tris(hydroxymethyl)aminomethane.

[0131] In some embodiments, the salt is selected from the group consisting of aluminum chloride, aluminum phosphate, aluminum carbonate, magnesium chloride, magnesium phosphate, and magnesium carbonate.

[0132] In some embodiments, the salt is selected from the group consisting of KCl, NH4Cl, Na3PO4, K3PO4, Na2SO4, K2HPO4, KH2PO4, Na2HPO4, NaH2PO4, (NH4)3PO4, (NH4)2HPO4, NH4H2PO4, potassium citrate, (NH4)2SO4, sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate, and ammonium acetate.

[0133] In some embodiments, the salt is selected from the group consisting of (NH4)3PO4, sodium formate, ammonium formate, K2CO3, KHCO3, Na2CO3, NaHCO3, MgSO4, MgCO3, CaCO3, CsOH, Cs2CO3, Ba(OH)2, and BaCO3.

[0134] In some embodiments, the salt is selected from the group consisting of NH4Cl, NH4OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

[0135] In some embodiments, the surfactant is soluble in aqueous solution at a concentration of 0.05% to 10% (w / w).

[0136] In some embodiments, the surfactant is selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, wherein the anionic surfactant is a carboxylate, a sulfonate, a petroleum sulfonate, an alkylbenzene sulfonate, a naphthalene sulfonate, an olefin sulfonate, an alkyl sulfate, a sulfate, a sulfated natural oil, a sulfated natural fat, a sulfated ester, a sulfated alkanolamide, a sulfated alkylphenol, an ethoxylated alkylphenol, or sodium N-lauroyl sarcosinate (NLS), and wherein the nonionic surfactant is an ethoxylated fatty alcohol, a polyoxyethylene surfactant, a carboxylic acid ester ... The cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n'-tetrasubstituted ethylenediamine, or a 2-alkyl 1-hydroxyethyl 2-imidazoline; and the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9 octadecenyl ammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

[0137] In some embodiments, the surfactant is selected from the group consisting of Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, Brij L23, Pluronic L-61, Pluronic F-127, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, N-lauroyl sarcosine sodium salt, cetyltrimethylammonium bromide, and span 80.

[0138] In some embodiments, at least one chaotropic agent of the binding buffer comprises an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0139] In some embodiments, the at least one chaotropic agent of the binding buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0140] In some embodiments, the binding buffer comprises guanidine and, optionally, further comprises at least one polymer.

[0141] In some embodiments, at least one chaotropic agent of the target binding buffer comprises an anion selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0142] In some embodiments, the at least one chaotropic agent of the target binding buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0143] In some embodiments, the target binding buffer comprises guanidine and, optionally, further comprises at least one polymer.

[0144] In some embodiments, the sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, cervical swab, semen, or breast milk.

[0145] In some embodiments, step (a) comprises preparing a DNA library from a sample to obtain a sample solution.

[0146] Some embodiments provide a method of diagnosing or determining risk of cancer in a subject, comprising: (i) isolating a target nucleic acid from said subject's biological sample using a method described herein; (ii) determining the presence of the target nucleic acid and determining whether the subject has or is at risk for having the cancer.

[0147] In some embodiments, the target nucleic acids are free DNA and circulating tumor DNA, whereby the method increases the ratio of circulating tumor DNA:free DNA and / or the variant allele frequency (VAF) in the sample to perform a cancer diagnostic assay.

[0148] Some embodiments provide a method of treating cancer in a subject, the method comprising diagnosing the cancer in the subject by steps (i) and (ii) above, and further comprising treating the subject if the subject is determined to have or be at risk of having the cancer.

[0149] Some embodiments provide a method of diagnosing or determining risk of a genetic disease or disorder in a fetus, comprising: (i) isolating a target nucleic acid from a biological sample of the mother of said fetus using a method described herein; (ii) determining the presence of the target nucleic acid and determining whether the fetus has or is at risk for a genetic disease or disorder.

[0150] In some embodiments, the target nucleic acid is circulating fetal DNA, whereby the method enriches the fetal fraction in the sample for non-invasive prenatal detection. For clarity, the fetal fraction refers to the fraction of all DNA of fetal origin circulating in the maternal blood.

[0151] Some embodiments provide a method of treating a genetic disease or disorder in a fetus, the method comprising diagnosing the genetic disease or disorder in the fetus, the method comprising steps (i) and (ii) above, and further comprising treating the fetus or the mother of the fetus if the fetus is determined to have or be at risk for the genetic disease or disorder.

[0152] Methods for determining the presence of target nucleic acids and determining the presence, risk, or absence of disease (eg, cancer and fetal genetic diseases and disorders) are known to those of skill in the art.

[0153] In some embodiments, a kit for isolating a target nucleic acid smaller than a target size from a sample containing nucleic acid components is provided, the kit including: (a) at least one ATPS component selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof; (b) a plurality of beads; (c) a fractionation buffer including at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide; and (d) a binding buffer including at least one chaotropic agent selected from the group consisting of thiocyanate, isothiocyanate, perchlorate, acetate, trichloroacetate, trifluoroacetate, chloride, and iodide.

[0154] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

[0155] In some embodiments, the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

[0156] In some embodiments, the at least one chaotropic agent has a concentration of about 1.5-8 M in the fractionation buffer.

[0157] In some embodiments, the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylic acid ester.

[0158] In some embodiments, the at least one polymer is present in the fractionation buffer at a concentration of about 0.1% to 15% (w / w).

[0159] In some embodiments, the at least one polymer has an average molecular weight ranging from 100 Da to 35,000 Da.

[0160] In some embodiments, the fractionation buffer further comprises one or more of a pH buffer, a metal chelator, or a combination thereof.

[0161] In some embodiments, a method for isolating a smaller-than-target size target nucleic acid from a sample containing nucleic acid components is provided, the method comprising: (a) preparing a sample solution from the sample; (b) contacting the sample solution with a solid phase medium configured to selectively bind the nucleic acid components, thereby binding the nucleic acid components to the solid phase medium and forming a medium-analyte complex; (c) adding a fractionation buffer containing at least one chaotropic agent to the medium-analyte complex to form a bulk fraction solution, wherein the smaller-than-target size target nucleic acid is released from the solid phase medium into the bulk fraction solution; and (d) separating the bulk fraction solution containing the isolated smaller-than-target size target nucleic acid from the solid phase medium.

[0162] In some embodiments, the solid phase medium is a solid phase extraction column.

[0163] In some embodiments, the solid phase extraction column is a spin column.

[0164] In some embodiments, the solid phase medium is a plurality of beads.

[0165] In some embodiments, the plurality of beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

[0166] In some embodiments, the concentration of the polymer is 0.5% to 80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 0.5% to 30% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 5% to 60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the concentration of the polymer is 12% to 50% (w / v) of the first ATPS and / or the second ATPS.

[0167] In some embodiments, the salt concentration is 0.1% to 80% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 5% to 60% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 0.1% to 50% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 0.1% to 20% (w / v) of the first ATPS and / or the second ATPS. In some embodiments, the salt concentration is 0.01% to 30% (w / v). In some embodiments, the salt concentration is 0.01% to 10% (w / v) of the first ATPS and / or the second ATPS.

[0168] In some embodiments, the concentration of the surfactant is 0.1% to 50% (w / v) of the first ATPS and / or the second ATPS, hi some embodiments, the concentration of the surfactant is 0.01% to 10% (w / v) of the first ATPS and / or the second ATPS.

[0169] In some embodiments, the first ATPS composition is polymer-salt based, comprising at least one polymer at a concentration of 5% to 80% (w / v) and at least one salt at a concentration of 0.1% to 80% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 5% to 60% (w / v) and at least one salt at a concentration of 0.5% to 50% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 12% to 50% (w / v) and at least one salt at a concentration of 0.1% to 20% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0170] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 0.5% to 30% (w / v) and at least one salt at a concentration of 5% to 60% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 1% to 6% (w / v) and at least one salt at a concentration of 10% to 50% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0171] In some embodiments, the first ATPS composition is polymer-salt based, comprising at least one polymer at a concentration of 0.5% to 30% (w / v) and at least one salt at a concentration of 5% to 60% (w / v). In some embodiments, the first ATPS composition comprises at least one polymer at a concentration of 1% to 6% (w / v) and at least one salt at a concentration of 10% to 50% (w / v). In some embodiments, the first ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0172] In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5% to 80% (w / v) and at least one salt at a concentration of 0.1% to 80% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 5% to 60% (w / v) and at least one salt at a concentration of 0.5% to 50% (w / v). In some embodiments, the second ATPS composition comprises at least one polymer at a concentration of 12% to 50% (w / v) and at least one salt at a concentration of 0.1% to 20% (w / v). In some embodiments, the second ATPS composition further comprises at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0173] In some embodiments, the first ATPS composition is polymer-polymer based and includes at least one polymer at a concentration of 0.2% to 50% (w / v). In some embodiments, the first ATPS composition further includes at least one salt at a concentration of 0.01% to 10% (w / v). In some embodiments, the first ATPS composition further includes at least one surfactant at a concentration of 0.01% to 10% (w / v).

[0174] In some embodiments, the first ATPS composition is surfactant-based and includes at least one surfactant at a concentration of 0.1% to 50% (w / v). In some embodiments, the first ATPS composition further includes at least one salt at a concentration of 0.01% to 30% (w / v).

[0175] Although the description refers to particular embodiments, the present disclosure should not be construed as being limited to the embodiments set forth herein. example

[0176] This specification provides examples that more fully describe some embodiments of the present disclosure. The examples provided herein are for purposes of clarity only and are not intended to limit the scope of the present invention in any way. All references given below and elsewhere in this application are incorporated herein by reference. Example 1: DNA concentration method without fractionation buffer

[0177] Below is an exemplary method of how to concentrate and isolate a target analyte based on the present disclosure: In this example, the target analyte is DNA.

[0178] The steps of the scheme were carried out as follows: 1. A required volume of processed biological sample (e.g., plasma) (e.g., 2 mL to 3 mL) was added to the first ATPS (Solution B) to form Solution B'. The biological sample processing method includes, but is not limited to, cleavage to form sample cleavage products. 2. Solution B' was vortexed thoroughly (eg, for about 10 seconds) until homogenous, and then centrifuged at 2,300 xg for 6 minutes. 3. The bottom phase of solution B' was transferred to a second ATPS (solution C) to form solution C'. 4. Solution C' was vortexed thoroughly for 10 seconds until homogeneous, and then centrifuged at 7,000 xg for 1 minute. 5. 800 μL of Binding Buffer (e.g., Binding Buffer BB1, BB2, or BB3) was added to a new 2 mL microcentrifuge tube. 6. The upper phase of Solution C' containing the concentrated target analytes from step 5 was transferred to a microcentrifuge tube. 7. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table 1, 12 μL) before use and add to the microcentrifuge tube from step 6 to allow the magnetic beads to bind to the target analyte, forming a bead-analyte complex. 8. The microcentrifuge tube was tilted and incubated for 5 minutes, then placed on a magnetic stand for 2 minutes to immobilize the bead-analyte complexes onto the tube wall. 9. Without disturbing the bead-analyte complex, the supernatant in the microcentrifuge tube was pipetted and discarded, and the microcentrifuge tube was removed from the magnetic stand. 10. 800 μL of binding buffer (e.g., Binding Buffer BB1, BB2, or BB3) was added to the microcentrifuge tube. The microcentrifuge tube was vortexed for 20 seconds and placed on a magnetic stand for 2 minutes to immobilize the bead-analyte complexes on the tube wall. 11. The supernatant in the microcentrifuge tube was pipetted and discarded without disturbing the bead-analyte complex. 12. Add an appropriate wash buffer (e.g., 800 μL) known to those skilled in the art to the microcentrifuge tube and spin on the magnetic stand, rotating once 120 degrees for a total of 720 degrees. After spinning, pipette and discard the supernatant in the microcentrifuge tube without disturbing the bead-analyte complex. 13. Step 12 was repeated at least once. 14. The microcentrifuge tube was then briefly rotated on its hinge outwards to collect any remaining wash buffer in the tube. 15. The microcentrifuge tube was placed back on the magnetic stand for 1 minute to immobilize the bead-analyte complexes onto the tube wall. 16. Carefully discard all supernatant (e.g., using a 10 μl pipette tip) without disturbing the bead-analyte complex. 17. The lid was opened and the bead-analyte complexes were allowed to dry on the magnetic stand for 7 minutes. 18. After drying, the microcentrifuge tube was removed from the magnetic stand. 19. An appropriate elution buffer known to those skilled in the art (eg, 40 μL) was added directly to the bead-analyte complex (in the microcentrifuge tube). 20. The bead-analyte complex was resuspended by continuous agitation using a pipette and pipetting up and down 5 times. 21. Briefly vortex the microcentrifuge tube (e.g., for 15 seconds). 22. The microcentrifuge tube was incubated at room temperature for 3 minutes. 23. The microcentrifuge tube was placed on the magnetic stand for 1 minute. 24. Carefully collect the supernatant containing the purified target analyte into a clean max collection tube without disturbing the magnetic beads. 25. The purified target analyte was used immediately or stored long term at -20°C or below. Example 2: DNA concentration method when using fractionation buffer

[0179] The following is another exemplary method of how to concentrate and isolate a target analyte based on the present disclosure: In this example, the target analyte is DNA.

[0180] The steps of the scheme were carried out as follows: 1. A required volume of processed biological sample (e.g., plasma) (e.g., 2 mL to 3 mL) was added to the first ATPS (Solution B) to form Solution B'. The biological sample processing method includes, but is not limited to, cleavage to form sample cleavage products. 2. Solution B' was vortexed thoroughly for 10 seconds until homogeneous, and then centrifuged at 2,300 xg for 6 minutes. 3. The bottom phase of solution B' was transferred to a second ATPS (solution C) to form solution C'. 4. Solution C' was vortexed thoroughly for 10 seconds until homogeneous, and then centrifuged at 7,000 xg for 1 minute. 5. 800 μL of Binding Buffer (e.g., Binding Buffer BB1, BB2, or BB3) was added to a new 2 mL microcentrifuge tube. 6. The upper phase of Solution C' containing the concentrated target analyte was transferred from step 5 to the tube containing binding buffer. 7. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table 1, 12 μL) before use and add to the microcentrifuge tube from step 6 to allow the magnetic beads to bind to the target analyte, forming a bead-analyte complex. 8. The microcentrifuge tube was tilted and incubated for 5 minutes, then placed on a magnetic stand for 2 minutes to immobilize the bead-analyte complexes onto the tube wall. 9. Without disturbing the bead-analyte complex, the supernatant in the microcentrifuge tube was pipetted and discarded, and the microcentrifuge tube was removed from the magnetic stand. 10. 300 μL of fractionation buffer (e.g., fractionation buffer F1, F2, or F3) was added to the microcentrifuge tube. The microcentrifuge tube was vortexed for 20 seconds, incubated with tilt rotation for 5 minutes, and placed on a magnetic stand for 2 minutes to immobilize the bead-analyte complexes to the tube wall and release target analytes smaller than the target size from the bead-analyte complexes into the supernatant. 11. Add 600 μL of the second binding buffer (e.g., binding buffer BB1, BB2, or BB3) to a new 2 mL microcentrifuge tube. 12. The supernatant from step 10 was transferred to the tube containing the second binding buffer from step 11. 13. Pipette the mixture up and down to ensure all the supernatant was transferred and mixed thoroughly with the second binding buffer. 14. Vortex the provided magnetic beads (e.g., magnetic beads selected from Table 1, 6 μL) before use and add to the microcentrifuge tube from step 13, allowing the magnetic beads to bind target analytes smaller than the target size, forming a second bead-analyte complex. 15. The microcentrifuge tube was tilted and incubated for 5 minutes, then placed on a magnetic stand for 4 minutes to immobilize the second bead-analyte complex onto the tube wall. 16. Without disturbing the second bead-analyte complex, the supernatant in the microcentrifuge tube was pipetted and discarded. 17. An appropriate wash buffer (e.g., 800 μL) known to those skilled in the art was added to the microcentrifuge tube and spun on a magnetic stand, rotating once 120 degrees for a total of 720 degrees. After spinning, the supernatant in the microcentrifuge tube was pipetted and discarded without disturbing the second bead-analyte complex. 18. Step 17 was repeated. 19. The lid was opened and the beads were allowed to dry on the magnetic stand for 15 minutes. 20. After drying, the tube was removed from the magnetic stand. 21. An appropriate elution buffer known to those skilled in the art (eg, 40 μL) was added directly to the second bead-analyte complex (in the microcentrifuge tube). 22. The second bead-analyte complex was resuspended by continuous agitation using a pipette and pipetting up and down five times. 23. The microcentrifuge tube was briefly vortexed for 10 seconds. 24. The microcentrifuge tube was incubated at room temperature for 3 minutes. 25. The microcentrifuge tube was placed on the magnetic stand for 1 minute. 26. Without interfering with the magnetic beads, the supernatant containing the purified target analytes below the target size was carefully collected into a clean max collection tube. 27. Purified target analytes below target size were used immediately or stored long term at -20°C or below. Example 3: Performance evaluation of exemplary methods

[0181] The performance of the methods and kits disclosed below may be evaluated according to the following steps. (i) Multiple magnetic bead extraction kit components were prepared by varying the following components: a. Solution B i. Polymer ii. Salt iii. Surfactants b. Solution C i. Polymer ii. Salt c. Magnetic beads d. Binding buffer i. Chaotropic Agents ii. Polymers e. Fractionation buffer i. Chaotropic Agents ii. Polymers (ii) Sample solutions were prepared and spiked with known amounts of target DNA to assess. (iii) Extraction was performed using the variant of the magnetic bead extraction kit prepared in step 1 above and an industry standard extraction kit according to their designated procedures. (iv) Target DNA was quantified by standard qPCR or ddPCR procedures.

[0182] In various exemplary embodiments, Solution B, Solution C, Binding Buffer and Fractionation Buffer were selected from the examples shown below in various combinations. [Table 2] Example 4a: Direct and reverse fractionation

[0183] In some embodiments, the present specification provides two different approaches / methods for using a fractionation buffer to separate target nucleic acids (e.g., DNA) into large and small fragments based on their size: direct fractionation and reverse fractionation.

[0184] Direct fractionation

[0185] Referring to FIG. 1A, a general operational flow of a direct fractionation method is shown based on one exemplary embodiment. In this embodiment, in a first optional cleavage step 111, an appropriate cleavage buffer is added to a sample (e.g., blood, plasma, serum, cerebrospinal fluid, urine, saliva, feces, tears, sputum, nasopharyngeal mucus, vaginal secretions, or penile secretions) to cleave cells in the sample and release biomolecules into the cleavage buffer solution. The cleaved sample is then subjected to DNA isolation and enrichment using two sequential aqueous two-phase systems (ATPS). In step 112, the cleaved sample is mixed with the first ATPS. The mixture is centrifuged to separate the top and bottom phases. In step 113, all of the bottom phase (also referred to as the "first target-rich phase"), into which DNA may be partitioned, is transferred to a second ATPS (second ATPS) and thoroughly mixed. The mixture is centrifuged to separate the top and bottom phases. In step 114, the upper phase from the second ATPS into which the target DNA was partitioned (also referred to as the "second target-rich phase") was extracted into an empty microcentrifuge tube. A direct fractionation buffer (e.g., as described in Examples 5-6 below) was added to the tube and thoroughly mixed with the upper phase from the second ATPS and the magnetic beads also added to the tube, allowing the magnetic beads to bind only the larger DNA fragments. The mixture was incubated for a period of time, then spun down and placed on a magnetic stand to immobilize the beads on the tube wall. Large DNA fragments bound to the beads, while small DNA fragments remained in the supernatant. The supernatant was then extracted without disturbing the beads and transferred to a new microcentrifuge tube (see step 115). Further in step 115, a binding buffer (e.g., Binding Buffer A described in Example 5 below) was added to the extracted supernatant. Magnetic beads were then added to the mixture of the supernatant and binding buffer and incubated for a period of time to bind the small DNA fragments. The tube was then spun down and placed on a magnetic stand to immobilize the bead-analyte complexes. The supernatant was discarded without disturbing the bead-analyte complexes. In step 116, the bead-analyte complexes may be further purified by several optional washing steps using an appropriate wash buffer.The bead-analyte complex was then resuspended in an appropriate elution buffer, mixed thoroughly, and placed on a magnetic stand to immobilize the magnetic beads. The supernatant containing the purified DNA sample was collected for further use.

[0186] Reverse fractionation

[0187] Referring to Figure 1B, a general operational flow of the reverse fractionation scheme is shown based on one exemplary embodiment. In this example, steps 121, 122, and 123 are the same as or similar to steps 111, 112, and 113, respectively, described in the direct fractionation scheme. In step 124, the upper phase (also referred to as the "second target-rich phase") from the second ATPS, into which the target DNA is partitioned, is extracted into a microcentrifuge tube containing a binding buffer (e.g., Binding Buffer A described in Example 5 below). Magnetic beads are added to the mixture and incubated for a period of time. The tube is then spun down and placed on a magnetic stand to immobilize the beads on the tube wall. DNA of all sizes should bind to the magnetic beads, while proteins and contaminants remain in the supernatant, which is discarded. In step 125, a reverse fractionation buffer (e.g., the buffer described in Examples 5-7 below) is added to the beads and mixed thoroughly. After incubation for a period of time, the tube is spun down and placed on a magnetic stand. Large DNA fragments bind to the beads, while small DNA fragments remain in the supernatant. The supernatant was extracted and transferred to a new microcentrifuge tube without disturbing the beads (see step 126). Further, in step 126, a target binding buffer (e.g., Binding Buffer B in Example 5 below) and magnetic beads were added to the supernatant to capture the fractionated DNA fragments onto the beads. The mixture was incubated for a period of time, spun down, and placed on a magnetic stand to immobilize the bead-analyte complexes. After pipetting, the supernatant was discarded. In step 127, the bead-analyte complexes may be further purified by several optional washing steps using an appropriate wash buffer. The bead-analyte complexes were then resuspended in an appropriate elution buffer, mixed thoroughly, and placed on a magnetic stand to immobilize the magnetic beads. The supernatant containing the purified DNA sample was collected for further use.

[0188] In some embodiments, possible magnetic beads that can be used include, but are not limited to, those listed in Table 1.2 below.

[0189] [Table 1.2] Example 4b: Calculating DNA cutoff values

[0190] In some examples, the DNA cutoff value for a given sample was calculated by the method described below.

[0191] A sample containing a DNA ladder was extracted using a size fractionation procedure according to any of the methods described in this disclosure. To estimate the DNA cutoff value, the extracted DNA sample and a positive control condition (representing a 100% recovery condition for all sizes of DNA) were analyzed using the Agilent Bioanalyzer High Sensitivity DNA Kit (catalog number 5067-4626). The concentration of each peak was measured using the Agilent 2100 analysis software and exported to a spreadsheet. First, for the positive control condition, the concentration of all DNA fragments larger than 100 bp was divided by the concentration of 100 bp DNA fragments to calculate the ratio (Conc / Conc_a) (as shown in Table I). The reason for selecting this 100 bp DNA size fragment (also referred to as the "non-size-selected internal 100 bp control") is that it is not affected by size fractionation and can therefore serve as a normalization value for the 100% recovery condition in each experiment.

[0192] [Table I]

[0193] The same ratio calculation was performed on the extracted DNA sample (referred to as "Example A") and is shown in Table II. The percent recovery was then calculated by dividing the "Ratio" for "Example A" in Table II by the "Ratio" for the "Positive Control" in Table I. Bioanalyzer artifacts and baseline signal fluctuations can result in values ​​greater than 100%. All values ​​greater than 100% were assumed to be 100% recovery. By finding two percent recovery values ​​that straddle 70% and performing a linear regression between these two points, the bp size that gives 70% recovery compared to the non-size-selected internal 100 bp control can be estimated. All values ​​were considered the DNA cutoff values ​​for the previous example.

[0194] [Table II] Example 5

[0195] The following study was conducted to compare the stability and robustness of direct and reverse fractionation protocols when used to extract DNA from different volumes of plasma samples (specifically, different volumes of the upper phase of a second ATPS added to the fractionation step). The ability of the direct and reverse fractionation protocols to accommodate sample-to-sample variation was evaluated.

[0196] Materials and Methods

[0197] Plasma cell cleavage

[0198] Each 2 mL of plasma was spiked with 100 fg of a 145 bp dsDNA oligonucleotide, 80 ng of a 20 bp ladder (Jena Bioscience, Cat. No. M212), and 40 ng of a 50 bp ladder (Jena Bioscience, Cat. No. M-213). Each 2 mL of spiked plasma was spiked with 160 μL of the appropriate cleavage buffer and 60 μL of proteinase K (28.5 mg / mL). The mixture was vortexed thoroughly and cleaved in a preheated 60°C heating block for 15 min.

[0199] Two-phase system

[0200] DNA was isolated and concentrated from cleaved plasma samples using two sequential aqueous two-phase systems (ATPS). 2.22 mL of the cleaved sample was transferred to the first ATPS (polymer, salt, and / or surfactant) and vortexed to mix. The mixture was centrifuged at 2,300 rcf for 6 minutes. The entire salt-rich bottom phase (approximately 1 mL), into which DNA could potentially partition (also referred to as the "first target-rich phase"), was transferred to the second ATPS (polymer, salt, and / or surfactant) and mixed thoroughly. The mixture was then centrifuged at 7,000 x g for 1 minute. The upper phase (approximately 150 μL), into which target DNA partitioned (also referred to as the "second target-rich phase"), was carefully extracted into an empty microcentrifuge tube and used for further purification. Different upper phase volumes (100 μL, 150 μL, and 180 μL) of the second ATPS were extracted three times. One set of samples with each upper phase volume underwent direct fractionation and another set underwent the reverse fractionation scheme.

[0201] Direct fractionation

[0202] For each upper phase volume of ATPS, two direct fractionation buffer formulations with different expected DNA cutoff sizes were used: (i) fractionation buffer D1 for a small (approximately 150 bp) expected cutoff size: 3–7 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator; and (ii) fractionation buffer D2 for a large (approximately 300 bp) expected cutoff size: 3–7 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator.

[0203] For each sample, the upper phase of the second ATPS was transferred to an empty microcentrifuge tube. 80 μL of direct fractionation buffer (Fraction Buffer D1 or D2) was added to the tube and thoroughly mixed with 12 μL of magnetic beads, which had also been added to the tube. Magnetic beads (Cat# MF-SIL-5024) were purchased from MagQu Co. Ltd. The mixture was incubated with tilted rotation for 5 minutes. The tube was then briefly spun to settle and placed on a magnetic stand for 2 minutes to immobilize the beads on the tube wall. Large DNA fragments bound to the beads, while small DNA fragments remained in the supernatant. The supernatant was then extracted and transferred to a new microcentrifuge tube without disturbing the beads. Six hundred microliters of binding buffer A (3–7 M guanidine salt and 0.1%–15% (w / v) polymer) was added to the extracted supernatant, providing the necessary discrete salts for salt bridge formation between the DNA and the solid-phase magnetic beads. Six microliters of magnetic beads were then added to the supernatant-binding buffer mixture and incubated for an additional 5 minutes with tilted rotation. The tube was then spun to settle and placed on a magnetic stand for 4 minutes. The supernatant was discarded without disturbing the bead-analyte complex. Six hundred microliters of binding buffer A was then added to the bead-analyte complex, and the tube was rotated a total of 720° on the stand. The supernatant was discarded, and the bead-analyte complex was then subjected to further purification.

[0204] Reverse fractionation

[0205] For different expected DNA cutoff sizes, two reverse fractionation buffer formulations were used for each upper phase volume: (i) fractionation buffer R1 for a small (approximately 150 bp) expected cutoff size: 3–7 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator; and (ii) fractionation buffer R2 for a large (approximately 300 bp) expected cutoff size: 0.5–5.0 M guanidine, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator.

[0206] Each upper phase volume from the second ATPS was transferred to a microcentrifuge tube containing 800 μL of binding buffer A (3–7 M guanidine salt and 0.1%–15% (w / v) polymer). 12 μL of magnetic beads was added to the mixture and incubated for 5 minutes with tilted rotation. The tube was spun to settle and placed on a magnetic stand for 2 minutes to immobilize the beads on the tube wall. DNA of all sizes should bind to the magnetic beads, while proteins and contaminants remained in the supernatant, which was discarded. 300 μL of reverse fractionation buffer (fractionation buffer R1 or R2) was then added to the beads. After vortex mixing, the mixture was incubated for 5 minutes on a rotary. The tube was spun to settle and placed on the magnetic stand for an additional 2 minutes. Large DNA fragments bound to the beads, while small DNA fragments remained in the supernatant. The supernatant was then extracted and transferred to a new microcentrifuge tube. 600 μL of binding buffer B (3-7 M guanidine salt and 0.1%-15% (w / v) polymer, also referred to in some examples as "target binding buffer") and 6 μL of magnetic beads were added to the supernatant to capture the fractionated DNA fragments onto the beads. The mixture was incubated on a rotary for an additional 5 minutes, then spun down and placed on a magnetic stand for 4 minutes. After pipetting again, the supernatant was discarded. The bead-analyte complex was then subjected to further purification.

[0207] DNA purification

[0208] 800 μL of wash buffer (70% ethanol, 0.001 M EDTA, 0.01 M Tris-HCl) was added to each sample processed using the direct or reverse fractionation protocol described above, and the tube was rotated a total of 720° on the magnetic stand. The supernatant was discarded. This wash step was performed twice. The tube was briefly centrifuged using a tabletop microcentrifuge with the hinge facing outward to collect any remaining wash buffer. The bead-analyte complex was then allowed to dry on the magnetic stand with the lid open for 7 minutes. The bead-analyte complex was resuspended in 40 μL of elution buffer (0.01 M Tris-HCl, 0.001 M EDTA) by repeated pipetting and briefly vortexed. The tube was placed on the magnetic stand for 1 minute to immobilize the magnetic beads. The supernatant containing the purified DNA sample was carefully collected into a low-binding tube without disturbing the magnetic beads, and detection was performed.

[0209] DNA detection

[0210] Recovery of DNA oligonucleotides of different sizes in extracted samples quantitatively by electrophoresis. Gel mixtures were obtained from Agilent TM The gel was prepared using a high-sensitivity DNA kit (Agilent, 5067-4626). 9 μL of the gel dye mixture was dispensed into each well on the microfluidic chip, and 1 μL of purified DNA sample was added. Electrophoresis on the chip was performed using an Agilent TM The analysis was performed using the Agilent 2100 Bioanalyzer software. TM The fluorescent signal of the reaction was collected and analyzed using a Technologies 2100 Expert. The actual DNA cutoff value was estimated using the calculation method described in Example 4b, i.e., the base pair value of a purified DNA sample with a % DNA recovery of 70%.

[0211] The sample set conditions and composition of the fractionation buffer formulations (used for direct and reverse fractionation) used in this study are summarized in Table 3 below.

[0212] [Table 3]

[0213] result

[0214] DNA recovery

[0215] Differences in sample volume, such as the upper phase volume extracted from the second ATPS used directly in subsequent fractionation (direct or reverse fractionation), can affect the efficiency of DNA fractionation. To address these differences, we used different upper phase volumes (100 μL, 150 μL, and 180 μL) of the second ATPS in the direct and reverse fractionation protocols to study the stability and robustness of the two fractionation types.

[0216] Figures 2A and 2B show electropherograms of DNA oligonucleotide recovery from plasma extracted from different volumes of the upper phase in the second ATPS using direct fractionation (using fractionation buffer D1) and reverse fractionation (using fractionation buffer R1), respectively, where the expected DNA cutoff value is small (approximately 150 bp). The actual DNA cutoff values ​​of purified DNA extracted from different upper phase volumes of the second ATPS using direct fractionation (using fractionation buffer D1) or reverse fractionation (using fractionation buffer R1) are shown in Table 4.

[0217] [Table 4]

[0218] As shown in Figure 2A and Table 4, when direct fractionation was used and the upper phase volume was small (condition 1, 100 μL), the recovery of large DNA fragments (>100 bp) was significantly reduced, whereas when the upper phase volume was large (condition 3, 180 μL), the recovery of large DNA oligonucleotides (100 bp to 200 bp) significantly increased. These results indicate that when direct fractionation was used, the cutoff value shifted significantly when the upper phase volume deviated from the standard (condition 2, 150 μL), indicating that changes in sample volume have a significant impact on the stability and efficiency of DNA fractionation when using direct fractionation.

[0219] Referring to Figure 2B and Table 4, the differences between the DNA fragment sizes recovered using reverse fractionation with different upper phase volumes (100 μL, 150 μL, and 180 μL) were obviously smaller than those using direct fractionation, and the shift in the DNA cutoff value was the smallest, i.e., within the acceptable range of 131 bp to 175 bp. As shown in Figure 2B, the electrophoresis patterns using reverse fractionation conditions 4 to 6 with different upper phase volumes were significantly overlapped.

[0220] Figures 3A and 3B show electropherograms of DNA oligonucleotide recovery from plasma extracted from different volumes of the upper phase in the second ATPS using direct fractionation (using fractionation buffer D2) and reverse fractionation (using fractionation buffer R2), respectively, where the expected DNA cutoff value is large (approximately 300 bp). The actual DNA cutoff values ​​of purified DNA extracted from different upper phase volumes of the second ATPS using direct fractionation (using fractionation buffer D2) or reverse fractionation (using fractionation buffer R2) are shown in Table 5.

[0221] [Table 5]

[0222] Referring to Figure 3A and Table 5, when direct fractionation was used and the upper phase volume was small (condition 7, 100 μL), the cutoff value shifted significantly to a lower value (i.e., 229 bp). Of note, when the upper phase volume was large (condition 9, 180 μL), the recovery of small DNA fragments (<100 bp) was low (i.e., 31% relative to the 150 μL condition), even though the recovery of large DNA fragments was similar to that using an upper phase volume of 150 μL (condition 8).

[0223] Referring to Figure 3B and Table 5, in the case of reverse fractionation, the difference between the DNA fragment sizes recovered using reverse fractionation with different upper phase volumes (100 μL, 150 μL, and 180 μL) was obviously smaller than that of direct fractionation, and the shift in the DNA cutoff value was the smallest, i.e., within the acceptable range of 281 bp to 356 bp. As shown in Figure 3B, the electrophoresis patterns using reverse fractionation conditions 10 to 12 with different upper phase volumes were significantly overlapped.

[0224] Overall, the results showed that reverse fractionation was unexpectedly more stable than direct fractionation, both in terms of DNA cutoff value and DNA recovery, especially for small DNA fragments, at different upper-phase volumes. The high stability and efficiency of DNA fractionation using the reverse fractionation method are advantageous for isolating small DNA fragments below the target size. Example 6

[0225] The following study was conducted to compare the stability and robustness of direct and reverse fractionation methods for DNA extracted from different sample types (i.e., plasma and urine). Fractionation stability and robustness are critical for a wide range of applications. One variable here is sample type, as different components in the sample can affect fractionation performance to different degrees. To investigate the resistance of direct and reverse fractionation to different sample types, studies were conducted on plasma and urine samples using the same direct and reverse fractionation buffers and methods described in Example 5 to investigate the differences in their performance.

[0226] Materials and Methods

[0227] Plasma cell cleavage

[0228] Each 2 mL of plasma was spiked with 100 fg of a 145 bp dsDNA oligonucleotide, 80 ng of a 20 bp ladder (Jena Bioscience, Cat. No. M212), and 40 ng of a 50 bp ladder (Jena Bioscience, Cat. No. M-213). 160 μL of the appropriate cleavage buffer and 60 μL of proteinase K (28.5 mg / mL) were added per 2 mL of spiked plasma. The mixture was vortexed thoroughly and cleaved in a preheated 60°C heating block for 15 min.

[0229] Urine pretreatment and cleavage

[0230] Urine samples were pretreated with 200 μL of 0.1 M EDTA per 10 mL of urine sample, vortexed thoroughly, and centrifuged at 3000 x g for 10 minutes. This preserves free DNA (cfDNA) present in the sample and prevents its degradation over time. The supernatant was transferred to a new tube, and the precipitate was discarded. Each 2 mL of urine was spiked with 100 fg of a 145-bp dsDNA oligonucleotide, 80 ng of a 20-bp ladder (Jena Bioscience, catalog no. M212), and 40 ng of a 50-bp ladder (Jena Bioscience, catalog no. M-213). Unwanted proteins and cells present in the pretreated urine sample were cleaved by adding 1200 μL of proteinase K (28.57 mg / mL) and 4 mL of the appropriate cleavage buffer to the 40 mL sample. The sample was then vortexed thoroughly until homogenous and placed in a preheated 37°C water bath for 15 minutes.

[0231] Two-phase system

[0232] DNA was isolated and concentrated from cleaved plasma and urine samples using two sequential aqueous two-phase systems (ATPS). 2.22 mL of cleaved plasma or 2.26 mL of cleaved urine was transferred to the first ATPS (polymer, salt, and / or surfactant) and vortexed to mix. The mixture was centrifuged at 2,300 rcf for 6 minutes. The entire salt-rich bottom phase (approximately 1 mL), into which DNA may partition (also referred to as the "first target-rich phase"), was transferred to the second ATPS (polymer, salt, and / or surfactant) and mixed thoroughly. The mixture was then centrifuged at 7,000 x g for 1 minute. The polymer-rich upper phase (approximately 150 μL), into which polymer-target DNA partitions (also referred to as the "second target-rich phase"), was carefully extracted into an empty microcentrifuge tube and used for further purification. One sample set containing plasma or urine samples underwent direct fractionation, while the other set underwent a reverse fractionation scheme.

[0233] Direct fractionation

[0234] For each sample type, two direct fractionation buffer formulations with different expected DNA cutoff sizes were used: (i) fractionation buffer D1 for a small (approximately 150 bp) expected cutoff size: 3–7 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator; and (ii) fractionation buffer D2 for a large (approximately 300 bp) expected cutoff size: 1–5 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator.

[0235] The steps for performing direct fractionation on plasma and urine samples to separate and isolate small and large DNA fragments are the same or similar to those discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the direct fractionation steps will not be repeated here.

[0236] Reverse fractionation

[0237] Two reverse fractionation buffer formulations were used for each sample type for different expected DNA cutoff sizes: (i) fractionation buffer R1 for a small (approximately 150 bp) expected cutoff size: 1–5 M guanidine salt, 0.1%–15% (w / v) polymer, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator; and (ii) fractionation buffer R2 for a large (approximately 300 bp) expected cutoff size: 0.5–5.0 M guanidine, 0.01 M–0.5 M pH buffer, and 0.01 M–0.5 M metal chelator.

[0238] The steps for performing reverse fractionation on plasma and urine samples to separate and isolate small and large DNA fragments are the same or similar to those discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, a discussion of the reverse fractionation steps will not be repeated here.

[0239] DNA purification

[0240] The steps for DNA purification for each sample are the same or similar to those discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the purification steps will not be repeated here.

[0241] DNA detection

[0242] Recovery of DNA oligonucleotides of different sizes in extracted samples quantitatively by electrophoresis. Gel mixtures were obtained from Agilent TM The gel was prepared using a high-sensitivity DNA kit (Agilent, 5067-4626). 9 μL of the gel dye mixture was dispensed into each well on the microfluidic chip, and 1 μL of purified DNA sample was added. Electrophoresis on the chip was performed using an Agilent TM The analysis was performed using the Agilent 2100 Bioanalyzer software. TMThe fluorescent signal of the reaction was collected and analyzed using a Technologies 2100 Expert. The actual DNA cutoff value was estimated using the calculation method described in Example 4b, i.e., the base pair value of a purified DNA sample with a % DNA recovery of 70%.

[0243] The sample set conditions and composition of the fractionation buffer formulations (used for direct and reverse fractionation) used in this study are summarized in Table 6 below.

[0244] [Table 6]

[0245] result

[0246] DNA recovery

[0247] Figures 4A and 4B show electropherograms of DNA oligonucleotide recovery from different sample types (i.e., plasma and urine) using direct fractionation (using fractionation buffer D1) and reverse fractionation (using fractionation buffer R1), respectively, where the expected DNA cutoff value is small (approximately 150 bp). The actual DNA cutoff values ​​for purified DNA extracted from different sample types using direct fractionation (using fractionation buffer D1) or reverse fractionation (using fractionation buffer R1) are shown in Table 7.

[0248] [Table 7]

[0249] Referring to Figure 4A and Table 7, it was found that direct fractionation was successful for plasma samples when the expected cutoff value was small (approximately 150 bp), but only small amounts of small (<50 bp) DNA were recovered in urine samples, indicating that direct fractionation cannot be adapted to the input of various sample types.

[0250] Referring to Figure 4B and Table 7, in the case of reverse fractionation, the difference between the DNA fragment sizes recovered using reverse fractionation with plasma and urine was clearly smaller than that of direct fractionation, and the difference in DNA cutoff values ​​(147 bp for plasma and 125 bp for urine) was also smaller, indicating the stability of reverse fractionation and its broader applicability to various sample types.

[0251] Figures 5A and 5B show electropherograms of DNA oligonucleotide recovery from plasma extracted from different sample types using direct fractionation (using fractionation buffer D2) and reverse fractionation (using fractionation buffer R2), respectively, where the expected DNA cutoff value is large (approximately 300 bp). The actual DNA cutoff values ​​for purified DNA extracted from different sample types using direct fractionation (using fractionation buffer D2) or reverse fractionation (using fractionation buffer R2) are shown in Table 8.

[0252] [Table 8]

[0253] Referring to Figure 5A and Table 8, when urine was used instead of plasma, the cutoff value shifted to a much smaller value (i.e., 138 bp) when direct fractionation was used, indicating that direct fractionation when changing sample types can result in over-removal of target DNA, which can be detrimental to downstream assays.

[0254] Referring to Figure 5B, in the case of reverse fractionation, the electrophoretic patterns of DNA fragments recovered using reverse fractionation with different sample types (i.e., plasma and urine) were significantly overlapping, indicating that the size fractions and extracted target DNA were generally consistent across different sample types. The DNA cutoff value for urine samples was shifted to a slightly higher value, which does not result in over-removal of target DNA (compared to direct fractionation) and is therefore more suitable for use in downstream assays.

[0255] Overall, these results indicated that reverse fractionation is more stable for different sample types and has a wider range of applications compared to direct fractionation. Example 7

[0256] The following study was performed to evaluate the estimated DNA cut-off size using reverse fractionation with different fractionation buffer formulations.

[0257] Materials and Methods

[0258] Plasma cell cleavage

[0259] 100 fg of a 145-bp dsDNA oligonucleotide and 80 ng of a 20-bp ladder (Jena Biosciences, catalog number M212) were spiked into 2 mL of plasma. 160 μL of the appropriate cleavage buffer and 60 μL of proteinase K (28.5 mg / mL) were added per 2 mL of spiked plasma. The mixture was vortexed thoroughly and cleaved in a preheated 60°C heating block for 15 min.

[0260] Two-phase system

[0261] The cleaved plasma samples were subjected to DNA isolation and enrichment using two sequential aqueous two-phase systems (ATPS). The steps for performing sequential ATPS on plasma samples were the same as or similar to those discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the sequential ATPS steps will not be repeated here. Samples collected after the ATPS steps were subjected to reverse fractionation schemes with different reverse fractionation buffer formulations.

[0262] Reverse fractionation

[0263] Different reverse fractionation buffer formulations were used and are summarized below in Table 9. The steps for performing reverse fractionation on plasma samples to separate and isolate small and large DNA fragments are the same or similar to those discussed above in connection with Example 5. For the sake of brevity and simplicity of this disclosure, a discussion of the reverse fractionation steps will not be repeated here.

[0264] [Table 9]

[0265] DNA purification

[0266] The steps for DNA purification for each sample are the same or similar to those discussed above with respect to Example 5. For the sake of brevity and simplicity of this disclosure, the discussion of the purification steps will not be repeated here.

[0267] DNA detection

[0268] The recovery of DNA oligonucleotides of different sizes in samples quantitatively extracted by electrophoresis. The steps for DNA detection for each sample were the same or similar to those discussed in Example 5 above. For the sake of brevity and simplicity of this disclosure, the discussion of the detection steps will not be repeated here. The percent recovery and estimated DNA cutoff value (also referred to in some examples as "actual DNA cutoff value") were calculated, for example, as described in Example 4b, i.e., the base pair value of a purified DNA sample with a percent DNA recovery of 70% was calculated.

[0269] result

[0270] DNA recovery

[0271] Figures 6A-6H show electropherograms of DNA oligonucleotide recovery from plasma samples using reverse fractionation with different reverse fractionation buffer formulations R-015 to R-021. The estimated DNA cutoff values ​​and % recovery of different sizes of DNA extracted using different reverse fractionation formulations are shown in Table 10.

[0272] [Table 10]

[0273] 6A-6H and Table 10, the results showed that the reverse fractionation method can be performed using a wide range of reverse fractionation buffer formulations. The results further showed that varying the concentrations of chaotropic agents, pH buffers, and / or metal chelators in the fractionation buffer can allow precise size selection of recovered DNA molecules by controlling the DNA cutoff value (e.g., from about 100 bp to about 500 bp). Numbered Examples Group 1: [Example]

[0274] 1. A method for concentrating and purifying one or more target analytes from a sample solution, comprising: (a) adding a sample solution containing one or more target analytes to a first aqueous two-phase system (ATPS) to form a mixture that separates into a first phase and a second phase, and the one or more target analytes are concentrated in the first phase; (b) isolating the first phase containing the concentrated one or more target analytes to obtain an enriched solution; (c) adding magnetic beads to the enriched solution and allowing the magnetic beads to bind to the one or more target analytes to form bead-analyte complexes; and (d) recovering the one or more target analytes from the bead-analyte complexes to obtain a final solution containing the concentrated and purified one or more target analytes. [Example]

[0275] The method of example 1, wherein step (b) further comprises: (i) adding the isolated first phase containing the concentrated target analytes to a second ATPS to form a second mixture that is separated into a third phase and a fourth phase, wherein the one or more target analytes are concentrated in the third phase; and (ii) isolating the third phase containing the concentrated target analytes in step (b) to form a concentrated solution used in step (c). [Example]

[0276] The method of any one of the preceding Examples, wherein the concentrated solution of step (b) is mixed with a binding buffer, wherein the binding buffer contains at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea, to obtain the concentrated solution used in step (c). [Example]

[0277] The method of any one of the preceding examples, wherein step (d) further comprises: (i) mixing the bead-analyte complexes with a fractionation buffer comprising a polymer, a salt, a surfactant, a chaotropic agent, or a combination thereof to form a fractionation solution, thereby releasing the one or more target analytes smaller than the target size from the bead-analyte complexes into the fractionation solution; (ii) immobilizing the bead-analyte complexes using a magnetic stand; and (iii) isolating the one or more target analytes smaller than the target size in the fractionation solution from the immobilized bead-analyte complexes. [Example]

[0278] The method of example 4, wherein step (d) further comprises: (iv) adding the one or more isolated target analytes smaller than the target size to a second binding buffer, wherein the second binding buffer comprises at least one chaotropic agent selected from n-butanol, ethanol, guanidine chloride, guanidine thiocyanate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea; (v) adding magnetic beads to the mixture of the one or more isolated target analytes smaller than the target size and the second binding buffer, wherein the magnetic beads bind to the one or more target analytes smaller than the target size to form second bead-analyte complexes; and (vi) recovering the one or more target analytes from the second bead-analyte complexes. [Example]

[0279] The method of any one of the preceding Examples, further comprising the step of (e) performing a diagnostic assay on the final solution to detect and quantify the one or more target analytes. [Example]

[0280] The method of any one of the preceding examples, wherein the one or more target analytes are selected from the group consisting of nucleic acids, proteins, antigens, biomolecules, sugar moieties, lipids, sterols, and combinations thereof. [Example]

[0281] The method of any one of the preceding Examples, wherein the one or more target analytes is DNA. [Example]

[0282] The method of any one of the preceding Examples, wherein the one or more target analytes is free DNA or circulating tumor DNA. [Example]

[0283] The method of any one of the preceding examples, wherein the first ATPS comprises a first ATPS component capable of forming a first phase and a second phase when dissolved in an aqueous solution, wherein the first ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof. [Example]

[0284] 11. The method of any one of Examples 2-10, wherein the second ATPS comprises a second ATPS component, the second ATPS component being capable of forming a third phase and a fourth phase when dissolved in an aqueous solution, and wherein the second ATPS component is selected from the group consisting of a polymer, a salt, a surfactant, and combinations thereof. [Example]

[0285] 12. The method of any one of examples 10 to 11, wherein the polymer is soluble in aqueous solution at a concentration of 4% to 84% (w / w). [Example]

[0286] The method of any one of Examples 10-12, wherein the polymer is selected from the group consisting of polyalkylene glycols (PEGs), e.g., hydrophobically modified polyalkylene glycols, poly(oxyalkylene) polymers, poly(oxyalkylene) copolymers, e.g., hydrophobically modified poly(oxyalkylene) copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactants, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers, and poly-N-isopropylacrylamide and copolymers thereof. The method of any one of the preceding Examples, wherein the polymer is selected from the group consisting of polyethers, polyimides, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, polyether-modified silicones, polyacrylamides, polyacrylic acids, and copolymers thereof. 2. The method of any one of the preceding Examples, wherein the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethyl cellulose, polyacrylic acid, hydroxypropyl cellulose, methyl cellulose, ethyl hydroxyethyl cellulose, maltodextrin, polyethyleneimine, poly N-isopropylacrylamide, and copolymers thereof.The method of any one of the preceding examples, wherein the polymer is selected from the group consisting of dipropylene glycol, tripropylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, and poly N-isopropylacrylamide. The method of any one of the preceding examples, wherein the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid, and copolymers thereof. The method of any one of the preceding examples, wherein the polymer is selected from the group consisting of dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch. The method of any one of the preceding examples, wherein the polymer has an average molecular weight in the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 5,000,000 Da. The method of any one of the preceding examples, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:90. [Example]

[0287] [Example]

[0288] The method according to any one of Examples 10 to 14, wherein the salt is dissolved in an aqueous solution at a concentration of 1% to 80% (w / w). [Example]

[0289] 16. The method according to any one of Examples 10 to 15, wherein the salt is dissolved in aqueous solution at a concentration of 8% to 80% (w / w). [Example]

[0290] The method according to any one of Examples 10 to 16, wherein the salt is selected from the group consisting of lyophilic salts, discrete salts, inorganic salts containing a cation and an anion, 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, borates, phosphate salts, potassium phosphate, and ammonium sulfate, wherein the cation is, for example, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium, and the anion is, for example, phosphate ion, sulfate ion, nitrate ion, chloride ion, and bicarbonate ion. [Example]

[0291] The method according to any one of Examples 10 to 17, wherein the surfactant is dissolved in aqueous solution at a concentration of 0.05% to 10% (w / w). [Example]

[0292] The method according to any one of Examples 10 to 18, wherein the surfactant is dissolved in aqueous solution at a concentration of 0.05% to 9.8% (w / w). [Example]

[0293] The surfactants include Triton-X, Triton-114, Igepal CA-630 and Nonidet P-40, anionic surfactants (e.g., carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils, sulfated natural fats, sulfated esters, sulfated alkanolamides, ethoxylated alkylphenols and sulfated alkylphenols), nonionic surfactants (e.g., ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylic acid esters, polyethylene glycol esters, sorbitan esters, fatty acid ethylene glycol esters, carboxylic acid amides, monoalkanolamine concentrates, polyoxyethylene fatty acid amides), cationic surfactants (e.g., quaternary ammonium salts, amines with amide bonds, polyoxyethylene alkyl and alicyclic amines, n,n,n',n' tetrasubstituted ethylenediamines, 2-alkyl 1-hydroxyethyl 2-imidazoline), and amphoteric surfactants (e.g., n-cocoyl 3-aminopropionic acid / sodium salt, n-tallow 3-iminodipropionate disodium salt, n-carboxymethyl n dimethyl n-9 octadecenyl ammonium hydroxide, n-cocamidoethyl n hydroxyethylglycine and its sodium salt). [Example]

[0294] The method of any one of the preceding examples, wherein step (a) further comprises: (i) embedding components capable of forming a first ATPS in a porous material; and (ii) contacting a sample solution with the porous material with the embedded components, wherein the components form a first phase and a second phase as the sample solution passes through the porous material.

[0295] Illustrative embodiments of the present invention have now been fully described. Although described with reference to specific embodiments, it will be apparent to those skilled in the art that the present invention may be practiced with variations of these specific details. Therefore, the present invention should not be construed as being limited to the embodiments set forth herein.

Claims

1. A method for isolating target nucleic acids smaller than the target size from a sample containing nucleic acid components, (a) A step of preparing a sample solution from the sample, (b) A step of bringing a plurality of beads into contact with the sample solution so that the nucleic acid component binds to the plurality of beads to form a bead-analyte complex, (c) Mixing the bead-analyte complex with a fractionation buffer containing at least one chaotropic agent to form a bulk fractionation solution, and releasing the target nucleic acid, which is smaller than the target size, from the bead-analyte complex into the bulk fractionation solution; (d) The step of fixing the bead-analyte composite, (e) A method comprising the step of separating the bulk fractionated solution containing the target nucleic acid, which is smaller than the isolated target size, from the immobilized bead-analyte complex.

2. Step (a) is, (a1) The sample is added to a first aqueous two-phase system (ATPS) to form a mixture that is separated into a first target-rich phase and a first target-deficient phase, and the nucleic acid component is concentrated in the first target-rich phase, (a2) The method according to claim 1, further comprising the step of isolating the first target-rich phase containing concentrated nucleic acid components to obtain the sample solution.

3. Step (a) is performed after step (a2), (a3) Step (a2) Adding the sample solution from step (a2) to the second ATPS to form a second mixture which is divided into a second target-rich phase and a second target-deficient phase, and the nucleic acid component is concentrated in the second target-rich phase, (a4) The method according to claim 2, further comprising the step of isolating the second target-rich phase containing concentrated nucleic acid components to form the sample solution in step (a).

4. The method according to claim 1, wherein, prior to step (b), the plurality of beads and the sample solution from step (a) are mixed with a binding buffer, wherein the binding buffer comprises at least one chaotropic agent.

5. Step (e) is, (e1) Mix the bulk fraction with a target binding buffer and a plurality of second beads, and bind the plurality of second beads to the target nucleic acid which is smaller than the target size to form a second bead-analyte complex, wherein the target binding buffer contains at least one chaotropic agent, (e2) The method according to claim 1, further comprising the step of recovering the target nucleic acid, which is smaller than the target size, from the second bead-analyte complex.

6. The method according to claim 1, wherein the plurality of beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

7. The method according to claim 5, wherein the plurality of second beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

8. The plurality of beads are magnetic beads, and step (b) is, (b1) A step of fixing the bead-analyte complex by applying a magnetic field and separating the bead-analyte complex from the bulk supernatant, (b2) The step of removing the bulk supernatant, (b) The method according to claim 1, further comprising the step of removing the magnetic field and proceeding to step (c).

9. The plurality of second beads are magnetic beads, and the target nucleic acid recovery in step (e2) is (i) The step of fixing the second bead-analyte complex by applying a first magnetic field and separating the second bead-analyte complex from the first supernatant, (ii) The step of removing the first supernatant liquid, (iii) A step of washing the second bead-analyte complex fixed with washing buffer, (iv) The step of discarding the washing buffer, (v) The step of removing the first magnetic field, (vi) The second bead-analyte complex is mixed with an elution buffer to form a bulk elution solution, the target nucleic acid which is smaller than the target size and the magnetic beads in the second bead-analyte complex are separated and released into the bulk elution solution, (vii) The step of fixing the magnetic beads by applying a second magnetic field, (viiii) The method of claim 5, further comprising the step of collecting the bulk elution solution containing the target nucleic acid which is smaller than the isolated target size.

10. (f) The method according to claim 1, further comprising the step of performing a diagnostic assay on the isolated target nucleic acid for detection, quantification, characterization, or a combination thereof.

11. The method according to claim 1, wherein the at least one chaotropic agent of the fractionated buffer is selected from the group consisting of thiocyanates, isothiocyanates, perchlorates, acetates, trichloroacetates, trifluoroacetates, chlorides, and iodides.

12. The method according to claim 1, wherein the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

13. The method according to claim 1, wherein the at least one chaotropic agent has a concentration of about 1.5 to 8 M in the fractionation buffer.

14. The method according to claim 1, wherein the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 to 3.9 M.

15. The method according to claim 1, wherein the at least one chaotropic agent is present in the fractionation buffer at a concentration of about 1.8 to 3.0 M.

16. The method according to claim 1, wherein the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylic acid esters.

17. The method according to claim 16, wherein the at least one polymer is present in the fractionation buffer at a concentration of about 0.1% to 15% (w / w).

18. The method according to claim 16, wherein the at least one polymer is present in the fractionation buffer at a concentration of about 1.0% to 5.0% (w / w).

19. The method according to claim 16, wherein at least one polymer has an average molecular weight in the range of 100 to 35,000 Da.

20. The method according to claim 1, wherein the fractionated buffer further comprises one or more of a pH buffer, a metal chelating agent, or a combination thereof.

21. The method according to claim 1, wherein the nucleic acid component and / or the target nucleic acid is DNA, RNA, or a combination thereof.

22. The method according to claim 1, wherein the nucleic acid component and / or the target nucleic acid is cDNA, plasmid DNA, free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating fetal DNA, microRNA (miRNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or a combination thereof.

23. The method according to claim 2, wherein the first ATPS comprises a first ATPS component, which, when dissolved in an aqueous solution, can form a first target-rich phase and a first target-deficient phase, wherein the first ATPS component is selected from the group consisting of polymers, salts, surfactants and combinations thereof.

24. The method according to claim 3, wherein the second ATPS comprises a second ATPS component, which, when dissolved in an aqueous solution, can form a second target-rich phase and a second target-deficient phase, wherein the second ATPS component is selected from the group consisting of polymers, salts, surfactants and combinations thereof.

25. The method according to claim 23, wherein the polymer is dissolved in the aqueous solution at a concentration of 0.5% to 80% (w / v).

26. The method according to claim 23, wherein the polymer is selected from the group consisting of polyethers, polyimides, polyalkylene glycols, vinyl polymers, alkoxylated surfactants, polysaccharides, alkoxylated starch, alkoxylated cellulose, alkylhydroxyalkylcellulose, polyether-modified silicones, polyacrylamides, polyacrylic acid, and copolymers thereof.

27. The method according to claim 23, wherein the polymer is selected from the group consisting of dipropylene glycol, tripylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, starch, carboxymethylcellulose, polyacrylic acid, hydroxypropylcellulose, methylcellulose, ethyl hydroxyethylcellulose, maltodextrin, polyethyleneimine, poly-N-isopropylacrylamide, and copolymers thereof.

28. The method according to claim 23, wherein the polymer is selected from the group consisting of dipropylene glycol, tripylene glycol, polyethylene glycol, polypropylene glycol, poly(ethylene glycol-propylene glycol), poly(ethylene glycol-random-propylene glycol), polyvinylpyrrolidone, polyvinyl alcohol, polyvinylcaprolactam, polyvinyl methyl ether, and poly-N-isopropylacrylamide.

29. The method according to claim 23, wherein the polymer is selected from the group consisting of polyacrylamide, polyacrylic acid, and copolymers thereof.

30. The method according to claim 23, wherein the polymer is selected from the group consisting of dextran, carboxymethyl dextran, dextran sulfate, hydroxypropyl dextran, and starch.

31. The method according to claim 23, wherein the polymer has an average molecular weight in the range of 200 to 1,000 Da, 200 to 35,000 Da, 425 to 2,000 Da, 400 to 35,000 Da, 980 to 12,000 Da, or 3,400 to 5,000,000 Da.

32. The method according to claim 23, wherein the polymer comprises ethylene oxide and propylene oxide units, and the polymer has an EO:PO ratio of 90:10 to 10:

90.

33. The method according to claim 23, wherein the salt is dissolved in the aqueous solution at a concentration of 0.1% to 80% (w / w).

34. The method according to claim 33, wherein the salt is dissolved in the aqueous solution at a concentration of 0.1% to 50% (w / w).

35. The method according to claim 33, wherein the salt comprises a cation selected from the group consisting of sodium, potassium, calcium, ammonium, lithium, magnesium, aluminum, cesium, barium, linear or branched trimethylammonium, triethylammonium, tripropylammonium, tributylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium, and tetrabutylammonium.

36. The method according to claim 33, wherein the salt comprises an anion selected from the group consisting of phosphate ions, hydrogen phosphate ions, dihydrogen phosphate ions, sulfate ions, sulfide ions, sulfite ions, bisulfate ions, carbonate ions, bicarbonate ions, acetate ions, nitrate ions, nitrite ions, sulfite ions, chloride ions, fluoride ions, chlorate ions, perchlorate ions, chlorite ions, hypochlorite ions, bromide ions, bromate ions, hypobromite ions, iodide ions, iodate ions, cyanate ions, thiocyanate ions, isothiocyanate ions, oxalate ions, formate ions, chromate ions, dichromate ions, permanganate ions, hydroxide ions, hydrogen ions, citrate ions, borate ions, and tris(hydroxymethyl)aminomethane.

37. The method according to claim 33, wherein the salt is selected from the group consisting of aluminum chloride, aluminum phosphate, aluminum carbonate, magnesium chloride, magnesium phosphate, and magnesium carbonate.

38. The salt is NaCl, KCl, NH 4 Cl, Na 3 PO 4 、K 3 PO 4 、Na 2 SO 4 、K 2 HPO 4 、KH 2 PO 4 、Na 2 HPO 4 、NaH 2 PO 4 、(NH 4 ) 3 PO 4 、(NH 4 ) 2 HPO 4 、NH 4 H 2 PO 4 、 potassium citrate, (NH 4 ) 2 SO 4 、 sodium citrate, sodium acetate, magnesium acetate, sodium oxalate, sodium borate and ammonium acetate, and is selected from the group consisting of, the method according to claim 33.

39. The aforementioned salt is (NH 4 ) 3 PO 4 Sodium formate, ammonium formate, K 2 CO 3 , KHCO 3 Na 2 CO 3 NaHCO 3 MgSO 4 , MgCO 3 CaCO 3 , CsOH, Cs 2 CO 3 , Ba(OH) 2 and BaCO 3 The method according to claim 33, selected from the group consisting of the following.

40. The aforementioned salt is NH 4 Cl, NH 4 The method according to claim 33, selected from the group consisting of OH, tetramethylammonium chloride, tetrabutylammonium chloride, tetramethylammonium hydroxide, and tetrabutylammonium hydroxide.

41. The method according to claim 23, wherein the surfactant is dissolved in the aqueous solution at a concentration of 0.05% to 10% (w / w).

42. The surfactant is selected from the group consisting of anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. Here, the anionic surfactant is a carboxylate salt, sulfonate, petroleum sulfonate, alkylbenzene sulfonic acid, naphthalene sulfonate, olefin sulfonate, alkyl sulfate, sulfate, sulfated natural oil, sulfated natural fat, sulfated ester, sulfated alkanolamide, sulfated alkylphenol, ethoxylated alkylphenol, or sodium N-lauroyl sarcosinate (NLS). Here, the nonionic surfactant is an ethoxylated aliphatic alcohol, a polyoxyethylene surfactant, a carboxylic acid ester, a polyethylene glycol ester, a sorbitan ester, a fatty acid glycol ester, a carboxyamide, a monoalkanolamine condensate, or a polyoxyethylene fatty acid amide. Here, the cationic surfactant is a quaternary ammonium salt, an amine having an amide bond, a polyoxyethylene alkylamine, a polyoxyethylene alicyclic amine, an n,n,n',n' tetrasubstituted ethylenediamine, or a 2-alkyl1-hydroxyethyl2-imidazoly n and The method according to claim 41, wherein the amphoteric surfactant is n-cocoyl 3-aminopropionic acid or its sodium salt, n-beef tallow 3-iminodipropionic acid ester or its disodium salt, n-carboxymethyl n-dimethyl n-9-octadecenylammonium hydroxide, or n-cocamidoethyl n-hydroxyethylglycine or its sodium salt.

43. The method according to claim 41, wherein the surfactant is selected from the group consisting of Triton X-100, Triton X-114, Triton X-45, Tween 20, Igepal CA630, Brij 58, Brij O10, Brij L23, Pluronic L-61, Pluronic F-127, sodium dodecyl sulfate, sodium cholate, sodium deoxycholate, sodium N-lauroyl sarcosinate, cetyltrimethylammonium bromide, and span 80.

44. The method according to claim 4, wherein the at least one chaotropic agent of the binding buffer comprises an anion selected from the group consisting of thiocyanate ions, isothiocyanate ions, perchlorate ions, acetate ions, trichloroacetate ions, trifluoroacetate ions, chloride ions, and iodide ions.

45. The method according to claim 4, wherein the at least one chaotropic agent of the binding buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

46. The method according to claim 5, wherein the at least one chaotropic agent of the target binding buffer comprises an anion selected from the group consisting of thiocyanate ions, isothiocyanate ions, perchlorate ions, acetate ions, trichloroacetate ions, trifluoroacetate ions, chloride ions, and iodide ions.

47. The method according to claim 5, wherein the at least one chaotropic agent of the target binding buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

48. The method according to claim 1, wherein the sample is blood, plasma, urine, saliva, feces, cerebrospinal fluid (CSF), lymph, serum, sputum, peritoneal fluid, sweat, tears, nasal swab, vaginal swab, cervical swab, semen, or breast milk.

49. The method according to claim 1, wherein step (a) comprises the step of preparing a DNA library from the sample and obtaining the sample solution.

50. The method according to claim 1, wherein the target nucleic acid is free DNA and circulating tumor DNA, and thereby the method increases the ratio of circulating tumor DNA to free DNA and / or the variant allele frequency (VAF) in the sample to perform a cancer diagnostic assay.

51. The method according to claim 1, wherein the target nucleic acid is circulating fetal DNA, and thereby the method enriches the fetal fraction in the sample to perform non-invasive prenatal detection.

52. A kit for isolating target nucleic acids smaller than the target size from a sample containing nucleic acid components, (a) At least one ATPS component selected from the group consisting of polymers, salts, surfactants and combinations thereof, (b) Multiple beads and (c) A fractionation buffer comprising at least one chaotropic agent selected from the group consisting of thiocyanates, isothiocyanates, perchlorates, acetates, trichloroacetates, trifluoroacetates, chlorides, and iodides, (d) A kit comprising a binding buffer containing at least one chaotropic agent selected from the group consisting of thiocyanates, isothiocyanates, perchlorates, acetates, trichloroacetates, trifluoroacetates, chlorides, and iodides.

53. The kit according to claim 52, wherein the plurality of beads are magnetic beads, silica-based beads, carboxy beads, hydroxy beads, amine-coated beads, or any combination thereof.

54. The kit according to claim 52, wherein the at least one chaotropic agent of the fractionation buffer is selected from the group consisting of guanidine hydrochloride (GHCl), guanidine thiocyanate, guanidine isothiocyanate (GITC), sodium thiocyanate, sodium iodide, sodium perchlorate, sodium trichloroacetate, sodium trifluoroacetate, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, thiourea, and urea.

55. The kit according to claim 52, wherein the at least one chaotropic agent has a concentration of about 1.5 to 8 M in the fractionation buffer.

56. The kit according to claim 52, wherein the fractionation buffer further comprises at least one polymer selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polypropylene glycol, dextran, poly(ethylene glycol-random-propylene glycol), pluronic, polyvinylpyrrolidone, and polyacrylic acid esters.

57. The kit according to claim 56, wherein at least one polymer is present in the fractionation buffer at a concentration of about 0.1% to 15% (w / w).

58. The kit according to claim 52, wherein the fractionated buffer further comprises one or more of a pH buffer, a metal chelating agent, or a combination thereof.

Images