Compositions and methods for nucleic acid normalization

Abstract

Methods, compositions and kits of the present disclosure are provided for use in nucleic acid analysis methods to normalize the mass of nucleic acid in each of a plurality of test samples such that substantially equal amounts of nucleic acid are present in each sample analyzed. The present invention uses magnetic particles having hydroxyl functional groups.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 282,469, filed November 23, 2021, the entire contents of which are incorporated herein by reference.

[0002] Aspects described herein relate to methods, compositions, and kits for normalizing the mass of nucleic acids in each of multiple test samples, thereby allowing a user to avoid manual quantification of each individual test sample. [Background technology]

[0003] Highly sophisticated nucleic acid analysis, such as next generation sequencing (NGS), is powerful for many reasons, not the least of which is the ability to simultaneously analyze thousands of samples. However, in practice, the effectiveness of such cutting-edge technology may be limited by the current lack of equally sophisticated methods to ensure that substantially the same amount of nucleic acid is present in all samples to be analyzed. Current methods require manual quantification of each individual sample, which requires considerable time and effort.

[0004] There is a continuing need for methods, compositions, and kits for normalizing the mass of nucleic acids in each of multiple test samples. Summary of the Invention

[0005] According to an aspect of the present disclosure, there is provided a method for normalizing a mass of a nucleic acid in each of a plurality of test samples, the method including: 1) providing a plurality of input samples comprising nucleic acids in an aqueous liquid, each of the plurality of input samples being in a separate container; 2) adding a binding mixture to each vessel to generate a normalized mixture in each vessel; The binding mixture comprises: i) a quantity of magnetic particles comprising pendant hydroxyl functional groups; ii) a chelating agent; iii) binding buffer, and iv) alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of said plurality of input samples comprises a mass of nucleic acid greater than the binding capacity of said quantity of magnetic particles; 3) incubating each normalization mixture under binding conditions, thereby reversibly and non-specifically binding a portion of the nucleic acids to the magnetic particles; 4) separating the magnetic particles with reversibly and non-specifically bound nucleic acids from unbound nucleic acids by application of a magnetic field; and 5) eluting the reversibly and non-specifically bound nucleic acid from the magnetic particles to generate a plurality of test samples, wherein each of the plurality of test samples comprises an isolated nucleic acid having a mass, wherein the mass of the isolated nucleic acid is approximately equal to the binding capacity of the magnetic particles, thereby providing a normalized nucleic acid mass in each of the plurality of test samples.

[0006] According to an aspect of the disclosed method for normalizing the mass of a nucleic acid in each of a plurality of test samples, no internal standard is added to the vessel.

[0007] According to aspects of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the mass of a nucleic acid in the input sample is not quantified.

[0008] According to an aspect of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the magnetic particles do not include a binding partner that specifically binds to the nucleic acid.

[0009] According to an aspect of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the magnetic particles do not include a binding partner that is a nucleic acid, biotin, avidin, an antibody, an aptamer, a receptor, or a receptor ligand.

[0010] According to an embodiment of the present disclosure, the method for normalizing the mass of a nucleic acid in each of a plurality of test samples further includes: 6) pooling the plurality of test samples to generate a pooled test sample, and sequencing at least a portion of the nucleic acids in the pooled test sample.

[0011] According to an embodiment of the method of normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, the chaotrope comprises one or more of the following: urea, guanidinium bromide (guanidinium hydrobromide or guanidinium monohydrobromide), guanidinium iodide (guanidinium hydroiodide), guanidinium chloride (guanidinium hydrochloride), guanidinium thiocyanate (guanidinium thiocyanate), guanidinium nitrate (guanidinium nitrate), guanidinium sulfate (guanidinium sulfate), guanidinium carbonate (guanidinium carbonate), sodium iodide, and sodium perchlorate.

[0012] According to an embodiment of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the binding buffer does not contain PEG.

[0013] According to an embodiment of the method of normalizing the mass of nucleic acids in each of a plurality of test samples of the present disclosure, the magnetic particles do not include carboxyl and / or amine functional moieties.

[0014] According to an embodiment of the present disclosure of a method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the magnetic particles comprising pendant hydroxyl functional groups include a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group.

[0015] According to an embodiment of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the chelating agent is one or more of the following: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA).

[0016] According to an embodiment of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the alcohol is one or more of the following: methanol, ethanol, and isopropanol.

[0017] According to an embodiment of the disclosed method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the binding buffer comprises an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate and has a pH in the range of about pH 4 to about pH 6.

[0018] According to an embodiment of the method of the present disclosure for normalizing the mass of nucleic acids in each of a plurality of test samples, further comprising: 7) recovering the unbound nucleic acids of one or more normalization mixtures. According to an embodiment of the method of the present disclosure, recovering the unbound nucleic acids of one or more normalization mixtures comprises: 7a) transferring said unbound nucleic acids of one or more normalization mixtures to corresponding containers; 7b) adding the binding mixture to each corresponding container; 7c) generating a recovery mixture in each corresponding vessel; The binding mixture comprises: a quantity of magnetic particles comprising pendant hydroxyl functional groups; Chelating agents, A binding buffer, and alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of said recovery mixtures comprises a mass of nucleic acid greater than, less than, or equal to the binding capacity of said quantity of magnetic particles; 7d) incubating each recovery mixture under binding conditions, thereby reversibly and non-specifically binding all or a portion of the nucleic acid to the magnetic particles to produce magnetic particles reversibly and non-specifically bound to the recovered nucleic acid; 7e) separating the magnetic particles reversibly and non-specifically bound to the recovered nucleic acid by application of a magnetic field; and 7f) Eluting the reversibly and non-specifically bound recovered nucleic acid from the magnetic particles.

[0019] According to an aspect of the present disclosure of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples, eluting the nucleic acid from the magnetic particles includes incubating the beads in an elution buffer.

[0020] According to an embodiment of the disclosed method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA.

[0021] According to an embodiment of the present disclosure, the method for normalizing the mass of a nucleic acid in each of a plurality of test samples further includes: washing the magnetic particles that are reversibly and non-specifically bound to the nucleic acid with a washing solution after application of a magnetic field and before elution.

[0022] According to an embodiment of the method for normalizing the mass of nucleic acid in each of a plurality of test samples of the present disclosure, the wash solution comprises about 80% ethanol. According to an embodiment of the method for normalizing the mass of nucleic acid in each of a plurality of test samples of the present disclosure, the volume to volume ratio of the alcohol to the binding buffer is in the range of about 0.25:1 to about 1.75:1. According to an embodiment of the method for normalizing the mass of nucleic acid in each of a plurality of test samples of the present disclosure, the volume to volume ratio of the alcohol to the binding buffer is in the range of about 1:1 to about 1.25:1. According to an embodiment of the method for normalizing the mass of nucleic acid in each of a plurality of test samples of the present disclosure, the volume to volume ratio of the alcohol to the binding buffer is about 1.125:1.

[0023] A kit according to an aspect of the present disclosure includes: magnetic particles containing pendant hydroxyl functional groups; a binding buffer containing a buffered aqueous solution of a chaotrope; cleaning fluid; and Elution buffer.

[0024] According to an aspect of the present disclosure, the magnetic particles having pendant hydroxyl functional groups include a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group.

[0025] According to an embodiment of the present disclosure, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution comprises about 60% to about 100% ethanol.

[0026] A kit according to an aspect of the present disclosure includes: magnetic particles containing pendant hydroxyl functional groups; a binding buffer containing a buffered aqueous solution of a chaotrope; Cleaning fluid; Elution buffer; Chelating agents; and alcohol.

[0027] According to an aspect of the present disclosure, the magnetic particles having pendant hydroxyl functional groups include a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group.

[0028] According to an embodiment of the present disclosure, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution comprises about 80% ethanol.

[0029] A kit according to an aspect of the present disclosure includes: magnetic particles containing pendant hydroxyl functional groups; a binding buffer comprising an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate, the binding buffer having a pH in the range of about pH 4 to about pH 6; cleaning solution; and Elution buffer.

[0030] According to an aspect of the present disclosure, the magnetic particles having pendant hydroxyl functional groups include a spacer having a hydroxyl functional group covalently bonded to the magnetic particle and / or the coating of the magnetic particle and extending from the magnetic particle and / or the coating of the magnetic particle, the spacer including a chain of at least three atoms covalently bonded to the hydroxyl functional group.

[0031] According to an embodiment of the present disclosure, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution comprises ethanol at a concentration of about 80%.

[0032] A kit according to an aspect of the present disclosure includes: magnetic particles containing pendant hydroxyl functional groups; a binding buffer comprising an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate, the binding buffer having a pH in the range of about pH 4 to about pH 6; Cleaning fluid; Elution buffer; Chelating agents; and alcohol.

[0033] According to an aspect of the present disclosure, the magnetic particles having pendant hydroxyl functional groups include a spacer having a hydroxyl functional group covalently bonded to the magnetic particle and / or the coating of the magnetic particle and extending from the magnetic particle and / or the coating of the magnetic particle, the spacer including a chain of at least three atoms covalently bonded to the hydroxyl functional group.

[0034] According to an embodiment of the present disclosure, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution comprises ethanol at a concentration of about 80%.

[0035] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, comprising: an input sample or a plurality of input samples, each input sample comprising nucleic acid in an aqueous liquid, each of the plurality of samples being in a separate container; and the combined mixture in each vessel, which produces a normalized mixture in each vessel; The binding mixture comprises: a quantity of magnetic particles comprising pendant hydroxyl functional groups; Chelating agents, A binding buffer, and alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and Each of the plurality of input samples comprises a mass of nucleic acid greater than the binding capacity of the quantity of magnetic particles.

[0036] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a spacer covalently bonded to the magnetic particles and / or the coating of the magnetic particles and having a hydroxyl functional group extending from the magnetic particles and / or the coating of the magnetic particles, the spacer comprising a chain of at least three atoms covalently bonded to the hydroxyl functional group.

[0037] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein no internal standard is present in said container.

[0038] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the magnetic particles do not comprise a binding partner that specifically binds to the nucleic acid.

[0039] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the magnetic particles do not comprise a binding partner that is a nucleic acid, biotin, avidin, an antibody, an aptamer, a receptor, or a receptor ligand.

[0040] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the chaotrope comprises one or more of the following: urea, guanidinium bromide (guanidinium hydrobromide or guanidinium monohydrobromide), guanidinium iodide (guanidinium hydroiodide), guanidinium chloride (guanidinium hydrochloride), guanidinium thiocyanate (guanidinium thiocyanate), guanidinium nitrate (guanidinium nitrate), guanidinium sulfate (guanidinium sulfate), guanidinium carbonate (guanidinium carbonate), sodium iodide, and sodium perchlorate.

[0041] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the binding buffer does not comprise PEG.

[0042] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the magnetic particles do not comprise carboxyl and / or amine functional moieties.

[0043] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the chelating agent is one or more of the following: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA).

[0044] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein the alcohol is one or more of the following: methanol, ethanol, and isopropanol.

[0045] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an embodiment of the present disclosure, wherein the binding buffer comprises an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate, and has a pH in the range of about pH 4 to about pH 6.

[0046] Provided according to an aspect of the present disclosure is a composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples, wherein a volume to volume ratio of the alcohol to the binding buffer is within the range of about 0.25:1 to about 1.75:1.

[0047] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein a volume to volume ratio of the alcohol to the binding buffer is within the range of about 1:1 to about 1.25:1.

[0048] A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, wherein a volume to volume ratio of the alcohol to the binding buffer is about 1.125:1. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 is a schematic diagram of a method according to an embodiment of the present disclosure. [Figure 2A] FIG. 2A is a graph showing that varying the ratio of ethanol to binding buffer changes the size range of DNA fragments that bind to and elute from magnetic particles with pendant hydroxyl functional groups, with increasing ethanol resulting in increased isolation of smaller DNA fragments. [Figure 2B] FIG. 2B is an image of a gel analysis of the size of the isolated DNA, showing that depending on the ratio of ethanol to binding buffer, DNA fragments of different size ranges are isolated. [Diagram 3]Figure 3 shows the results of a gel analysis of varying ratios of ethanol to binding buffer. The DNA ladder (Gene Ladder 50 bp) in the leftmost lane shows the change in the size range of DNA fragments that bind to and elute from magnetic particles with pendant hydroxyl functional groups when compared to samples of DNA isolated using various ratios of ethanol to binding buffer. Increasing ethanol resulted in increased isolation of smaller DNA fragments. [Figure 4] Figure 4 is an image of a gel analysis of the size of isolated DNA, showing that DNA fragments of different size ranges are isolated depending on the ratio of ethanol to binding buffer. When the ratio of ethanol to binding buffer was 1.125, the size distribution became bell-shaped. [Diagram 5] Figure 5 shows the results of a gel analysis of varying ratios of ethanol to binding buffer. A comparison of the size distribution of the DNA ladder (Gene Ladder 50 bp) in the leftmost lane to samples of DNA isolated using various ratios of aqueous component (Aqu) to binding buffer (BB) of a "normalized mixture" containing DNA in an aqueous liquid showed no significant effect on the size range of DNA fragments bound to and eluted from magnetic particles containing pendant hydroxyl functional groups. [Figure 6] Figure 6 shows the results of a gel analysis of changing pH of the normalization mixture. The DNA ladder in the leftmost lane (Gene Ladder 50 bp) was compared in size distribution to samples of DNA isolated using the indicated pH, and it was found that increasing the pH had only a minor effect on the size range of DNA fragments that bound to and eluted from magnetic particles with pendant hydroxyl functional groups, with the exception of fragments below 150 bp. [Figure 7]Figure 7 is a graph showing results from the disclosed method using magnetic particles with pendant hydroxyl functional groups at various DNA input amounts of 25 ng, 50 ng, 100 ng, 250 ng, 350 ng, 500 ng, 625 ng, and 750 ng by two separate operators. Figure 10 shows that equal amounts of DNA were recovered when the same amount of magnetic particles with pendant hydroxyl functional groups was added to each sample containing different amounts of DNA, suggesting that the disclosed method provides normalization by total DNA mass. [Figure 8] FIG. 8 is a graph showing library recovery and percent cluster balancing achieved using the method of the present disclosure, demonstrating replicability and reproducibility. Various estimated DNA input amounts were employed, ranging from 500 ng to 1100 ng. The magnetic particles used, with pendant hydroxyl functional groups, allowed normalization at 500 ng or higher after PCR. 100% normalization was achieved across 88 libraries by recovered DNA amount (+ / -1.35-fold difference; total average recovered DNA: approx. 100 ng (approx. 11.5 nM)). Approximately 94% normalization was achieved by clustering. With the exception of five libraries below the % cluster threshold, there was a + / -1.38-fold difference, and across 88 libraries there was a + / -1.48-fold difference. [Figure 9] FIG. 9 is an image of the results of gel electrophoresis using samples from each of the 24 normalized amplified libraries. [Figure 10] FIG. 10 is a graph of the results of a fragment size analysis using samples from each of the 24 normalized amplified libraries. [Figure 11] FIG. 11 is an image of the results of gel electrophoresis using samples of each of the eight normalized amplified libraries. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Such terms shall be understood as defined in J. Sambrook and DW Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; FM Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002;Next Generation Sequencing: Methods and Protocols (Methods in Molecular Biology), 2018, Humana Press, Springer Nature; DL Nelson and MM Cox, Lehninger Principles of Biochemistry, 4th Ed., WH Freeman & Company, 2004; and Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, It can be found defined and used illustratively in context in various standard texts, including the International Standards Board of Education (ISBN 2004).

[0051] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless expressly stated otherwise or the context clearly indicates otherwise.

[0052] The terms "includes / comprises / including / comprising", "has / having", and grammatical variations thereof, when used herein, are not intended to be limiting and specify the presence of stated features, elements, and / or components, but do not exclude the presence or addition of one or more other features, elements, components, and / or groups thereof.

[0053] The term "about" and grammatical equivalents as used herein in connection with a reference numerical value refer to the reference numerical value and to numerical values ​​within 10% of the reference numerical value, including values ​​that are plus or minus (+ / -) 1%, + / - 2%, + / - 3%, + / - 4%, + / - 5%, + / - 6%, + / - 7%, + / - 8%, + / - 9%, or + / - 10% of the reference numerical value. The term "about" and its grammatical equivalents, as used herein, in the context of stated ranges of reference numerical values, refers to ranges that include numerical values ​​that are + / -1%, + / -2%, + / -3%, + / -4%, + / -5%, + / -6%, + / -7%, + / -8%, + / -9%, or + / -10% of the reference lower limit of that numerical range, and numerical values ​​that are + / -1%, + / -2%, + / -3%, + / -4%, + / -5%, + / -6%, + / -7%, + / -8%, + / -9%, or + / -10% of the reference upper limit of that numerical range. Additionally, unless otherwise specified, numerical entries herein are understood to include all intermediate and fractional values ​​of the listed numerical values, e.g., 50%, 60%, 75% are understood to include 55%, 64.5%, 74%.

[0054] A method for normalizing the mass of a nucleic acid in each of a plurality of test samples is provided according to an aspect of the present disclosure, comprising: 1) providing a plurality of input samples comprising nucleic acids in an aqueous liquid, each of the plurality of samples being in a separate container; 2) adding a binding mixture to each vessel, which produces a normalization mixture in each vessel, wherein: The binding mixture comprises: i) a quantity of magnetic particles comprising pendant hydroxyl functional groups; ii) a chelating agent; iii) binding buffer, and iv) alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; said amount of magnetic particles being capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of the plurality of input samples comprises a mass of nucleic acid greater than a binding capacity of the quantity of magnetic particles; 3) incubating each normalization mixture under binding conditions, thereby reversibly and non-specifically binding a portion of the nucleic acids to the magnetic particles; 4) separating the magnetic particles with reversibly and non-specifically bound nucleic acids from unbound nucleic acids by application of a magnetic field; 5) eluting the reversibly and non-specifically bound nucleic acids from the magnetic particles to generate a plurality of test samples; wherein each of the plurality of test samples comprises an isolated nucleic acid having a particular mass; The mass of the isolated nucleic acid is approximately equal to the binding capacity of the magnetic particles, thereby providing a normalized mass of nucleic acid in each of the multiple test samples.

[0055] FIG. 1 illustrates a method for normalizing the mass of a nucleic acid in each of a plurality of test samples according to an embodiment of the present disclosure, the method including: providing a plurality of input samples comprising a nucleic acid in an aqueous liquid, each of the plurality of input samples being present in a separate container, referred to in FIG. 1 as "input DNA."

[0056] At 2 in FIG. 1, it is shown diagrammatically that nucleic acids bind to magnetic particles containing pendant hydroxyl functional groups after adding a binding mixture to each vessel to generate a normalization mixture in each vessel and incubating each normalization mixture under binding conditions, thereby reversibly and non-specifically binding a portion of the nucleic acid to the magnetic particles. At 3 in FIG. 1, it is shown diagrammatically that the magnetic particles with the reversibly and non-specifically bound nucleic acid are separated from the unbound nucleic acid by application of a magnetic field. Removal of the supernatant containing the unbound nucleic acid, now shown as "unbound DNA", is also shown at *. Once the supernatant containing the unbound nucleic acid has been removed, the nucleic acid reversibly and non-specifically bound to the magnetic particles is washed with a wash solution, in this case an ethanol wash solution, as shown at 4.

[0057] At 5 in FIG. 1, a step of eluting the reversibly and non-specifically bound nucleic acid from the magnetic particles to generate a plurality of test samples, each of the plurality of test samples containing an isolated nucleic acid having a specific mass, the mass of the isolated nucleic acid being approximately equal to the binding capacity of the magnetic particles, thereby providing a normalized mass of nucleic acid, i.e., "normalized DNA" in this figure, in each of the plurality of test samples. Furthermore, FIG. 1 shows an optional recovery of the unbound nucleic acid, which includes at 2B adding a binding mixture to each vessel and incubating under binding conditions, followed by binding of the previously unbound nucleic acid to magnetic particles containing pendant hydroxyl functional groups, thereby reversibly and non-specifically binding some or all of the previously unbound nucleic acid to the magnetic particles. At 3B, separation of the magnetic particles with reversibly and non-specifically bound nucleic acid from the supernatant by application of a magnetic field is shown. At 4B, a washing step is shown, and at 5B, elution of the nucleic acid is shown, generating purified unbound nucleic acid. As used herein, the term "normalization" refers to a process that includes the generation of a plurality of test samples, each individual test sample in the plurality containing substantially the same amount of nucleic acid compared to each other test sample in the plurality. The plurality of test samples is derived from a corresponding plurality of input samples containing nucleic acid. The nucleic acid in the input samples may or may not be quantified prior to the generation of the corresponding plurality of test samples. The nucleic acid in the test samples may or may not be quantified. An advantage of the method of normalizing the mass of a nucleic acid in each of a plurality of test samples according to aspects of the present disclosure is that quantification of the nucleic acid in the test samples is not required.

[0058] As used herein, the term "substantially equal" with respect to the amount of nucleic acid in the test sample refers to an amount that differs by no more than 40%, e.g., no more than 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less.

[0059] The term "nucleic acid" as used herein refers to a polymer of nucleotides, such as ribonucleotides and / or deoxyribonucleotides and / or nucleotide analogs, covalently linked together. Nucleic acids include, but are not limited to, DNA, such as genomic DNA, cDNA, DNA amplification products, and / or RNA, such as mRNA, rRNA, tRNA, siRNA, miRNA, piRNA, and small RNA.

[0060] A "nucleic acid" may include standard nucleotides, typically referred to as adenine (A), guanine (G), cytidine (C), thymidine (T), and uridine (U), in reference to the bases involved. The term "nucleotide" as used herein refers to a molecule that includes a base moiety, a sugar moiety, and a phosphate moiety. A nucleic acid may include one or more non-standard nucleotides, i.e., nucleotide analogs. The term "nucleotide analog" refers to a nucleotide that includes one or more modifications to the base moiety and / or sugar moiety, and / or phosphate moiety that modify at least one aspect of the chemical nature of the nucleotide analog, while retaining other properties that allow the nucleotide analog to perform its intended function, as compared to a reference standard nucleotide. Nucleotide analogs, and nucleic acids that include them, are well known in the art and may be synthesized according to standard procedures and / or obtained commercially.

[0061] According to aspects of the present disclosure, the nucleic acid in the input sample is derived from a biological sample obtained from any organism, any cell, or any cell derived from any organism, including unicellular organisms, multicellular organisms, prokaryotes, eukaryotes, invertebrates, vertebrates, or any nucleic acid-containing organism, such as viruses or mycoplasma. According to aspects of the present disclosure, the nucleic acid in the input sample is derived from a biological sample obtained from plants, bacteria, archaea, or fungi. According to aspects of the present disclosure, the nucleic acid in the input sample is derived from a mammalian subject or a non-mammalian subject. The biological sample obtained from the subject can be, but is not limited to, saliva, blood, plasma, serum, mucus, urine, feces, nasal material, cerebrospinal fluid, ventricular fluid, pleural effusion, lung and bronchial lavage samples, sweat, tears, semen, bladder washing samples, amniotic fluid, lymph, hair, skin, tumor, and peritoneal fluid samples.

[0062] The mammalian subject may be any mammal, including, but not limited to: humans; non-human primates; rodents, such as mice, rats, or guinea pigs; domesticated pets, such as cats or dogs; cows, pigs, sheep, goats, camels, vicunas, or rabbits; whales; and marine mammals, such as dolphins, seals, or sea lions.

[0063] The non-mammalian subject may be a non-mammalian, including, but not limited to, birds, reptiles, amphibians, insects, fish, nematodes, and the like.

[0064] The subject can be of any gender and age. According to aspects of the methods of the present disclosure, the subject is a human.

[0065] According to aspects of the present disclosure, the nucleic acids in the input sample are from an environmental sample, which may be a liquid, gaseous, or solid sample, including, but not limited to, a water sample, a sewage sample, an air sample, a surface swab, a food sample, a beverage sample, a clothing sample, and a soil sample.

[0066] In certain embodiments, the input sample and the test sample comprise amplified DNA, such as DNA obtained by an amplification reaction. In certain embodiments, the input sample and the test sample comprise amplified DNA produced by polymerase chain reaction (PCR). In certain embodiments, the input sample and the test sample comprise amplified DNA produced by isothermal amplification. The template nucleic acid for the amplification reaction can be nucleic acid of any origin, including a biological sample or an environmental sample.

[0067] Amplification of a nucleic acid is achieved using an in vitro amplification method. The term "amplification method" refers to a method for copying a template target nucleic acid, thereby generating a nucleic acid that includes a copy of all or a portion of the template target nucleic acid.

[0068] Amplification methods encompassed by embodiments of the present invention include amplification methods that involve template-specific primer extension catalyzed by a nucleic acid polymerase using a pair of primers flanking a target nucleic acid, and include, but are not limited to, polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), ligation-mediated PCR (LM-PCR), pHi-29 PCR, real-time quantitative PCR (qPCR), whole genome amplification, and other nucleic acid amplification methods, e.g., as described in C.W. Dieffenbach et al., PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2003; V. Demidov et al., DNA Amplification: Current Technologies and Applications, Taylor & Francis, 2004; and Kroneis, T. (Ed.), Whole Genome Amplificationmethods and Protocols (Methods inmolecular Biology), 2015, Humana Press ISBN-10: 1493929895.

[0069] The term "isothermal amplification" refers to nucleic acid amplification that includes single temperature amplification and thus does not require thermal cycling (as in PCR). Examples of isothermal amplification include, but are not limited to, circular helicase-dependent amplification (cHDA), genomic exponential amplification reaction (GEAR), helicase-dependent amplification (HDA), isothermal multiple displacement amplification (IMDA), loop-mediated isothermal amplification (LAMP), multiple displacement amplification (MDA), nicking enzyme amplification reaction (NEAR), nucleic acid sequence-based amplification (NASBA), divergent amplification (RAM), recombinase polymerase amplification (RPA), rolling circle amplification (RCA), autonomous sequence replication (3SR), RNA signal-mediated amplification technology (SMART), strand displacement amplification (SDA), single primer isothermal amplification (SPIA), and transcription-mediated amplification (TMA).

[0070] The terms "amplified nucleic acid" and "amplified DNA" and their plurals refer to the products of the process of replicating a target nucleic acid template.

[0071] Amplified nucleic acids optionally contain additional material that is present in the primer but not in the original nucleic acid template, such as, but not limited to, nucleic acid sequences, functional groups for chemical reactions, and detectable labels, etc. Such primer-derived material provides additional functionality, such as a primer binding site for further amplification reactions and / or functional groups for chemical attachment to a substrate.

[0072] As used herein, the term "mass" refers to the amount of DNA expressed in mass units, e.g., micrograms or nanograms.

[0073] Any container can be used in the methods of the present disclosure. In certain embodiments, the container can be a tube, a vial, a chamber, a well, or a cavity. In certain embodiments, the container can be a multi-compartment container that can separately hold multiple individual samples, each sample in a separate compartment, such as, but not limited to, a slide, chip, or tray with multiple cavities, or a multi-well plate.

[0074] According to an embodiment of the method of normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, no internal standard is used. According to an embodiment of the method of normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, no exogenous internal standard is added to or included in the vessel in which the input sample or test sample resides.

[0075] In accordance with aspects of the disclosed method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the mass of the nucleic acid in the input sample is not quantified, and thus, for example, the nucleic acid in the input sample is not quantified and / or the nucleic acid in the test sample is not quantified, thereby eliminating a time-consuming and laborious step in the generation of the test sample.

[0076] A plurality of test samples having mass-normalized nucleic acids in each of the plurality of test samples are useful in a variety of nucleic acid analysis procedures, such as, but not limited to, sequencing, including high-throughput sequencing, methylation analysis, single-stranded conformation polymorphism analysis, mass spectrometry, capillary electrophoresis, high-resolution melt analysis, and restriction fragment length polymorphism.

[0077] A plurality of test samples having mass-normalized nucleic acids in each of the plurality of test samples are useful in a variety of nucleic acid analysis procedures, such as, but not limited to, massively parallel sequencing (also known as next generation sequencing (NGS)), polony sequencing, ion semiconductor sequencing, pyrosequencing, single molecule real-time sequencing, sequencing by synthesis, sequencing by ligation, combinatorial probe anchor synthesis, nanopore sequencing, and chain termination sequencing.

[0078] Magnetic particles capable of reversibly and non-specifically binding nucleic acids are included in the methods, compositions, and kits according to aspects of the present disclosure.

[0079] As disclosed herein, magnetic particles with pendant hydroxyl functional groups are included in the methods, compositions, and kits according to aspects of the present disclosure and can reversibly and nonspecifically bind to nucleic acids. With respect to magnetic particles with pendant hydroxyl functional groups, the term "nonspecific binding" as used herein refers to the association of the magnetic particles with nucleic acids by nonspecific interactions, such as, for non-limiting examples, ionic interactions and / or hydrogen bonds in the binding mixture under binding conditions as described herein. The term "nonspecific binding" as used herein further refers to the association of the magnetic particles with nucleic acids to such an extent that the nucleic acids remain nonspecifically bound to the magnetic particles when separated from the unbound nucleic acids by applying a magnetic field, removing the supernatant, and washing with a washing solution, but the association is easily reversed by elution of the nucleic acids from the magnetic particles.

[0080] The term "magnetic" as used herein includes magnetic materials, including paramagnetic, superparamagnetic, ferromagnetic, and ferrimagnetic materials. Thus, a magnetic particle capable of reversibly and non-specifically binding to nucleic acids may include one or more magnetic materials, including paramagnetic, superparamagnetic, ferromagnetic, and ferrimagnetic materials. Examples of such magnetic materials include Fe, Co, CrO2, Dy, EuO, Gd, Ni, MnAs, MnBi, NiO / Fe, NiFe2O4, and Fe3O4.

[0081] The magnetic particles can be of various shapes, regular, irregular, or a mixture of regular and irregular, including, but not limited to, spheres, spheroids, ellipsoids, ellipsoids, rods, and rods. The magnetic particles can be beads.

[0082] The magnetic particles typically have an average diameter or average longest dimension of less than 50 microns. The magnetic particles may be nanoparticles, microparticles, or mixtures thereof, where the average diameter or average longest dimension of the microparticles ranges from about 1 nm to about 1000 nm, and the average diameter or average longest dimension of the microparticles ranges from about 1 micron to about 50 microns. According to an embodiment of the present disclosure, the magnetic particles have an average diameter or average longest dimension in the range of about 1 nm to about 1000 nm, such as about 1 nm to about 10 nm, such as about 1 nm to about 100 nm, such as about 10 nm to about 50 nm, such as about 10 nm to about 100 nm, such as about 50 nm to about 200 nm, such as about 100 nm to about 250 nm, such as about 200 nm to about 500 nm, such as about 300 nm to about 600 nm, such as about 500 nm to about 750 nm, such as about 700 nm to about 1000 nm, such as about 1 micron to 10 microns, such as about 10 microns to about 20 microns, or such as about 25 microns to about 50 microns. According to an embodiment of the present disclosure, the magnetic particles have an average diameter or average longest dimension in the range of about 0.5 microns to about 5 microns. According to an embodiment of the present disclosure, the magnetic particles have an average diameter or average longest dimension in the range of about 1 micron to about 3 microns.

[0083] According to an aspect of the method of normalizing the mass of nucleic acids in each of a plurality of test samples of the present disclosure, the magnetic particles include a functionalized surface and contain hydroxyl functional groups pending from the surface, i.e., pendant hydroxyl functional groups. The magnetic particles may include a solid magnetic core encapsulated in one or more non-magnetic layers that include, or can be functionalized to include, hydroxyl functional groups.

[0084] According to an aspect of the present disclosure, the magnetic particles may include a core of a fragment of a magnetic material encapsulated in a polymeric material and surface functionalized to include hydroxyl functional groups. According to an aspect of the present disclosure, the magnetic particles may include a core of a fragment of a magnetic material encapsulated in polyvinyl alcohol (PVA) and the surface of the particles includes hydroxyl functional groups, such as by silanization.

[0085] According to an aspect of the present disclosure, the magnetic particles include silica.

[0086] According to an aspect of the present disclosure, the magnetic particles are silica coated magnetic particles.

[0087] According to an aspect of the present disclosure, the spacer having a hydroxyl functional group is covalently attached to and extends from the magnetic particle surface and / or the magnetic particle surface coating, such as a silica or polymer coating, such that the hydroxyl functional group is spaced from the magnetic particle surface and / or the magnetic particle surface coating. According to an aspect of the present disclosure, the spacer can be a polyatomic group or an atomic chain. A non-limiting example of a spacer that is a polyatomic group is C(O).

[0088] According to an embodiment of the present disclosure, the spacer is or comprises a chain of atoms, such as a branched or linear chain, of 3 to 20 or more atoms. According to an embodiment of the present disclosure, the spacer is or comprises a linear chain of 4 to 20 or more atoms. According to an embodiment of the present disclosure, the spacer is or comprises a linear chain of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. According to an embodiment of the present disclosure, the spacer is or comprises a linear chain of 6, 7, 8, 9, 10, 11, or 12 atoms. According to an embodiment of the present disclosure, the spacer is or comprises a linear chain of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. According to an embodiment of the present disclosure, the spacer is or comprises a linear chain of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. According to an embodiment of the present disclosure, the spacer is or comprises a linear chain of 10, 11, 12, 13, 14, or 15 atoms.

[0089] According to an embodiment of the present disclosure, the spacer is a substituted or unsubstituted C3-C 20 Alkyl, substituted or unsubstituted C3-C 20 Alkenyl, substituted or unsubstituted C3-C 20Alkynyl, substituted or unsubstituted C6-C 12 Aryl, substituted or unsubstituted C3-C 12 Cycloalkyl 、 Substituted or unsubstituted C5-C 12 Heteroaryl or substituted or unsubstituted C5-C 12 The spacer may be, or may include, but is not limited to, a chain of atoms such as heterocyclyl, where the chain includes at least one hydroxyl functional group, preferably at least one terminal hydroxyl functional group. As used herein, the term "terminal hydroxyl functional group" refers to a hydroxyl group on the final atom of the spacer chain of atoms, i.e., the atom of the chain of atoms farthest from the magnetic particle, or a hydroxyl group on an atom that is within 2, 3, or 4 atoms of the final atom of the spacer chain of atoms.

[0090] According to an embodiment of the present disclosure, the spacer is selected from the group consisting of substituted or unsubstituted C3 alkyl, substituted or unsubstituted C4 alkyl, substituted or unsubstituted C5 alkyl, substituted or unsubstituted C6 alkyl, substituted or unsubstituted C7 alkyl, substituted or unsubstituted C8 alkyl, substituted or unsubstituted C9 alkyl, substituted or unsubstituted C 10 Alkyl, substituted or unsubstituted C 11 Alkyl, substituted or unsubstituted C 12 Alkyl, substituted or unsubstituted C 13 Alkyl, substituted or unsubstituted C 14 Alkyl, substituted or unsubstituted C 15 Alkyl, substituted or unsubstituted C 16 Alkyl, substituted or unsubstituted C 17 Alkyl, substituted or unsubstituted C 18 Alkyl, substituted or unsubstituted C 19 Alkyl or substituted or unsubstituted C 20The magnetic particles may be or include a chain of atoms such as an alkyl, where the chain includes at least one hydroxyl functional group, preferably at least one terminal hydroxyl functional group. Magnetic particles including hydroxyl functional groups pendant from the surface, i.e., pendant hydroxyl functional groups, may be produced by known techniques or purchased commercially, see, for example, U.S. Patent No. 7,129,308 and Yang, H., et al., ACS Appl. Mater. Interfaces, 2015, 7, 1, 774-781.

[0091] According to an embodiment of the method of normalizing the mass of nucleic acid in each of a plurality of test samples of the present disclosure, the binding mixture includes an amount of magnetic particles capable of reversibly and non-specifically binding nucleic acid with a binding capacity ranging from about 1 nanogram to about 5 micrograms, where each of the plurality of input samples includes a mass of nucleic acid that is greater than the binding capacity of the amount of magnetic particles. The binding capacity of the amount of magnetic particles can be determined using any of a variety of methods, including, but not limited to, empirical determination and / or estimation and / or prediction based on the properties of the magnetic particles. Thus, for example, the binding capacity of the magnetic particles can be determined empirically by determining the amount of nucleic acid that binds to the magnetic particles using a known amount of magnetic particles with a known amount of nucleic acid under defined binding conditions. In another example, the binding capacity can be calculated by using properties of the magnetic particles, including the surface area of ​​the magnetic particles, the density of functional groups on the magnetic particles, and the number of functional groups associated with each individual nucleic acid molecule that is non-specifically bound to the magnetic particles. Thus, for example, the surface area of ​​a magnetic particle with a diameter of 1 micron is 3.14×10 6 nm 2 Assuming that one hydroxyl functional group is pendant from the surface of the magnetic particle, the surface area of ​​the magnetic particle is 1 nm 2Assuming a density of 1000 nm per 1000 nm and estimating that the number of hydroxyl functional groups pendant from the surface of the magnetic particle that are involved in non-specific binding of nucleic acid molecules is between 1 and 10, the binding capacity of a single magnetic particle is predicted to be in the range of about 30,000 to 300,000 nucleic acid molecules per 1 micron diameter magnetic particle. Similar estimates of binding capacity can be made using different magnetic particle sizes, different functional group densities, and different nucleic acid molecule sizes.

[0092] According to an embodiment of the method of normalizing the mass of nucleic acids in each of a plurality of test samples of the present disclosure, the magnetic particles do not include pendant carboxyl functional moieties and / or pendant amine functional moieties.

[0093] According to an embodiment of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, the magnetic particles do not include a binding partner that specifically binds to the nucleic acid. According to an embodiment, the term "binding partner that specifically binds to the nucleic acid" refers to a protein, peptide, and / or nucleic acid that can specifically bind to the nucleic acid. For example, the magnetic particles do not include a binding partner that is a nucleic acid, avidin, antibody, aptamer, receptor, or receptor ligand that is complementary to the nucleic acid in the input sample or test sample. According to an embodiment of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, the magnetic particles do not include biotin.

[0094] According to an aspect of the present disclosure, a method for normalizing the mass of nucleic acids in each of a plurality of test samples includes adding a binding mixture to each container to generate a normalization mixture in each container.

[0095] The binding mixture includes: i) a quantity of magnetic particles having pendant hydroxyl functional groups, ii) a chelating agent, iii) a binding buffer, and iv) an alcohol.

[0096] The amount of magnetic particles with pendant hydroxyl functional groups contained in the binding mixture is sufficient to reversibly and non-specifically bind nucleic acid with a binding capacity ranging from about 1 nanogram to about 5 micrograms. The amount of magnetic particles with pendant hydroxyl functional groups contained in the binding mixture can be calculated, for example, by determining the binding capacity of the magnetic particles with pendant hydroxyl functional groups and considering the amount of nucleic acid desired in the test sample.

[0097] The chelating agent included in the conjugation mixture according to embodiments of the present disclosure is present in the conjugation mixture at a concentration of about 0.5 mM to about 5 mM. The chelating agent included in the conjugation mixture according to embodiments of the present disclosure is one or more of the following: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA).

[0098] The binding buffer included in the binding mixture comprises a buffered aqueous solution of a chaotrope, which may be one or more of the following: urea, guanidinium bromide (guanidinium hydrobromide or guanidinium monohydrobromide), guanidinium iodide (guanidinium hydroiodide), guanidinium chloride (guanidinium hydrochloride), guanidinium thiocyanate (guanidinium thiocyanate), guanidinium nitrate (guanidinium nitrate), guanidinium sulfate (guanidinium sulfate), guanidinium carbonate (guanidinium carbonate), sodium iodide, and sodium perchlorate. The chaotrope included in the binding buffer according to embodiments of the present disclosure is present in the binding buffer at a concentration of about 0.1 M to about 6 M.

[0099] The buffer contained in the binding buffer may be an acetate buffer containing potassium acetate or sodium acetate adjusted with acetic acid to have a pH in the range of about pH 4 to about pH 6.

[0100] According to an embodiment of the present disclosure, the binding buffer does not contain PEG.

[0101] According to an embodiment of the present disclosure, the binding buffer comprises an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate, and has a pH ranging from about pH 4 to about pH 6.

[0102] The alcohols included in the bonding mixture may include one or more of the following: methanol, ethanol, and isopropanol.

[0103] According to an embodiment of the present disclosure, the volume to volume ratio of the alcohol to the binding mixture is in the range of about 0.25:1 to about 1.75:1. According to an embodiment of the present disclosure, the volume to volume ratio of the alcohol to the binding buffer is in the range of about 1:1 to about 1.25:1. According to an embodiment of the present disclosure, the volume to volume ratio of the alcohol to the binding buffer is about 1.125:1.

[0104] According to aspects of the present disclosure, the average size of the nucleic acids bound to the magnetic particles can be controlled by varying the volume to volume ratio of alcohol to binding buffer.

[0105] As shown in Figures 3 and 4, a larger volume to volume ratio of alcohol to binding buffer selects smaller nucleic acids (enriched to an average size of about 100 bp to about 300 bp in length at a ratio of alcohol to binding buffer of 1.75:1) for binding to the magnetic beads, and thus selectively recovers smaller nucleic acids in the final test sample. Conversely, a smaller volume ratio of alcohol to binding buffer selects larger nucleic acids (enriched to an average size of about 500 bp to about 1000 bp in length at a ratio of alcohol to binding buffer of 0.25:1) for binding to the magnetic beads, and thus selectively recovers larger nucleic acids in the final test sample. Thus, by appropriately selecting a volume to volume ratio of alcohol to binding buffer in the binding mixture in the range of 0.25:1 to 1.75:1, nucleic acids of desired sizes can be selectively recovered.

[0106] As described above, a binding mixture is added to each container to generate a normalization mixture in each container. The normalization mixture in each container is then incubated under binding conditions to promote binding of the nucleic acids to the magnetic particles in the normalization mixture. The binding conditions include a temperature in the range of about 15° C. to about 30° C., a time in the range of about 5 minutes to about 2 hours, such as a temperature in the range of about 20° C. to about 25° C., and a time in the range of about 5 minutes to about 15 minutes. The temperature may be higher or lower, such as in the range of about 4° C. to about 40° C., and the incubation time may be adjusted accordingly to be longer or shorter.

[0107] Separation of the magnetic particles with reversibly and non-specifically bound nucleic acids from the unbound nucleic acids in each vessel may be achieved by application of a magnetic field. The supernatant can then be removed, leaving the magnetic particles with reversibly and non-specifically bound nucleic acids in each vessel remaining in each vessel, i.e., separated from the unbound nucleic acids.

[0108] The magnetic particles with reversibly and non-specifically bound nucleic acids are separated from the unbound nucleic acids, whereby the reversibly and non-specifically bound nucleic acids are eluted from the magnetic particles to generate a plurality of test samples. According to an aspect of the present disclosure, elution of the nucleic acids from the magnetic particles includes incubating the beads in an elution buffer. According to an aspect of the present disclosure, the "elution buffer" is an aqueous liquid that promotes dissociation of the non-specifically bound nucleic acids from the magnetic particles, so that the nucleic acids are not substantially degraded and are released into the elution buffer. The elution buffer can be an aqueous liquid such as, but not limited to, 10 mM Tris-HCl, pH 7.0-8.5, 10 mM Tris-Acetate, pH 7.0-8.5, saline, phosphate buffered saline, water, and may further include EDTA, such as 0.1 mM EDTA. According to an aspect of the present disclosure, the elution buffer includes 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA.

[0109] After elution, each of the multiple test samples thereby produced contains an isolated nucleic acid having a particular mass, the mass of the isolated nucleic acid being substantially equal to the binding capacity of the magnetic particles, thereby providing a normalized mass of nucleic acid in each of the multiple test samples.

[0110] According to an embodiment of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, further comprising: washing the magnetic particles reversibly and non-specifically bound to the nucleic acid with a wash solution after application of a magnetic field and before elution. According to an embodiment of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, the wash solution comprises ethanol in a range of about 60% to about 100%. According to an embodiment of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, the wash solution comprises an aqueous ethanol solution containing about 60% ethanol, about 70% ethanol, about 80% ethanol, about 90% ethanol, or more. According to an embodiment of the method for normalizing the mass of a nucleic acid in each of a plurality of test samples of the present disclosure, the wash solution comprises an aqueous solution of about 80% ethanol.

[0111] According to aspects of the present disclosure, once test samples are obtained, two or more of the test samples can be pooled prior to analysis. According to aspects of the present disclosure, once test samples are obtained, two or more of the test samples can be pooled, if desired, in equal or unequal volumes prior to analysis. One or more nucleic acid analysis methods can be performed on the pooled samples.

[0112] According to aspects of the present disclosure, the unbound nucleic acids of the one or more normalized mixtures can be recovered for later use. The recovered unbound nucleic acids of the one or more normalized mixtures can be stored and / or used in any of a variety of nucleic acid analyses.

[0113] According to aspects of the disclosed methods, recovering unbound nucleic acids of one or more normalized mixtures comprises: transferring the unbound nucleic acids of one or more normalization mixtures to a corresponding container; adding the combined mixture to each corresponding vessel to generate a recovery mixture in each corresponding vessel; wherein the binding mixture comprises: a quantity of magnetic particles having pendant hydroxyl functional groups; Chelating agents, A binding buffer, and alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acids with a binding capacity in the range of 1 nanogram to 5 micrograms; and each of said recovery mixtures comprises a mass of nucleic acid greater than, less than, or equal to the binding capacity of said aliquot amount of magnetic particles; incubating each recovery mixture under binding conditions, thereby reversibly and non-specifically binding all or a portion of the nucleic acid to the magnetic particles to produce magnetic particles reversibly and non-specifically bound to recovered nucleic acid; Separating the magnetic particles reversibly and non-specifically bound to the recovered nucleic acid by applying a magnetic field; and and eluting the reversibly and non-specifically bound recovered nucleic acid from the magnetic particles.

[0114] A kit according to an embodiment of the present disclosure includes: magnetic particles having pendant hydroxyl functional groups; a binding buffer containing a buffered aqueous solution of a chaotrope; a wash solution; and an elution buffer. According to an embodiment of the present disclosure, the elution buffer includes 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution includes about 80% ethanol.

[0115] A kit according to an embodiment of the present disclosure includes: magnetic particles having pendant hydroxyl functional groups; a binding buffer containing a buffered aqueous solution of a chaotrope; a wash solution; an elution buffer; a chelating agent, and an alcohol. According to an embodiment of the present disclosure, the elution buffer includes 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution includes about 80% ethanol.

[0116] A kit according to an embodiment of the present disclosure includes: magnetic particles having pendant hydroxyl functional groups; a binding buffer comprising guanidine hydrochloride and an aqueous buffer of potassium acetate or sodium acetate, the pH of the binding buffer being in the range of about pH 4 to about pH 6; cleaning fluid; and Elution buffer.

[0117] According to an embodiment of the present disclosure, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the wash solution comprises about 80% ethanol.

[0118] A kit according to an aspect of the present disclosure includes: magnetic particles having pendant hydroxyl functional groups; a binding buffer comprising guanidine hydrochloride and an aqueous buffer of potassium acetate or sodium acetate, the pH of the binding buffer being in the range of about pH 4 to about pH 6; Cleaning fluid; Elution buffer; Chelating agents; and alcohol.

[0119] According to an embodiment of the present disclosure, the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. According to an embodiment of the present disclosure, the washing solution comprises about 60% to about 100% ethanol. According to an embodiment of the present disclosure, the washing solution comprises an aqueous solution of ethanol, the ethanol being about 60%, about 70%, about 80%, about 90%, about 95% or more in the aqueous solution. According to an embodiment of the present disclosure, the washing solution is an aqueous solution containing about 80% ethanol.

[0120] Embodiments of the compositions and methods of the present invention are described in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the compositions and methods of the present invention. EXAMPLES

[0121] Materials and methods used in the examples are with modifications where indicated.

[0122] To make 36 mL of pH adjusted potassium acetate: [Table 1]

[0123] Transfer approximately 10 mL of the solution to a clean tube and measure the pH, which should be between 5.1 and 5.3.

[0124] To make 50 mL of Binding Buffer: [Table 2]

[0125] In this example, the final concentrations of the binding buffer components were: 4.35 M guanidine hydrochloride, 0.58 M potassium acetate, final pH adjusted to pH 4.1-4.4 using acetic acid.

[0126] Transfer approximately 10 mL of the solution to a clean tube and measure the pH, which should be between 4.15 and 4.35.

[0127] Washing buffer 80% ethanol

[0128] Elution buffer 10mM Tris-HCl, pH8.0, 0.1mM EDTA.

[0129] Master binding mix for each reaction vessel containing 50 µL of sample with nucleic acid to be normalized: 25 μL 20 mM EDTA 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL) - 22.5 μg total Binding buffer 80 μL 90 μL 100% ethanol

[0130] [Example 1]

[0131] For each reaction vessel containing 50 μL of PCR reaction to be normalized, Make a master binding mix containing: 25 μL 20 mM EDTA 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL) - 22.5 μg total Binding buffer 80 μL 90μL of 100% ethanol.

[0132] Once the master binding mix is ​​prepared, vortex the tube containing it thoroughly (>5 seconds) to ensure that the beads are evenly distributed.

[0133] Add 196 μL (single channel) of the master ligation mixture to each reaction vessel (tube) containing 50 microliters of DNA-containing aqueous solution (input sample) and perform three cycles of pipetting (cycle = aspirate / dispense).

[0134] Once the master binding mix has been aliquoted into each reaction vessel to form the normalization mix, set the multichannel pipette to 150 µL and pipette the mix for 10 cycles to completely disperse the beads in the normalization mix.

[0135] Additional mixing may be required to properly disperse the beads.

[0136] The normalization mixture is incubated stationary at room temperature for 10 minutes.

[0137] After the 10 minute incubation, place the tube on a magnet for 1 minute. The time may vary depending on the strength of the magnet. Leave the beads on the magnet until the solution is clear.

[0138] Remove the supernatant with a multichannel pipette set to 200 µL - NOTE: The total volume of the supernatant is approximately 246 µL. Two aspirations are required to remove all the supernatant.

[0139] If recovery of unbound DNA is anticipated, the supernatant is transferred to a clean tube, 3 μL of 50 mg / mL magnetic particles containing pendant hydroxyl functional groups is added, pipette mixed for 10 cycles to completely disperse the beads, and then incubated stationary for 10 minutes at room temperature.

[0140] If recovery of unbound DNA is not required, discard the supernatant.

[0141] Once the solution in the tube becomes clear, indicating that the magnetic particles containing pendant hydroxyl functional groups and the bound nucleic acid have been separated from the unbound nucleic acid, a magnetic field is applied and, while the tube is still in contact with the magnet, 200 μL of freshly made 80% ethanol is added to wash the magnetic particles containing pendant hydroxyl functional groups and the bound nucleic acid.

[0142] Incubate on magnet at room temperature for 30 seconds.

[0143] The supernatant is removed and discarded.

[0144] Repeat the 80% ethanol wash step by adding 200 μL of 80% ethanol, incubate on the magnet for 30 seconds at room temperature, remove and discard the supernatant. Using a set of clean p20 multichannel pipette tips, set the p20 volume to 15 μL and carefully remove any residual ethanol from the bottom of the tube.

[0145] The pellet containing the nucleic acid-binding pendant hydroxyl functionalized magnetic particles is allowed to air dry by placing the tube on a magnet for 3 minutes, leaving the cap of the tube open.

[0146] Remove the tube from the magnet and add 22 µL of elution buffer to the pellet, pipetting repeatedly until all beads are sufficiently resuspended (10 pipette cycles).

[0147] Remove magnet and incubate at room temperature for 2 minutes.

[0148] Place the tube on the magnet for 2 minutes. This time may vary depending on the strength of the magnet. Leave the beads on the magnet until the solution becomes clear.

[0149] Transfer 20 μL of the supernatant to a new tube.

[0150] The expected total DNA recovery is approximately 100 to 150 ng (approximately 5 to 6.5 ng / μL).

[0151] If sequencing, pool equal volumes of all libraries, a minimum of 2 µL per library.

[0152] The combined library is mixed thoroughly. The final pool is quantified on Qubit. The size of the final pool is assessed on the LabChip.

[0153] [Example 2]

[0154] Effect of different ratios of ethanol to binding buffer on the size distribution of captured DNA

[0155] The ratio of ethanol to binding buffer was varied and the procedure of Example 1 was followed. An equal amount of 400 ng of sheared DNA was included in each input sample.

[0156] The master binding mix used contained the following: For each reaction vessel containing a sample of 400 ng of sheared DNA in 50 μL of aqueous liquid, 25 μL 20 mM EDTA 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL) - 22.5 μg total Binding buffer 80 μL 10μL, 20μL, 30μL, or 40μL of 100% ethanol.

[0157] As shown in the graph of Figure 2A, varying the ratio of ethanol to binding buffer results in a change in the size range of DNA fragments that bind to and elute from magnetic particles containing pendant hydroxyl functional groups. Increasing the amount of ethanol increases the separation of smaller DNA fragments. Figure 2B is an image of a gel analysis of the size of isolated DNA, showing that different size ranges of DNA fragments are isolated depending on the ratio of ethanol to binding buffer.

[0158] [Example 3]

[0159] Effect of different ratios of ethanol to binding buffer on the size distribution of captured DNA

[0160] The ratios of ethanol (EtOH) and binding buffer (BB) were varied and the procedure of Example 1 was followed. An equal amount of 400 ng of sheared DNA was included in each input sample.

[0161] The master binding mix used contained the following: For each reaction vessel containing 400 ng of sheared DNA sample in 50 μL of aqueous liquid, 25 μL 20 mM EDTA, 1μ of magnetic particles containing pendant hydroxyl functional groups (22.5μg / μL) – 22.5μg total, 80 μL Binding Buffer (BB), Volumes of 100% ethanol to achieve the following ratios: 0.25:1 ethanol:binding buffer, 0.50:1 ethanol:binding buffer, 0.75:1 ethanol:binding buffer, 1.00:1 ethanol:binding buffer, 1.25:1 ethanol:binding buffer, 1.50:1 ethanol:binding buffer, 1.75:1 ethanol:binding buffer. As a control, ethanol was omitted.

[0162] FIG. 3 shows the results of a gel analysis of varying ethanol to binding buffer ratios. Compared to DNA samples isolated using various ratios of ethanol to binding buffer, the DNA ladder in the leftmost lane (Gene Ladder 50 bp) shows the variation in the size range of DNA fragments that bind to and elute from the magnetic particles containing pendant hydroxyl functional groups. Increasing ethanol results in increased separation of smaller DNA fragments. FIG. 4 shows an image of a gel analysis of the size of the isolated DNA, showing that DNA fragments of different sizes are isolated depending on the ratio of ethanol to binding buffer. An ethanol to binding buffer ratio of 1.125 results in a bell-shaped size distribution. Table 3 shows the average size in base pairs (bp) of the sheared DNA fragments isolated using various ratios of ethanol to binding buffer. [Table 3]

[0163] [Example 4]

[0164] Effect of binding buffer (BB) versus the aqueous component (Aqu) of the "normalized mixture" containing DNA in an aqueous liquid on the size distribution of captured DNA.

[0165] Following the method of Example 1, each input sample contained an equal amount of 400 ng of sheared DNA substituted for the ratio of binding buffer to the aqueous component.

[0166] The master binding mix used contained the following: For each reaction vessel containing 400 ng of sheared DNA sample in 50 μL of aqueous liquid, 25 μL 20 mM EDTA, 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL) – 22.5 μg total, a volume of binding buffer to achieve a ratio of 2.55:1 binding buffer:Aqu, 2.35:1 binding buffer:Aqu, 2.16:1 binding buffer:Aqu, 1.96:1 binding buffer:Aqu, 1.76:1 binding buffer:Aqu, 1.57:1 binding buffer:Aqu, 1.37:1 binding buffer:Aqu, 1.18:1 binding buffer:Aqu; 90μL of 100% ethanol.

[0167] Figure 5 shows the results of a gel analysis of varying ratios of ethanol to binding buffer. Size distribution shows that the DNA ladder in the leftmost lane (Gene Ladder 50 bp) does not significantly affect the size range of DNA fragments that bind to and elute from magnetic particles containing pendant hydroxyl functional groups, as compared to isolated DNA samples using various ratios of aqueous component (Aqu) to binding buffer (BB) of a "normalized mixture" containing DNA in an aqueous liquid.

[0168] [Example 5]

[0169] Effect of pH of normalization mixture on size distribution of captured DNA.

[0170] The pH was varied and the procedure of Example 1 was followed. An equal amount of sheared DNA, 400 ng, was included in each input sample.

[0171] The master binding mix used contained: For each reaction vessel containing 400 ng of sheared DNA sample in 50 μL of aqueous liquid, 25 μL 20 mM EDTA 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL) - 22.5 μg total Binding buffer 80 μL 90μL of 100% ethanol.

[0172] The final normalized mixtures had a pH of 4.15 to 4.35 ("standard"), pH 5.0, pH 5.5, or pH 6.

[0173] Figure 6 shows the results of a gel analysis of varying pH of the normalization mixture. The DNA ladder in the leftmost lane (Gene Ladder 50 bp) is compared in terms of size distribution to DNA samples isolated using the indicated pH. Increasing the pH has a minor effect on the size range of DNA fragments that bind to and elute from magnetic particles containing pendant hydroxyl functional groups, eliminating fragments below 150 bp.

[0174] [Example 6]

[0175] The disclosed method was performed by two separate operators with various amounts of input DNA: 25ng, 50ng, 100ng, 250ng, 350ng, 500ng, 625ng, and 750ng. However, as can be seen in Figure 7, when magnetic particles containing pendant hydroxyl functional groups were added to each sample containing the same amount of different amounts of DNA, equal amounts of DNA were recovered, indicating that this product and procedure achieves normalization by total mass of DNA.

[0176] XP beads are carboxylate beads in a PEG:NaCl solution that are designed to bind to all the DNA in the tube, then washed to remove the salt and eluted. These are the standard bead and buffer types used to purify DNA; a commercially available example is Beckman AgencourtBeads.

[0177] FIG. 8 is a graph showing library recovery and percent cluster balancing, illustrating the replicability and reproducibility achieved using the method of the present disclosure. Various estimated input DNA amounts were used, between 500 ng and 1100 ng. The magnetic particles used, containing pendant hydroxyl functional groups, were normalized after PCR at 500 ng or higher. The amount of DNA recovered achieved 100% normalization across 88 libraries (+ / - 1.35-fold difference. Total average of DNA recovered: approx. 100 ng (approx. 11.5 nm). Clustering achieved approx. 94% normalization; + / - 1.38-fold difference, excluding 5 libraries below the % cluster threshold; + / - 1.48-fold difference across 88 libraries).

[0178] [Example 7]

[0179] Bead-based library normalization

[0180] In this example, 24 genomic DNA samples were used to generate 24 amplified libraries. For this, 10 ng of genomic DNA input material was used, and the fragmentation time was 10 minutes. Amplification included 7 cycles of PCR, generating 24 amplified libraries, which were normalized as follows:

[0181] Normalized magnetic beads containing pendant hydroxyl functional groups were suspended in 100% ethanol to yield a concentration of 22.5 μg / μL of magnetic beads.

[0182] The normalized magnetic beads were then mixed with binding buffer by adding 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL, i.e., 22.5 μg total) to 108 μL of binding buffer, which in this example contained 3.31 M guanidine HCl, 0.44 M potassium acetate, and 4.76 M EDTA.

[0183] The ethanol:binding buffer master mix containing the magnetic beads was generated by mixing 75 μL of 100% ethanol with 109 μL of binding buffer / magnetic beads. The volume ratio of 100% ethanol to binding buffer was 0.69:1.

[0184] For normalization, 25 μL of each amplified library was placed into separate wells of a 96-well PCR plate, and then 184 μL of master mix containing normalization magnetic beads was added to each well. The final concentration of EDTA was 2.79 mM. The material in each well was mixed thoroughly until homogenized and then incubated at room temperature for 8 minutes.

[0185] The 96-well PCR plate was then placed on a magnetic stand for 5 minutes at room temperature until the supernatant was completely clear and the normalization magnetic beads had settled to the bottom of the wells.

[0186] The clear supernatant was then removed without disturbing the pellet of normalized magnetic beads. Once removed, the supernatant, containing the non-normalized library, was transferred to a clean tube and frozen. If desired, the tube may be thawed and purified for further recovery.

[0187] With the 96-well plate containing the pellets of magnetic normalized beads still on the magnetic stand, 200 μL of 80% ethanol was added to each normalized magnetic bead pellet and the plate was incubated at room temperature for 30 seconds. The ethanol supernatant was then carefully removed with a pipette. This was repeated for a total of two washes with 80% ethanol. Once the ethanol was removed, the washed pellets of normalized magnetic beads were allowed to air dry at room temperature for 3 minutes.

[0188] The dried normalized magnetic beads were then resuspended in 23 μL of elution buffer in each well and mixed thoroughly until homogenized. The elution buffer used in this example was a solution of 10 mM Tris-HCl, pH 8.0 and 0.1 mM EDTA. The resuspended normalized magnetic beads were then incubated at room temperature for 2 minutes.

[0189] After incubation, the 96-well PCR plate was placed on the magnetic stand for 2 minutes at room temperature, after which the supernatant was completely clear.

[0190] 20 μL of the cleared supernatant (elution sample) was transferred from each well to the corresponding wells of a new 96-well plate.

[0191] Five microliters of each eluted sample was removed and pooled with the other samples into a single 1.5 mL tube to create a pooled library. Quantification of the pooled library can be performed using fluorometric methods to determine concentration. qPCR can also be used to quantify DNA library templates and obtain optimal cluster density.

[0192] A sample of the pooled library was examined by electrophoresis to confirm that the desired library sizing was achieved: 1.20 fold difference across the 24 amplified libraries after normalization, see Figures 9 and 10. Figure 9 is an image of the gel electrophoresis results using a sample of each of the 24 normalized amplified libraries. Figure 10 is a graph of the fragment size analysis results using each of the 24 normalized amplified libraries.

[0193] At this stage, the pooled library was used for cluster generation using standard Illumina® protocols.

[0194] [Example 8]

[0195] Bead-based library normalization

[0196] In this example, eight genomic DNA samples were used to generate eight amplified libraries. For this, 10 ng of genomic DNA input material was used, and the fragmentation time was 10 minutes. The amplification included 7 cycles of PCR, generating eight such amplified libraries. These were then normalized as follows:

[0197] Normalized magnetic beads containing pendant hydroxyl functional groups were suspended in 100% ethanol to yield a concentration of 22.5 μg / μL of magnetic beads.

[0198] The normalized magnetic beads were then mixed with binding buffer by adding 1 μL of magnetic particles containing pendant hydroxyl functional groups (22.5 μg / μL, i.e., 22.5 μg total) to 108 μL of binding buffer, which in this example contained 3.31 M guanidine HCl, 0.44 M potassium acetate, and 4.76 M EDTA.

[0199] A master mix of ethanol:binding buffer containing magnetic beads was generated by mixing 75 μL of 100% ethanol with 109 μL of binding buffer / magnetic beads. The volume to volume ratio of 100% ethanol to binding buffer was 0.69:1.

[0200] For normalization, 25 μL of each amplified library was placed into separate wells of a 96-well PCR plate, and then 184 μL of master mix containing normalization magnetic beads was added to each well. The final concentration of EDTA was 2.79 mM. The material in each well was then thoroughly mixed until homogenized, and then incubated at room temperature for 8 minutes.

[0201] The 96-well PCR plate was then placed on a magnetic stand at room temperature for 5 minutes until the supernatant was completely clear and the normalization magnetic beads had settled to the bottom of the wells.

[0202] The clear supernatant was then removed without disturbing the pellet of normalized magnetic beads. Once removed, the supernatant containing the non-normalized library was transferred to a clean tube and frozen. If desired, the tube may be thawed and purified for further recovery.

[0203] With the 96-well plate containing the pellets of normalized magnetic beads still on the magnetic stand, 200 μL of 80% ethanol was added to each normalized magnetic bead pellet, and the plate was then incubated at room temperature for 30 seconds. The ethanol supernatant was then carefully removed with a pipette. This was repeated for a total of two washes with 80% ethanol. Once the ethanol was removed, the washed pellets of normalized magnetic beads were air-dried at room temperature for 3 minutes.

[0204] The dried normalized magnetic beads were then resuspended in 23 μL of elution buffer in each well and mixed thoroughly until homogenized. The elution buffer used in this example was a solution of 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. The resuspended normalized magnetic beads were then incubated at room temperature for 2 minutes.

[0205] After incubation, the 96-well PCR plate was placed on a magnetic stand at room temperature for 2 minutes, after which the supernatant became completely clear.

[0206] 20 μL of the cleared supernatant (elution sample) was transferred from each well to the corresponding wells of a new 96-well plate.

[0207] 5 μL of each eluted sample was removed and pooled with the other samples into a single 1.5 mL tube to create a pooled library. Quantification of the pooled library can be performed using a fluorescent assay to determine concentration. qPCR may be used to quantify the DNA library templates and obtain optimal cluster density.

[0208] Samples of the pooled libraries were examined by electrophoresis to confirm that the desired library sizing was achieved: 1.38 fold difference across the eight amplified libraries after normalization, see Figure 11. Figure 11 is an image of the gel electrophoresis results using samples of each of the eight normalized amplified libraries.

[0209] At this stage, the pooled library was used for cluster generation using standard Illumina® protocols.

[0210] item

[0211] Item 1. A method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the method comprising: providing a plurality of input samples comprising nucleic acids in an aqueous liquid, each of the plurality of input samples being in a separate container; adding a binding mixture to each container to generate a normalized mixture in each container; The binding mixture comprises: a quantity of magnetic particles comprising pendant hydroxyl functional groups; Chelating agents, A binding buffer, and alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of said plurality of input samples comprises a mass of nucleic acid greater than the binding capacity of said quantity of magnetic particles; incubating each normalization mixture under binding conditions, thereby reversibly and non-specifically binding a portion of the nucleic acids to the magnetic particles; separating the magnetic particles with reversibly and non-specifically bound nucleic acids from unbound nucleic acids by application of a magnetic field; and eluting the reversibly and non-specifically bound nucleic acids from the magnetic particles to generate a plurality of test samples; wherein each of the plurality of test samples comprises an isolated nucleic acid having a mass; The mass of the isolated nucleic acid is approximately equal to the binding capacity of the magnetic particles, thereby providing a normalized mass of nucleic acid in each of the plurality of test samples. Item 2. The method according to Item 1, wherein no internal standard is added to the container. Item 3. The method of any one of items 1 to 2, wherein the mass of nucleic acid in the input sample is not quantified. Item 4. The method according to any one of Items 1 to 3, wherein the magnetic particles do not contain a binding partner that specifically binds to the nucleic acid. Item 5. The method of item 4, wherein the magnetic particles do not contain a binding partner that is a nucleic acid, biotin, avidin, an antibody, an aptamer, a receptor, or a receptor ligand. Item 6. The method of any one of items 1 to 5, further comprising: pooling the plurality of test samples to generate a pooled test sample; and sequencing at least a portion of the nucleic acids in the pooled test sample. Item 7. The method according to any one of Items 1 to 6, wherein the chaotrope comprises one or more of the following: urea, guanidinium bromide (guanidinium hydrobromide or guanidinium monohydrobromide), guanidinium iodide (guanidinium hydroiodide), guanidinium chloride (guanidinium hydrochloride), guanidinium thiocyanate (guanidinium thiocyanate), guanidinium nitrate (guanidinium nitrate), guanidinium sulfate (guanidinium sulfate), guanidinium carbonate (guanidinium carbonate), sodium iodide, and sodium perchlorate. Item 8. The method according to any one of items 1 to 7, wherein the binding buffer does not contain PEG. Item 9. The method according to any one of items 1 to 8, wherein the magnetic particles do not contain carboxyl functional groups and / or amine functional groups. Item 10. The method according to any one of Items 1 to 9, wherein the chelating agent is one or more of the following: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA). Item 11. The method according to any one of Items 1 to 10, wherein the alcohol is one or more of the following: methanol, ethanol, and isopropanol. Item 12. The method according to any one of Items 1 to 11, wherein the binding buffer comprises an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate and has a pH in the range of about pH 4 to about pH 6. Item 13. The method according to any one of items 1 to 12, further comprising: recovering the unbound nucleic acids of one or more normalization mixtures. Item 14. The method according to any one of items 1 to 13, comprising: transferring the unbound nucleic acids of one or more normalization mixtures to corresponding containers; adding the binding mixture to each corresponding vessel to generate a recovery mixture in each corresponding vessel; The binding mixture comprises: a quantity of magnetic particles comprising pendant hydroxyl functional groups; Chelating agents, A binding buffer, and alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of said recovery mixtures comprises a mass of nucleic acid greater than, less than, or equal to the binding capacity of said quantity of magnetic particles; incubating each recovery mixture under binding conditions, thereby reversibly and non-specifically binding all or a portion of said nucleic acid to said magnetic particles to produce magnetic particles reversibly and non-specifically bound to said recovered nucleic acid; Separating the magnetic particles reversibly and non-specifically bound to the recovered nucleic acid by application of a magnetic field; and Eluting the reversibly and non-specifically bound recovered nucleic acid from the magnetic particles. Item 15. The method of any one of Items 1 to 14, wherein eluting the nucleic acid from the magnetic particles comprises incubating the beads in an elution buffer. Item 16. The method according to any one of Items 1 to 15, wherein the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. Item 17. The method according to any one of Items 1 to 16, further comprising: washing the magnetic particles reversibly and non-specifically bound to the nucleic acid with a washing solution after application of a magnetic field and before elution. Item 18. The method according to any one of Items 1 to 17, wherein the cleaning solution comprises about 60% to about 100% ethanol. Item 19. The method of any one of items 1 to 18, wherein the volume to volume ratio of the alcohol to the binding buffer is within the range of about 0.25:1 to about 1.75:1. Item 20. The method of any one of items 1 to 19, wherein the volume to volume ratio of the alcohol to the binding buffer is within the range of about 1:1 to about 1.25:1. Item 21. The method of any of items 1 to 20, wherein the volume to volume ratio of the alcohol to the binding buffer is about 1.125:1. Item 22. The method of any one of claims 1 to 21, wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group. Item 23. A kit comprising: magnetic particles comprising pendant hydroxyl functional groups; a binding buffer comprising a buffered aqueous solution of a chaotrope; a wash solution; and an elution buffer. Item 24. The kit according to Item 23, further comprising a chelating agent and an alcohol. Item 25. The kit according to Item 23 or 24, wherein the binding buffer comprises an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate and has a pH in the range of about pH 4 to about pH 6. Item 26. The kit according to any one of Items 23 to 25, wherein the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA. Item 27. The kit according to any one of Items 23 to 26, wherein the washing solution contains about 60% to about 100% ethanol. Item 28. The kit of any of Items 23 to 27, wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group. Item 29. A composition for use in a method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the composition comprising: an input sample or a plurality of input samples, each input sample comprising nucleic acid in an aqueous liquid, each of said plurality of samples being in a separate container; generating a combined mixture in each vessel, a normalized mixture in each vessel, The binding mixture comprises: a quantity of magnetic particles comprising pendant hydroxyl functional groups; Chelating agents, A binding buffer, and alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and non-specifically binding to nucleic acid with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and Each of the plurality of input samples comprises a mass of nucleic acid greater than the binding capacity of the quantity of magnetic particles. Item 30. The composition of item 29, wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group. Item 31. The composition according to Item 29 or Item 30, wherein no internal standard is present in the container. Item 32. The composition according to any one of Items 29 to 31, wherein the magnetic particles do not contain a binding partner that specifically binds to the nucleic acid. Item 33. The composition according to any one of Items 29 to 32, wherein the magnetic particles do not contain a binding partner that is a nucleic acid, biotin, avidin, an antibody, an aptamer, a receptor, or a receptor ligand. Item 34. The composition according to any one of Items 29 to 33, wherein the chaotrope comprises one or more of the following: urea, guanidinium bromide (guanidinium hydrobromide or guanidinium monohydrobromide), guanidinium iodide (guanidinium hydroiodide), guanidinium chloride (guanidinium hydrochloride), guanidinium thiocyanate (guanidinium thiocyanate), guanidinium nitrate (guanidinium nitrate), guanidinium sulfate (guanidinium sulfate), guanidinium carbonate (guanidinium carbonate), sodium iodide, and sodium perchlorate. Item 35. The composition according to any one of Items 29 to 34, wherein the binding buffer does not contain PEG. Item 36. The composition according to any one of Items 29 to 35, wherein the magnetic particles do not contain carboxyl functional groups and / or amine functional groups. Item 37. The composition according to any one of Items 29 to 36, wherein the chelating agent is one or more of the following: diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA). Item 38. The composition according to any one of Items 29 to 37, wherein the alcohol is one or more of the following: methanol, ethanol, and isopropanol. Item 39. The composition according to any of Items 29 to 38, wherein the binding buffer comprises an aqueous buffer solution of guanidine hydrochloride and potassium acetate or sodium acetate and has a pH in the range of about pH 4 to about pH 6. Item 40. The composition of any of items 29 to 39, wherein the volume to volume ratio of the alcohol to the binding buffer is within the range of about 0.25:1 to about 1.75:1. Item 41. The composition of any of Items 29 to 40, wherein the volume to volume ratio of the alcohol to the binding buffer is within the range of about 1:1 to about 1.25:1. Item 42. The composition of any of Items 29 to 41, wherein the volume to volume ratio of the alcohol to the binding buffer is about 1.125:1.

[0212] Any patents or publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0213] The compositions and methods described herein represent presently preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications and other uses can be made without departing from the scope of the invention as set forth in the claims. [Explanation of symbols]

[0214] [Figure 1] Input DNA: Binding: Binding Separation: Separation Ethanol Wash: Ethanol wash Elution: Transfer: Normalized DNA: Normalized DNA 60 Minutes: 60 minutes Unbound DNA: Unbound DNA Purified Unbound DNA: Purified Unbound DNA Optional Unbound DNA Recovery Protocol (B): Optional Unbound DNA Recovery Protocol (B) [Figure 2A] Fluorescence: Aligned Time (sec): Aligned time (sec) [Figure 2B] +10uL EtOH: +10μL Ethanol +20uL EtOH: +20μL Ethanol +30uL EtOH: +30μL Ethanol +40uL EtOH: +40μL Ethanol 400ng input: 400ng input Promega Sheared DNA [Figure 3] GeneRuler 50 bp: GeneRuler 50bp [Figure 4] Promega Sheared DNA EtOH:BB: Ethanol:Binding Buffer (Sheared): (Sheared) [Figure 5] GeneRuler 50 bp: GeneRuler 50bp BB:Aqu: Binding buffer: aqueous component [Figure 6] Ladder: Ladder Standard: Standard [Figure 7] Normalization Beads Comparison: Normalization Beads Comparison Replicate: Replicate Beads: Beads DNA Recovered: Recovered DNA DNA Input into Library Prep: DNA input into library preparation [Figure 8] Library Recovery Ratio and % Cluster Balancing: Library Recovery Ratio and % Cluster Balancing Cluntering: Clustering Lib Yield Ratio: Library yield rate [Figure 10] Fluorescence: Size:

Claims

1. 1. A method for normalizing the mass of a nucleic acid in each of a plurality of test samples, the method comprising: (I) providing a plurality of input samples comprising nucleic acids in an aqueous liquid, each of the plurality of input samples being in a separate container; (II) adding a binding mixture to each container to generate a normalization mixture in each container, said binding mixture comprising: (i) a quantity of magnetic particles comprising pendant hydroxyl functional groups; (ii) a chelating agent, (iii) binding buffer, and (iv) alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and nonspecifically binding to nucleic acids with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of the plurality of input samples comprises a mass of nucleic acid greater than the binding capacity of the quantity of magnetic particles; (III) incubating each normalization mixture under binding conditions, thereby reversibly and non-specifically binding a portion of the nucleic acids to the magnetic particles; (IV) separating the magnetic particles with reversibly and non-specifically bound nucleic acids from unbound nucleic acids by applying a magnetic field; and (V) eluting the reversibly and non-specifically bound nucleic acids from the magnetic particles to produce a plurality of test samples, wherein each of the plurality of test samples comprises isolated nucleic acids having a mass approximately equal to the binding capacity of the magnetic particles, thereby providing a normalized nucleic acid mass in each of the plurality of test samples.

2. The method of claim 1, wherein no internal standard is added to the container.

3. 3. The method of claim 1, wherein the mass of nucleic acid in the input sample is not quantified.

4. The method of claim 1 , wherein the magnetic particles do not contain a binding partner that specifically binds to the nucleic acid.

5. The method of claim 4 , wherein the magnetic particles do not comprise a binding partner that is a nucleic acid, biotin, avidin, an antibody, an aptamer, a receptor, or a receptor ligand.

6. 10. The method of claim 1 or 4, further comprising: (VI) pooling the plurality of test samples to produce a pooled test sample; and sequencing at least some of the nucleic acids in the pooled test sample.

7. 10. The method of claim 1, wherein the chaotrope comprises one or more of the following: Urea, guanidinium bromide (guanidine hydrobromide or guanidine monohydrobromide), guanidinium iodide (guanidine hydroiodide), guanidinium chloride (guanidine hydrochloride), guanidine thiocyanate (guanidinium thiocyanate), guanidine nitrate (guanidinium nitrate), guanidine sulfate (guanidinium sulfate), guanidine carbonate (guanidinium carbonate), sodium iodide, and sodium perchlorate

8. 8. The method of claim 1, wherein the binding buffer is PEG-free.

9. The method of claim 1 , wherein the magnetic particles do not contain carboxyl and / or amine functional moieties.

10. 2. The method of claim 1, wherein the magnetic particles comprising pendant hydroxyl functional groups comprise a spacer covalently bonded to the magnetic particle and / or the coating of the magnetic particle and having a hydroxyl functional group extending from the magnetic particle and / or the coating of the magnetic particle, wherein the spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group.

11. 11. The method of claim 1, 4, 7 or 10, wherein the chelating agent is one or more of the following: Diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and N,N-bis(carboxymethyl)glycine (NTA)

12. 11. The method of claim 1, 4, 7 or 10, wherein the alcohol is one or more of the following: Methanol, ethanol, and isopropanol

13. 11. The method of claim 1, wherein the binding buffer comprises an aqueous buffer of guanidine hydrochloride and potassium acetate or sodium acetate, and has a pH in the range of about pH 4 to about pH 6.

14. 10. The method of claim 1, further comprising: (VII) recovering the unbound nucleic acids of one or more normalized mixtures.

15. 15. The method of claim 14, wherein the recovering step comprises: (a) transferring the unbound nucleic acids of one or more normalization mixtures to corresponding containers; (b) adding the binding mixture to each corresponding container; (c) generating a combined mixture in each corresponding container, the combined mixture comprising: (i) a quantity of magnetic particles comprising pendant hydroxyl functional groups; (ii) a chelating agent, (iii) binding buffer, and (iv) alcohol, wherein the binding buffer comprises a buffered aqueous solution of a chaotrope; the quantity of magnetic particles is capable of reversibly and nonspecifically binding to nucleic acids with a binding capacity in the range of about 1 nanogram to about 5 micrograms; and each of the recovery mixtures comprises a mass of nucleic acid greater than, less than, or equal to the binding capacity of the quantity of magnetic particles; (d) incubating each recovery mixture under binding conditions, thereby reversibly and non-specifically binding all or a portion of the nucleic acids to the magnetic particles to produce magnetic particles reversibly and non-specifically bound to recovered nucleic acids; (e) reversibly and non-specifically separating the magnetic particles bound to the recovered nucleic acids by applying a magnetic field; and (f) eluting the reversibly and non-specifically bound recovered nucleic acids from the magnetic particles.

16. 16. The method of claim 1, wherein eluting the nucleic acid from the magnetic particles comprises incubating the beads in an elution buffer.

17. 17. The method of claim 16, wherein the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA.

18. 16. The method of claim 1 or 4 or 7 or 10 or 15, further comprising: (VIII) After application of the magnetic field and before elution, the magnetic particles reversibly and nonspecifically bound to the nucleic acid are washed with a washing solution.

19. 20. The method of claim 18, wherein the cleaning solution comprises about 60% to about 100% ethanol.

20. 16. The method of claim 1, wherein the alcohol and binding buffer are present in a volume to volume ratio of alcohol to binding buffer in the range of about 0.25:1 to about 1.75:

1.

21. 16. The method of claim 1, wherein the alcohol and binding buffer are present in a volume to volume ratio of alcohol to binding buffer in the range of about 1:1 to about 1.25:

1.

22. 16. The method of claim 1, wherein the alcohol and binding buffer are present in a volume to volume ratio of alcohol to binding buffer of about 1.125:

1.

23. Kit including: magnetic particles containing pendant hydroxyl functional groups; a binding buffer containing a buffered aqueous solution of a chaotrope; cleaning solution; and Elution buffer 24. The kit of claim 23, further comprising a chelating agent and an alcohol.

25. 25. The kit of claim 23 or 24, wherein the binding buffer comprises an aqueous buffer of guanidine hydrochloride and potassium acetate or sodium acetate, and has a pH in the range of about pH 4 to about pH 6.

26. 25. The kit of claim 23 or 24, wherein the elution buffer comprises 10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA.

27. 25. The kit of claim 23 or 24, wherein the washing solution comprises about 60% to about 100% ethanol.

28. 25. The kit of claim 23 or 24, wherein: the magnetic particles containing pendant hydroxyl functional groups include spacers covalently bonded to and extending from the magnetic particles and / or coatings thereof, the spacers having hydroxyl functional groups; The spacer comprises a chain of at least three atoms covalently bonded to the hydroxyl functional group.

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