Nucleic acid isolation and related methods

Abstract

Solid supports modified with pectin derivatives are provided. The solid carrier is used in nucleic acid isolation, separation and detection methods.

Description

[0001] This application is a divisional of PCT International Application PCT / US2019 / 046912, filed August 16, 2019, which entered the National Phase in the People’s Republic of China on April 2, 2021, as Chinese Patent Application No. 201980065458.6, entitled “Nucleic Acid Isolation and Related Methods,” which claims priority to U.S. Provisional Application No. 62 / 765,149, filed August 17, 2018, the entire contents of which are incorporated herein by reference.

[0002] Cross Reference to Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 765,149, filed August 17, 2018, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0004] The present invention relates to solid supports comprising modified pectin and methods of use thereof. BACKGROUND

[0005] In contrast to traditional diagnostic methods, molecular diagnostic assays that employ nucleic acid amplification and / or detection with the aid of various automated analysis techniques, such as polymerase chain reaction (PCR), can provide rapid and accurate results in a shorter time and can be easily automated. However, in order to perform a molecular diagnostic assay on a biological sample, nucleic acids must be isolated from the biological material to remove components that can affect the accuracy of the assay, for example, by inhibiting polymerase activity. Even though a variety of methods for nucleic acid extraction exist, currently available methods often involve lengthy procedures and are not easily automated. Thus, the preparation of nucleic acid samples prior to amplification and detection of a particular target is the most challenging step in molecular diagnostics.

[0006] There is a need for a simple and rapid method of nucleic acid isolation to prepare quality nucleic acids free of amplification inhibitors that does not require extensive sample handling and can accommodate clinical laboratory automation. There is a need for reagents that can facilitate the isolation of nucleic acids from biological samples containing nucleic acids in a manner that is compatible with rapid, automated methods of nucleic acid detection. The present invention satisfies this need and provides further related advantages. SUMMARY

[0007] In one aspect, provided herein is a solid support comprising a plurality of modified pectin molecules covalently bound to the solid support. In some embodiments, the modified pectin comprises a plurality of amino groups. In some embodiments, the modified pectin is amidated pectin. In some embodiments, the amidated pectin comprises one or more units represented by the following formula, isomers, salts, or tautomers thereof:

[0008] ,

[0009] wherein

[0010] n is 0, 1, 2, or 3;

[0011] R1is H or C1-C3alkyl;

[0012] X is independently at each occurrence C2-C4alkylene or C4-C6heteroalkylene;

[0013] Y is C2-C3alkylene or C4-C6heteroalkylene; and

[0014] R2and R3are independently H or C1-C3alkyl.

[0015] In some embodiments, the amidated pectin is a pectin amidated with a C4-C20 polyamine. In some embodiments, the polyamine is ethylenediamine, putrescine, cadaverine, spermine, or spermidine.

[0016] In some embodiments, the amidated pectin comprises one or more units having the structure:

[0017] ,

[0018] wherein

[0019] n is 0, 1, 2, or 3;

[0020] m is 2, 3, or 4;

[0021] p is 2, 3, or 4; and

[0022] R1, R2, and R3are independently H or C1-C3alkyl.

[0023] In some embodiments, the amidated pectin comprises one or more units having the structure:

[0024] , .

[0025] In some embodiments, the amidated pectin is an amidated citrus pectin or an amidated apple pectin. In some embodiments, the amidated pectin has a molecular weight of about 4,000 Da to about 500,000 Da, about 5,000 Da to about 300,000 Da, about 100,000 Da to about 300,000 Da, or about 50,000 Da to about 200,000 Da.

[0026] In some embodiments, the solid support comprises a material selected from the group consisting of polystyrene, glass, ceramic, polypropylene, polyethylene, silica, zirconium oxide, titanium dioxide, aluminum oxide, polycarbonate, latex, polyethersulfone, PMMA, carboxymethylcellulose, zeolite, and cellulose.

[0027] In some embodiments, the solid support is a magnetic bead, a glass bead, a polystyrene bead, a polystyrene filter, a polycarbonate filter, a polyethersulfone filter, or a glass filter.

[0028] In another aspect, provided herein is a method of isolating nucleic acid from a sample comprising nucleic acid, comprising:

[0029] (a) contacting the sample with a solid support disclosed herein, thereby binding the nucleic acid to the solid support;

[0030] (b) optionally washing the nucleic acid bound to the solid support; and

[0031] (c) eluting the nucleic acid from the solid support with an eluent.

[0032] In some embodiments, the eluent comprises ammonia or an alkali metal hydroxide. In some embodiments, the eluent has a pH of about 9 or higher, about 10 or higher, or about 11 or higher. In some embodiments, the eluent has a pH of about 9 to about 12, about 9.5 to about 12, about 10 to about 12, or about 9 to about 11. In some embodiments, the eluent comprises a polyanion. In some embodiments, the polyanion is carrageenan or a carrier nucleic acid. In some embodiments, the eluent comprises a polyanion and a base, such as an alkali metal hydroxide. In some embodiments, the eluent comprises i-carrageenan and KOH.

[0033] In some embodiments, the method comprises contacting the sample with a lysis solution prior to contacting the sample with the solid support, thereby releasing nucleic acid into solution. In some embodiments, the lysis solution comprises a chaotropic agent. In some embodiments, the chaotropic agent is selected from the group consisting of guanidinium thiocyanate, guanidinium hydrochloride, an alkali metal perchlorate, an alkali metal iodide, urea, formamide, or a combination thereof. In some embodiments, the chaotropic agent is guanidinium thiocyanate or guanidinium hydrochloride. In some embodiments, the lysis solution comprises a salt. In some embodiments, the salt is sodium chloride or calcium chloride. In some embodiments, the lysis solution does not comprise a chaotropic agent. In some embodiments, the lysis solution comprises a buffer. In some embodiments, the buffer is Tris. In some embodiments, the lysis solution comprises a surfactant. In some embodiments, the lysis solution comprises an antifoam agent.

[0034] In some embodiments, contacting the sample with the solid support is performed in the absence of a chaotropic agent.

[0035] In some embodiments, the sample is selected from blood, plasma, serum, semen, a tissue biopsy, urine, fecal matter, saliva, a smear specimen, a bacterial culture, a cell culture, a viral culture, a PCR reaction mixture, or an in vitro nucleic acid modification reaction mixture. In some embodiments, the tissue biopsy is a paraffin-embedded tissue. In some embodiments, the nucleic acid comprises genomic DNA. In some embodiments, the nucleic acid comprises total RNA. In some embodiments, the nucleic acid comprises microbial nucleic acid or viral nucleic acid. In some embodiments, the viral nucleic acid is HBV DNA. In some embodiments, the nucleic acid is a circulating nucleic acid.

[0036] In some embodiments, the method is performed in an automated cartridge.

[0037] In another aspect, provided herein is a method for detecting a nucleic acid in a sample, comprising:

[0038] (a) contacting a sample comprising a nucleic acid with a solid support disclosed herein, thereby binding the nucleic acid to the solid support;

[0039] (b) optionally washing the nucleic acid bound to the solid support;

[0040] (c) eluting the nucleic acid; and

[0041] (d) detecting the nucleic acid.

[0042] In some embodiments, detecting the nucleic acid comprises amplifying the nucleic acid by polymerase chain reaction (PCR). In some embodiments, the polymerase chain reaction is nested PCR, isothermal PCR, or RT-PCR.

[0043] In another aspect, provided herein is a separation material for chromatography, comprising a solid support comprising amidated pectin covalently bonded thereto.

[0044] In some embodiments, the amidated pectin has one or more units represented by the following formula, isomers, salts, or tautomers thereof:

[0045] ,

[0046] R 2 and R 3 are independently selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C20 heteroalkyl.

[0047] In some embodiments, the solid support is silica, alumina, titania, zirconia, or a mixed silica material. DETAILED DESCRIPTION

[0048] In one aspect, provided herein is a solid support for purifying nucleic acids from a sample comprising nucleic acids, comprising one or more modified pectin molecules covalently bound to a surface. In some embodiments, the modified pectin comprises a plurality of amino groups. In some embodiments, the modified pectin is amidated pectin. As used herein, the term "solid support" refers to any matrix comprising paramagnetic particles, gels, controlled-pore glass, magnetic beads, microspheres, nanospheres, capillaries, filters, columns, cloths, wipes, paper, planar supports, multi-well plates, multi-well membranes, multi-well monoliths, wafers, combs, or any combination thereof. The solid support can comprise any suitable material, including but not limited to glass, silica, titania, iron oxide, ethylenic backbone polymers, polypropylene, polyethylene, polystyrene, ceramic, cellulose, nitrocellulose, and divinylbenzene. Preferably, the solid support comprises a material selected from the group consisting of polystyrene, glass, ceramic, polypropylene, polyethylene, silica, polycarbonate, latex, PMMA, zeolite, polyethersulfone, carboxymethylcellulose, cellulose, and combinations thereof. In some embodiments, the solid support is not pectin, e.g., unmodified pectin or modified pectin.

[0049] In some embodiments, the solid support is a magnetic bead, a glass bead, a polystyrene bead, a polystyrene filter, a polycarbonate filter, a polyethersulfone filter, or a glass filter. Preferably, the materials suitable for making the solid supports disclosed herein, e.g., without the modification of pectin described herein, have low non-specific binding that do not bind nucleic acids, proteins, or other components in a sample for which nucleic acid isolation is desired.

[0050] Modified pectin

[0051] In some embodiments, the modified pectin is amidated pectin. Pectin is a naturally occurring complex polysaccharide commonly found in plant cell walls. Pectin generally comprises an alpha 1-4 linked polygalacturonic acid backbone interspersed with rhamnose residues and modified by neutral sugar side chains and non-sugar components such as acetyl, methyl, and ferulic acid groups. The galacturonic acid residues in pectin are partially esterified and present as methyl esters. The degree of esterification is defined as the percentage of carboxyl groups that are esterified. Pectins with a degree of esterification, e.g., above 50% are classified as high methoxyl pectin ("HM") or high ester pectin, and pectins with a degree of esterification below 50% are referred to as low methoxyl ("LM") pectin or low ester pectin. Most pectins found in fruits and vegetables are HM pectins.

[0052] As used herein, "amidated pectin" refers to any naturally occurring pectin that has been structurally modified, for example, by chemical, physical, or biological (including enzymatic) means, or by some combination thereof, in which some of the ester or acid groups have been converted to amide groups. Amidated pectin can be prepared by contacting unmodified pectin with a suitable amine solution, thereby converting ester groups of the unmodified pectin to amides.

[0053]

[0054] Alternatively, unmodified pectin or hydrolyzed pectin (including partially hydrolyzed pectin) can be reacted with an amine in the presence of a suitable coupling agent to form an amidated pectin. Non-limiting examples of suitable coupling agents include carbodiimide coupling agents, such as DCC and EDCI, and phosphonium and iminium-based reagents, such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH.

[0055]

[0056] In some embodiments, the modified pectin is a modified pectin obtained by reductive amination of a periodate-oxidized pectin. Methods of reductive amination of carbohydrates, such as pectin, are known in the art.

[0057] The modified pectin can be obtained from unmodified pectin by any of the methods described herein. Particularly useful starting materials for the synthesis of modified pectin are apple and citrus pectins. In some embodiments, the starting pectin has a molecular weight of about 4,000 Da to about 500,000 Da, about 5,000 Da to about 300,000 Da, about 10,000 Da to about 150,000 Da, or about 10,000 Da to about 100,000 Da.

[0058] In some embodiments, the amidated pectin comprises a plurality of uronic acid units and one or more additional monomeric units. Uronic acids include sugar acids that comprise both a carbonyl (e.g., an aldehyde or ketone group) and a carboxylic acid (-COOH) functional group. Generally, uronic acids are derived from sugars in which a terminal hydroxyl group has been oxidized to a carboxylic acid, and are generally named according to their parent sugar, e.g., glucuronic acid is a uronic acid derived from glucose. A uronic acid derived from a hexose sugar is referred to as a hexuronic acid, and a uronic acid derived from a pentose sugar is referred to as a penturonic acid.

[0059] In some embodiments, the amidated pectin comprises one or more units, in addition to one or more uronic acid units, selected from the group consisting of:

[0060] (I), (II),

[0061] wherein R 1 is selected from the group consisting of optionally substituted C1-C8alkyl, optionally substituted C3-C8cycloalkyl, optionally substituted C3-C8heterocycloalkyl, and optionally substituted C2-C20heteroalkyl; and

[0062] R 2 and R 3 are independently selected from the group consisting of H, optionally substituted C1-C6alkyl, optionally substituted C3-C6cycloalkyl, and optionally substituted C2-C20heteroalkyl.

[0063] In some embodiments, R3is optionally substituted C1-C6alkyl. In some embodiments, R3is optionally substituted C4-C20heteroalkyl, such as a short PEG chain optionally substituted with one or more amines. In some embodiments,

[0064] In some embodiments, each of R1, R2, and R3comprises no more than one amine. In some embodiments, each of R1, R2, and R3comprises no amine. In some embodiments, each of R2and R3comprises one or more amines. In some embodiments, R2is H and R3is optionally substituted C4-C20heteroalkyl, such as a polyamine or an oligoethylene glycol comprising 2-6 ethylene glycol units, optionally substituted with one or more amines.

[0065] In some embodiments, R1is methyl, ethyl, or propyl. In some embodiments, R2and R3are both H. In some embodiments, R2is H and R3is optionally substituted C1-C8alkyl. In some embodiments, R2is H and R3is H, CH3, CH2CH2NH2, CH2CH2N(CH3)2, CH2CH2OH, or CH2CH2NHCH2CH2NH2. In some embodiments, R 2 and R 3 are both CH3.

[0066] In some embodiments, the amidated pectin further comprises one or more units of Formula (III), or isomers, salts, tautomers, or combinations thereof:

[0067] (III)

[0068] wherein:

[0069] R3is H, CH3, CH2CH2NH2, CH2CH2N(CH3)2, CH2CH2OH, (CH2)2O(CH2)2NH2, or CH2CH2NHCH2CH2NH2.

[0070] It is understood that if the polysaccharide comprises two or more units of formula (II) or (III), R3 within the polysaccharide can be the same or different.

[0071] In some embodiments, the amidated pectin disclosed herein comprises one or more monomeric units having at least one amino group. In some embodiments, the amidated pectin comprises one or more monomeric units having the structure of VI, isomers, salts, tautomers, or combinations thereof:

[0072] (IV),

[0073] wherein:

[0074] n is 0, 1, 2, or 3;

[0075] R4 is H or C1-C3 alkyl;

[0076] X is independently at each occurrence C2-C4 alkylene or C4-C6 heteroalkylene;

[0077] Y is C2-C3 alkylene or C4-C6 heteroalkylene; and

[0078] R5 and R6 are independently H or C1-C3 alkyl.

[0079] In some embodiments, the amidated pectin disclosed herein comprises one or more monomeric units having the structure of V, isomers, salts, tautomers, or combinations thereof:

[0080] (V),

[0081] wherein:

[0082] n is 0, 1, 2, or 3;

[0083] m is independently at each occurrence 2, 3, or 4;

[0084] p is 2, 3, or 4;

[0085] R4 is H or C1-C3 alkyl; and

[0086] R5 and R6 are independently H or C1-C3 alkyl.

[0087] In some embodiments, the amidated pectin comprises one or more monomeric units comprising a primary amino group. In some embodiments, the amidated pectin comprises one or more monomeric units comprising a quaternary ammonium group. In some embodiments, the amidated pectin is amidated with a polyamine. As used herein, a polyamine is a compound comprising two or more amino groups. Polyamines that can be used in the modification of pectin for the solid supports disclosed herein include both synthetic polyamines and naturally occurring polyamines (e.g., spermidine, spermine, putrescine). In some embodiments, the polyamine is selected from the group consisting of spermine, spermidine, putrescine, ethylenediamine, and cadaverine. In some embodiments, the polyamine is spermine or spermidine.

[0088] In some embodiments, the amidated pectin comprises one or more units having the formula VI, VII, or VIII, including isomers, salts, and tautomers thereof:

[0089] (VI), (VII), or (VIII).

[0090] In some embodiments, the amidated pectin comprises a plurality of additional monomeric units represented by the structures of formulas I-VIII. As used herein, the term "plurality" means more than one. For example, a plurality of monomeric units means at least two monomeric units, at least three monomeric units, or at least monomeric units, etc. If embodiments of the present application include more than one monomeric unit, they can also be referred to as a first monomeric unit, a second monomeric unit, a third monomeric unit, etc.

[0091] As used herein, the terms "alkyl," "alkenyl," and "alkynyl" include straight chain, branched chain, and cyclic monovalent hydrocarbon radicals and combinations thereof, comprising only C and H when they are unsubstituted. Examples include methyl, ethyl, iso-butyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. Sometimes the total number of carbon atoms in each such group is described herein, e.g., when the group can comprise up to ten carbon atoms, it can be denoted as 1-10C, C1-C10, C1-C10, C1-10, or C1-10. The terms "heteroalkyl," "heteroalkenyl," and "heteroalkynyl" as used herein refer to the corresponding hydrocarbon in which one or more of the chain carbon atoms have been replaced with a heteroatom. Exemplary heteroatoms include N, O, S, and P. When a heteroatom is allowed to replace a carbon atom, e.g., in a heteroalkyl group, the numbers describing the group are written as before, e.g., C3-C10, indicating the sum of the number of carbon atoms in the ring or chain plus the number of such heteroatoms included as replacements for carbon atoms in the described ring or chain.

[0092] A single radical can contain more than one type of multiple bond or more than one multiple bond; when these radicals contain at least one carbon-carbon double bond, they are included within the definition of the term "alkenyl," and when they contain at least one carbon-carbon triple bond, they are included within the definition of the term "alkynyl."

[0093] Alkyl, alkenyl, and alkynyl groups can optionally be substituted to the extent chemically possible. Typical substituents include, but are not limited to, halogen (F, CI, Br, I), =0, =NCN, =NOR, =NR, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl, and each R is optionally substituted with halogen (F, CI, Br, I), =0, =NCN, =NOR', =NR', OR', NR'2, SR', SO2R', SO2NR'2, NR'SO2R', NR'CONR'2, NR'C(O)OR', NR'C(O)R', CN, C(O)OR', C(O)NR'2, OC(O)R', C(O)R', and NO2, where each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, or C5-C10 heteroaryl. Alkyl, alkenyl, and alkynyl groups can also be substituted with C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, or C5-C10 heteroaryl, each of which can be substituted with substituents appropriate for the particular group.

[0094] While "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl, the term "cycloalkyl" is used herein to describe a carbocyclic non-aromatic group connected via ring carbon atoms, while "cycloalkylalkyl" is used to describe a carbocyclic non-aromatic group connected to a molecule through an alkyl linker. Similarly, "heterocyclyl" is used to denote a non-aromatic cyclic group containing at least one heteroatom as a ring member and connected to a molecule through a ring atom, which can be C or N; "heterocyclylalkyl" can be used to describe a group connected to another molecule through an alkylene linker. As used herein, these terms also include rings containing one or two double bonds, as long as the ring is not aromatic.

[0095] "Aromatic" or "aryl" substituents or moieties refer to monocyclic or fused bicyclic moieties having well-known aromatic character; examples of aryl groups include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to monocyclic or fused bicyclic ring systems containing one or more heteroatoms as ring members. Suitable heteroatoms include N, O, and S, including combinations thereof that allow for aromaticity in 5-membered rings as well as 6-membered rings. Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as pyridyl, pyrimidinyl, pyrazinyl, thienyl, furanyl, pyrrolyl, pyrazolyl, thiazolyl, oxazolyl, and imidazolyl, as well as fused bicyclic moieties formed by fusing one of these monocyclic groups to a phenyl ring or to any of the heteroaromatic monocyclic groups to form C8-C10 bicyclic groups such as indolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolyl, quinolinyl, benzothiazolyl, benzofuranyl, pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like. Any monocyclic or fused ring bicyclic system having aromatic character in terms of electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups in which at least the ring directly attached to the remainder of the molecule has aromatic character. Typically, the ring system contains 5-14 ring member atoms. Typically, monocyclic heteroaryl groups contain 5-6 ring members, while bicyclic heteroaryl groups contain 8-10 ring members.

[0096] Aryl and heteroaryl moieties can be substituted with a variety of substituents, including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms thereof, each of which can be further substituted itself; other substituents for aryl and heteroaryl moieties include halogen (F, Cl, Br, I), OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRC(O)OR, NRC(O)R, CN, C(O)OR, C(O)NR2, OC(O)R, C(O)R, and NO2, where each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each R is optionally substituted as described above for alkyl. Substituents on aryl or heteroaryl groups can also be substituted with groups described herein as appropriate for each type of such substituent or component of the substituent. Thus, for example, an arylalkyl substituent can be substituted on the aryl portion with typical substituents for aryl described herein, and it can be further substituted on the alkyl portion with typical or appropriate substituents for alkyl described herein.

[0097] As used herein, "optionally substituted" means that the particular group described can have one or more hydrogen substituents replaced by non-hydrogen substituents. In some optionally substituted groups or moieties, all hydrogen substituents are replaced by non-hydrogen substituents (e.g., polyfluoroalkyl groups, such as trifluoromethyl). If not otherwise specified, the total number of such substituents that can be present is equal to the number of H atoms present on the unsubstituted form of the group in question. In cases where an optional substituent is attached by a double bond, e.g., carbonyl oxygen or oxo (=0), the group takes up two available valences, and thus, the total number of substituents that can be included is reduced according to the number of available valences.

[0098] As used herein, unless otherwise indicated, the term "amino" includes primary, secondary, and tertiary amino groups.

[0099] Covalent attachment of amidated pectin to a solid support can be achieved in any suitable manner, for example, by reacting a polyamine amidated pectin with a solid support comprising an amine-reactive group, such as an epoxide, aldehyde, ketone, or activated ester. Amidated pectins comprising primary or secondary amino groups can also be attached to a solid support by cross-linking, for example, an amino-modified solid surface. As used herein, cross-linking refers to the process of chemically linking two or more molecules through covalent bonds. In some cases, a cross-linking agent can be used to attach the amidated pectin to the solid support, thereby forming a pectin-modified solid support. As used herein, a cross-linking reagent (or cross-linker) is a molecule comprising two or more reactive termini that are capable of chemically linking to specific functional groups (e.g., primary amines, carboxyls, thiols, etc.) on molecules and / or solid supports. Methods of covalently attaching amino-containing molecules to functionalized surfaces and solid supports are known in the art.

[0100] In some embodiments, the amidated pectin of the present application is covalently attached to a solid support through an amide bond (e.g., an amide bond formed between a carboxyl group of the solid support and an amino group of the amidated pectin). Formation of an amide bond can be carried out by any suitable method. For example, an amidated pectin comprising one or more primary amino groups can be reacted with a substrate comprising one or more carboxylic acid groups in the presence of a suitable coupling agent. Non-limiting examples of suitable coupling agents include carbodiimide coupling agents, such as DCC and EDCI, and phosphonium and iminium-based reagents, such as BOP, PyBOP, PyBrOP, TBTU, HBTU, HATU, COMU, and TFFH. In some preferred embodiments, the carboxylic acid groups of the solid substrate can be converted to activated esters, which are then reacted with the amino groups of the amidated pectin.

[0101] In some embodiments, the solid support comprises an amidated pectin having one or more units represented by any one of formulas (II)-(VIII), wherein the amidated pectin is covalently attached to the solid support.

[0102] In another aspect, provided herein are methods for isolating nucleic acids from a sample comprising nucleic acids, comprising:

[0103] (a) contacting the sample with a solid support disclosed herein, thereby binding the nucleic acids to the solid support;

[0104] (b) optionally washing the nucleic acids bound to the solid support; and

[0105] (c) eluting the nucleic acids from the solid support by contacting the nucleic acids bound to the solid support with an elution reagent.

[0106] Lysis solution

[0107] In some embodiments, the sample comprising nucleic acids is contacted with a lysis solution prior to contacting with the solid support, thereby lysing cells contained in the sample and releasing the nucleic acids into solution. After lysis of the sample, the nucleic acids can be bound to a solid matrix, such as silica or glass matrices covalently modified with amidated pectin as described herein. In some embodiments, the solid support is incorporated into an automated cartridge, such as a GenXpert ® cartridge. After binding, the supernatant is then removed and the nucleic acids are eluted from the matrix with an elution buffer, such as an alkaline solution as described above. The eluate can then be processed in the cartridge to detect the target gene of interest. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents present in the cartridge in the form of lyophilized pellets. In some embodiments, the PCR uses a Taq polymerase with a hot start function, such as AptaTaq (Roche, Switzerland).

[0108] In some embodiments, the lysis solution comprises a chaotropic agent, such as guanidine thiocyanate, guanidine hydrochloride, alkali metal perchlorate, alkali metal iodide, urea, formamide, and combinations thereof. In some embodiments, the lysis solution comprises a salt. Preferably, the salt is sodium chloride or calcium chloride.

[0109] In some embodiments, the methods disclosed herein do not require the use of a chaotropic agent or high concentration of salt to bind the nucleic acids to the solid support of the present invention.

[0110] In some embodiments, the sample is lysed by contacting the sample with a lysis buffer prior to the addition of the polysaccharide reagent solution and subsequent precipitation of the nucleic acids. In some embodiments, a lysis reagent is added to the solution of the polysaccharide reagent that precipitates the nucleic acids. In some embodiments, the polysaccharide reagent described herein is dissolved in the lysis solution. In some embodiments, the lysis solution comprises one or more proteases. Suitable proteases include, but are not limited to, serine proteases, threonine proteases, cysteine proteases, aspartic proteases, metalloproteases, glutamic acid proteases, metalloproteases, and combinations thereof. Illustrative suitable proteases include, but are not limited to, proteinase k (a broad-spectrum serine protease), subtilisin, trypsin, chymotrypsin, pepsin, papain, and the like. Other proteases will be available to those skilled in the art using the teachings and examples provided herein.

[0111] In some embodiments, the methods described herein are used to isolate nucleic acids (e.g., DNA, RNA) from a fixed paraffin-embedded biological tissue sample according to any of the methods described herein; amplify the precipitated nucleic acids using a pair of oligonucleotide primers capable of amplifying a region of the target nucleic acid to obtain an amplified sample; and determine the presence and / or amount of the target nucleic acid. In some embodiments, the target nucleic acid is DNA (e.g., a gene). In some embodiments, the target nucleic acid is RNA (e.g., mRNA, non-coding RNA, etc.). In some embodiments, the nucleic acids isolated using the methods described herein are well suited for use in diagnostic methods, prognostic methods, methods of monitoring treatment (e.g., cancer treatment), and the like. Thus, in some illustrative, non-limiting embodiments, the nucleic acids extracted from a fixed paraffin-embedded sample (e.g., from an FFPET sample) can be used to determine the presence and / or expression level of a gene, and / or the mutational status of a gene. Such methods are particularly well suited for determining the presence and / or expression level and / or mutational status of one or more cancer markers. Thus, in some embodiments, the nucleic acids isolated using the methods described herein are used to detect the presence and / or copy number and / or expression level and / or mutational status of one or more cancer markers.

[0112] Washing and elution

[0113] The detection and isolation methods disclosed herein can optionally include a washing step, i.e., the precipitated nucleic acids can optionally be washed on the solid support, e.g., to remove components of the lysis buffer. Typically, the concentrated, e.g., precipitated, nucleic acids are solubilized prior to detection. In some embodiments, the concentrated nucleic acids are solubilized in a buffer that is compatible with a PCR reaction.

[0114] In some embodiments, for example, when using polyamine-modified polysaccharides to precipitate nucleic acids, the precipitated nucleic acids can be eluted from the polyamine by contact with a suitable eluent. In some embodiments, the eluent comprises ammonia or an alkali metal hydroxide. In some embodiments, the eluent has an alkaline pH. In some embodiments, the eluent has a pH of about 9 to about 12, about 9.5 to about 12, about 10 to about 12, or about 9 to about 11. Preferably, the pH of the eluent is above 10. Preferably, the eluent comprises ammonium hydroxide, NaOH, or KOH in a concentration sufficient to destroy the binding of the nucleic acid to the polysaccharide reagent. Exemplary eluents include 1% ammonia, 15mM KOH, or 15mM NaOH.

[0115] In some embodiments, the eluent comprises a polyanion. In some embodiments, the polyanion is a polymer comprising multiple anionic groups. In some embodiments, the anionic group is a phosphate, phosphonate, sulfate, or sulfonate group or a combination thereof. In some embodiments, the polyanion is a polymer that is negatively charged at a pH of about 7 or above. Both synthetic polyanions and naturally occurring polyanions can be used in the methods disclosed herein. In some embodiments, the polyanion is carrageenan. In other embodiments, the polyanion is a carrier nucleic acid. As used herein, a carrier nucleic acid is a nucleic acid that does not interfere with subsequent detection of concentrated nucleic acids, such as by PCR. Exemplary carrier nucleic acids include poly-rA, poly-dA, herring sperm DNA, salmon sperm DNA, and others well known to those skilled in the art. In some embodiments, the eluent comprises carrageenan and an alkali metal hydroxide, such as NaOH or KOH.

[0116] Nucleic acid

[0117] In some embodiments, the methods described herein are used to separate nucleic acids from a solution comprising nucleic acids. The solution comprising nucleic acids can be obtained by cracking the material comprising nucleic acids. The material comprising nucleic acids is generally selected from blood, tissue biopsy such as paraffin-embedded tissue, spread specimens, bacterial cultures, viral cultures, urine, semen, cell suspensions and adherent cells, PCR reaction mixtures and in vitro nucleic acid modification reaction mixtures. The material comprising nucleic acids can include human, bacterial, fungal, animal or plant materials. In other embodiments, a solution comprising nucleic acids can be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction. In other embodiments, a solution comprising nucleic acids can be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction.

[0118] As used herein, the term "nucleic acid" refers to any synthetic or naturally occurring nucleic acid, e.g., DNA or RNA, having any possible configuration, i.e., a form of double-stranded nucleic acid, a form of single-stranded nucleic acid, a form of aptamer, or any combination thereof. The nucleic acid can be DNA, e.g., genomic DNA. The nucleic acid can also be RNA, e.g., total RNA. The nucleic acid can be single-stranded or double-stranded nucleic acid, e.g., a short double-stranded DNA fragment. The nucleic acid can be synthetic nucleic acid. In some embodiments, the nucleic acid is a circulating nucleic acid.

[0119] Nucleic acids isolated using the methods and solid supports described herein have suitable quality to be amplified for detecting and / or quantifying one or more target nucleic acid sequences in a sample. The nucleic acid isolation methods and solid supports described herein can also be suitable for basic research aimed at discovering gene expression profiles relevant to the diagnosis and prognosis of diseases. The methods are also suitable for the diagnosis and / or prognosis of diseases, the determination of specific treatment regimens, and / or the monitoring of treatment efficacy.

[0120] In some embodiments, the methods described herein are used to precipitate nucleic acids from a sample comprising nucleic acids. The material comprising nucleic acids can be selected from the group consisting of blood, serum, tissue biopsies such as paraffin-embedded tissue, oral fluid, swabbed specimens, bacterial cultures, viral cultures, urine, semen, cell suspensions and adherent cells, PCR reaction mixtures, and in vitro nucleic acid modification reaction mixtures. The material comprising nucleic acids can include human, animal, or plant material. In some embodiments, the nucleic acids are in solution. Solutions comprising nucleic acids include solutions of extracellular nucleic acids and solutions obtained by lysing cells comprising nucleic acids. In other embodiments, the solution comprising nucleic acids can be obtained from a nucleic acid modification reaction or a nucleic acid synthesis reaction.

[0121] Amplification method

[0122] The methods described herein simplify the isolation of nucleic acids from biological samples and efficiently produce isolated nucleic acids that are well suited for use in RT-PCR systems. In some embodiments, the nucleic acids isolated from a sample comprising nucleic acids using the methods described herein can be detected by any suitable known nucleic acid detection method. Although in some embodiments, the extracted nucleic acids are used in amplification reactions, other uses are contemplated. Thus, for example, the isolated nucleic acids (or amplification product(s) thereof) can be used in various sequencing or hybridization schemes, including but not limited to nucleic acid-based microarrays and next-generation sequencing.

[0123] In one aspect, provided herein is a method for detecting a nucleic acid, comprising:

[0124] (a) contacting a sample comprising nucleic acids with a solid support disclosed herein, thereby binding the nucleic acids to the solid support;

[0125] (b) optionally washing the nucleic acids bound to the solid support;

[0126] (c) eluting the nucleic acids from the solid support by contacting the nucleic acids bound to the solid support with an elution reagent; and

[0127] (d) detecting the nucleic acids.

[0128] In some embodiments, the detection method comprises nucleic acid amplification. Suitable non-limiting exemplary amplification methods include polymerase chain reaction (PCR), reverse transcriptase PCR, real-time PCR, nested PCR, multiplex PCR, quantitative PCR (Q-PCR), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), rolling circle amplification (RCA), and strand displacement amplification (SDA).

[0129] In some embodiments, the amplification method comprises initial denaturation at about 90 °C to about 100 °C for about 1 to about 10 minutes, followed by cycles comprising denaturation at about 90 °C to about 100 °C for about 1 to about 30 seconds, annealing at about 55 °C to about 75 °C for about 1 to about 30 seconds, and extension at about 55 °C to about 75 °C for about 5 to about 60 seconds. In some embodiments, the cycle denaturation step is omitted for the first cycle following initial denaturation. The specific times and temperatures will depend on the specific nucleic acid sequence being amplified, and can be readily determined by one of ordinary skill in the art.

[0130] In some embodiments, the isolation and detection of nucleic acids is performed in an automated sample processing and / or analysis platform. In some embodiments, a commercially available automated analysis platform is utilized. For example, in some embodiments, the GeneXpert system (Cepheid, Sunnyvale, Calif.) is utilized. However, the present application is not limited to a particular detection method or analysis platform. One of skill in the art recognizes that any number of platforms and methods can be used.

[0131] The GeneXpert system uses a self-contained disposable cartridge. Sample extraction, amplification, and detection of nucleic acids can all be performed in this self-contained "cartridge lab." See, e.g., U.S. Patent No. 6,374,684, which is incorporated by reference herein in its entirety. Components of the cartridge include, but are not limited to, a processing chamber containing reagents, a filter, and capture technology for extracting, purifying, and amplifying target nucleic acids. Valves enable fluid transfer from one chamber to another and contain nucleic acid lysis and filtration components. An optical window enables real-time optical detection. Reaction tubes enable very rapid thermal cycling. In some embodiments, the GenXpert system includes multiple modules for scalability. Each module includes multiple cartridges, as well as sample processing and analysis components.

[0132] Solid phase for chromatography

[0133] In some embodiments, disclosed herein are separation materials for chromatography comprising a solid support having a polysaccharide bound thereto. In some embodiments, the polysaccharide is a polyuronide or an amidated pectin. In some embodiments, the polysaccharide is an amidated pectin adsorbed on the surface of the solid support. In other embodiments, the amidated pectin is covalently, non-covalently, or by a combination of covalent bonds and non-covalent interactions, immobilized on the surface of the solid support.

[0134] In some embodiments, the separation material comprises a polysaccharide bound to a solid support, wherein the polysaccharide comprises one or more units represented by Formula II, isomers, salts, tautomers, or combinations thereof:

[0135] (II),

[0136] wherein

[0137] R 2 and R 3 are independently selected from the group consisting of H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C20 heteroalkyl.

[0138] In some embodiments, the amidated pectin is a pectin comprising one or more units having the structure of Formula II-VIII.

[0139] Suitable solid supports for making the separation materials include silica gel and other inorganic materials, such as AI2O3 (alumina), TiO2 (titania), or ZrO2 (zirconia). Organic polymeric resins can also be used to make the separation materials disclosed herein. Certain materials using hybrid particle technology (HPT) are suitable for making the separation materials disclosed herein, such as hybrid organic / inorganic materials, such as Waters BEH technology™ materials. HPT materials retain the key advantages of silica, such as purity, mechanical strength, high sphericity, tunable particle size, pore size, surface area, and surface chemistry. At the same time, such hybrid materials are stable at basic pH, such as stable at pH above 8.

[0140] Preferably, the solid support used to make the separation materials is porous. In some embodiments, the separation material is a porous particle having amidated pectin bound thereto by covalent bonds or non-covalent interactions. In other embodiments, the separation material is a porous monolith having amidated pectin bound thereto by covalent bonds or non-covalent interactions.

[0141] In some embodiments, the solid support used to prepare the isolated materials disclosed herein is silica gel or silica. Silica is characterized by pore size, particle size, and / or specific surface area. Silica-based isolated materials preferably have a pore size of about 30 to about 1000 Angstroms, a particle size of about 2 to about 300 microns, and a specific surface area of about 35 m2 / g to about 1000 m2 / g. In some embodiments, the silica gel has a pore size of about 40 Angstroms to about 500 Angstroms, about 60 Angstroms to about 500 Angstroms, about 100 Angstroms to about 300 Angstroms, and about 150 Angstroms to about 500 Angstroms. In some embodiments, the silica gel has a particle size of about 2 to about 25 microns, about 5 to about 25 microns, about 15 microns, about 63 to about 200 microns, about 75 to about 200 microns; and a specific surface area of about 100 m2 / g to about 350 m2 / g, about 100 m2 / g to about 500 m2 / g, about 65 m2 / g to about 550 m2 / g, about 100 m2 / g to about 675 m2 / g, about 35 to about 750 m2 / g.

[0142] In some embodiments, the chromatographic material according to the present application comprises magnetic silica particles. Magnetic silica particles include a superparamagnetic core coated with a hydrous silica adsorption surface (i.e., a surface having silanol or Si-OH groups). Suitable commercially available magnetic silica particles include MagneSil™ particles available from Promega Corporation (Madison, Wis.).

[0143] In some embodiments, the solid support is alumina. Exemplary alumina solid supports include, but are not limited to, about 150 mesh and 58 Angstroms Brockmann alumina.

[0144] In some embodiments, the amidated pectin is chemically bonded to the solid support via a linker. The linker between the solid support and the amidated pectin can comprise an alkylene or heteroalkylene chain. Preferably, the linker comprises 2-20 carbon atoms, and in addition to carbon atoms, can comprise nitrogen and oxygen atoms. In some embodiments, the linker is an oligoethylene linker, such as a PEG oligomer.

[0145] Preparation of the isolated materials can be achieved in any suitable manner. For example, the solid support can be reacted with a surface modifier. As used herein, a surface modifier is a moiety that imparts certain chromatographic functionality to the underlying solid support. The surface modifier, such as the amidated pectin disclosed herein, can be attached to the underlying solid support through a derivatization reaction, non-covalent coating, or a combination thereof. In some embodiments, the organic groups of the underlying solid support form covalent bonds with the surface modifier, such as the amidated pectin comprising a reactive group. This covalent attachment of the amidated pectin can be achieved through a variety of mechanisms well known in the art, such as cycloaddition, as well as nucleophilic and electrophilic substitution.

[0146] In some embodiments, the base solid support is a silica gel comprising silanol groups. Such silica gel solid supports can be reacted with a modifying agent comprising a silylating group to obtain the isolated materials disclosed herein. For example, the silanol groups are surface modified with a silylating reagent having the formula XaRbSi-L-Z, wherein X is CI, Br, I, C1-C5 alkoxy, dialkylamino, or triflate; a and b are each an integer from 0 to 3, wherein the sum of a and b equals 3; R is a C1-C6 linear, branched, or cyclic alkyl group; L is an optional C1-C20 alkylene or heteroalkylene linking group, which can be optionally substituted; and Z is a functional group.

[0147] In some embodiments, Z comprises amidated pectin. In other embodiments, Z comprises a functional group that can be further functionalized by amidated pectin, such as an amino group, a carbonyl group, or a carboxyl group. Examples of silylating agents include aminosilylating agents, such as 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, aminoalkylsilatrane, 3-(2-aminoethyl)aminopropyltriethoxysilane, and 3-(2-aminoethyl)aminopropyltriethoxysilane. Reaction of silica gel with aminosilylating agents provides silica gel comprising surface amino groups that can be further modified and / or reacted with amidated pectin comprising one or more reactive groups. In other embodiments, silica gel is reacted with an amidated pectin derivative comprising a silica reactive group, such as a silatrane or trialkoxysilane derivative.

[0148] In some embodiments, disclosed herein are columns, capillaries, or cartridges comprising a solid support comprising a surface and one or more amidated pectin molecules bound to the surface as adsorbents or carriers.

[0149] In some embodiments, the isolated materials and chromatography columns disclosed herein are useful for separating, partitioning, and purifying nucleic acids, for example, from biological samples or chemical reaction mixtures. In some embodiments, separation is achieved by high performance liquid chromatography (HPLC), size exclusion chromatography, or electrophoresis.

[0150] In some embodiments, the isolated materials disclosed herein are suitable for the isolation of nucleic acids, including but not limited to dsDNA, ssDNA, RNA, and hybrids thereof. Elution of nucleic acids from the isolated materials and their separation can be achieved by increasing the ionic strength of the eluent mobile phase or by increasing the concentration of the eluent in a stepwise or gradient fashion. The mobile phase can optionally comprise an organic solvent suitable for HPLC separation, such as acetonitrile or methanol. The increase in ionic strength can be achieved by increasing the concentration of a suitable salt, such as sodium chloride or guanidinium salt.

[0151] While each element of the application is described herein as containing multiple embodiments, it should be understood that each embodiment of a given element of the application can be used with each embodiment of the other elements of the application, unless otherwise indicated, and each such use is intended to form a different embodiment of the application.

[0152] The application is further illustrated by the following examples, which are merely for further illustration and should not be construed as limiting. Example

[0153] Example 1: Preparation of amide pectin-modified solid support (EDC route)

[0154] A. Preparation of amide pectin-modified beads

[0155] Unless otherwise indicated, all reagents were from commercial sources.

[0156] Pectin amidated with spermine was prepared according to the following procedure. Other amidated pectins were prepared in a similar manner.

[0157] (A) Apple pectin (2.5 g) was added in portions to 250 mL of deionized water under magnetic stirring until all was dissolved. To this solution was added 2.5 mL of 5 M NaOH, stirred for 20 minutes, and then 1 M HC1 was added until the pH stabilized at ~4.5 (then ~12 mL of 1 M HC1 was added). Then 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC.HC1, 2.5 g) and 0.75 g of N-hydroxysuccinimide (NHS) were added and stirred for 1 hour for activation. Spermine (Sigma, 18.63 g, 7 equivalents) was then immediately added. The solution became gelled and was shaken until all material was dissolved and further incubated at room temperature for 20 hours.

[0158] (B) The reaction mixture from (A) was poured into 500 mL of MeOH under stirring, forming a gelled precipitate. The mixture was then stirred for 30 minutes and filtered through a 500 mL disposable plastic filter using polyethylene frits (Opti-Chem, OP-6602-18). The collected gelled filter cake was then rinsed with methanol (100 mL) and further filtered overnight, resulting in a dry brown gelled mass. This material was then washed with an additional 150 mL of MeOH and dried at 50 °C in a vacuum oven for 18 hours. The resulting hard granules were pulverized into a powder using a pestle in a mortar.

[0159] (C) Washing

[0160] Materials:

[0161] A. Acidic wash. Prepare the following mixture in a 1000 mL bottle: IPA (550 mL, graduated cylinder), deionized water (345 mL), and concentrated hydrochloric acid (105 mL)

[0162] B. Neutral wash. Prepare the following mixture in a 1000 mL flask: 590 mL IPA and 410 mL deionized water.

[0163] Charge the product from step (B) into a 125 mL flask and add 110 mL of wash solution to the powdered material. Stir the suspension at room temperature for 30 minutes, filter over a glass funnel, and wash with 5 x 15-20 mL of acidic wash followed by 5 x 15-20 mL of neutral wash followed by 2 x 35 mL of MeOH. Further air dry the material for 60 minutes and then air dry at 0.15 mbar for 17 hours.

[0164] B. Preparation of amide pectin-modified beads

[0165] The following solid support (bead) materials were modified with amidated pectin according to the following procedure:

[0166] Silica microspheres, carboxyl, 1.0 μm (Polysciences, Warrington, PA, 24754-1)

[0167] Carboxyl-polystyrene particles, 5.11 μm (Spherotec, Germany, CP-50-10)

[0168] NHS activated Sepharose 4 Fast Flow (Sepharose beads, GE healthcare, Chicago, IL, 17-0906-01); and

[0169] Carboxyl modified magnetic beads, 5.7 μm (Spherotec, Germany).

[0170] For the Sepharose beads, the NHS activated bead form was used, so the EDC / NHS activation step was omitted. Hydrolyzed NHS-Sepharose beads were used for the non-modified bead measurements.

[0171] In this example, a procedure for functionalizing carboxyl modified beads with amidated pectin containing amines, such as the product from Example 1, is provided.

[0172] Carboxyl modified polystyrene beads (about 5 microns, 2 mL 5wt% suspension) (Spherotec, CP-50-10) were diluted with deionized (DI) water (4 mL) and sonicated for 15 minutes. To the bead suspension was added 40 mg EDC.HC1 and 40 mg NHS. The suspension was stirred for 24 hours to activate, centrifuged briefly at 4000 rpm for 5 minutes, and the supernatant was decanted. The beads were resuspended in 5 ml of deionized water and to this was added a 1% solution of amidated pectin (5 mL). The amidated pectin solution was prepared by stirring the amidated pectin in deionized water for 18 hours and then centrifuging at 9000 rpm for 30 minutes to remove any undissolved material. The resulting suspension was stirred for 18 hours and then centrifuged at 9000 rpm for 30 minutes, diluted with 45 mL of water and rinsed in the same manner. This process was repeated with 0.1 M NaOH (1x), 0.1 M HC1 (1x), and deionized water (2x). The beads were resuspended in 5 ml of DI H20, sonicated for 30 minutes, and the concentration was measured by the amount of beads left after drying a 150 μΐ aliquet in a Speedvac under vacuum.

[0173] Example 2: Preparation of amide pectin-modified solid support (reductive amination route)

[0174] In this example, a general procedure for modifying polysaccharides such as pectin with various polyamines by oxidation followed by reductive amination is provided.

[0175] (A) Oxidation. Apple pectin (2.5 g) was added in portions to 250 mL of deionized water with magnetic stirring until all was dissolved. To this was added potassium periodate (2.43 g) in portions with stirring and stirred for 18 hours. The reaction mixture was then dialyzed against water through an 8 kDa MWCO dialysis tubing over three days. The resulting desalted polymer was then lyophilized to yield oxidized pectin as an off-white solid. The concentration of aldehyde can be easily measured by hydroxylamine titration (as described by Zhao, H.; Heindel, N.D. J. Pharm. Res. 8(3), 400-402.). The aldehyde content was determined to be 4.9 mmol / g (about 1 equivalent of aldehyde per polymer unit).

[0176] (B) Reductive Amination. The oxidized pectin from Step A (1.0 g) was suspended in 100 mL of deionized water, spermine (1.32 g, 1.25 equivalents) was added, and the mixture was stirred at room temperature for 18 hours. Sodium borohydride pellets (1.0 g) were added to the reaction and the reaction mixture was stirred for 18 hours. The reaction mixture was then dialyzed against water through an 8 kDa MWCO dialysis tubing over three days and then lyophilized to yield 200 mg of amidated pectin as an off-white fluffy solid.

[0177] The product of the above reaction was used to modify a solid support as described above in Example 1.

[0178] Example 3: Evaluation of nucleic acid capture of modified beads on filters

[0179] This experiment shows that an exemplary solid support, such as the amidated pectin modified beads prepared as described in Example 1, can capture DNA or RNA on a filter.

[0180] Materials

[0181] The following materials were used in the examples: genomic DNA (Promega Cat# G3041 ~202 ng / µL); RNA control (Life Tech Cat# 4307281, 50 ng / µL); Quantitative fluorescent Picogreen DNA dye (Thermo); Quantitative fluorescent Ribogreen RNA dye (Thermo); Biotek fluorometer and black assay plates suitable for fluorescent quantitation of nucleic acids; calibrated pipettes and pipette tips; lx TE buffer (Thermo: EnzChek® Reverse Transcriptase Assay Kit, P / N E22064 used according to manufacturer’s instructions) or 20 mM Tris, pH ~8.5; Whatman GF / F filters and Pall Supor 0.2 micron filters; filter holder.

[0182] Methods

[0183] Test solutions of DNA or RNA in lx TE buffer were prepared at a desired final concentration (e.g., 100 ng / mL). Modified beads were added to the test solution (DNA or RNA in TE). As a control, a solution of DNA or RNA was prepared without the addition of beads. Exemplary test solutions:

[0184] TE buffer with nucleic acid, with 0.1-1.5 mg of modified beads;

[0185] TE buffer with nucleic acid (negative control), no beads;

[0186] TE buffer with nucleic acid, no beads, unfiltered.

[0187] Mix a 1 mL sample of nucleic acid solution with the modified beads for 15 seconds to promote mixing and binding of nucleic acids to the bead surface. Draw the sample into a 1 mL syringe and pass it through a GF / F or other filter of interest using a syringe filter assembly or prefabricated filter. Collect the eluate into a 2 mL Eppendorf tube. Because captured nucleic acids are retained on the beads on the filter, the amount of captured nucleic acids can be indirectly assessed by the absence of nucleic acids in the eluate, as described below.

[0188] Prepare a DNA or RNA standard curve according to the manufacturer's instructions; prepare 500 µL of each standard and blank in a total of eight tubes. Prepare a working dye solution by diluting the dye 1:200 in TE buffer and store in the dark. Measure the fluorescence of the standard curve samples and each eluate sample using a Biotek microplate reader according to the manufacturer's instructions. Calculate the concentration of nucleic acids in the eluate samples using the standard curve, and calculate the percent capture relative to the theoretical concentration. Compare test samples to a 100% unfiltered control to determine the percent recovery of nucleic acids. Filter a no-bead control sample to assess background filter capture, which is minimal. The 100% control was not filtered.

[0189] Tables 1-5 show the results of the filtration experiments, indicating that the solid supports modified with amidated pectin can effectively capture nucleic acids.

[0190] Table 1. Capture of hgRNA and hgDNA using modified glass beads on Pall Supor 0.2 μm filters.

[0191]

[0192] Table 2. Capture of hgRNA and hgDNA using modified agarose beads on Whatman GF / F filters.

[0193]

[0194] Table 3. Capture of hgRNA and hgDNA using modified polystyrene beads on Whatman GF / F filters.

[0195]

[0196] Table 4. Capture of hgRNA and hgDNA using modified polystyrene beads on Pall Supor 0.2 μm filters.

[0197]

[0198] Table 5. Capture of hgRNA and hgDNA on Modified Polystyrene Beads on Pall Supor 0.2 µm Filters

[0199]

[0200] Example 4 Extraction of nucleic acid from urine and feces

[0201] This experiment demonstrates that the solid support disclosed herein can be used to extract nucleic acids from fecal and urine samples and that the isolated DNA can be detected by PCR amplification.

[0202] Preparation of urine or feces samples

[0203] As shown below, fragmented MTB DNA (fMTB DNA 200-400bp) was spiked into different volumes of urine or feces. Controls for this experiment were prepared by spiking equal amounts of fMTB DNA directly into separate RT-PCR reactions to have a comparison representing 100% extraction and recovery efficiency.

[0204] Extraction of fragmented MTB DNA from urine or feces using exemplary amide pectin-modified microparticles. Example 5: Extraction of nucleic acid from urine and feces using amide pectin-modified microparticles Preparation of urine or feces samples Extraction of fragmented MTB DNA from urine or feces using exemplary amide pectin-modified microparticles. Example 6: Extraction of nucleic acid from urine and feces using amide pectin-modified microparticles Preparation of urine or feces samples Extraction of fragmented MTB DNA from urine or feces using exemplary amide pectin-modified microparticles. Example 7: Extraction of nucleic acid from urine and feces using amide pectin-modified microparticles Preparation of urine or feces samples Extraction of fragmented MTB DNA from urine or feces using exemplary amide pectin-modified microparticles. Example 8: Extraction of nucleic acid from urine and feces using amide pectin-modified microparticles Preparation of urine or feces samples Extraction of fragmented MTB DNA from urine or feces using exemplary amide pectin-modified microparticles. Example 9: Extraction of nucleic acid from urine and feces using

[0205] Add 1-10 mL urine / fecal sample to a centrifuge tube or Eppendorf tube of appropriate size. Add the exemplary amidated pectin-modified microparticles to the sample. The optimal amount to add depends on the batch of beads chosen for each experiment, the sample type, and the sample volume. Mix the sample well, optionally incubate for up to 60 minutes to increase nucleic acid binding, then centrifuge at high speed in a benchtop centrifuge for 2 minutes to pellet the microparticles. Carefully decant the supernatant to avoid disturbing the microparticle pellets. Wash the bead pellets with one milliliter of water, mix gently to wash the pellets, and centrifuge at high speed in a benchtop centrifuge for two minutes to pellet the microparticles. Carefully decant the supernatant to avoid disturbing the microparticle pellets. Add 100 μΐ of low-salt elution buffer to the bead pellets, which consists of 10 mM KOH and 0.01% i-carrageenan (Sigma). Mix the pellets gently and optionally incubate for up to 60 minutes to increase elution from the microparticles. Carefully remove the supernatant containing the eluted nucleic acids to avoid disturbing the bead pellets. The eluate is then used directly in an RT-PCR reaction. PCR was performed as described by Chakravorty et al. mBio, J 2017 July / August, vol. 8, no. 4 e00812-17 for Xpert MTB / RIF Ultra analysis.

[0206] The results are shown in Tables 6-8 below.

[0207] Table 6 PCR analysis of DNA extracted from 10 mL urine samples. Performance of microparticles modified with spermine amidated pectin (EDC coupling or reductive amination) to extract MTB DNA from 10 mL urine is shown. ΔCt was calculated as the difference in Ct for the 100% spiked extract in the control.

[0208]

[0209] Table 7 PCR analysis of DNA extracted from 1 mL urine samples. Performance of microparticles modified with spermine amidated pectin (EDC coupling or reductive amination) for extraction of MTB DNA from 1 mL of urine. ΔCt was calculated as the difference in Ct for the extract from the 100% spike-in control.

[0210]

[0211] Table 8 PCR analysis of DNA extracted from 1 mL fecal samples. Different microparticle modification formulations and their performance for extraction of MTB DNA from 1 mL of fecal sample. The presence of a reducing agent indicates modification of the polymer via the reductive amination route. The absence of a reducing agent (N / A) indicates modification of the polymer via the EDC / NHS route. ΔCt was calculated as the difference in Ct for the extract from the 100% spike-in control.

[0212]

[0213] While the illustrative embodiments have been illustrated and described, it will be the objective of various changes without departing from the spirit and scope of the application.

Claims

1. A solid support comprising a plurality of modified pectin molecules covalently bound to the solid support, wherein the modified pectin is an amidated pectin having one or more units represented by the following formula, or stereoisomers, salts, tautomers or combinations thereof: ， where R 2 and R 3 are independently selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C20 heteroalkyl.

2. The solid support of claim 1, wherein the amidated pectin comprises one or more units represented by the following formula, or a stereoisomer, salt, tautomer, or combination thereof: ， in, n is 0-3; R1 is H or C1-C3 alkyl; X is independently at each occurrence C2-C4 alkylene or C4-C6 heteroalkylene; Y is C2-C3 alkylene or C4-C6 heteroalkylene; and R2 and R3 are independently H or C1-C3 alkyl.

3. The solid support of claim 1, wherein the amidated pectin is pectin amidated with a C4-C20 polyamine. The solid support according to claim 3 , wherein the polyamine is ethylenediamine, putrescine, cadaverine, spermine or spermidine.

5. The solid support of claim 1, wherein the amidated pectin comprises one or more units having the following structure, or a stereoisomer, salt, tautomer, or combination thereof: ， in n is 0, 1, 2, or 3; m is 2, 3 or 4; p is 2, 3, or 4; and R1, R2 and R3 are independently H or C1-C3 alkyl.

6. The solid support of claim 1, wherein the amidated pectin comprises one or more units having the following structure, or a stereoisomer, salt, tautomer, or combination thereof: 、 、 。 7. The solid support of claim 1, wherein the amidated pectin has a molecular weight of 4,000 Da to 500,000 Da, 5,000 Da to 300,000 Da, 100,000 Da to 300,000 Da, or 50,000 Da to 200,000 Da.

8. The solid support of claim 1, wherein the solid support comprises a material selected from the group consisting of polystyrene, glass, ceramic, polypropylene, polyethylene, silica, zirconia, titania, alumina, polycarbonate, latex, polymethyl methacrylate, zeolite, polyethersulfone, carboxymethyl cellulose, and cellulose.

9. The solid support of claim 1, wherein the solid support is magnetic beads, glass beads, polystyrene beads, polystyrene filters, polycarbonate filters, polyethersulfone filters, or glass filters.

10. A method for isolating nucleic acid from a sample containing nucleic acid, comprising: (a) contacting the sample with the solid support according to any one of claims 1 to 9, thereby binding the nucleic acid to the solid support; (b) optionally washing the nucleic acid bound to the solid support; and (c) eluting the nucleic acid from the solid support using an eluent.

11. The method of claim 10, wherein the eluent comprises ammonia or an alkali metal hydroxide.

12. The method of claim 10, wherein the eluent has a pH of 9 or above, 10 or above, or 11 or above.

13. The method of claim 10, wherein the eluent has a pH of 9 to 12, 9.5 to 12, 10 to 12, or 9 to 11.

14. The method of claim 10, wherein the eluent comprises a polyanion.

15. The method of claim 14, wherein the polyanion is carrageenan.

16. The method of claim 14, wherein the polyanion is a carrier nucleic acid.

17. The method of claim 10, wherein the eluent comprises carrageenan and KOH.

18. The method of any one of claims 10-17, wherein the method comprises contacting the sample with a lysis solution prior to contacting the sample with the solid support, thereby releasing nucleic acids into solution.

19. The method of claim 18, wherein the lysis solution comprises a chaotropic agent.

20. The method of claim 19, wherein the chaotropic agent is selected from guanidine thiocyanate, guanidine hydrochloride, alkali metal perchlorate, alkali metal iodide, urea, formamide, or a combination thereof.

21. The method of claim 19, wherein the chaotropic agent is guanidine thiocyanate or guanidine hydrochloride.

22. The method of claim 18, wherein the lysis solution comprises a salt.

23. The method of claim 22, wherein the salt is sodium chloride or calcium chloride.

24. The method of claim 18, wherein the lysis solution does not comprise a chaotropic agent.

25. The method of claim 18, wherein the lysis solution comprises a buffer.

26. The method of claim 25, wherein the buffer is Tris.

27. The method of claim 18, wherein the lysis solution comprises a surfactant.

28. The method of claim 18, wherein the lysis solution comprises an antifoaming agent.

29. The method of claim 10, wherein contacting the sample with a solid support is performed in the absence of a chaotropic agent.

30. The method of claim 10, wherein the sample is selected from blood, plasma, serum, semen, tissue biopsy, urine, stool, saliva, a smear specimen, a bacterial culture, a cell culture, a viral culture, a PCR reaction mixture, or an in vitro nucleic acid modification reaction mixture.

31. The method of claim 30, wherein the tissue biopsy is paraffin-embedded tissue.

32. The method of claim 10, wherein the nucleic acid comprises genomic DNA.

33. The method of claim 10, wherein the nucleic acid comprises total RNA.

34. The method of claim 10, wherein the nucleic acid comprises a microbial nucleic acid or a viral nucleic acid.

35. The method of claim 34, wherein the viral nucleic acid is HBV DNA.

36. The method of claim 10, wherein the nucleic acid is a circulating nucleic acid.

37. The method of any one of claims 10-17, wherein the method is performed in an automated cartridge.

38. A method for detecting nucleic acid in a sample, comprising: (a) contacting a sample containing nucleic acid with the solid support according to any one of claims 1 to 9, thereby binding the nucleic acid to the solid support; (b) optionally washing the nucleic acid bound to the solid support; (c) eluting the nucleic acid; and (d) detecting the nucleic acid.

39. The method of claim 38, wherein detecting the nucleic acid comprises amplifying the nucleic acid by polymerase chain reaction (PCR).

40. The method of claim 39, wherein the polymerase chain reaction is nested PCR, isothermal PCR, or RT-PCR.

41. A separation material for chromatography comprising a solid support comprising amidated pectin chemically bonded thereto, wherein the amidated pectin has one or more units represented by the formula: ， where R 2 and R 3 are independently selected from H, optionally substituted C1-C6 alkyl, optionally substituted C3-C6 cycloalkyl, and optionally substituted C2-C20 heteroalkyl.

42. The separation material of claim 41, wherein the solid support is silica, alumina, titania, zirconia or a mixed silica material.

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