Nucleic Acid Capture, Concentration, and Purification

Abstract

An example of a kit includes a flow cell assembly. The flow cell assembly includes a reaction chamber, a temperature-controlled flow path in selective fluid communication with an inlet of the reaction chamber, and a filter disposed within the temperature-controlled flow path. The reaction chamber includes recesses separated by gap regions and capture primers attached within each of the recesses. The filter i) blocks enriched biological sample-polymer complexes formed within the temperature-controlled flow path at a first temperature, and ii) allows the passage of enriched biological sample and polymers released from the complexes within the temperature-controlled flow path at a second temperature.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 047,103, filed July 1, 2020, and Dutch Application No. 2026080, filed July 17, 2020, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Two classes of nucleic acids exist in living organisms (e.g., humans, animals, etc.): ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Both RNA and DNA can be classified into different types, such as messenger RNA, ribosomal RNA, nuclear DNA, and cytoplasmic DNA. The various types of nucleic acids can be analyzed for various purposes, such as research, diagnostics, forensics, and genome sequencing.

[0003] Introduction A first aspect disclosed herein is a kit comprising a flow cell assembly, the flow cell assembly including: a reaction chamber having recesses separated by gap regions and capture primers attached within each of the recesses; a temperature-controlled flow channel in selective fluid communication with an inlet of the reaction chamber; and a filter disposed within the temperature-controlled flow channel, the filter i) blocking concentrated deoxyribonucleic acid (DNA)-methylcellulose complexes formed within the temperature-controlled flow channel at a first temperature, and ii) allowing passage of concentrated DNA and methylcellulose released from the complexes within the temperature-controlled flow channel at a second temperature.

[0004] In one example of the first embodiment, the kit further comprises a sample fluid, the sample fluid comprising an aqueous carrier, a DNA sample, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt. In one example, the polymer chemically inert to DNA hybridization is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and combinations thereof, having a weight-average molecular weight ranging from about 500 to less than about 200,000. In another example, the DNA sample is present in the sample fluid at a first molar concentration ranging from about 1 pM (picomolar concentration) to about 1 mM, the methylcellulose is present in the sample fluid in an amount ranging from about 0.5 wt % to about 20 wt % based on the total weight of the sample fluid, the polymer chemically inert to DNA hybridization is present in the sample fluid in an amount ranging from greater than 0 wt % to about 20 wt % based on the total weight of the sample fluid, and the salt is present in the sample fluid at a second molar concentration ranging from greater than 0 M to about 2 M.

[0005] In an example of the first embodiment, the flow cell assembly further comprises a bypass line in fluid communication with the inlet of the temperature-controlled channel and the outlet of the temperature-controlled channel, a first bypass valve that controls the flow of sample fluid to the inlet of the temperature-controlled channel, and a second bypass valve that controls the flow of concentrated DNA and methylcellulose to the reaction chamber.

[0006] In one example of the first embodiment, at least one surface of the temperature control channel comprises a heating plate.

[0007] It should be understood that any of the features of the first aspect disclosed herein may be combined together in any desired manner and / or configuration to achieve the benefits as described in this disclosure, including, for example, capturing and concentrating DNA prior to analysis, processing, etc.

[0008] A second aspect disclosed herein is a method, comprising: combining a DNA sample with a solution to form a sample fluid, the solution being comprised of an aqueous carrier, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt, the solution being at a temperature ranging from about 5°C to about 30°C; and heating the sample fluid to at least the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex in the aqueous carrier.

[0009] In one example of the second embodiment, the method further comprises cooling the sample fluid below the gelling temperature of the methylcellulose, thereby disentangling the DNA-methylcellulose complex and releasing the DNA sample and the methylcellulose.

[0010] In another example of the second embodiment, the method further comprises increasing the concentration of salt in the solution, thereby decreasing the gelation temperature of the methylcellulose.

[0011] In one example of the second embodiment, the DNA sample comprises cell-free DNA, library DNA, whole genome amplified DNA, or a combination thereof.

[0012] In one example of the second embodiment, the DNA sample contains a plurality of DNA inserts of different sizes, including small and large DNA inserts, wherein at least some of the large DNA inserts are entangled in the DNA-methylcellulose complexes and at least some of the small DNA inserts are unentangled in the DNA-methylcellulose complexes, and the method further comprises performing a purification process after heating to separate at least some of the small DNA inserts from the DNA-methylcellulose complexes. In one example, the purification process involves filtration, centrifugation, decantation, or a combination thereof. In another example, the method further comprises cooling the DNA-methylcellulose complexes below the gelling temperature of the methylcellulose, thereby unentangling the DNA-methylcellulose complexes and releasing at least some of the large DNA inserts and the methylcellulose.

[0013] It should be understood that any features of the second aspect may be combined together in any desired manner. Furthermore, it should be understood that any combination of features of the first and / or second aspects can be used together and / or combined with any of the examples disclosed herein to achieve the benefits described in this disclosure, including, for example, DNA capture and / or DNA purification.

[0014] A third aspect disclosed herein is a method, comprising: introducing a sample fluid into a temperature-controlled channel having a filter disposed therein, the sample fluid comprising an aqueous carrier, a DNA sample, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt; heating the temperature-controlled channel as the sample fluid is introduced to raise the temperature of the sample fluid contained therein to at least the gelation temperature of the methylcellulose, thereby forming DNA-methylcellulose complexes within the temperature-controlled channel; continuing the flow of the sample fluid through the temperature-controlled channel as the temperature-controlled channel is heated, thereby concentrating a plurality of DNA-methylcellulose complexes in the filter within the temperature-controlled channel; and cooling the temperature-controlled channel to lower the temperature of the sample fluid contained therein below the gelation temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complexes and releasing the DNA sample and methylcellulose, thereby allowing the DNA sample and methylcellulose to pass through the filter.

[0015] In an example of the third aspect, the method further comprises selecting a heating temperature for the temperature-controlled channel according to a concentration of salt in the sample fluid.

[0016] In another example of the third aspect, the method further comprises transporting the DNA sample and methylcellulose from the temperature-controlled channel to a flow cell.

[0017] It should be understood that any features of the third aspect may be combined together in any desired manner. Further, it should be understood that any combination of features of the first aspect and / or the second aspect and / or the third aspect can be used together and / or combined with any of the examples disclosed herein to achieve the benefits as described in this disclosure, including, for example, concentrating DNA prior to analysis, processing, etc.

[0018] A fourth aspect disclosed herein is a method, comprising: introducing a sample fluid into a flow cell, the sample fluid comprising an aqueous carrier, a DNA sample, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt; the flow cell comprising reaction chambers having recesses separated by gap regions and capture primers attached within each of the recesses; initiating seeding and hybridization of at least a portion of the DNA sample in at least some of the recesses; and heating the flow cell to slightly increase the temperature of the sample fluid contained therein. the flow cell to at least the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex with the unbound DNA sample; introducing an additional amount of sample fluid into the flow cell; cooling the flow cell to reduce the temperature of the sample fluid contained therein to below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complex and releasing the DNA sample and methylcellulose; and initiating seeding and hybridization of at least a portion of the DNA sample from the released DNA sample and the additional sample fluid in at least some of the recesses.

[0019] In an example of the fourth aspect, the method further comprises selecting a heating temperature for the flow cell according to a concentration of salt in the sample fluid.

[0020] It should be understood that any features of the fourth aspect may be combined together in any desired manner. Furthermore, it should be understood that any combination of features of the first aspect and / or the second aspect and / or the third aspect and / or the fourth aspect can be used together and / or combined with any of the examples disclosed herein to achieve the benefits described in this disclosure, including, for example, improved DNA seeding onto the flow cell surface.

[0021] A fifth aspect disclosed herein is a kit, the kit comprising a sample fluid, the sample fluid comprising an aqueous carrier, a deoxyribonucleic acid (DNA) sample, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt or solvent for adjusting the gelling temperature of the methylcellulose.

[0022] In one example of the fifth embodiment, the DNA sample is present in the sample fluid at a first molar concentration ranging from about 1 pM to about 1 mM, the methylcellulose is present in the sample fluid in an amount ranging from about 0.5 wt % to about 20 wt % based on the total weight of the sample fluid, the polymer that is chemically inert to DNA hybridization is present in the sample fluid in an amount ranging from greater than 0 wt % to about 20 wt % based on the total weight of the sample fluid, and the sample fluid comprises a salt, and the salt is present in the sample fluid at a second molar concentration ranging from greater than 0 M to about 2 M.

[0023] In one example of the fifth aspect, the kit further comprises a flow cell assembly, the flow cell assembly including a temperature-controlled flow path for receiving a sample fluid, a reaction chamber having recesses separated by gap regions, capture primers attached in each of the recesses, and an inlet in selective fluid communication with the temperature-controlled flow path, and a filter disposed in the temperature-controlled flow path, the filter i) blocking enriched DNA sample-methylcellulose complexes generated in the temperature-controlled flow path when the sample fluid is exposed to a first temperature, and ii) allowing passage of the enriched DNA sample and methylcellulose released from the complexes in the temperature-controlled flow path at a second temperature.

[0024] It should be understood that any features of the fifth aspect may be combined together in any desired manner. Furthermore, it should be understood that any combination of features of the first aspect and / or the second aspect and / or the third aspect and / or the fourth aspect can be used together, and / or the fifth aspect can be used together, and / or in combination with any of the examples disclosed herein to achieve the benefits as described in this disclosure, including, for example, improved DNA seeding onto the flow cell surface. [Brief explanation of the drawings]

[0025] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numbers correspond to similar, but perhaps not identical, components. For purposes of brevity, reference numbers or features having a previously mentioned function may or may not be described in conjunction with other drawings in which they appear. [Figure 1] FIG. 1 is a flow diagram illustrating an example of a method for deoxyribonucleic acid (DNA) capture. [Figure 2] FIG. 2 is a schematic diagram of the method of FIG. 1. [Figure 3] FIG. 2 is a schematic diagram of another example of a method involving the method of FIG. 1 and purification. [Figure 4] FIG. 1 is a flow diagram showing an example of a DNA concentration method. [Figure 5] Figure 5A is a schematic diagram of a flow cell assembly. Figure 5B is a cross-sectional view along line 5B-5B of Figure 5A, showing sample fluid flow and complex formation in the temperature-controlled channel as the temperature is increased. Figure 5C is a cross-sectional view along line 5C-5C of Figure 5A, showing a filter within the temperature-controlled channel and the concentration of DNA-methylcellulose complexes in the filter. Figure 5D shows the temperature-controlled channel of Figure 5C as the temperature is decreased. Figure 5E is an enlarged perspective view of an example of a reaction chamber with a patterned sequencing surface. [Figure 6] FIG. 1 is a flow diagram showing an example of a method for improving DNA seeding onto the flow cell surface. [Figure 7] 7A-7F illustrate the method of FIG. [Figure 8] 1 is a graph showing turbidity (Y-axis, optical density) versus salt concentration (X-axis, M) in an aqueous solution containing 1 wt % methylcellulose and 16 wt % polyethylene glycol when exposed to different temperatures. [Figure 9] 1 is a graph showing sample intensity (Y-axis, normalized fluorescence units) versus DNA insert size (X-axis, base pairs) for sample fluids exposed to example purification methods disclosed herein at different temperatures. [Figure 10] 1 is a graph showing sample intensity (Y-axis, normalized fluorescence units) versus DNA insert size (X-axis, base pairs) for a control fluid and a sample fluid exposed to an example capture method disclosed herein. [Figure 11] 1 is a graph showing sample intensity (Y-axis, normalized fluorescence units) versus DNA insert size (X-axis, base pairs) for a control fluid and two sample fluids exposed to an example capture method disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0026] DNA can be used in a variety of applications, such as replication and sequencing. Some sequencing systems utilize relatively low DNA loading inputs to produce high-quality data. Therefore, minimizing DNA sample loss is desirable. In the examples disclosed herein, DNA is captured and concentrated to maximize DNA loading available for purification, plating, and the like. More specifically, the examples disclosed herein utilize methylcellulose, a thermoreversible precipitation polymer, as a DNA complexing agent. Under controlled temperature conditions, methylcellulose forms complexes with or releases DNA from the complexes. The methods disclosed herein utilize methylcellulose-DNA complexes for DNA capture, concentration, purification, and / or plating.

[0027] definition Terms used herein should be understood to have their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are described below.

[0028] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0029] The terms "comprising," "including," "containing," and various forms of these terms are synonymous and intended to be equally broad. Furthermore, unless expressly stated otherwise, examples of comprising, including, or having an element or elements having a particular characteristic can include additional elements, regardless of whether the additional elements have that characteristic.

[0030] The term "adapter," as used herein, refers to a linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by ligation or tagging. Suitable adapter lengths can range from about 10 bases to about 100 bases, or from about 12 bases to about 60 bases, or from about 15 bases to about 50 bases. Adapters can comprise any combination of nucleotides and / or nucleic acids. In some examples, adapters can comprise a sequence complementary to at least a portion of a primer, for example, a primer comprising a universal nucleotide sequence (e.g., a P5 or P7 sequence). In some examples, adapters can comprise a sequencing primer sequence or a sequencing binding site. A combination of different adapters can be incorporated into a nucleic acid molecule, such as a DNA fragment.

[0031] As used herein, "cell-free DNA" (cfDNA) refers to DNA or RNA that is not contained within cells. By way of example, cfDNA can be cell-free fetal DNA or cell-free tumor DNA. Cell-free fetal DNA is fetal DNA or RNA that circulates freely in the maternal bloodstream. Cell-free tumor DNA is fragmented DNA derived from a tumor (i.e., circulating tumor DNA or ctDNA). The examples disclosed herein may also be suitable for capturing, concentrating, etc., viral and / or bacterial DNA.

[0032] As used herein, the term "complementary DNA" (cDNA) refers to DNA synthesized from a single-stranded RNA template, for example, in a reaction catalyzed by the enzyme reverse transcriptase.

[0033] As used herein, the term "deposition" refers to any suitable application technique, which may be manual or automated, and which in some cases results in the modification of surface properties. Generally, deposition may be carried out using evaporation techniques, coating techniques, grafting techniques, etc. Some specific examples include chemical vapor deposition (CVD), spray coating (e.g., ultrasonic spray coating), spin coating, dunk or dip coating, doctor blade coating, puddle dispensing, flow-through coating, aerosol printing, screen printing, microcontact printing, inkjet printing, etc.

[0034] As used herein, the term "recess" refers to a discrete recessed feature in a substrate having a surface opening at least partially surrounded by a gap region of the substrate. The recess can have a variety of shapes at the surface opening, such as, for example, a circle, an ellipse, a square, a polygon, a star (with any number of vertices), etc. The cross section of the recess taken perpendicular to the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc. By way of example, the recess can be a well or two interconnected wells. The recess can also have a more complex structure, such as a ridge, a stepped structure, etc.

[0035] As used herein, the term "DNA insert" refers to a DNA fragment in a sample. The size of the DNA can range from about 100 bases or base pairs to 100 million bases or base pairs, or more. The DNA inserts in any given sample can have a size distribution ranging from small to large. Small DNA inserts generally refer to DNA fragments having about 100 bases or base pairs to about 1,000 bases or base pairs (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or any number in between).Large DNA inserts generally refer to DNA fragments having more than 1,000 bases or base pairs (e.g., 1200, 1300, 1500, 2000, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7,500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12500, 13000, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,500, 26,000, 26,500, 27,000, 27,500 0, 28,000, 28,500, 29,500, 30,000, 30,500, 31,000, 31,500, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 42,000, 45,000 00, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160 ,000, 170,000, 180,000, 200,000, 225,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, "800,000, 850,000, 900,000, 1,000,000, 1,250,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 30,000,000, 40,000,000, 50,000,000, 75,000,000, 100,000,000 or more, or any number in between."

[0036] As used herein, the terms "fluid communication" and "fluidically connected" refer to two spatial regions being connected to one another such that a liquid or gas can flow between the two spatial regions. For example, a temperature-control channel may be in fluid communication with a reaction chamber such that fluid can flow freely or in a controlled manner from the temperature-control channel into the reaction chamber. The terms "fluid communication" or "fluidically connected" allow two spatial regions to be in fluid communication through one or more valves, restrictors, or other fluidic components configured to control or regulate the flow of fluid through the system.

[0037] The term "flow cell" refers to a vessel having a chamber in which a reaction can be carried out, an inlet for delivering reagents to the chamber, and an outlet for removing reagents from the chamber. In some cases, the chamber allows for detection of a reaction occurring within the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of arrays, optically labeled molecules, etc.

[0038] As used herein, a "fragment" refers to a portion or piece of genetic DNA material.

[0039] Also, as used herein, "genomic DNA" (gDNA) and "whole genome amplified DNA" refer to high molecular weight (>1000 base pairs (bp)) chromosomal DNA.

[0040] As used herein, "library DNA" refers to a collection of DNA fragments that contain adapters at both ends. In some examples, the DNA fragments may be fragments of gDNA or cDNA.

[0041] As used herein, a "nucleotide" comprises a nitrogenous heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of nucleic acid sequences. Examples of nucleotides include ribonucleotides and deoxyribonucleotides. In ribonucleotides (nucleotides of RNA), the sugar is ribose, while in deoxyribonucleotides (nucleotides of DNA), the sugar is deoxyribose, i.e., a sugar lacking the hydroxyl group at the 2' position of the ribose. The nitrogenous heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), as well as modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), as well as modified derivatives or analogs thereof. The C-1 atom of the deoxyribose is linked to the N-1 atom of the pyrimidine or the N-9 atom of the purine. By way of example, the phosphate groups can be in mono-, di-, tri-, tetra-, penta-, or hexa-phosphate form. It should further be understood that while these nucleotides are naturally occurring nucleotides, non-natural nucleotides, modified nucleotides, or analogs of the foregoing nucleotides can also be used.

[0042] As used herein, the phrase "a polymer that is chemically inert to DNA hybridization" refers to a polymer that does not participate in or otherwise interfere with DNA hybridization.

[0043] Also, as used herein, the term "primer" refers to a nucleic acid molecule (e.g., an adapter attached to a DNA fragment) that can hybridize to a target sequence. As an example, an amplification primer can serve as a starting point for template amplification and cluster generation. In another example, a synthesized nucleic acid (template) strand can contain a site to which a primer (e.g., a sequencing primer) can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid strand. Any primer can contain any combination of nucleotides or their analogs. In some examples, a primer is a single-stranded oligonucleotide or polynucleotide. The primer length can be any number of bases and can contain various natural or unnatural nucleotides. In one example, a sequencing primer is a short strand ranging from 10 to 60 bases, or from 20 to 40 bases.

[0044] "Reaction chamber" refers to an area within a flow cell where a reaction can be carried out. The reaction chamber may include a recess in which sequencing chemicals (e.g., amplification primers) are immobilized.

[0045] As used herein, a "temperature-controlled channel" is an enclosed area through which a liquid sample can flow and which can be heated or cooled by an internal component (e.g., a heating plate) or an external component (e.g., a laser).

[0046] The aspects and examples described and claimed herein can be understood in light of the above definitions.

[0047] Sample fluid In any of the examples disclosed herein, a sample fluid is used. The sample fluid includes a DNA sample in a solution of an aqueous carrier, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt or solvent for adjusting the gelation temperature of the methylcellulose. In some examples, the solution is composed of an aqueous carrier, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt or solvent for adjusting the gelation temperature of the methylcellulose. In one example, the sample fluid includes a DNA sample in a solution of an aqueous carrier, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt.

[0048] The DNA sample may include cell-free DNA, library DNA, whole genome amplified DNA, or a combination thereof. The DNA sample may be single-stranded or double-stranded DNA depending on the application for which the sample fluid is used. For example, single-stranded DNA may be contained in a sample fluid used for sequencing applications, and single-stranded or double-stranded DNA may be contained in a sample fluid to be purified.

[0049] In one example, the DNA sample is present in the sample fluid at a molar concentration ranging from about 1 pM to about 1 mM (1000 μM). In another example, the DNA sample is present in the sample fluid at a molar concentration ranging from about 10 pM to about 950 μM, e.g., from about 25 pM to about 750 μM, from about 500 pM to about 500 nM, from about 500 nM to about 500 μM, etc.

[0050] The aqueous carrier in the solution can be water, a salt solution, or a buffer solution (e.g., a weak acid and one of its salts (conjugate base) or a weak base and one of its salts (conjugate acid)). Examples of buffer solutions include tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl) buffer, tris(hydroxymethyl)aminomethane (TRIS) buffer, or saline sodium citrate (SSC) buffer. The aqueous carrier makes up the remainder of the sample fluid, and therefore its amount can vary depending on the amounts of other components.

[0051] Methylcellulose is a thermoreversible hydrogel that precipitates or gels in aqueous solution upon exposure to heat. For example, methylcellulose is commercially available from Dow Chemical Co. under the trade name METHOCEL®. In one example, the lower critical solution temperature (LCST) transition range of methylcellulose ranges from about 25°C to about 60°C, which may depend on the grade of methylcellulose and / or the concentration of methylcellulose in the sample fluid. Other factors, such as the pH of the solution, may also affect the LCST. Furthermore, at higher concentrations, the LCST is more measurable than at lower concentrations. Salt in the sample fluid may alter the LCST transition range. For example, higher salt concentrations can cause methylcellulose to precipitate at lower temperatures. For example, when the salt concentration is in the range of about 1.5M to about 2M, the LCST transition range of methylcellulose ranges from about 5°C to about 45°C. Below the LCST, methylcellulose dissolves in the aqueous carrier; above the LCST, methylcellulose precipitates from the aqueous carrier. This behavior is reversible, so methylcellulose can be controllably switched between dissolved and precipitated states.

[0052] In one example, the methylcellulose is present in the sample fluid in an amount ranging from about 0.5% to about 20% by weight, based on the total weight of the sample fluid, and in another example, the methylcellulose is present in the sample fluid in an amount ranging from about 1% to about 15% by weight, such as from about 1.5% to about 10% by weight, or from about 5% to about 7.5% by weight, based on the total weight of the sample fluid.

[0053] The sample fluid also includes a polymer that is chemically inert to DNA hybridization. In the examples disclosed herein, the polymer that is chemically inert to DNA hybridization is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and combinations thereof, having a weight average molecular weight (g / mol or Daltons) ranging from about 500 to less than about 200,000. Unlike methylcellulose, the chemically inert polymer does not precipitate from the sample fluid upon heat exposure.

[0054] In one example, the chemically inert polymer is present in the sample fluid in an amount ranging from greater than 0% to about 20% by weight, based on the total weight of the sample fluid. In another example, the chemically inert polymer is present in the sample fluid in an amount ranging from about 0.5% to about 18% by weight, such as from about 1.5% to about 16% by weight, or from about 5% to about 15% by weight, based on the total weight of the sample fluid.

[0055] The sample fluid also includes a salt or solvent that can be included to adjust the gelation temperature of the methylcellulose. When a salt solution or buffer is used as the aqueous solution, additional salts or solvents may or may not be added. Examples of suitable salts include sodium chloride (NaCl), sodium bromide (NaBr), and sodium iodide (NaI). In one example, salts containing potassium, calcium, magnesium, or ammonium cations can be used. In another example, salts containing carbonate, sulfate, phosphate, or nitrate anions can be used. In another example, solvents such as ethylene glycol, propylene glycol, and / or glycerol can be used to shift the gelation temperature.

[0056] In one example, the total salt concentration in the sample fluid ranges from greater than 0 M to about 2 M. In other examples, the salt is present in the sample fluid at a molar concentration ranging from about 0.25 M to about 1.75 M, e.g., from about 0.5 M to about 1.5 M, from about 1 M to about 2 M, from about 0.1 M to about 1 M, etc.

[0057] To create the sample fluid, a solution can be prepared by mixing together an aqueous carrier, methylcellulose, a chemically inert polymer, and a salt or solvent. The temperature of the solution can be maintained below the precipitation / gelation temperature of the methylcellulose. This helps prevent premature precipitation of the methylcellulose and can also prevent any salts from precipitating out of solution. A DNA sample can be added to the solution to form the sample fluid. Some examples of the methods disclosed herein can include increasing the concentration of salt or the amount of solvent in the solution, thereby lowering the gelation temperature of the methylcellulose.

[0058] Any example of a sample fluid may be included in the kit. The components of the kit may depend on the application for which the sample fluid will be used. For example, the kit may include a flow cell if the sample fluid will be used for sequencing.

[0059] Some exemplary methods are described herein that utilize exemplary sample fluids. The sample fluids discussed in connection with the methods specifically refer to salts. However, it should be understood that any of the sample fluids disclosed herein (e.g., containing a solvent instead of a salt) can be used in any of the methods disclosed herein.

[0060] DNA capture An example of a DNA capture method 100 is shown in FIG. 1. The method 100 includes combining a DNA sample with a solution comprising an aqueous carrier, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt to form a sample fluid, the solution being at a temperature ranging from about 5° C. to about 30° C. (reference numeral 102). The sample fluid is heated to at least the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex in the aqueous carrier (reference numeral 104). It should be understood that any example of a sample fluid can be used in the DNA capture method 100.

[0061] Method 100 is shown schematically in Figure 2. Sample fluid 12, to the left of the arrow in Figure 2, is below the gelation temperature of methylcellulose 14. At this temperature, methylcellulose 14, chemically inert polymer 18, and salt 22 are dissolved in aqueous carrier 20. Salt 22 in aqueous carrier 20 reduces the solubility of methylcellulose and serves to reduce charge interactions with DNA, thus inducing DNA to adhere to the precipitated methylcellulose.

[0062] The sample fluid 12 to the right of the arrow in Figure 2 is above the gelation temperature of methylcellulose 14. At this temperature, the chemically inert polymer 18 and salt 22 remain dissolved in the aqueous carrier 20. However, the methylcellulose 14 precipitates from solution. The precipitated methylcellulose polymer chains physically interact with the DNA sample 16 to form DNA-methylcellulose complexes 10. The DNA molecules become entangled in the precipitated methylcellulose polymer chains. The interaction between the DNA molecules and the methylcellulose polymer chains is non-covalent.

[0063] The temperature to which the sample fluid 12 is heated depends on the gelation temperature of the methylcellulose 14. The gelation temperature of the methylcellulose 14 depends, in part, on the concentration of the methylcellulose 14 in the sample fluid 12. Thus, some examples of the method 100 may involve selecting a heating temperature according to the concentration of the methylcellulose 14 in the sample fluid 12. Generally, the temperature to which the sample fluid 12 is heated ranges from about 25°C to about 60°C. As a specific example, when the concentration of the methylcellulose is in the range of about 1% to about 6% by weight, the heating temperature ranges from about 40°C to about 60°C. As another specific example, when the concentration of the methylcellulose is in the range of about 5% to about 12% by weight, the heating temperature ranges from about 25°C to about 50°C. As described herein, the gelation temperature of the methylcellulose 14 may also depend on the concentration of the salt 22 in the sample fluid 12. Thus, some examples of the method 100 may involve selecting a heating temperature according to the concentration of the salt in the sample fluid. As a specific example, when the salt concentration is in the range of about 0.5 M to about 1.5, the heating temperature is in the range of about 40° C. to about 60° C. As another specific example, when the salt concentration is in the range of about 1.8 M to about 2 M, the heating temperature is in the range of about 5° C. to about 60° C.

[0064] Heating may be performed using any suitable heat source, which may be an internal component of the fluidic device containing the sample fluid 12, or an external component that is not part of the fluidic device containing the sample fluid. An example of an internal component may include a heating plate integrated into the flow path (see, e.g., FIG. 5B). An example of an external component may include a heating plate on which the fluidic device is placed, or a laser directed at the fluidic device.

[0065] The duration for which the sample fluid 12 is heated depends on the application for which the method 100 is being performed. For example, if the method 100 is used to capture and concentrate a DNA sample in a particular region of a flow path, heating may be performed until a desired amount of sample fluid has been introduced into the flow path.

[0066] If or when it is desirable to release the DNA captured in the DNA-methylcellulose complex 10, the method 100 may further include cooling the sample fluid 12 below the gelation temperature of the methylcellulose 14, thereby disentangling the DNA-methylcellulose complex 10 and releasing the DNA sample 16 and the methylcellulose 14. Below the gelation temperature, the methylcellulose 14, the chemically inert polymer 18, and the salt 22 are dissolved in the aqueous carrier 20.

[0067] In some examples, cooling is passive, as the sample fluid 12 cools naturally to room temperature. Alternatively, cooling may be performed using any suitable cooling source, which may be an internal component of the fluidic device containing the sample fluid 12 or an external component that is not part of the fluidic device containing the sample fluid. An example of an internal component may include a thermoelectric cooler integrated into a flow path or reaction chamber. An example of an external component may include a fan directed toward the fluidic device.

[0068] 2, methylcellulose 14 acts as a DNA capture or release agent depending on the temperature to which sample fluid 12 is exposed, which may be desirable for a variety of applications including DNA capture, purification, or concentration.

[0069] DNA purification An example of the DNA capture method 100 can be used for capturing and purifying DNA. In one example, a purification method may be desirable when the DNA sample in the sample fluid 12 contains multiple DNA inserts of different sizes, including small and large DNA inserts. In one example, the DNA inserts are single-stranded DNA inserts. When multiple DNA inserts of different sizes are present in the sample fluid 12, the larger DNA inserts tend to interact with the precipitated methylcellulose polymer chains more than the smaller DNA inserts. Thus, at least some of the larger DNA inserts become entangled in the DNA-methylcellulose complex 10, and at least some of the small DNA inserts do not become entangled in the DNA-methylcellulose complex 10. This may be due to the size of the larger DNA inserts, which may physically prevent the smaller DNA inserts from entangling with the precipitated methylcellulose polymer chains.

[0070] An example of a purification method is shown schematically in Figure 3. This example of the method includes combining a DNA sample containing both small DNA inserts 16A and large DNA inserts 16B with a solution to form a sample fluid 12, heating the sample fluid 12 to at least the gelling temperature of the methylcellulose 14, thereby forming large DNA insert-polymer complexes 10' in the aqueous carrier 20, and performing a purification process after heating to separate at least a portion of the small DNA inserts 16A from the large DNA insert-polymer complexes 10'.

[0071] 3 is below the gelling temperature of methylcellulose 14. At this temperature, methylcellulose 14, chemically inert polymer 18, and salt 22 are dissolved in aqueous carrier 20. Small DNA insert 16A and large DNA insert 16B are dispersed in aqueous carrier 20.

[0072] Upon heating to at least the gelation temperature of methylcellulose 14, chemically inert polymer 18 and salt 22 remain dissolved in aqueous carrier 20, while methylcellulose 14 precipitates from solution. The precipitated methylcellulose polymer chains physically interact with at least some of the large DNA inserts 16B to form large DNA insert-polymer complexes 10'. At least some of the smaller DNA inserts 16A are not entangled with the methylcellulose polymer chains and are therefore not part of the large DNA insert-polymer complexes 10'.

[0073] The sample fluid 12 with the large DNA insert-polymer complexes 10' and the free small DNA inserts 16A therein may then be subjected to a purification process. The purification process may involve filtration, centrifugation, decantation, or a combination thereof. Filtration may be used to separate the large DNA insert-polymer complexes 10' from the small DNA insert 16A fluid and aqueous carrier 20 (and any components dissolved therein). Centrifugation may be used to separate the large DNA insert-polymer complexes 10' from the small DNA inserts 16A. In any instance where the fluid and small DNA inserts 16A are separated from the large DNA insert-polymer complexes 10' in the same container, decantation may be used to remove the aqueous carrier 20, any components dissolved therein, and the small DNA inserts 16A. In one example, centrifugation may be followed by decantation.

[0074] Fresh solution (without additional DNA sample) can be added to the separated complexes 10' before cooling to release the large DNA insert 16B. If or when it is desirable to release the large DNA insert 16B trapped in the DNA-methylcellulose complexes 10', this example can further include cooling the DNA-methylcellulose complexes 10' below the gelling temperature of the methylcellulose 14, thereby unentangling the complexes 10' and releasing the large DNA insert 16B and the methylcellulose 14. Below the gelling temperature, the methylcellulose 14, the chemically inert polymer 18, and the salt 22 are dissolved in the aqueous carrier 20.

[0075] DNA enrichment Another exemplary method is shown in Figure 4 at reference numeral 200. This exemplary method 200 can be used for DNA capture and enrichment. The method 200 includes introducing a sample fluid into a temperature-controlled flow channel having a filter disposed therein, the sample fluid including an aqueous carrier, a DNA sample, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt (reference numeral 202); heating the temperature-controlled flow channel as the sample fluid is introduced to raise the temperature of the sample fluid contained therein to at least the gelling temperature of the methylcellulose, thereby forming DNA-methylcellulose complexes within the temperature-controlled flow channel (reference numeral 204); continuing the flow of the sample fluid through the temperature-controlled flow channel as the temperature-controlled flow channel is heated, thereby concentrating a plurality of DNA-methylcellulose complexes in the filter within the temperature-controlled flow channel (reference numeral 206); and cooling the temperature-controlled flow channel to lower the temperature of the sample fluid contained therein below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complexes and releasing the DNA sample and methylcellulose, thereby allowing the DNA sample and methylcellulose to pass through the filter (reference numeral 208).

[0076] The method 200 is illustrated generally in Figures 5A-5E.

[0077] 5A illustrates a flow cell assembly 24 that can be used in method 200. Flow cell assembly 24 includes a flow cell 26, which includes a reaction chamber 28 (shown in FIG. 5E) having recesses separated by gap regions and a capture primer attached within each of the recesses, a temperature-controlled channel 30 in selective fluid communication with an inlet 32 ​​of reaction chamber 28, and a filter 34 (shown in FIG. 5C) disposed within temperature-controlled channel 30, which i) blocks concentrated deoxyribonucleic acid (DNA)-polymer complexes 10 formed within temperature-controlled channel 30 at a first temperature and ii) allows passage of concentrated DNA 16 and polymer (methylcellulose 14) released from complexes 10 within temperature-controlled channel 30 at a second temperature.

[0078] In the flow cell assembly 24, the temperature control channel 30 is positioned upstream of the flow cell 26. This positioning allows for capture and concentration of the DNA sample 16 from the sample fluid 12 before the DNA sample 16 is introduced into the reaction chamber 28 of the flow cell 26.

[0079] The temperature-controlled flow channel 30 may be a tube, a pipe, a microfluidic channel, etc. The temperature-controlled flow channel 30 may have any shape, volume, and length suitable for the purpose of capturing and concentrating a desired amount of DNA sample 16 in the sample fluid 12 before the sample fluid 12 is introduced into the reaction chamber 28 of the flow cell 26. The exemplary flow channel 30 shown in Figure 5A has a serpentine shape.

[0080] The temperature-controlled flow channel 30 can be heated and cooled such that the sample fluid 12 flowing through the flow channel 30 is also heated or cooled. In one example, the temperature-controlled flow channel 30 includes an internal heating component 36 (FIG. 5B), such as a heating plate, disposed within the flow channel 30 or defining one or more interior walls of the flow channel 30. In another example, the flow cell assembly 24 includes an external heating component 38 (FIG. 5B), such as a laser, disposed outside the flow channel 30. The external heating component 38 may be operatively positioned to direct heat toward the flow channel 30. Some examples of the temperature-controlled flow channel 30 also include an internal cooling component (not shown), such as a thermoelectric cooler, disposed within the flow channel 30 or defining one or more interior walls of the flow channel 30. Other examples of the temperature-controlled flow channel 30 include an external cooling component (not shown), such as a fan, disposed outside the flow channel 30. The internal or external component may also be a combination device capable of active heating and active cooling.

[0081] Although not shown, the internal or external heating component 36 or 38, and the internal or external cooling component (if used), may be operably connected to a temperature control unit that operates and controls the temperature of the component 36 or 38. Additionally, a temperature sensor may be positioned within the flow path 30 to provide the temperature control unit with real-time data of the temperature within the flow path 30 so that a desired internal temperature can be achieved and / or maintained.

[0082] The flow cell assembly 24 may also include a bypass line 40 in fluid communication with an inlet 48 of the temperature control channel 30 and an outlet 50 of the temperature control channel 30, a first bypass valve 42 for controlling the flow of sample fluid 12 to the inlet of the temperature control channel 30, and a second bypass valve 44 for controlling the flow of concentrated DNA 16 and polymer (methylcellulose 14) to the reaction chamber 28.

[0083] The bypass line 40 may be a tube, a pipe, a microfluidic channel, or the like. The bypass line 40 allows fluid to be directed from a reservoir 46 or other storage unit to the reaction chamber 28 without passing through the temperature-controlled flow path 30. The bypass line 40 may be used, for example, when the fluid delivered to the reaction chamber 28 does not contain the DNA sample 16, when heating the fluid is not desired, or when concentration of the DNA sample 16 is not desired. Examples of fluids that may be directed through the bypass line 40 rather than through the temperature-controlled flow path 30 include wash fluids, deblocking agent fluids, reaction fluids including polymerase, sequencing primers, nucleotides, etc.

[0084] The first bypass valve 42 is switchable between two positions: one position directs flow through the temperature control flow path 30 (thus closing the bypass line 40), and the other position directs flow through the bypass line 40 (thus closing the temperature control flow path 30). The second bypass valve 44 is also switchable between two positions: one position directs flow from the temperature control flow path 30 (not allowing backflow through the bypass line 40), and the other position directs flow through the bypass line 40 (thus not allowing backflow through the temperature control flow path 30). The arrows in FIG. 5A indicate the fluid flow when the valves 42, 44 are positioned to open the temperature control flow path 30 and close the bypass line 40. Any suitable valves can be used for the first and second bypass valves 42, 44.

[0085] The temperature control channel 30 may be connected to the inlet 32 ​​of the reaction chamber 28 through a manifold 70 or other fluid connector.

[0086] The temperature control channel 30 also includes a filter 34. The filter 34 can be secured within the temperature control channel 30 using adhesives, mechanical attachment mechanisms, or the like. In other examples, the filter 34 may be incorporated into the temperature control channel 30 during manufacturing. The filter 34 may be attached to the entire inner circumference of the temperature control channel 30. Thus, the filter 34 covers a cross-sectional area (e.g., parallel to the inlet 48 of the temperature control channel 30).

[0087] The filter 34 in the temperature-controlled flow channel 30 can have any suitable pore size that allows the aqueous carrier and any components dissolved therein to flow through, while blocking the DNA-methylcellulose complex 10 from flowing through. In one example, the pore size of the filter 34 ranges from about 1 μm to about 100 μm, allowing unbound DNA to migrate therethrough. Examples of suitable filter materials include nitrocellulose, nylon (polyamide), and the like.

[0088] In addition to the temperature-controlled flow channel 30, the flow cell assembly 24 also includes a flow cell 26. An example of a flow cell 26 will now be described in more detail with reference to Figures 5A and 5E.

[0089] In the example shown in FIG. 5A , the flow cell 26 includes eight reaction chambers 28. While eight reaction chambers 28 are shown, it should be understood that any number of reaction chambers 28 (e.g., a single reaction chamber 28, four reaction chambers 28, etc.) may be included in the flow cell 26. Each reaction chamber 28 is an area defined between two joined components (e.g., a substrate 52 and a lid or two substrates 52) to which a fluid (e.g., as described herein) can be introduced and removed. Each reaction chamber 28 may be isolated from each other reaction chamber 28 such that fluid introduced into any particular reaction chamber 28 does not flow into any adjacent reaction chambers 28. Some examples of fluids introduced into the reaction chambers 28 may introduce reaction components (e.g., a DNA sample 16, a polymerase, sequencing primers, nucleotides, etc.), wash solutions, deblocking agents, etc.

[0090] The reaction chamber 28 is at least partially defined by a substrate 52. The substrate 52 may be a single layer structure or may be a multi-layer structure (as shown in FIG. 5E).

[0091] Examples of suitable monolayer structural materials include epoxy siloxanes, glass, modified or functionalized glass, plastics (including acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefin / cycloolefin polymers (COP) (such as ZEONOR® from Zeon), polyimides, etc.), nylon (polyamide), ceramic / ceramic oxide, silica, fused silica, or silica-based materials, aluminum silicate, silicon and modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), silicon oxide (SiO2), tantalum pentoxide (Ta2O5) or other tantalum oxides (TaO x ), hafnium oxide (HfO2), carbon, metals, inorganic glass, etc.

[0092] Some examples of multilayer structures include glass or silicon having a coating layer of tantalum oxide or another ceramic oxide on its surface. Another example of a multilayer structure may include a silicon-on-insulator (SOI) substrate. Yet another example of a multilayer structure, as shown in FIG. 5E, includes an underlying support 54 (e.g., glass or silicon) having a patterned material 56 thereon. It should be understood that any material that can be selectively deposited or deposited and patterned to form recesses 58 and void regions 60 can be used for patterned material 56.

[0093] As an example of patterned material 56, an inorganic oxide may be selectively applied via vapor deposition, aerosol printing, or inkjet printing to support 52. Examples of suitable inorganic oxides include tantalum oxide (e.g., TaO), aluminum oxide (e.g., AlO), silicon oxide (e.g., SiO), hafnium oxide (e.g., HfO), etc.

[0094] As another example of the patterned material 56, a resin may be applied to the support 52 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, puddle dispensing, ultrasonic spray coating, doctor blade coating, aerosol printing, screen printing, microcontact printing, and the like. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, embossing techniques, molding techniques, microetching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane-based resins (e.g., POSS® from Hybrid Plastics), non-polyhedral oligomeric silsesquioxane epoxy resins, poly(ethylene glycol) resins, polyether resins (e.g., ring-opened epoxies), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (e.g., CYTOP® from Bellex), and combinations thereof.

[0095] As used herein, the term "polyhedral oligomeric silsesquioxane" refers to a hybrid intermediate between silica (SiO) and silicone (RSiO) (e.g., RSiO 1.5) refers to a chemical composition having the formula [RSiO 3 / 2 ] n where the R groups can be the same or different. Exemplary R groups of the polyhedral oligomeric silsesquioxanes include epoxy, azide / azido, thiol, poly(ethylene glycol), norbornene, tetrazine, acrylate, and / or methacrylate, or further, for example, alkyl, aryl, alkoxy, and / or haloalkyl groups.

[0096] In one example, the substrate 52 (single layer or multilayer) can have a rectangular sheet or panel having a diameter ranging from about 2 mm to about 300 mm, or a maximum dimension of up to about 10 feet (about 3 meters). In one example, the substrate 52 is a wafer having a diameter ranging from about 200 mm to about 300 mm, and in another example, the substrate 52 is a die having a width ranging from about 0.1 mm to about 10 mm. While exemplary dimensions are provided, it should be understood that a substrate 52 having any suitable dimensions can be used. As another example, a panel can be used that is a rectangular support having a surface area larger than a 300 mm round wafer.

[0097] In some examples, the reaction chamber 28 is etched into a glass substrate. In other examples, the reaction chamber 28 is patterned into a multilayer structure of patterned material 56 using photolithography, nanoimprint lithography, etc. In yet other examples, a separate material (not shown) can be applied to the substrate 52, such that the separate material defines the walls of the reaction chamber 28 and the substrate 52 defines the bottom of the reaction chamber 28.

[0098] In one example, the reaction chambers 28 have a substantially rectangular configuration. The length and width of the reaction chambers 28 may be smaller than the length and width, respectively, of the substrate 52 so that a portion of the substrate surface surrounding the reaction chamber 28 is available for attachment to a lid (not shown) or another substrate 52. In some cases, the width of each reaction chamber 28 may be at least about 1 mm, at least about 2.5 mm, at least about 5 mm, at least about 7 mm, at least about 10 mm, or more. In some cases, the length of each reaction chamber 28 may be at least about 10 mm, at least about 25 mm, at least about 50 mm, at least about 100 mm, or more. The width and / or length of each reaction chamber 28 may be greater than, less than, or between the values ​​specified above. In another example, the reaction chambers 28 are square (e.g., 10 mm x 10 mm).

[0099] The depth of the reaction chamber 28 can be as small as a monolayer thickness when microcontact printing, aerosol printing, or inkjet printing is used to deposit the distinct materials that define the walls of the reaction chamber 28. In other examples, the depth of the reaction chamber 28 can be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or greater. In one example, the depth can range from about 10 μm to about 100 μm. In another example, the depth can range from about 10 μm to about 30 μm. In yet another example, the depth is about 5 μm or less. It should be understood that the depth of the reaction chamber 28 can be greater than, less than, or between the values ​​specified above.

[0100] Each reaction chamber 28 is in fluid communication with an inlet and an outlet (not shown). The inlet and outlet of each reaction chamber 28 may be located at opposite ends of the flow cell 26. Alternatively, the inlet and outlet of each reaction chamber 28 may be located anywhere along the length and width of the reaction chamber 28 that allows for the desired fluid flow.

[0101] The inlets allow fluids to be introduced into the reaction chamber 28, and the outlets allow fluids to be extracted from the reaction chamber 28. Each of the inlets and outlets is fluidly connected to a fluid control system (e.g., including a reservoir 46, a pump, valves 42, 44, a waste container, etc.) that controls the introduction and removal of fluids.

[0102] 5E shows an example of a structure within reaction chamber 28. The structure includes recesses 58 separated by gap regions 60. The recesses 58 may be defined in substrate 52. In one example, the recesses 58 are defined in a single layer structure (e.g., etched in glass) or in a multi-layer structure of patterned material 56, as shown in FIG.

[0103] Many different layouts of the recesses 58 are possible, including regular, repeating, and irregular patterns. In one example, the recesses 58 are arranged in a hexagonal grid to provide close packing and improve density. Other layouts may include, for example, a rectilinear (rectangular) layout, a triangular layout, etc. In some examples, the layout or pattern may be an xy format of the recesses 58 in rows and columns. In some other examples, the layout or pattern may be a repeating arrangement of the recesses 58 and / or the interstitial regions 60. In yet other examples, the layout or pattern may be a random arrangement of the recesses 58 and / or the interstitial regions 60.

[0104] The layout or pattern of recesses 58 may be characterized in terms of the density of recesses 58 (number of recesses 58) in a defined area. For example, recesses 58 may be arranged at intervals of 1 mm 2 For example, they may be present at a density of about 2 million per mm 2 Approximately 100, 1mm per 2 Approximately 1,000 per 1mm 2 Approximately 100,000 per 1mm 2 Approximately 1 million per 1mm 2 Approximately 2 million per 1mm 2 Approximately 5 million per 1mm 2 Approximately 10 million per mm 2The density of the recesses 58 in the patterned material 56 can be adjusted to different densities, including densities of about 50 million per recess, or less. It should further be understood that the density of the recesses 58 in the patterned material 56 can be between one of the lower limit values ​​and one of the upper limit values ​​selected from the ranges above. By way of example, a high-density array can be characterized as having recesses 58 separated by less than about 100 nm, a medium-density array can be characterized as having recesses 58 separated by about 400 nm to about 1 μm, and a low-density array can be characterized as having recesses 58 separated by more than about 1 μm. While example densities are provided, it should be understood that any suitable density can be used. The density of the recesses 58 can depend in part on the depth of the recesses 58. In some cases, it may be desirable for the spacing between the recesses 58 to be even greater than the examples listed herein.

[0105] The layout or pattern of the recesses 58 may additionally or alternatively be characterized in terms of an average pitch, or the spacing from the center of a recess 58 to the center of an adjacent recess 58 (center-to-center spacing), or the spacing from the right edge of one recess 58 to the left edge of an adjacent recess 58 (edge-to-edge spacing). The pattern may be regular so that the coefficient of variation around the average pitch is small, or the pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch may be, for example, approximately about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, or about 100 μm. The average pitch of a particular pattern of recesses 58 may be between one of the lower and upper limits selected from the ranges above. In one example, the pitch (center-to-center spacing) of the recesses 58 is about 1.5 μm. While example average pitch values ​​are provided, it should be understood that other average pitch values ​​may also be used.

[0106] The size of each recess 58 may be characterized by its volume, open area, depth, and / or diameter.

[0107] Each recess 58 can have any volume capable of confining fluid. The minimum or maximum volume can be selected to accommodate, for example, the throughput (e.g., multiplexing), resolution, nucleotide, or analyte reactivity expected for downstream use of flow cell 26. For example, the volume can be at least about 1×10 -3 μm 3 , at least about 1 x 10 -2 μm 3 , at least about 0.1 μm 3 , at least about 1 μm 3 , at least about 10 μm 3 , at least about 100 μm 3 Alternatively or additionally, the volume may be at most about 1×10 4 μm 3 , up to about 1 × 10 3 μm 3 , up to about 100 μm 3 , up to about 10 μm 3 , up to about 1 μm 3 , up to about 0.1 μm 3 , or even less.

[0108] The area occupied by the opening of each recess can be selected based on the same criteria as the volume described above. For example, the area of ​​each recess opening is at least about 1×10 -3 μm 2 , at least about 1 x 10 -2 μm 2 , at least about 0.1 μm 2 , at least about 1 μm 2 , at least about 10 μm 2 , at least about 100 μm 2 Alternatively or additionally, the area may be at most about 1×10 3 μm 2 , up to about 100 μm 2 , up to about 10 μm 2 , up to about 1 μm 2 , up to about 0.1 μm 2 , up to about 1 × 10 -2 μm 2The area occupied by the opening of each recess may be greater than, less than, or anything in between.

[0109] The depth of each recess 58 may be large enough to accommodate the polymer hydrogel 66 and capture primers 62, 64. In one example, the depth may be at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or additionally, the depth may be at most about 1×10 3 The depth of each recess 58 may be greater than, less than, or between the values ​​specified above.

[0110] In some cases, the diameter or length and width of each recess 58 may be at least about 50 nm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, or more. Alternatively or additionally, the diameter or length and width may be at most about 1×10 3 The diameter or length and width of each recess 58 may be greater than, less than, or equal to the above-specified values.

[0111] A polymer hydrogel 66 resides in each of the recesses 58. Examples of polymer hydrogels 66 include acrylamide copolymers represented by the following structure (I):

[0112] [ka] During the ceremony, R Ais selected from the group consisting of azide, optionally substituted amino, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxy, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone, sulfate, and thiol; R B is H or optionally substituted alkyl; R C , R D , and R E are each independently selected from the group consisting of H and optionally substituted alkyl; -(CH2) p - may be optionally replaced by p is an integer ranging from 1 to 50; n is an integer ranging from 1 to 50,000, and m is an integer ranging from 1 to 100,000.

[0113] One specific example of an acrylamide copolymer represented by structure (I) is poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM).

[0114] Those skilled in the art will recognize that the arrangement of the repeating features "n" and "m" in structure (I) is representative and that the monomer subunits may be present in any order in the polymer structure (e.g., random, block, patterned, or combinations thereof).

[0115] The molecular weight of PAZAM and other forms of acrylamide copolymers can range from about 5 kDa to about 1500 kDa, or from about 10 kDa to about 1000 kDa, or in a particular example, can be about 312 kDa.

[0116] In some instances, PAZAM and other forms of acrylamide copolymers are linear polymers. In other instances, PAZAM and other forms of acrylamide copolymers are lightly crosslinked polymers.

[0117] In another example, the polymer hydrogel 66 may be a variation of structure (I). In one example, the acrylamide units are N,N-dimethylacrylamide.

[0118] [ka] In this example, the acrylamide unit of structure (I) can be replaced by

[0119] [ka] where R D , R E , and R F are each H or C1-C6 alkyl, and R G and R H are each C1-C6 alkyl (instead of H as in acrylamide). In this example, q may be an integer ranging from 1 to 100,000. In another example, in addition to the acrylamide units, N,N-dimethylacrylamide may be used. In this example, structure (I) contains, in addition to the repeating features "n" and "m",

[0120] [ka] where R D , R E , and R F are each H or C1-C6 alkyl, and R G and R H is a C1-C6 alkyl. In this example, q can be an integer ranging from 1 to 100,000.

[0121] As another example of polymer hydrogel 66, the repeating "n" feature in structure (I) can be replaced with a heterocyclic azide group-containing monomer having structure (II):

[0122] [ka] In the formula, R 1 is H or C1-C6 alkyl, R2 is H or C1-C6 alkyl, L is a linker comprising a linear chain having 2-20 atoms selected from the group consisting of carbon, oxygen, and nitrogen, with 10 optional substituents on the carbons and an optional nitrogen atom in the chain, E is a linear chain comprising 1-4 atoms selected from the group consisting of carbon, oxygen, and nitrogen, with optional substituents on the carbons and any nitrogen atoms in the linear chain, A is an N-substituted amide with H or C1-C4 alkyl attached to the N, and Z is a nitrogen-containing heterocycle. Examples of Z include 5- to 10-membered rings that exist as single ring structures or fused structures. Some specific examples of Z include pyrrolidinyl, pyridinyl, or pyrimidinyl.

[0123] As yet another example, polymer hydrogel 66 may include repeat units of each of structures (III) and (IV):

[0124] [ka] where R 1a , R 2a , R 1b and R 2b each independently selected from hydrogen, optionally substituted alkyl, or optionally substituted phenyl; R 3a and R 3b each is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7-C14 aralkyl; 1 and L 2 are independently selected from an optionally substituted alkylene linker or an optionally substituted heteroalkylene linker.

[0125] It should be understood that other molecules can be used to form the polymer hydrogel 66, so long as they are functionalized to graft oligonucleotide primers 62, 64. Other examples of suitable polymer layers include those with colloidal structures, such as agarose, or polymer mesh structures, such as gelatin, or cross-linked polymer structures, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA), or azide-decomposed versions of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or vinyl-containing acrylic acid, or from monomers that form a [2+2] photocycloaddition reaction. Still other examples of suitable polymer hydrogels 66 include mixed copolymers of acrylamide and acrylate. Various polymer structures containing acrylic monomers (e.g., acrylamide, acrylate, etc.) can be utilized in the embodiments disclosed herein, including star polymers, star-shaped or star-block polymers, branched polymers, including dendrimers, etc. For example, monomers (eg, acrylamide, acrylamide containing a catalyst, etc.) may be incorporated into the branches (arms) of a star polymer, either randomly or in blocks.

[0126] In yet another example, the acrylamide copolymer is formed using nitroxide-mediated polymerization, and therefore, at least a portion of the copolymer chain has an alkoxyamine end group. In the copolymer chain, the term "alkoxyamine end group" refers to a dormant species -ONR1R2, where each of R1 and R2 can be the same or different and can independently be a straight or branched chain alkyl or a ring structure, and the oxygen atom is attached to the remainder of the copolymer chain. In some examples, the alkoxyamine also is attached to some of the repeating acrylamide monomers, such as the R A Thus, in one example, structure (I) includes an alkoxyamine end group, and in another example, structure (I) includes an alkoxyamine end group and an alkoxyamine group on at least a portion of the side chain.

[0127] In the example shown in FIG. 5E, polymer hydrogel 66 is disposed within each of the recesses 58 and not over the surrounding gap regions 60 .

[0128] To introduce the polymer hydrogel 66 into the recesses 58, a mixture of the polymer hydrogel 66 can be created and then applied to the substrate 52. In one example, the polymer hydrogel 66 can be present in a mixture (e.g., with water, or with ethanol and water). The mixture can then be applied to the substrate surface (including within the recesses 58) using spin coating, or dipping or dip coating, spray coating, or flowing the material under positive or negative pressure, or another suitable technique. These types of techniques result in blanket deposition of the polymer hydrogel 66 over the patterned material 56 (e.g., within the recesses 58 and over the void regions 60). Other selective deposition techniques (e.g., involving masks, controlled printing techniques, etc.) can be used to specifically deposit the polymer hydrogel 66 within the recesses 58 and not over the void regions 60.

[0129] In some examples, the substrate surface may be activated, and then the mixture (including polymer hydrogel 66) may be applied to the substrate surface. In one example, a silane or silane derivative (e.g., norbornene silane) may be deposited on the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface may be exposed to plasma ashing to generate surface-activating agents (e.g., —OH groups) that can attach to the polymer hydrogel 66.

[0130] Depending on the polymer hydrogel 66, the applied mixture may be subjected to a curing process. In one example, curing may occur at temperatures ranging from room temperature (e.g., about 25° C.) to about 95° C. for times ranging from about 1 millisecond to about several days.

[0131] In some cases, polishing may then be performed to remove the polymer hydrogel 66 from the gap regions 60 while leaving the polymer hydrogel 66 within the recesses 58 at least substantially intact.

[0132] The flow cell 26 also contains capture primers 62,64.

[0133] A grafting process can be performed to graft the capture primers 62, 64 to the polymer hydrogel 66 within the recesses 58. In one example, the capture primers 62, 64 can be immobilized to the polymer hydrogel 66 by a single-point covalent bond at or near the 5' end of the primers 62, 64. This bond i) renders the adapter-specific portion of the primers 62, 64 free to anneal to their cognate nucleic acid fragments and ii) leaves the 3' hydroxyl group free for primer extension. Any suitable covalent bond can be used for this purpose. Examples of terminal primers that can be used include alkyne-terminated primers, which can be bound to an azide moiety on the polymer hydrogel 66. Specific examples of suitable primers 62, 64 include the P5 and P7 primers used on the surface of commercially available flow cells from Illumina Inc. for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, GEXOME ANALYZER™, ISEQ™, and other instrument platforms.

[0134] In one example, grafting may involve flow-through deposition (e.g., using a temporarily or permanently attached lid), dunk coating, spray coating, paddle dispensing, or another suitable method of attaching the primers 62, 64 to the polymer hydrogel 66 within the recesses 58. Each of these exemplary techniques may utilize a primer solution or mixture, which may include the primers 62, 64, water, a buffer, and a catalyst. With any grafting method, the primers 62, 64 react with the reactive groups of the polymer hydrogel 66 and have no affinity for the surrounding interstitial regions 60. Thus, the primers 62, 64 selectively graft to the polymer hydrogel 66 and not to the interstitial regions 60.

[0135] 5A or 5E, it should be understood that the flow cell 26 can also include a lid attached to the substrate 52. In one example, the lid may be bonded to at least a portion of the substrate 52, such as a portion of the gap region 60. In one example, the lid is bonded around the periphery between the reaction chambers 28. The bond formed between the lid and the substrate 52 can be a chemical bond or a mechanical bond (e.g., using fasteners, etc.).

[0136] The lid may be any material that is transparent to the excitation light directed at the substrate 52. By way of example, the lid may be glass (e.g., borosilicate, fused silica, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is D 263® available from Schott North America, Inc. Commercially available examples of suitable plastic materials, i.e., cycloolefin polymers, are ZEONOR® products available from Zeon Chemicals LP.

[0137] The lid may be bonded to the substrate 52 using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, glass frit bonding, or other methods known in the art. In one example, a spacer layer may be used to bond the lid to the substrate 52. The spacer layer may be any material that seals at least a portion of the substrate 52 and the lid together. In some examples, the spacer layer may be a radiation absorbing material that aids in bonding the substrate 52 and the lid.

[0138] In another example, instead of a lid, the flow cell 26 may include two substrates 52 bonded together such that the reaction chambers 28 face each other and a flow path is defined therebetween. The two substrates 52 may be bonded together in a portion of the gap region 60 (e.g., around the periphery and between the reaction chambers 28). The bond formed between the substrates 52 may be a chemical bond or a mechanical bond (e.g., using fasteners, etc.).

[0139] Referring back to method 200, sample fluid 12 (shown in FIG. 5B) is introduced into temperature control flow path 30. Sample fluid 12 may be stored in or introduced into reservoir 46 and then pumped through an inlet line 68, which is fluidly connected to first bypass valve 42, bypass line 40, and temperature control flow path 30. First bypass valve 42 may be positioned to direct sample fluid 12 into temperature control flow path 30.

[0140] When sample fluid 12 is introduced into temperature-controlled channel 30, the temperature of channel 30 and sample fluid 12 is below the gelation temperature of methylcellulose 14 in sample fluid 12. At this temperature, methylcellulose 14, chemically inert polymer 18, and salt 22 are dissolved in aqueous carrier 20, while DNA sample 16 is dispersed in aqueous carrier 20. This is shown schematically to the left of temperature-controlled channel 30 in FIG. 5B.

[0141] The method 200 also involves heating the temperature-controlled channel 30 when the sample fluid 12 is introduced. The temperature to which the temperature-controlled channel 30 is heated depends on the gelation temperature of the methylcellulose 14 in the sample fluid 12. The temperature-controlled channel 30 may be brought to a temperature at or above the gelation temperature to allow the temperature of the sample fluid 12 to reach the gelation temperature.

[0142] Internal or external heating components 36 or 38 may be used to heat the temperature controlled flow path 30. Sensor feedback may be used to achieve and / or maintain the desired temperature.

[0143] As shown to the right of the temperature-controlled channel 30 in Figure 5B, heat causes the methylcellulose 14 to precipitate from the aqueous carrier 20. The precipitated methylcellulose polymer chains physically interact with the DNA sample 16 to form a DNA-methylcellulose complex 10 within the temperature-controlled channel 30.

[0144] Method 200 also involves continuing the flow of sample fluid 12 through temperature-controlled channel 30 as the temperature-controlled channel is heated, causing DNA-methylcellulose complexes 10 to flow toward filter 34, where further flow is blocked, thereby concentrating the plurality of DNA-methylcellulose complexes 10 at filter 34 within temperature-controlled channel 30, as shown in FIG.

[0145] It should be understood that aqueous carrier 20 and any components dissolved therein (e.g., salt 22 and chemically inert polymer 18) may continue to flow through filter 34 even if DNA-methylcellulose complex 10 is blocked. Aqueous carrier 20 and any components dissolved therein may be directed directly to a waste container (not shown) or may be directed to flow cell 26 and through flow cell 26 to a waste container.

[0146] The method 200 further includes cooling the temperature-controlled channel 30 such that the temperature of the sample fluid 12 contained within the temperature-controlled channel 30 is reduced below the gelation temperature of the methylcellulose 14. In some examples, the internal or external heating component 36 or 38 may be turned off to cool the temperature-controlled channel 30. This involves passive cooling, as the sample fluid is allowed to cool on its own. In other examples, the temperature-controlled channel 30 may include a cooling component or a heating / cooling component that can be controlled to actively reduce the temperature of the temperature-controlled channel 30 and the sample fluid 12 contained therein.

[0147] Cooling can begin after the sample fluid 12 has flowed for a predetermined time, and / or when a desired amount of sample fluid 12 has been introduced into the temperature-controlled channel 30, and / or when a desired number of complexes 10 have been concentrated in the filter 34. By way of example, the volume of fluid passing through the filter 34 can be calculated or a specific counter can be incorporated into the line of the filter 34.

[0148] The temperature to which the temperature control channel 30 is cooled depends on the gelation temperature of the methylcellulose 14 in the sample fluid 12. The temperature control channel 30 can be lowered below the gelation temperature so that the temperature of the sample fluid 12 can also be cooled below the gelation temperature.

[0149] 5D, cooling disentangles the DNA-methylcellulose complexes 10 in the temperature-controlled channel 30, concentrating them, for example, in a filter 34. More specifically, cooling dissolves the methylcellulose 14 into the aqueous carrier 20, disentangling the DNA-methylcellulose complexes 10 and releasing the DNA sample 16. Both the dissolved methylcellulose 14 and the DNA sample 16 can flow out of the temperature-controlled channel 30 through the filter 34.

[0150] The method 200 may further include transporting the DNA sample 16 and methylcellulose 14 to a flow cell 26 and into a reaction chamber 28 for analysis (e.g., sequencing).

[0151] The flow cell assembly 24 described in connection with the method 200 can be part of a kit that can also include an example of the sample fluid 12 .

[0152] DNA seeding Another exemplary method is shown in Figure 6 at reference numeral 300. This exemplary method 300 can be used to improve DNA seeding for sequencing. The method 300 includes introducing a sample fluid into a flow cell (reference numeral 302), the sample fluid including an aqueous carrier, a DNA sample, methylcellulose, a polymer that is chemically inert to DNA hybridization, and a salt, the flow cell including a reaction chamber having recesses separated by gap regions and a capture primer attached within each of the recesses; initiating seeding and hybridization of at least a portion of the DNA sample in at least some of the recesses (reference numeral 304); and heating the flow cell to raise the temperature of the sample fluid contained therein to at least the gelling temperature of the methylcellulose. the temperature of the flow cell to a temperature below the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex with the unbound DNA sample (reference numeral 306); introducing an additional amount of sample fluid into the flow cell (reference numeral 308); cooling the flow cell to reduce the temperature of the sample fluid contained therein below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complex and releasing the DNA sample and methylcellulose (reference numeral 310); and initiating seeding and hybridization of at least a portion of the released DNA sample and DNA sample from the additional sample fluid in at least some of the recesses (reference numeral 312).

[0153] The method 300 is illustrated generally in Figures 7A-7F.

[0154] In this example of method 300, the DNA sample 16 in the sample fluid 12 contains DNA library fragments. The DNA library fragments contain adapters at opposite ends. The DNA library fragments can be prepared using any library preparation technique that fragments longer pieces of genetic material and incorporates desired adapters at the ends of the fragments. Some exemplary library preparation techniques include tagging or ligation.

[0155] 7A, sample fluid 12 is introduced into flow cell 26. More specifically, sample fluid 12 is introduced into reaction chamber 28, which includes recesses 58 separated by gap regions 60 and capture primers 62, 64 deposited within recesses 58.

[0156] Once the sample fluid 12 is introduced into the reaction chamber 28, seeding (e.g., immobilization) of the DNA sample 16 can begin, as shown in Figure 7B. Seeding is achieved via hybridization between one of the adapters on the DNA sample 16 and a complementary one of the capture primers 62, 64. Seeding can be performed at a hybridization temperature appropriate for the DNA sample 16 and the capture primers 62, 64. The seeded DNA sample 16 is designated by reference numeral 16', which herein refers to the seeded DNA library fragments 16'.

[0157] The method 300 then involves heating the flow cell 26. The temperature to which the flow cell 26 is heated depends on the gelling temperature of the methylcellulose 14 in the sample fluid 12. The flow cell 26 may be brought to a temperature at or above the gelling temperature to allow the temperature of the sample fluid 12 to reach the gelling temperature.

[0158] Throughout method 300, heating and cooling of flow cell 26 (e.g., to a suitable hybridization temperature, at, above, or below the gelation temperature, etc.) may involve internal or external heating components similar to components 36 or 38 described herein. The internal heating component may be located within the reaction chamber 28, or the external heating component may be part of the sequencing device in which the flow cell 26 is located. The internal or external heating component may be controlled to heat the flow cell 26 or its particular reaction chamber 28 to a desired temperature. Sensor feedback may be used to achieve and / or maintain the desired temperature.

[0159] As shown in Figure 7C, heat precipitates methylcellulose 14 from the aqueous carrier 20, and the precipitated methylcellulose polymer chains physically interact with unbound DNA samples 16 (e.g., unseeded DNA library fragments) to form DNA-methylcellulose complexes 10 within the reaction chamber 28. Any DNA samples 16 that are not seeded / immobilized can become entangled in the DNA-methylcellulose complexes 10. The seeded DNA library fragments 16' are hydrogen bonded to their respective capture primers 62, 64 and therefore do not become entangled in the complexes 10.

[0160] Method 300 also involves introducing an additional amount of sample fluid 12 into the reaction chamber 28 of the flow cell 26. This introduction occurs as the flow cell 26 is heated. This introduction can occur relatively quickly so that complex formation occurs within the reaction chamber 28 and not in the fluid lines leading to the reaction chamber 28. Furthermore, the rapid introduction of the additional sample fluid 12 can help balance DNA complexation and DNA hybridization. The introduction of the additional sample fluid may occur within a few minutes, e.g., within 5 minutes. The newly introduced methylcellulose 14 is thermally precipitated and complexes with unbound DNA sample 16 to form additional DNA-methylcellulose complexes 10. This concentrates multiple DNA-methylcellulose complexes 10 within the reaction chamber 28, as shown in FIG. 5D.

[0161] The method 300 further includes cooling the flow cell 26 (or its reaction chamber 28) to reduce the temperature of the sample fluid 12 contained therein below the gelling temperature of the methylcellulose 14. The internal or external heating component may be turned off to cool the flow cell 26 or reaction chamber 28. This involves passive cooling, as the sample fluid 12 is allowed to cool on its own. In other examples, the flow cell 26 may include a cooling component or a heating / cooling component that can be controlled to actively reduce the temperature of the reaction chamber 28 and the sample fluid 12 contained therein.

[0162] Cooling may be initiated when a desired amount of sample fluid 12 has been introduced into reaction chamber 28 and / or when a desired number of complexes 10 have been concentrated within reaction chamber 28 .

[0163] The temperature to which the flow cell 26 (or its reaction chamber 28) is cooled depends on the gelling temperature of the methylcellulose 14 in the sample fluid 12. The flow cell 26 (or its reaction chamber 28) can be lowered below the gelling temperature so that the temperature of the sample fluid 12 can also be cooled below the gelling temperature.

[0164] 7E, cooling disentangles the DNA-methylcellulose complex 10 within the reaction chamber 28. More specifically, cooling dissolves the methylcellulose 14 into the aqueous carrier 20, disentangling the DNA-methylcellulose complex 10 and releasing the DNA sample 16. In this manner, the DNA sample 16 is concentrated within the reaction chamber 28.

[0165] Once the DNA sample 16 is released from the complex 10, seeding (e.g., immobilization) of the DNA sample 16 can begin again, as shown in Figure 7F. The higher the concentration of DNA sample 16 in the reaction chamber 28, the more effective the seeding can be in that a greater number of DNA library fragments (sample 16) are immobilized within the recesses 58, as compared to the seeding event occurring in Figure 7B, for example. As described herein, seeding can be performed at a hybridization temperature appropriate for the DNA sample 16 and capture primers 62, 64.

[0166] The method 300 may further include introducing a wash solution to remove any remaining unbound DNA sample 16 (e.g., unseeded DNA library fragments) as well as the sample fluid 12, and then initiating a sequencing cycle. The following example describes a sequencing by synthesis (SBS) cycle.

[0167] The SBS cycle can begin with the amplification of the seeded DNA library fragments 16' using cluster generation. In one example of cluster generation, the seeded DNA library fragments 16' are copied from the hybridized primers 62, 64 by 3' extension using a high-fidelity DNA polymerase. The original seeded DNA library fragments 16' are denatured, leaving copies immobilized on the capture primers 62, 64. Isothermal bridge amplification or some other form of amplification can be used to amplify the immobilized copies. For example, the copied template loops over to hybridize to adjacent complementary primers 62, 64, and the polymerase copies the copied template, forming a double-stranded bridge, which is denatured to form two single strands. These two strands loop over, hybridize to, and are extended again to form two new double-stranded loops on adjacent complementary primers 62, 64. This process is repeated for each template copy through cycles of isothermal denaturation and amplification to create dense clonal clusters. Each cluster of double-stranded bridge structures is denatured. In one example, the reverse strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. Clustering results in the formation of multiple template polynucleotide strands along the reaction chamber 28. An example of this clustering is bridge amplification, which is one example of amplification that can be performed. It should be understood that other amplification techniques, such as the exclusion amplification (Examp) workflow (Illumina Inc.), can also be used.

[0168] A sequencing primer may be introduced that hybridizes to a complementary sequence on the template polynucleotide strand, rendering the template polynucleotide strand ready for sequencing. The 3' ends (not bound to the copy) of the template and any flow cell-bound primers 62, 64 may be blocked to prevent interference with the sequencing reaction, particularly to prevent undesired priming.

[0169] To initiate sequencing, an incorporation mixture can be added to the flow cell 26. In one example, the incorporation mixture includes a liquid carrier, a polymerase, and fluorescently labeled nucleotides. The fluorescently labeled nucleotides can include a 3'OH blocking group. When the incorporation mixture is introduced into the flow cell 26, the fluid enters the reaction chamber 28, and in some examples, into the recess 58 (where the template polynucleotide strand resides).

[0170] Fluorescently labeled nucleotides are added to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner, and detection of the order and type of nucleotides added to the sequencing primer can be used to sequence the template. More specifically, one of the nucleotides is incorporated by each polymerase into a nascent strand that extends the sequencing primer and is complementary to the template polynucleotide strand. In other words, for at least a portion of the template polynucleotide strand traversing flow cell 26, each polymerase extends the hybridized sequencing primer with one of the nucleotides in the incorporation mixture.

[0171] The incorporation of the nucleotide can be detected through an imaging event during which an illumination system (not shown) can provide excitation light to the reaction chamber 28.

[0172] In some instances, the nucleotides may further comprise a reversible termination feature (e.g., a 3'OH blocking group) that terminates further primer extension once the nucleotide is added to the sequencing primer. For example, a nucleotide analog having a reversible end portion may be added to the sequencing primer such that further extension cannot occur until a deblocking agent is delivered to remove the portion. Thus, in instances using reversible ends, a deblocking reagent can be delivered to flow cell 26 after detection has occurred.

[0173] One or more washes can be performed between the various fluid delivery steps. The SBS cycle is then repeated n times to extend the sequencing primer by n nucleotides, thereby allowing a sequence of length n to be detected.

[0174] In some cases, the forward strand may be sequenced and removed, and then the reverse strand may be constructed and sequenced as described herein.

[0175] While SBS has been described in detail, it should be understood that the flow cell 26 described herein can be utilized with other sequencing protocols, for genotyping, or in other chemical and / or biological applications. In some cases, the primers 62, 64 of the flow cell 26 may be selected to enable simultaneous paired-end sequencing, with both the forward and reverse strands present on the polymer hydrogel 66, allowing simultaneous base calling of each read. Sequential and simultaneous paired-end sequencing facilitates the detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.

[0176] To further illustrate the present disclosure, examples are presented herein. It should be understood that these examples are provided for illustrative purposes and are not to be construed as limiting the scope of the present disclosure. [Example]

[0177] Example 1 Several sample fluids were prepared to test the gelation / precipitation temperature of methylcellulose versus salt. Each solution contained 1 wt. % methylcellulose (METHOCEL® E from Dow Chemical Co.), 16 wt. % polyethylene glycol (weight average molecular weight 1000 g / mol), different concentrations of sodium chloride (NaCl), and water. The NaCl concentrations ranged from 0.4 M to 2 M.

[0178] To achieve methylcellulose precipitation, different solutions were exposed to different temperatures (5°C, 25°C, 45°C, or 60°C). The optical density of each solution was measured using a plate reader. Optical density is a measure of light scattering, with higher numbers indicating more precipitated methylcellulose. An optical density of less than 0.1 meant that all of the methylcellulose was in solution. The results are shown in Figure 8. At lower temperatures (5°C and 25°C) and lower salt concentrations (1.25M and below), the methylcellulose was completely dissolved. As the salt concentration increased, the methylcellulose was able to precipitate from solution at lower temperatures. These results demonstrate that the salt concentration in the sample fluid disclosed herein can be used to adjust the gelation / precipitation temperature so that the complexes disclosed herein can be produced at the desired temperature.

[0179] Example 2 A sample fluid was prepared using a DNA ladder containing multiple DNA inserts of different sizes. The sample fluid contained water, methylcellulose (1% METHOCEL® E from Dow Chemical Co.), polyethylene glycol (5%, weight average molecular weight 1000 g / mol), and 1 M NaCl. The DNA inserts were added to the solution to form the sample fluid. The sample fluid was then incubated for 5 minutes with heating at 25°C. The sample fluid was then centrifuged and decanted. The first supernatant was collected and run on an Agilent TapeStation.

[0180] The precipitate was rinsed to remove unbound DNA and resuspended in sodium citrate-buffered saline. The reconstituted sample fluid was heated again, this time to 60°C for 5 minutes. The reconstituted sample fluid was then centrifuged and decanted. The second supernatant was collected and removed, leaving behind the precipitated solid. The precipitated solid was suspended in buffer and cooled (to release any bound DNA insert). This solution was run on the TapeStation.

[0181] The TapeStation results for the first supernatant and precipitate are shown in Figure 9 (sample intensity (normalized FU on the Y-axis) vs. size (number of base pairs on the X-axis)). The first supernatant had more smaller DNA inserts, while the precipitate had more larger DNA inserts. These results demonstrate the ability of the method disclosed herein to remove smaller DNA inserts via complexation of the larger DNA inserts with methylcellulose.

[0182] Example 3 One sample fluid (Example 1) and one comparative sample fluid (Comparative Example 2) were prepared with the PhiX library. Example 1 contained water and methylcellulose (1% METHOCEL® E from Dow Chemical Co.) and the PhiX library. Comparative Example 2 contained water and the PhiX library, but no methylcellulose.

[0183] Example 1 and Comparative Example 2 were incubated for 5 minutes with heating at 60°C. Example 1 and Comparative Example 2 were then centrifuged and decanted. Each pellet was rinsed to remove unbound DNA, resuspended in 1 M TRIS buffer, and chilled (to release the bound DNA insert). Each resuspended solution was run on an Agilent TapeStation.

[0184] The TapeStation results for Example 1 and Comparative Example 2 are shown in Figure 10 (sample intensity (normalized FU on the Y-axis) versus size (number of base pairs on the X-axis). Example 1 shows a sharper peak compared to Comparative Example 2, thus demonstrating that methylcellulose captured larger DNA inserts. Notably, Comparative Example 2 had a low, broad peak at 500 bp, while Example 1 had a much higher, sharper peak. This indicated that Example 1 was more concentrated and potentially purified.

[0185] The resuspended libraries from both Example 1 and Comparative Example 2 were then run on an iSeq™ sequencing system (Illumina Inc.) to determine whether the methylcellulose had any detrimental effects on the libraries themselves. Although not reproduced herein, the sequencing metrics for Example 1 and Comparative Example 2 were found to be similar, thus confirming that the methylcellulose did not have any detrimental effects on the libraries themselves.

[0186] Example 4 Two additional sample fluids (Examples 3 and 4) and one additional comparative sample fluid (Comparative Example 5) were prepared with the PhiX library. Example 3 contained water, methylcellulose (1% METHOCEL® E from Dow Chemical Co.), and the PhiX library. Example 4 contained water, methylcellulose (5% METHOCEL® E from Dow Chemical Co.), and the PhiX library. Comparative Example 5 contained water and the PhiX library, but no methylcellulose.

[0187] Examples 3, 4, and 5 were incubated for 5 minutes with heating at 60°C. Examples 3, 4, and 5 were then centrifuged and decanted. Each pellet was rinsed to remove unbound DNA, resuspended in 1 M TRIS buffer, and chilled (to release the bound DNA insert). Each resuspended solution was run on an Agilent TapeStation.

[0188] The TapeStation results for Examples 3, 4, and Comparative Example 5 are shown in Figure 11 (sample intensity (normalized FU on the Y-axis) versus size (number of base pairs on the X-axis)). As the methylcellulose concentration increased (Comparative Example 5 through Examples 3 and 4), the peaks around 25 bp, 75 bp, and 170 bp became higher and sharper. The results demonstrate that high concentrations of methylcellulose did not adversely affect the capture efficiency of the polymer.

[0189] Additional Notes It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (unless such concepts are mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. Specifically, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. It should also be understood that terms used explicitly herein, and which may also appear in any disclosures incorporated by reference, should be given the meaning most consistent with the particular concepts disclosed herein.

[0190] References throughout this specification to "one example," "another example," "an example," etc. mean that a particular element (e.g., a feature, structure, and / or characteristic) described in connection with an example is included in at least one example described herein and may or may not be present in other examples. Furthermore, unless the context clearly dictates otherwise, it should be understood that the described elements with respect to any example may be combined in any suitable manner in the various examples.

[0191] Ranges provided herein should be understood to include the stated range and any value or subrange within that stated range, as if such value or subrange were explicitly recited. For example, a range of about 400 nm to about 1 μm (1000 nm) should be understood to include not only the explicitly recited limits of about 400 nm to about 1 μm, but also individual values, such as about 708 nm, about 945.5 nm, etc., and subranges, such as about 425 nm to about 825 nm, about 550 nm to about 940 nm, etc. Furthermore, when "about" and / or "substantially" are used to describe values, these are meant to encompass small variations (up to ±10%) of the stated value.

[0192] While certain embodiments have been described in detail, it should be understood that the disclosed examples may be modified, and therefore the foregoing description should be considered non-limiting.

Claims

1. A kit comprising: a flow cell assembly, the flow cell assembly comprising: A reaction chamber comprising: recesses separated by gap regions; a reaction chamber having a capture primer attached within each of the recesses; a temperature control channel in selective fluid communication with an inlet of the reaction chamber; and a filter disposed within the temperature-controlled flow path, the filter i) blocking concentrated deoxyribonucleic acid (DNA)-methylcellulose complexes formed within the temperature-controlled flow path at a first temperature, and ii) allowing passage of concentrated DNA and methylcellulose released from the complexes within the temperature-controlled flow path at a second temperature.

2. an aqueous carrier; a DNA sample; Methylcellulose, a polymer that is chemically inert to DNA hybridization; 10. The kit of claim 1, further comprising a sample fluid comprising:

3. 3. The kit of claim 2, wherein the polymer that is chemically inert to DNA hybridization is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and combinations thereof, having a weight average molecular weight ranging from about 500 to less than about 200,000.

4. the DNA sample is present in the sample fluid at a first molar concentration ranging from about 1 pM to about 1 mM; the methylcellulose is present in the sample fluid in an amount ranging from about 0.5% to about 20% by weight, based on the total weight of the sample fluid; the polymer that is chemically inert to DNA hybridization is present in the sample fluid in an amount ranging from greater than 0 wt % to about 20 wt % based on the total weight of the sample fluid; 3. The kit of claim 2, wherein the salt is present in the sample fluid at a second molar concentration ranging from greater than 0M to about 2M.

5. The flow cell assembly comprises: a bypass line in fluid communication with the inlet of the temperature control channel and the outlet of the temperature control channel; a first bypass valve for controlling the flow of sample fluid to the inlet of the temperature control channel; 10. The kit of claim 1, further comprising a second bypass valve controlling the flow of the concentrated DNA and the methylcellulose into the reaction chamber.

6. The kit of claim 1 , wherein at least one surface of the temperature-controlled channel comprises a heating plate.

7. The DNA sample is present in the sample fluid at a first molar concentration in a range of about 1 pM to about 1 mM; the methylcellulose is present in the sample fluid in an amount ranging from about 1% to about 15% by weight, based on the total weight of the sample fluid; the polymer that is chemically inert to DNA hybridization is present in the sample fluid in an amount ranging from about 1.5% to about 16% by weight, based on the total weight of the sample fluid; 3. The kit of claim 2, wherein the salt is present in the sample fluid at a second molar concentration ranging from 0M to 2M.

8. The kit described in claim 7, wherein the polymer that is chemically inert to DNA hybridization is polyethylene glycol having a weight average molecular weight in the range of about 500 to about 1,000.

9. The kit described in claim 7, wherein the salt is sodium chloride.

10. The kit described in claim 2, wherein the salt is selected from the group consisting of sodium chloride; sodium bromide; sodium iodide; salts containing potassium, calcium, magnesium, or ammonium cations; and salts containing carbonate anions, sulfate anions, phosphate anions, or nitrate anions.

11. The kit described in claim 2, wherein the DNA sample comprises cell-free DNA, library DNA, whole genome amplified DNA, or a combination thereof.

12. The kit described in claim 2, wherein the aqueous carrier comprises water, a salt solution, or a buffer containing tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl) buffer, tris(hydroxymethyl)aminomethane (TRIS) buffer, or sodium citrate saline (SSC).

13. The kit of claim 1, wherein the number of the recesses in a predetermined area is in the range of about 100 to about 50 million per mm 2 , the average pitch of each recess is in the range of about 50 nm to about 100 μm, and the average volume of each recess is in the range of about 1×10 −3 μm 3 to about 100 μm 3 .

14. The kit described in claim 1, wherein the reaction chamber further has a polymer hydrogel present in each recess, and the polymer hydrogel is an acrylamide copolymer.

15. The kit described in claim 14, wherein the capture primer is immobilized on a polymer hydrogel within each recess.

16. The kit described in claim 15, wherein the polymer hydrogel and the capture primer are not present in the gap region.

17. 1. A method comprising: introducing a sample fluid into a temperature-controlled flow path having a filter disposed therein, the sample fluid comprising: an aqueous carrier; a DNA sample; Methylcellulose, a polymer that is chemically inert to DNA hybridization; introducing a salt; heating the temperature-controlled channel when the sample fluid is introduced to raise the temperature of the sample fluid contained therein to at least the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex within the temperature-controlled channel; continuing the flow of the sample fluid through the temperature-controlled channel as the temperature-controlled channel is heated, thereby concentrating a plurality of the DNA-methylcellulose complexes at the filter within the temperature-controlled channel; cooling the temperature-controlled flow path to reduce the temperature of the sample fluid contained therein below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complex and releasing the DNA sample and the methylcellulose, thereby allowing the DNA sample and the methylcellulose to pass through the filter.

18. 18. The method of claim 17, further comprising selecting a heating temperature for the temperature-controlled channel according to the concentration of the salt in the sample fluid.

19. 18. The method of claim 17, further comprising transporting the DNA sample and the methylcellulose from the temperature-controlled channel to a flow cell.

20. 1. A method comprising: introducing a sample fluid into a flow cell, the sample fluid comprising: an aqueous carrier; a DNA sample; Methylcellulose, a polymer that is chemically inert to DNA hybridization; salt, The flow cell includes a reaction chamber, the reaction chamber comprising: recesses separated by gap regions; a capture primer attached within each of the recesses; initiating seeding and hybridization of at least a portion of the DNA sample in at least a portion of the wells; heating the flow cell to raise the temperature of the sample fluid contained therein to at least the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex with unbound DNA sample; introducing an additional amount of the sample fluid into the flow cell; cooling the flow cell to reduce the temperature of the sample fluid contained therein below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complex and releasing the DNA sample and the methylcellulose; and initiating seeding and hybridization of at least a portion of the DNA sample from the released DNA sample and the additional sample fluid in at least some of the wells.

21. 21. The method of claim 20, further comprising selecting a heating temperature for the flow cell according to the concentration of the salt in the sample fluid.

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