TW202217007A - Nucleic acid capture, concentration, and purification - Google Patents

Abstract

An example of a kit includes a flow cell assembly. The flow cell assembly includes a reaction chamber, a temperature controlled flow channel in selective fluid communication with an inlet of the reaction chamber, and a filter positioned in the temperature controlled flow channel. The reaction chamber includes depressions separated by interstitial regions and capture primers attached within each of the depressions. The filter is i）to block concentrated biological sample-polymer complexes generated in the temperature controlled flow channel at a first temperature, and ii）to allow passage of concentrated biological sample and polymer released from the complexes in the temperature controlled flow channel at a second temperature.

Description

Nucleic acid capture, concentration and purification

Cross-references to related applications

This application claims U.S. Provisional Application Serial No. 63/047,103, filed July 1, 2020, the contents of which are incorporated herein by reference in their entirety.

Two types of nucleic acids are found in living organisms (such as humans, animals, etc.), namely ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Both RNA and DNA can be grouped into different types, such as messenger RNA, ribosomal RNA, nuclear DNA, cytoplasmic DNA, and the like. Nucleic acids of various types can be analyzed for a variety of purposes, such as research, diagnostic, forensics, genome sequencing, and the like.

A first aspect disclosed herein is a kit comprising: a flow cell assembly including a reaction chamber having recesses separated by a gap region and connected within each of the recesses a capture primer; a temperature-controlled flow channel in selective fluid communication with the inlet of the reaction chamber; and a filter disposed in the temperature-controlled flow channel, the filter i) being used to block particles generated in the temperature-controlled flow channel at the first temperature Concentrating the deoxyribonucleic acid (DNA)-methylcellulose complex, and ii) for passing the concentrated DNA and methylcellulose released from the complex in the temperature-controlled flow channel at the second temperature.

In an example of the first aspect, the kit further comprises a 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 having a weight average molecular weight ranging from about 500 to less than about 200,000 and its combinations. In another example, the DNA sample is present in the sample fluid at a first molar concentration in the range of about 1 pM (picomolar concentration) to about 1 mM; methylcellulose is about 0.5 wt based on the total weight of the sample fluid % to about 20 wt % is present in 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 in the range of greater than 0 M to about 2 M.

In an example of the first aspect, the flow cell assembly further comprises: a bypass line in fluid communication with the temperature-controlled flow channel inlet and with the temperature-controlled flow channel outlet; a first bypass valve used to control the sample fluid flow to the inlet of the temperature-controlled flow channel; and a second bypass valve to control the flow of concentrated DNA and methylcellulose to the reaction chamber.

In an example of the first aspect, at least one surface of the temperature-controlled flow channel includes a heating plate.

It will be appreciated 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 prior to analysis, processing or the like Capture and concentrate DNA.

A second aspect disclosed herein is a method comprising combining a DNA sample with a solution to form a sample fluid, the solution consisting of an aqueous carrier, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt , and the solution is at a temperature in the range of about 5°C to about 30°C; and the sample fluid is heated to at least the gelling temperature of methylcellulose, thereby forming a DNA-methylcellulose complex in the aqueous carrier .

In an example of the second aspect, the method further comprises cooling the sample fluid below the gelling temperature of methylcellulose, thereby disentangling the DNA-methylcellulose complex to release the DNA sample and methylcellulose cellulose.

In another example of the second aspect, the method further comprises increasing the concentration of the salt in the solution, thereby reducing the gelling temperature of the methylcellulose.

In an example of the second aspect, the DNA sample includes free-cell DNA, library DNA, whole genome amplified DNA, or a combination thereof.

In an example of the second aspect, the DNA sample includes a plurality of DNA inserts of different sizes, the inserts including small DNA inserts and large DNA inserts; at least some of the large DNA inserts are entangled in in the DNA-methylcellulose complex; at least some of the small DNA inserts are not entangled in the DNA-methylcellulose complex; and the method further comprises performing a purification process after heating to insert the small DNA inserts At least some of the fragments are isolated from DNA-methylcellulose complexes. In an example, the purification process involves filtration, centrifugation, decantation, or a combination thereof. In another example, the method further comprises cooling the DNA-methylcellulose complex below the gelling temperature of methylcellulose, thereby disentangling the DNA-methylcellulose complex to release the large At least some of the DNA inserts and methylcellulose.

It will be appreciated that any of the features of the second aspect may be combined in any desired manner. Furthermore, it is to be understood that any combination of features of the first aspect and/or the second aspect may be used together, and/or may be combined with any of the examples disclosed herein, to achieve as described in this disclosure Benefits include, for example, capturing DNA and/or purifying DNA.

A third aspect disclosed herein is a method comprising introducing a sample fluid into a temperature-controlled flow channel having a filter disposed therein, the sample fluid comprising an aqueous carrier, a DNA sample, methylcellulose, chemically reactive to DNA hybridization Inert polymers and salts; heating the temperature-controlled flow channel upon introduction of the sample fluid so that the temperature of the sample fluid contained therein increases to at least the gelation temperature of methylcellulose, thereby forming DNA in the temperature-controlled flow channel - methylcellulose complexes; while heating the temperature-controlled flow channel, continue to flow the sample fluid through the temperature-controlled flow channel, thereby concentrating the plurality of DNA-methylcellulose complexes at the filter in the temperature-controlled flow channel and cooling the temperature-controlled flow channel such that the temperature of the sample fluid contained therein is lowered below the gelling temperature of methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complex to release the DNA sample and Methylcellulose, whereby DNA samples and methylcellulose can pass through the filter.

In an example of the third aspect, the method further includes selecting a heating temperature of the temperature-controlled flow channel based on the concentration of salt in the sample fluid.

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

It will be appreciated that any of the features of the third aspect may be combined 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 may be used together, and/or may be combined with any of the examples disclosed herein, to achieve Benefits as described in the present invention include, for example, concentration of DNA prior to analysis, processing or the like.

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; and the flow cell includes a reaction chamber having recesses separated by gap regions and capture primers connected within each of the recesses; inoculating and hybridizing at least some of the DNA samples in at least some of the recesses ; heating the flow cell so that the temperature of the sample fluid contained therein increases to at least the gelling temperature of methylcellulose, thereby forming a DNA-methylcellulose complex with the unbound DNA sample; introducing an additional amount of the sample fluid to the flow cell; cooling the flow cell so that the temperature of the sample fluid contained therein is lowered below the gelling temperature of methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complex to release the DNA sample and methylcellulose; and inoculating and hybridizing at least some of the released DNA samples in at least some of the wells and at least some of the DNA samples from the additional sample fluid.

In an example of the fourth aspect, the method further comprises selecting a heating temperature of the flow cell based on the concentration of salt in the sample fluid.

It will be appreciated that any of the features of the fourth aspect may be combined 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 may be used together, and/or may be used with the examples disclosed herein. Any combination, to achieve the benefits as described in the present invention, includes, for example, improved DNA seeding on the surface of the flow cell.

A fifth aspect disclosed herein is a kit comprising a sample fluid consisting of: an aqueous carrier; a deoxyribonucleic acid (DNA) sample; methylcellulose; a polymer chemically inert to DNA hybridization substances; and salts or solvents used to adjust the gelling temperature of methylcellulose.

In an example of the fifth aspect, the DNA sample is present in the sample fluid at a first molar concentration in the range of about 1 pM to about 1 mM; the methylcellulose is about 0.5 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 sample fluid includes a salt, and the salt is present in the sample fluid at a second molar concentration in the range of greater than 0 M to about 2 M.

In an example of the fifth aspect, the kit further comprises: a flow cell assembly including a temperature-controlled flow channel to receive the sample fluid; a reaction chamber having recesses separated by gap regions; connected to each of the recesses a capture primer within it; and an inlet in selective fluid communication with a temperature-controlled flow channel; and a filter disposed in the temperature-controlled flow channel, the filter i) for blocking when the sample fluid is exposed to a first temperature The concentrated DNA sample-methylcellulose complexes produced in the temperature-controlled flow channel, and ii) the concentrated DNA sample and methylcellulose used to be released from the complexes in the temperature-controlled flow channel at a second temperature base cellulose through.

It will be appreciated that any of the features of the fifth aspect may be combined 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 may be used together, and/or the fifth aspect may be used together, and /or can be combined with any of the examples disclosed herein to achieve benefits as described in this disclosure, including, for example, improved DNA seeding on the surface of a flow cell.

DNA can be used in a variety of applications, such as replication and sequencing. Some sequencing systems utilize relatively low DNA load input in order to output high quality data. Therefore, there is a need to minimize DNA sample loss. The examples disclosed herein capture and concentrate DNA in order to maximize the DNA load available for purification, seeding, and the like. More specifically, the examples disclosed herein utilize the thermally reversible precipitation polymer methylcellulose as a complexing agent for DNA. Under controlled temperature conditions, methylcellulose forms a complex with DNA or releases DNA from the complex. The methods disclosed herein utilize methylcellulose-DNA complexes for DNA capture, concentration, purification and/or seeding.

definition

It is to be understood that unless otherwise defined, terms used herein will have their ordinary meaning in the relevant art. Certain terms used herein and their meanings are set forth below.

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

The terms comprising, including, containing, and various forms of these terms are synonymous with each other and are intended to be equally broad. Furthermore, unless expressly stated to the contrary, instances of an element or elements comprising, comprising, or having a particular property may include additional elements, whether or not the additional elements have that property.

As used herein, the term "adapter" refers to a linear oligonucleotide sequence that can be fused to a nucleic acid molecule, eg, by ligation or tagmentation. Suitable adaptor 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. Adaptors can include any combination of nucleotides and/or nucleic acids. In some examples, an adaptor can include a sequence complementary to at least a portion of a primer, eg, a primer that includes a universal nucleotide sequence such as a P5 or P7 sequence. In some examples, an adaptor can include a sequencing primer sequence or a sequencing binding site. Combinations of different adaptors can be incorporated into nucleic acid molecules, such as DNA fragments.

As used herein, "cell-free DNA" (cfDNA) refers to DNA or RNA that is not contained within a cell. As an 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 tumor-derived fragment DNA (ie, circulating tumor DNA or ctDNA). The examples disclosed herein may also be suitable for capturing, concentrating, etc. viral DNA and/or bacterial DNA.

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

As used herein, the term "depositing" refers to any suitable application technique, which may be manual or automated, and which in some cases results in a change in surface properties. In general, deposition can be performed using vapor deposition techniques, coating techniques, grafting techniques, or the like. Some specific examples include chemical vapor deposition (CVD), spray coating (eg, ultrasonic spray), spin coating, dunk/dip coating, blade coating, puddle dispensing, flow-through Coating, aerosol printing, screen printing, microcontact printing, inkjet printing or similar techniques.

As used herein, the term "depression" refers to an individual concave feature in a substrate having surface openings at least partially surrounded by interstitial regions of the substrate. The recesses may have any of a variety of shapes at their openings in the surface, including, for example, circles, ovals, squares, polygons, stars (with any number of vertices), and the like. The cross-sections of the depressions obtained normal to the surface may be curvilinear, square, polygonal, hyperbolic, conical, angular, and the like. As an example, the recess may be a hole or two interconnected holes. The recesses can also have more complex structures, such as ridges, step features, and the like.

As used herein, the term "DNA insert" refers to a DNA fragment in a sample. DNA size can range from about one hundred bases or base pairs to one hundred million bases or base pairs or more. DNA inserts in any given sample can have a size distribution ranging from small to large. A small DNA insert generally refers to having from about 100 bases or base pairs to about 1,000 bases or base pairs (eg, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any number in between) of DNA fragments. A large DNA insert generally refers to having more than 1,000 bases or base pairs (eg, 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, 15500, 16000, 16500, 17000, 18,000, 18,500, 19,500, 20,000, 20,000, 20,000, 2000 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、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、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 fragments of DNA, or any number in between)

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

The term "flow cell" refers to a vessel having a chamber in which a reaction can occur, an inlet for delivering reagents to the chamber; and an outlet for removing reagents from the chamber. In some examples, the chamber enables detection of reactions occurring in the chamber. For example, the chamber may include one or more transparent surfaces that allow for optical detection of arrays, optically labeled molecules, or the like.

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

As also used herein, "genomic DNA" (gDNA) and "whole genome amplified DNA" refer to high molecular weight (>1000 base pair (bp)) chromosomal DNA.

As used herein, "library DNA" refers to a collection of DNA fragments that includes adaptors at both ends. In some examples, the DNA fragments can be fragments of gDNA or cDNA.

As used herein, "nucleotide" includes nitrogen-containing heterocyclic bases, sugars, and one or more phosphate groups. Nucleotides are the monomeric units of nucleic acid sequences. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (the nucleotides of RNA), the sugar is ribose, and in deoxyribonucleotides (the nucleotides of DNA), the sugar is deoxyribose, that is, the lack of sugar is present in ribose hydroxyl at the 2' position. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G) and their modified derivatives or analogs. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and modified derivatives or analogs thereof. The C-1 atom of deoxyribose is bonded to the N-1 of pyrimidine or the N-9 of purine. For example, the phosphate group can be in the form of a monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, or hexaphosphate. These nucleotides are natural nucleotides, but it should be further understood that non-natural nucleotides, modified nucleotides, or analogs of the foregoing nucleotides may also be used.

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

As also used herein, the term "primer" refers to a nucleic acid molecule that can hybridize to a target sequence, such as an adaptor attached to a DNA fragment. As one example, an amplification primer can serve as a starting point for template amplification and cluster generation. As another example, synthetic nucleic acid (template) strands can include sites to which primers (eg, sequencing primers) can hybridize to allow primer synthesis of new strands complementary to the synthetic nucleic acid strands. Any primer can include any combination of nucleotides or analogs thereof. In some examples, the primer is a single-stranded oligonucleotide or polynucleotide. Primer lengths can be any number of bases in length and can include a variety of natural or non-natural nucleotides. In one example, the sequencing primers are short strands ranging from 10 bases to 60 bases or 20 bases to 40 bases.

"Reaction chamber" refers to the area in a flow cell where a reaction can occur. The reaction chamber can include recesses in which sequencing chemical reactions (eg, amplification primers) are immobilized.

As used herein, a "temperature controlled flow channel" is an enclosed area through which a liquid sample can flow and which can be heated or cooled by internal components (eg, a heating plate) or external components (eg, a laser).

The aspects and embodiments set forth herein and recited within the scope of the claims can be understood in light of the above definitions.

sample fluid

In any of the examples disclosed herein, a sample fluid is used. The sample fluid includes a DNA sample in an aqueous carrier, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt or solvent to adjust the gelling temperature of methylcellulose. In some examples, the solution consists of an aqueous carrier, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt or solvent to adjust the gelling temperature of the methylcellulose. In one example, the sample fluid includes a DNA sample in an aqueous carrier, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt.

DNA samples can include cell-free DNA, pool DNA, whole genome amplified DNA, or a combination thereof. The DNA sample can be single-stranded or double-stranded DNA, depending on the application in which the sample fluid will be used. For example, single-stranded DNA can be included in sample fluids to be used in sequencing applications, and single-stranded or double-stranded DNA can be included in sample fluids to be purified.

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 other examples, the DNA sample is present in the sample fluid at a molar concentration in the range of about 10 pM to about 950 µM, eg, about 25 pM to about 750 µM, about 500 pM to about 500 nM, about 500 nM to about 500 µM et al.

The aqueous carrier in solution can be water, a saline solution, or a buffered solution (eg, one of a weak acid and its salt (conjugate base), or one of a weak base and its salt (conjugate acid)). Exemplary buffer solutions include pars(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl) buffer, pars(hydroxymethyl)aminomethane (TRIS) buffer, or saline sodium citrate (SSC) buffer liquid. The aqueous carrier constitutes the balance of the sample fluid, and thus the amount can vary depending on the amount of the other components.

Methylcellulose is a thermoreversible hydrogel that precipitates or gels in aqueous solution upon exposure to heat. As an example, methylcellulose is available from Dow Chemical Company under the trademark METHOCEL®. In one example, the lower critical solution temperature (LCST) transition range of methylcellulose is in the range of about 25°C to about 60°C, which can be seen in the grade of methylcellulose and/or in the sample fluid Depends on the concentration of methyl cellulose. Other factors such as solution pH can also affect the LCST. Additionally, the LCST is more measurable when the concentration is higher than when the concentration is lower. Salts in the sample fluid can alter the LCST transition range. For example, increased salt concentration can force methylcellulose to precipitate out at low temperatures. For example, the LCST transition of methylcellulose ranges from about 5°C to about 45°C when the salt concentration is in the range of about 1.5 M to about 2 M. Below the LCST, the methylcellulose dissolves in the aqueous carrier, and at or above the LCST, the methylcellulose precipitates out of the aqueous carrier. This state is reversible, and thus methylcellulose can be controllably exchanged between a dissolved state and a precipitated state.

In one example, 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. In another example, methylcellulose is present in the sample fluid in an amount ranging from about 1 wt% to about 15 wt%, eg, about 1.5 wt% to about 10 wt%, about 5 wt%, based on the total weight of the sample fluid % to about 7.5 wt%, etc.

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

In one example, the chemically inert polymer 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. In another example, the chemically inert polymer is about 0.5 wt% to about 18 wt%, eg, about 1.5 wt% to about 16 wt%, about 5 wt% to about 15 wt%, based on the total weight of the sample fluid An amount within the same range is present in the sample fluid.

The sample fluid also includes a salt or solvent, which can be included to adjust the gelling temperature of the methylcellulose. If a salt solution or buffer solution is used as the aqueous solution, additional salt or solvent 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 gelling temperature.

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 in the range of about 0.25 M to about 1.75 M, eg, about 0.5 M to about 1.5 M, about 1 M to about 2 M, about 0.1 M to about 1 M Wait.

When making sample fluids, solutions 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 kept below the precipitation/gelling temperature of methylcellulose. This helps prevent premature precipitation of methylcellulose and also prevents any salts from coming out of solution. A DNA sample can be added to the solution to form a sample fluid. Some examples of the methods disclosed herein may include increasing the concentration of salt or the amount of solvent in the solution, thereby reducing the gelling temperature of methylcellulose.

Any instance of a sample fluid can be included in the kit. The components of the kit may depend on the application in which the sample fluid will be used. For example, the kit can include a flow cell when the sample fluid is used in sequencing.

Several example methods utilizing examples of sample fluids are described herein. The sample fluids discussed with respect to these methods specifically mention salts. It should be understood, however, that any example of a sample fluid disclosed herein (eg, including a solvent rather than a salt) may be used in any example of the methods disclosed herein.

DNA capture

An example of a DNA capture method 100 is shown in FIG. 1 . The method 100 includes combining a DNA sample and a solution to form a sample fluid, the solution consisting of an aqueous carrier, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt, and the solution is at a temperature of about 5°C to about 30°C and heating the sample fluid to at least the gelling temperature of methylcellulose, thereby forming a DNA-methylcellulose complex in the aqueous carrier (ref. 104). It should be understood that any example of a sample fluid can be used in the DNA capture method 100 .

The method 100 is shown schematically in FIG. 2 . The sample fluid 12 to the left of the arrow in FIG. 2 is below the gelation temperature of methylcellulose 14 . At this temperature, methylcellulose 14, chemically inert polymer 18, and salt 22 dissolve in aqueous carrier 20. Salt 22 in aqueous carrier 20 reduces methylcellulose solubility and helps reduce DNA charge interactions, thereby inducing DNA adhesion to precipitated methylcellulose.

The sample fluid 12 to the right of the arrow in FIG. 2 is at or 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, methylcellulose 14 precipitated out of solution. The precipitated methylcellulose polymer chains physically interact with the DNA sample 16 to form the DNA-methylcellulose complex 10 . DNA molecules are entangled in the precipitated methylcellulose polymer chains. The interaction between DNA molecules and methylcellulose polymer chains is non-covalent.

The temperature to which the sample fluid 12 is heated depends on the gelling temperature of the methylcellulose 14 . The gelling temperature of methylcellulose 14 depends in part on the concentration of methylcellulose 14 in sample fluid 12 . Thus, some examples of method 100 may involve selecting a heating temperature based on the concentration of methylcellulose 14 in sample fluid 12 . Generally, the temperature to which the sample fluid 12 is heated is in the range of about 25°C to about 60°C. As a specific example, when the methylcellulose concentration is in the range of about 1 wt% to about 6 wt%, the heating temperature is in the range of about 40°C to about 60°C. As another specific example, when the methylcellulose concentration is in the range of about 5 wt% to about 12 wt%, the heating temperature is in the range of about 25°C to about 50°C. The gelation temperature of methylcellulose 14 may also depend on the concentration of salt 22 in sample fluid 12, as described herein. Thus, some examples of method 100 may involve selecting a heating temperature based on the concentration of 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.

Heating may be performed using any suitable heat source, which may be internal components of the fluidic device containing the sample fluid 12, or may be external components that are 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 channel (see eg, Figure 5B). Examples of external components include a heating plate on which the fluidic device is placed, or a laser, the laser being directed towards the fluidic device.

The duration of heating the sample fluid 12 depends on the application in which the method 100 is performed. For example, if method 100 is used to capture and concentrate a DNA sample in a particular region of a flow channel, heating may be performed until the desired amount of sample fluid is introduced into the flow channel.

If or when the DNA captured in the DNA-methylcellulose complex 10 needs to be released, the method 100 may further include cooling the sample fluid 12 below the gelling temperature of the methylcellulose 14 by This disentangles the DNA-methylcellulose complex 10 to release the DNA sample 16 and methylcellulose 14 . Below the gelling temperature, methylcellulose 14, chemically inert polymer 18 and salt 22 dissolve in aqueous carrier 20.

In some instances, cooling is passive because the sample fluid 12 is allowed to cool to room temperature on its own. 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 may be an external component that is not part of the fluidic device containing the sample fluid. Examples of internal components may include thermoelectric coolers integrated into flow channels or reaction chambers. Examples of external components include fans directed toward the fluid device.

As schematically shown in FIG. 2, depending on the temperature to which the sample fluid 12 is exposed, methylcellulose 14 acts as a DNA capture agent or a DNA release agent. This may be required for a variety of applications including DNA capture, purification or concentration.

DNA purification

An example of the DNA capture method 100 can be used for DNA capture and purification. In one example, purification methods may be required when the DNA sample in sample fluid 12 includes a plurality of DNA inserts of different sizes, including small DNA inserts and large DNA inserts. In one example, the DNA insert is a single-stranded DNA insert. When multiple DNA inserts of different sizes are present in the sample fluid 12, the larger DNA inserts tend to interact more with the precipitated methylcellulose polymer chains than the smaller DNA inserts. Thus, at least some of the large DNA inserts are entangled in the DNA-methylcellulose complex 10 and at least some of the small DNA inserts are not entangled in the DNA-methylcellulose complex 10 . This can be attributed to the size of the larger DNA inserts, which can physically prevent the smaller DNA inserts from becoming entangled with the precipitated methylcellulose polymer chains.

An example of a purification method is shown schematically in FIG. 3 . This example of the method includes combining a DNA sample including both small DNA inserts 16A and large DNA inserts 16B with a solution to form sample fluid 12; heating sample fluid 12 to at least a gel of methylcellulose 14 coagulation temperature, thereby forming a large DNA insert-polymer complex 10' in the aqueous carrier 20; and a purification process followed by heating to polymerize at least some of the small DNA insert 16A with the large DNA insert-polymer complex 10' was isolated.

The leftmost sample fluid 12 in FIG. 3 is below the gelation temperature of methylcellulose 14 . At this temperature, methylcellulose 14, chemically inert polymer 18, and salt 22 dissolve in aqueous carrier 20. The small DNA insert 16A and the large DNA insert 16B are dispersed in the aqueous carrier 20 .

When heated to at least the gelling temperature of methylcellulose 14, chemically inert polymer 18 and salt 22 remain dissolved in aqueous vehicle 20 while methylcellulose 14 precipitates out of solution. The precipitated methylcellulose polymer chains physically interact with at least some of the large DNA inserts 16B, forming large DNA insert-polymer complexes 10'. At least some of the smaller DNA inserts 16A are not entangled with methylcellulose polymer chains, and thus are not part of the large DNA insert-polymer complex 10'.

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

Fresh solution (no additional DNA sample) can be added to the isolated complex 10' prior to cooling to release the large DNA insert 16B. If it is desired to release the large DNA insert 16B captured in the DNA-methylcellulose complex 10' or when it is desired to release the DNA insert, this example may further include making the DNA-methylcellulose complex 10' Cooling to below the gelling temperature of methylcellulose 14 thereby unwinds complex 10' to release large DNA insert 16B and methylcellulose 14. Below the gelling temperature, methylcellulose 14, chemically inert polymer 18 and salt 22 dissolve in aqueous carrier 20.

concentrate DNA

Another example method is shown at reference numeral 200 in FIG. 4 . This exemplary method 200 can be used for DNA capture and concentration. The method 200 includes introducing a sample fluid including an aqueous carrier, a DNA sample, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt (reference number) into a temperature-controlled flow channel having a filter disposed therein. 202); heating the temperature-controlled flow channel when introducing the sample fluid, so that the temperature of the sample fluid contained therein is increased to at least the gelation temperature of the methylcellulose, thereby forming DNA in the temperature-controlled flow channel - Methylcellulose complex (ref. 204); while heating the temperature-controlled flow channel, continue to pass the sample fluid through the temperature-controlled flow channel, thereby concentrating the complex at the filter in the temperature-controlled flow channel each of the DNA-methylcellulose complexes (ref. 206); cooling the temperature-controlled flow channel so that the temperature of the sample fluid contained therein is lowered below the gelling temperature of the methylcellulose, thereby allowing The concentrated DNA-methylcellulose complexes disentangle to release the DNA sample and the methylcellulose, whereby the DNA sample and the methylcellulose can pass through the filter (ref. 208).

Method 200 is shown schematically in Figures 5A-5E.

FIG. 5A depicts a flow cell assembly 24 that may be used in method 200 . Flow cell assembly 24 includes flow cell 26 that includes reaction chamber 28 having recesses separated by gap regions and capture primers connected within each of the recesses (FIG. 5E); temperature controlled flow a channel 30 in selective fluid communication with the inlet 32 of the reaction chamber 28; and a filter 34 (shown in FIG. 5C ) disposed in the temperature-controlled flow channel 30, the filter 34 i) for blocking at the first temperature Concentrated deoxyribonucleic acid (DNA)-polymer complex 10 produced in temperature-controlled flow channel 30 at a second temperature, and ii) concentrated deoxyribonucleic acid (DNA)-polymer complex 10 for release from complex 10 in temperature-controlled flow channel 30 at a second temperature DNA 16 and polymer (methylcellulose 14) pass through.

In the flow tank assembly 24 , a temperature-controlled flow channel 30 is positioned upstream of the flow tank 26 . This arrangement enables the DNA sample 16 to be captured and concentrated from the sample fluid 12 prior to its introduction into the reaction chamber 28 of the flow cell 26 .

The temperature-controlled flow channel 30 may be a conduit, conduit, microfluidic channel, or the like. 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 the DNA sample 16 in the sample fluid prior to introducing the sample fluid 12 into the reaction chamber 28 of the flow cell 26. The example channel 30 shown in Figure 5A has a serpentine shape.

The temperature-controlled flow channel 30 may be heated and cooled such that the sample fluid 12 flowing through the 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 in the channel 30 or defining one or more inner walls of the flow channel 30 . In another example, the flow channel assembly 24 includes an external heating component 38 ( FIG. 5B ), such as a laser, disposed outside the channel 30 . An external heating assembly 38 may be operably positioned to direct heat toward the channel 30 . Some examples of temperature-controlled flow channels 30 also include internal cooling components (not shown), such as thermoelectric coolers, etc., disposed in the channel 30 or defining one or more inner walls of the flow channel 30 . Other examples of temperature-controlled flow channels 30 include external cooling components (not shown), such as fans, disposed outside of the channels 30 . The internal or external components can also be combined devices capable of active heating and active cooling.

Although not shown, the inner heating element 36 or outer heating element 38 and the inner cooling element or outer cooling element (when in use) may be operatively connected to a temperature control unit that operates the element 36 or 38 and controls the temperature of the element 36 or 38. In addition, a temperature sensor can be placed in the channel 30 to provide real-time data on the temperature in the channel 30 to the temperature control unit so that the desired internal temperature can be obtained and/or maintained.

The flow cell assembly 24 may also include: a bypass line 40 in fluid communication with the inlet 48 of the temperature-controlled flow channel 30 and with the outlet 50 of the temperature-controlled flow channel 30; a first bypass valve 42 for controlling the sample fluid 12 the flow to the inlet of the temperature controlled flow channel 30;

The bypass line 40 may be a conduit, pipe, microfluidic channel, or the like. Bypass line 40 allows fluid to be directed from reservoir 46 or other storage unit to reaction chamber 28 without passing through temperature-controlled flow channel 30 . For example, bypass line 40 may be used when the fluid to be delivered to reaction chamber 28 does not include DNA sample 16, when heating of the fluid is not required, or when the concentration of DNA sample 16 is undesirable. Examples of fluids that may be routed through bypass line 40 rather than through temperature-controlled flow channel 30 include wash fluids, deblocker fluids, reactive fluids (including polymerases, sequencing primers, nucleotides, etc.).

The first bypass valve 42 is switchable between two positions, one directing fluid through the temperature-controlled flow channel 30 (and thus closing the bypass line 40 ) and the other directing fluid through the bypass line 40 (and thus Close the temperature-controlled flow channel 30). The second bypass valve 44 is also switchable between two positions, one directing fluid out of the temperature-controlled flow channel 30 (and not allowing reverse flow through the bypass line 40 ) and the other directing fluid through Bypass line 40 (and thus not allowing reverse flow through temperature controlled flow channel 30). The arrows shown in FIG. 5A depict fluid flow when the valves 42 , 44 are positioned to open the temperature-controlled flow channel 30 and close the bypass line 40 . Any suitable valve may be used for the first bypass valve 42 and the second bypass valve 44 .

The temperature-controlled flow channel 30 may be connected to the inlet 32 of the reaction chamber 28 via a manifold 70 or other fluid connector.

The temperature-controlled flow channel 30 also includes a filter 34 . The filter 34 may be secured within the temperature-controlled flow channel 30 using adhesives, mechanical attachment mechanisms, or the like. In other examples, the filter 34 may be built into the temperature-controlled flow channel 30 during manufacture. The filter 34 may be attached to the entire inner periphery of the temperature-controlled flow channel 30 . Thus, the filter 34 covers a cross-sectional area (eg, it is parallel to the inlet 48 of the temperature-controlled flow channel 30).

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

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

In the example shown in FIG. 5A , the flow cell 26 includes eight reaction chambers 28 . Although eight reaction chambers 28 are shown, it should be understood that any number of reaction chambers 28 may be included in the flow cell 26 (eg, a single reaction chamber 28, four reaction chambers 28, etc.). Each reaction chamber 28 is an area defined between two bonded components (eg, a substrate 52 and a lid or two substrates 52 ) that may have fluids introduced therein and removed therefrom (eg, as described herein). of them). Each reaction chamber 28 may be separated from each other reaction chamber 28 such that fluid introduced into any particular reaction chamber 28 does not flow into any adjacent reaction chamber 28 . Some examples of fluids introduced into reaction chamber 28 may introduce reaction components (eg, DNA sample 16, polymerase, sequencing primers, nucleotides, etc.), wash solutions, deblockers, and the like.

The reaction chamber 28 is at least partially bounded by the substrate 52 . Substrate 52 may be a single-layer structure, or may be a multi-layer structure (as shown in FIG. 5E ).

Examples of suitable single-layer construction materials include epoxy siloxanes, glass, modified or functionalized glass, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, Polybutene, polyurethane, polytetrafluoroethylene (such as TEFLON® from Chemours), cyclic olefin/cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon), polyimide, etc. ), nylon (polyamide), ceramics/ceramic oxides, silica, fused silica or silica-based materials, aluminum silicates, silicon and modified silicon (such as boron-doped p+ silicon), nitrogen Silicon oxide (Si ₃ N ₄ ), silicon oxide (SiO ₂ ), tantalum pentoxide (Ta ₂ O ₅ ) or other tantalum oxide (TaO _x ), hafnium oxide (HfO ₂ ), carbon, metal, inorganic glass or its analog.

Some examples of multilayer structures include glass or silicon with tantalum oxide or another ceramic oxide coating on the surface. Other examples of multilayer structures may include silicon-on-insulator (SOI) substrates. Other examples of multilayer structures, such as the example shown in Figure 5E, include an underlying support 54 (eg, glass or silicon) with 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 gap regions 60 can be used to pattern material 56 .

As one example of patterning material 56, inorganic oxides may be selectively applied to support 52 via vapor deposition, aerosol printing, or ink jet printing. Examples of suitable inorganic oxides include tantalum oxide (eg, _Ta2O5 ), aluminum _oxide (eg, _Al2O3 ), silicon oxide (eg, _SiO2 ), hafnium oxide (eg, _{HfO2 )} , and the like.

As another example of patterning material 56, a resin may be applied to support 52 and then patterned. Suitable deposition techniques include chemical vapor deposition, dip coating, dunk coating, spin coating, spray coating, liquid dispensing, ultrasonic spray coating, blade coating, aerosol printing, screen printing, Micro-contact printing, etc. Suitable patterning techniques include photolithography, nanoimprint lithography (NIL), stamping techniques, imprinting techniques, molding techniques, micro-etching techniques, printing techniques, and the like. Some examples of suitable resins include polyhedral oligomeric silsesquioxane resins (eg, POSS® from Hybrid Plastics), non-polyhedral oligomeric silsesquioxane epoxy resins, poly(ethylene glycol) resins, polyethers Resins (eg, ring-opening epoxy resins), acrylic resins, acrylate resins, methacrylate resins, amorphous fluoropolymer resins (eg, CYTOP® from Bellex), and combinations thereof.

As used herein, the term "polyhedral oligomeric silsesquioxane" refers to a chemical composition that is a mixed intermediate between silicon dioxide ( _SiO2 ) and polysiloxane ( _R2SiO ). substance (RSiO _1.5 ). Examples of polyhedral oligomeric silsesquioxanes may be the polyhedral oligomeric silsesquioxanes described in Kehagias et al., Microelectronic Engineering 86 (2009) pp. 776-778, which reference is incorporated by reference in its entirety . In one example, the composition is an organosilicon compound having the formula [RSiO _3/2 ]n, wherein the R groups may be the same or different. Example R groups of polyhedral oligomeric silsesquioxanes include epoxy, azide/azido, thiol, poly(ethylene glycol), norbornene, tetrakis, acrylate and/or methacrylic acid Esters, or otherwise such as alkyl, aryl, alkoxy and/or haloalkyl.

In one example, the substrate 52 (single or multi-layer) may have a diameter in the range of about 2 mm to about 300 mm, or a rectangular sheet or plate with a maximum dimension of up to 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. In another example, the substrate 52 is a die having a width in the range of about 0.1 mm to about 10 mm. Although example dimensions have been provided, it should be understood that any suitable dimensions of substrate 52 may be used. As another example, a plate in the form of a rectangular support can be used, which has a larger surface area than a 300 mm round wafer.

In some examples, the reaction chamber 28 is etched into the glass substrate. In other examples, reaction chamber 28 is patterned into patterned material 56 having a multilayer structure using photolithography, nanoimprint lithography, or the like. In yet other examples, a separate material (not shown) may 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 .

In one example, the reaction chamber 28 has a substantially rectangular configuration. The length and width of reaction chamber 28 may be less than the length and width of substrate 52 , respectively, such that a portion of the substrate surface surrounding reaction chamber 28 may be 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, less than, or between the values specified above. In another example, the reaction chamber 28 is square (eg, 10 mm x 10 mm).

When microcontact, aerosol or ink jet printing is used to deposit the individual materials that define the walls of the reaction chamber 28, the depth of the reaction chamber 28 can be as small as a single layer thickness. For other examples, the depth of reaction chamber 28 may be about 1 μm, about 10 μm, about 50 μm, about 100 μm, or more. 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 another example, the depth is about 5 μm or less. It should be understood that the depth of reaction chamber 28 may be greater, less than, or between the values specified above.

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 disposed at both ends of the flow channel 26 . The inlet and outlet of the corresponding reaction chamber 28 may alternatively be positioned anywhere along the length and width of the reaction chamber 28 that achieves the desired fluid flow.

The inlet allows the introduction of fluid into the reaction chamber 28 and the outlet allows fluid to be extracted from the reaction chamber 28 . Each of the inlet and outlet is fluidly connected to a fluid control system (including, for example, reservoir 46, pumps, valves 42, 44, waste containers, etc.) that controls fluid introduction and discharge.

FIG. 5E depicts an example of the architecture within reaction chamber 28 . The framework includes recesses 58 separated by gap regions 60 . Recesses 58 may be defined in substrate 52 . In one example, recesses 58 are defined in a single-layer structure (eg, etched into glass), or as shown in FIG. 5E , in patterned material 56 of a multi-layer structure.

Numerous different arrangements of recesses 58 are contemplated, including regular, repeating, and irregular patterns. In one example, the recesses 58 are arranged in a hexagonal grid for tight packing and improved density. Other layouts may include, for example, rectilinear (rectangular) layouts, triangular layouts, and the like. In some examples, the layout or pattern may be columns and rows of recesses 58 in an x-y format. In some other examples, the layout or pattern may be a repeating arrangement of recesses 58 and/or gap regions 60 . In yet other examples, the layout or pattern may be a random arrangement of recesses 58 and/or gap regions 60 .

The layout or pattern of recesses 58 may be characterized with respect to the density of recesses 58 (the number of recesses 58) in a defined area. For example, the recesses 58 may exist at a density of approximately 2 million per square millimeter. The density can be adjusted to different densities including, for example, the following densities about 100/mm, about 1,000/mm, about 100,000/mm, about 1 million/mm, about 2 million/mm mm, about 5 million per square millimeter, about 1 million per square millimeter, about 50 million per square millimeter or more or less. It should be further understood that the density of recesses 58 in patterned material 56 may be selected between one of the lower limit values and one of the upper limit values of the above ranges. As an example, high density arrays can be characterized to have recesses 58 spaced apart by less than about 100 nm, medium density arrays can be characterized to have recesses 58 spaced from about 400 nm to about 1 μm apart, and low density arrays can be characterized to have recesses 58 spaced apart by about 400 nm to about 1 μm 58 are separated by more than about 1 μm. Although example densities have been provided, it should be understood that any suitable densities can be used. The density of recesses 58 may depend in part on the depth of recesses 58 . In some cases, the spacing between recesses 58 may be required to be even greater than the examples listed herein.

The layout or pattern of the recesses 58 can also or alternatively be at an average spacing or a spacing from the center of a recess 58 to the center of an adjacent recess 58 (center-to-center space) or from the right edge of one recess 58 to the left of an adjacent recess 58 The aspect of the distance between edges (space between edges) is characterized. The pattern may be regular such that the coefficient of variation with respect to the average spacing 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 can be, for example, about 50 nm, about 0.1 μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, or longer or shorter. The average pitch of a particular pattern of recesses 58 may be between one of the lower values and one of the upper values selected from the above ranges. In one example, the recesses 58 are spaced apart (center-to-center) by about 1.5 μm. Although example average pitch values have been provided, it should be understood that other average pitch values may be used.

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

Each recess 58 may have any volume capable of confining fluid. The minimum or maximum volume may be selected, eg, to accommodate the expected throughput (eg, multiplexity), resolution, nucleotide or analyte reactivity for downstream use of the flow cell 26 . For example, the volume can be at least about 1×10 ⁻³ μm ³ , at least about 1×10 ⁻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 Or more. Alternatively or additionally, the volume may be at most about 1×10 ⁴ μm ³ , at most about 1×10 ³ μm ³ , at most about 100 μm ³ , at most about 10 μm ³ , at most about 1 μm ³ , at most about 0.1 μm ³ , or smaller.

The area occupied by each recess opening can be selected based on similar criteria as set forth above for volume. For example, the area of each recess opening may be at least about 1×10 ⁻³ μm ² , at least about 1×10 ⁻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 10 μm 2 About 100 μm ² or more. Alternatively or additionally, the area may be at most about 1×10 ³ μm ² , at most about 100 μm ² , at most about 10 μm ² , at most about 1 μm ² , at most about 0.1 μm ² , at most about 1×10 ⁻² μm ² or less. The area occupied by each recess opening can be greater, less than, or between the values specified above.

The depth of each recess 58 may be large enough to accommodate the polymeric hydrogel 66 and capture primers 62,64. In one example, the depth can 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 deeper. Alternatively or additionally, the depth may be at most about 1×10 ³ μm, at most about 100 μm, at most about 10 μm or less. In some examples, the depth is about 0.4 μm. The depth of each recess 58 may be greater, less than, or between the values specified above.

In some cases, the diameter or length and width of each recess 58 can 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 longer. Alternatively or additionally, the diameter or length and width may be at most about 1×10 ³ μm, at most about 100 μm, at most about 10 μm, at most about 1 μm, at most about 0.5 μm, at most about 0.1 μm or less (eg, about 50 nm). In some examples, the diameter or length and width are about 0.4 μm. The diameter or length and width of each recess 58 can be greater, less than, or between the values specified above.

其中： R ^A選自由以下組成之群：疊氮基、視需要經取代之胺基、視需要經取代之烯基、視需要經取代之炔、鹵素、視需要經取代之腙、視需要經取代之肼、羧基、羥基、視需要經取代之四唑、視需要經取代之四𠯤、氧化腈、硝酮、硫酸酯及硫醇； R ^B為H或視需要經取代之烷基； R ^C、R ^D及R ^E各自獨立地選自由H及視需要經取代之烷基組成之群； -（CH ₂） _p-中之每一者可視需要經取代； p為1至50範圍內之整數； n為1至50,000範圍內之整數；且 m為1至100,000範圍內之整數。 A polymeric hydrogel 66 is present in each of the recesses 58 . Examples of polymeric hydrogels 66 include acrylamide copolymers represented by the following structure (I):

wherein: R ^A is selected from the group consisting of: azido, optionally substituted amine, optionally substituted alkenyl, optionally substituted alkyne, halogen, optionally substituted hydrazone, optionally substituted Substituted hydrazine, carboxyl, hydroxyl, optionally substituted tetrazole, optionally substituted tetrazole, nitrile oxide, nitrone, sulfate and thiol; R ^B is H or optionally substituted alkyl; R ^C , ^RD , and RE are each independently selected from the group consisting of ^H and optionally substituted alkyl; each of -( _CH2 ) _p- is optionally substituted; p is in the range of 1 to 50 Integer; n is an integer in the range of 1 to 50,000; and m is an integer in the range of 1 to 100,000.

A specific example of an acrylamide copolymer represented by structure (I) is poly(N-(5-azidoacetamidopentyl) acrylamide-co-acrylamide (PAZAM).

One of ordinary skill in the art will recognize that the configuration of repeating "n" and "m" features in formula (I) is representative, and that the monomeric subunits may be present in the polymer structure in any order (eg, no random, block, patterned, or a combination thereof).

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

In some examples, PAZAM and other forms of acrylamide copolymers are linear polymers. In some other examples, PAZAM and other forms of acrylamide copolymers are lightly cross-linked polymers.

）置換。在此實例中，結構（I）中之丙烯醯胺單元可經

置換，其中R ^D、R ^E及R ^F各自為H或C1-C6烷基，且R ^G及R ^H各自為C1-C6烷基（而非如同丙烯醯胺之情況為H）。在此實例中，q可為1至100,000範圍內之整數。在另一個實例中，除丙烯醯胺單元之外，可使用N,N-二甲基丙烯醯胺。在此實例中，除重複的「n」及「m」個特徵以外，結構（I）可包括

，其中R ^D、R ^E及R ^F各自為H或C1-C6烷基，且R ^G及R ^H各自為C1-C6烷基。在此實例中，q可為1至100,000範圍內之整數。 In other examples, polymeric hydrogel 66 can be a variant of structure (I). In one example, the acrylamide units can be treated with N,N-dimethylacrylamide (

) replacement. In this example, the acrylamide units in structure (I) can be

Substitutions wherein ^RD , RE, and ^RF are each ^H or C1-C6 alkyl, and ^RG and ^RH are each C1-C6 alkyl (rather than H as in the case of acrylamide). In this example, q can be an integer ranging from 1 to 100,000. In another example, N,N-dimethylacrylamide can be used in addition to the acrylamide units. In this example, in addition to the repeated "n" and "m" features, structure (I) may include

, wherein R ^D , R ^E and R ^F are each H or C1-C6 alkyl, and R ^G and ^RH are each C1-C6 alkyl. In this example, q can be an integer ranging from 1 to 100,000.

其中R ¹為H或C1-C6烷基；R ₂為H或C1-C6烷基；L為包括直鏈之連接子，該直鏈具有2至20個選自由碳、氧及氮組成之群的原子及10個在鏈中之碳及任何氮原子上視需要存在之取代基；E為直鏈，其包括1至4個選自由碳、氧及氮組成之群的原子及鏈中之碳及任何氮原子上視需要存在之取代基；A為經N取代之醯胺，其中H或C1-C4烷基連接於N；且Z為含氮雜環。Z之實例包括以單一環狀結構或稠合結構存在之5至10個環成員。Z之一些特定實例包括吡咯啶基、吡啶基或嘧啶基。 As another example of polymeric hydrogel 66, the repeating "n" feature in structure (I) can be replaced with a monomer including a heterocyclic azide group having structure (II):

wherein R ¹ is H or C1-C6 alkyl; R ₂ is H or C1-C6 alkyl; L is a linker including a straight chain, the straight chain has 2 to 20 selected from the group consisting of carbon, oxygen and nitrogen E is a straight chain including 1 to 4 atoms selected from the group consisting of carbon, oxygen and nitrogen and carbon in the chain and any optional substituent on the nitrogen atom; A is an N-substituted amide, wherein H or C1-C4 alkyl is attached to N; and Z is a nitrogen-containing heterocycle. Examples of Z include 5 to 10 ring members in a single ring structure or a fused structure. Some specific examples of Z include pyrrolidinyl, pyridyl, or pyrimidinyl.

及

其中R ^1a、R ^2a、R ^1b及R ^2b中之各者獨立地選自氫、視需要經取代之烷基或視需要經取代之苯基；R ^3a及R ^3b中之各者獨立地選自氫、視需要經取代之烷基、視需要經取代之苯基或視需要經取代之C7-C14芳烷基；且各L ¹及L ²獨立地選自視需要經取代之伸烷基連接子或視需要經取代之伸雜烷基連接子。 As another example, polymeric hydrogel 66 may include repeating units of each of structures (III) and (IV):

and

wherein each of R ^1a , R ^2a , R ^1b and R ^2b is independently selected from hydrogen, optionally substituted alkyl, or optionally substituted phenyl; each of R ^3a and R ^3b is independently selected from hydrogen, optionally substituted alkyl, optionally substituted phenyl, or optionally substituted C7-C14 aralkyl; and each L ¹ and L ² are independently selected from optionally substituted alkylene linker or optionally substituted heteroalkyl linker.

It should be understood that other molecules may be used to form the polymeric hydrogel 66, so long as the molecules are functionalized to graft the oligonucleotide primers 62, 64 thereon. Other examples of suitable polymer layers include molecules with a colloidal structure, such as agarose; or molecules with a polymer network structure, such as gelatin; or molecules with a cross-linked polymer structure, such as polyacrylamide polymers and Copolymer, silane free acrylamide (SFA) or the azide form of SFA. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or vinyl-containing acrylic acid, or from monomers forming [2+2] photocycloaddition reactions. Still other examples of suitable polymeric hydrogels 66 include hybrid copolymers of acrylamide and acrylate. Various polymer frameworks containing acrylic monomers (eg, acrylamide, acrylates, etc.) can be used in the examples disclosed herein, such as branched polymers, including star polymers, star or star block copolymers, Dendrimers, etc. For example, monomers (eg, acrylamide, acrylamide with catalyst, etc.) can be incorporated randomly or in blocks into the branches (arms) of the star polymer.

In another example, nitroxide-mediated polymerization is used to form acrylamide copolymers, and thus at least some of the copolymer chains have alkoxyamine end groups. In the copolymer chain, the term "alkoxyamine end group" refers to the dormant species ONR ₁ R ₂ , wherein each of R ₁ and R ₂ may be the same or different, and may independently be A straight or branched chain alkyl or cyclic structure in which the oxygen atom is attached to the remainder of the copolymer chain. In some examples, alkoxyamines can also be incorporated into some repeating acrylamide monomers, such as at position ^RA in structure (I). Thus, in one example, structure (I) includes alkoxyamine end groups; and in another example, structure (I) includes alkoxyamine end groups and alkoxyamine groups in at least some of the side chains.

In the example shown in FIG. 5E , the polymeric hydrogel 66 is disposed within each of the recesses 58 and not over the surrounding gap region 60 .

To introduce polymeric hydrogel 66 into recess 58 , a mixture of polymeric hydrogel 66 may be produced and then applied to substrate 52 . In one example, the polymeric hydrogel 66 may be present in a mixture (eg, with water or with ethanol and water). The mixture may then be applied to the substrate surface (including the recesses 58 ) using spin coating or dipping or dipping, spraying or flowing the material under positive or negative pressure, or another suitable technique. These types of techniques deposit polymeric hydrogel 66 entirely on patterned material 56 (eg, in recesses 58 and on gap regions 60). Other selective deposition techniques (eg, involving masking, controlled printing techniques, etc.) may be used to deposit the polymeric hydrogel 66 exclusively in the recesses 58 and not on the interstitial regions 60 .

In some examples, the substrate surface can be activated, and then the mixture, including the polymeric hydrogel 66, can be applied to the substrate surface. In one example, silanes or silane derivatives (eg, norbornene silanes) can be deposited on the substrate surface using vapor deposition, spin coating, or other deposition methods. In another example, the substrate surface can be exposed to plasma ashing to produce one or more surfactants (eg, -OH groups) that can adhere to the polymeric hydrogel 66 .

Depending on the polymerized hydrogel 66, the applied mixture may be exposed to a curing process. In one example, curing can be performed at a temperature ranging from room temperature (eg, about 25°C) to about 95°C for a period ranging from about 1 millisecond to about several days.

In some examples, polishing may then be performed to remove the polymeric hydrogel 66 from the interstitial regions 60 while leaving the polymeric hydrogel 66 in the recesses 58 at least substantially intact.

The flow cell 26 also includes capture primers 62,64.

A grafting process may be performed to graft the capture primers 62 , 64 to the polymeric hydrogel 66 in the recess 58 . In one example, the capture primers 62, 64 can be immobilized to the polymeric hydrogel 66 by a single point of covalent attachment at or near the 5' end of the primers 62, 64. This ligation leaves i) the adaptor-specific portions of primers 62, 64 free for attachment to their cognate nucleic acid fragments and ii) free of the 3' hydroxyl group of the primer extension. Any suitable covalent linkage can be used for this purpose. Examples of capping primers that can be used include alkyne capping primers, which can be attached to the azide moiety of polymeric hydrogel 66. Specific examples of suitable primers 62, 64 include the P5 and P7 primers used on the surface of commercial flow cells sold by Illumina, Inc. for use in HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, Sequencing on NOVASEQ™, GENOME ANALYZER™, ISEQ™ and other instrument platforms.

In one example, grafting can involve polymeric hydrogels via deposition (eg, using temporarily bonded or permanently bonded caps), dip coating, spray coating, liquid dispensing, or by attaching primers 62, 64 in recesses 58 Another suitable method of 66 is flow. Each of these example techniques may utilize a primer solution or mixture, which may include primers 62, 64, water, buffers, and catalysts. In the case of either grafting method, the primers 62 , 64 react with the reactive groups of the polymeric hydrogel 66 and have no affinity for the surrounding interstitial region 60 . Thus, the primers 62 , 64 are selectively grafted to the polymeric hydrogel 66 and not to the interstitial region 60 .

Although not shown in FIG. 5A or FIG. 5E , it should be understood that the flow channel 26 may also include a cover attached to the substrate 52 . In one example, the cover may be bonded to at least a portion of the substrate 52 , such as some of the gap regions 60 . In one example, the cover is bonded at the perimeter and between the reaction chambers 28 . The bond formed between the lid and the substrate 52 may be chemical bonding or mechanical bonding (eg, using fasteners, etc.).

The cover can be any material that is transparent to the excitation light that guides the substrate 52 . As an example, the cover may be glass (eg, borosilicate, fused silica, etc.), plastic, or the like. A commercially available example of a suitable borosilicate glass is D 263®, available from Schott North America. A commercially available example of a suitable plastic material, ie, a cyclic olefin polymer, is the ZEONOR® product, available from Zeon Chemicals L.P.

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, frit bonding, or other methods known in the art. In one example, a spacer layer can be used to bond the lid to the substrate 52 . The spacer layer can be any material that seals at least some of the substrates 52 and the lid together. In some examples, the spacer layer may be a radiation absorbing material that facilitates bonding of the substrate 52 to the lid.

In other examples, the flow channel 26, rather than the lid, may include two substrates 52 bonded together such that the reaction chambers 28 face each other and the fluid channels are defined therebetween. The two substrates 52 may be bonded together at some gap regions 60 (eg, at the perimeter and between the reaction chambers 28 ). The bonds formed between the substrates 52 may be chemical bonds or mechanical bonds (eg, using fasteners, etc.).

Referring back to method 200 , sample fluid 12 (shown in FIG. 5B ) is introduced into temperature-controlled flow channel 30 . The sample fluid 12 may be stored in the reservoir 46 or introduced into the reservoir and then pumped through the intake line 68, which is fluidly connected to the first bypass valve 42, the bypass line 40 and the temperature controlled flow channel 30. The first bypass valve 42 may be positioned such that the sample fluid 12 is directed to the temperature-controlled flow channel 30 .

When the sample fluid 12 is introduced into the temperature-controlled flow channel 30 , the temperature of the flow channel 30 and the sample fluid 12 is lower than the gelation temperature of the methylcellulose 14 in the sample fluid 12 . At this temperature, methylcellulose 14, chemically inert polymer 18 and salt 22 dissolve in aqueous carrier 20 while DNA sample 16 is dispersed in aqueous carrier 20. This is shown schematically at the left side of the temperature controlled flow channel 30 in Figure 5B.

The method 200 also involves heating the temperature-controlled flow channel 30 as the sample fluid 12 is introduced into the temperature-controlled flow channel. The temperature to which the temperature-controlled flow channel 30 is heated will depend on the gelation temperature of the methylcellulose 14 in the sample fluid 12 . The temperature-controlled flow channel 30 can reach the gel temperature or above, so that the temperature of the sample fluid 12 can reach the gel temperature.

Internal or external heating components 36 or 38 may be used to heat the temperature-controlled flow channel 30 . Sensor feedback can be used to achieve and/or maintain the desired temperature.

As shown on the right side of the temperature-controlled flow channel 30 in FIG. 5B , the heat precipitates the methylcellulose 14 from the aqueous carrier 20 . The precipitated methylcellulose polymer chains physically interact with the DNA sample 16 to form the DNA-methylcellulose complex 10 within the temperature-controlled flow channel 30 .

The method 200 also involves continuing to flow the sample fluid 12 through the temperature-controlled flow channel 30 while heating the temperature-controlled flow channel. This causes the DNA-methylcellulose complex 10 to flow towards the filter 34 where it is blocked from further flow. As shown in FIG. 5C , this concentrates the plurality of DNA-methylcellulose complexes 10 at the filter 34 in the temperature-controlled flow channel 30 .

It will be appreciated that even though the DNA-methylcellulose complex 10 is blocked, the aqueous carrier 20 and any components dissolved therein, such as the salt 22 and the chemically inert polymer 18, can continue to flow through the filter 34. Aqueous carrier 20 and any components dissolved therein may be directed to a waste sump (not shown), or may be directed to flow-through 26 and through it into the waste sump.

The method 200 further includes cooling the temperature-controlled flow channel 30 such that the temperature of the sample fluid 12 contained therein is lowered below the gelation temperature of the methylcellulose 14 . In some examples, the inner or outer heating components 36 or 38 may be turned off to cool the temperature-controlled flow channel 30 . This involves passive cooling by allowing the sample fluid to cool itself. In other examples, the temperature-controlled flow channel 30 can include a cooling component or a heating/cooling component that can be controlled to actively reduce the temperature of the temperature-controlled flow channel 30 and the sample fluid 12 contained therein.

Can begin after a predetermined duration of sample fluid 12 flow and/or when a desired amount of sample fluid 12 has been introduced into temperature-controlled flow channel 30, and/or when a desired amount of complex 10 is concentrated at filter 34 cool down. As an example, the volume of fluid passing through filter 34 may be calculated, or a specific counter may be incorporated into the line at filter 34 .

The temperature to which the temperature-controlled flow channel 30 is cooled will depend on the gelation temperature of the methylcellulose 14 in the sample fluid 12 . The temperature-controlled flow 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.

As shown in FIG. 5D , cooling causes the DNA-methylcellulose complex 10 to disentangle in the temperature-controlled flow channel 30 , eg, concentrate at the filter 34 . More specifically, cooling dissolves methylcellulose 14 in aqueous carrier 20 , which disentangles DNA-methylcellulose complex 10 and releases DNA sample 16 . Both the solubilized methylcellulose 14 and the DNA sample 16 can flow through the filter 34 and out of the temperature-controlled flow channel 30 .

Method 200 may further include delivering DNA sample 16 and methylcellulose 14 to flow cell 26 and reaction chamber 28 for analysis (eg, sequencing).

The flow cell assembly 24 described in connection with the method 200 may be part of a kit that may also include an instance of the sample fluid 12 .

DNA inoculate

Another example method is shown at reference numeral 300 in FIG. 6 . This example method 300 can be used to improve DNA seeding for sequencing. The method 300 includes introducing a sample fluid into a flow cell, the sample fluid including an aqueous carrier, a DNA sample, methylcellulose, a polymer chemically inert to DNA hybridization, and a salt; and the flow cell includes a region having a gap formed by Reaction chambers of spaced recesses and capture primers attached within each of the recesses (ref. 302); initiation of seeding and hybridization of at least some of the DNA samples in at least some of the recesses (ref. 304); heated flow cell , causing the temperature of the sample fluid contained therein to increase to at least the gelling temperature of methylcellulose, thereby forming a DNA-methylcellulose complex (ref. 306) with the unbound DNA sample; an additional amount of sample The fluid is introduced into the flow cell (ref. 308); the flow cell is cooled so that the temperature of the sample fluid contained therein is lowered to below the gelation temperature of methylcellulose, thereby dissolving the concentrated DNA-methylcellulose complex. entanglement to release DNA samples and methylcellulose (ref. 310); and inoculation and hybridization of at least some of the released DNA samples in at least some of the wells and at least some of the DNA samples from additional sample fluids (ref. 312) .

Method 300 is shown schematically in Figures 7A-7F.

In this example of method 300, DNA sample 16 in sample fluid 12 includes DNA library fragments. DNA library fragments include adapters at both ends. DNA library fragments can be prepared using any library preparation technique that yields longer fragments of genetic material and incorporates the desired adaptors into the ends of the fragments. Some example library preparation techniques include tagging or conjugation.

In FIG. 7A , sample fluid 12 is introduced into flow cell 26 . More specifically, sample fluid 12 is introduced into reaction chamber 28 that includes recess 58 separated by gap region 60 and capture primers 62 , 64 connected within recess 58 .

When the sample fluid 12 is introduced into the reaction chamber 28, seeding (eg, immobilization) of the DNA sample 16 can be initiated, as shown at FIG. 7B. Seeding is achieved via hybridization between one of the adaptors on the DNA sample 16 and the complement of the capture primers 62,64. Seeding can be performed at suitable hybridization temperatures for the DNA sample 16 and capture primers 62,64. Inoculated DNA sample 16 is shown with reference number 16' and is referred to herein as inoculated DNA library fragment 16'.

The method 300 then involves heating the flow cell 26 . The temperature to which the flow cell 26 is heated will depend on the gelation temperature of the methylcellulose 14 in the sample fluid 12. The flow cell 26 can be brought to a gel temperature or above, so that the temperature of the sample fluid 12 can reach the gel temperature.

Throughout method 300, heating and cooling flow cell 26, eg, to a suitable hybridization temperature, at, above or below gelation temperature, etc., may involve internal or external heating components, similar to components 36 or 38 described herein. The internal heating assembly may be positioned within the reaction chamber 28, or the external heating assembly may be part of the sequencing device into which the flow cell 26 is housed. Internal or external heating components can be controlled to heat the flow cell 26 or its specific reaction chamber 28 to a desired temperature. Sensor feedback can be used to achieve and/or maintain the desired temperature.

As shown in Figure 7C, the heat causes the precipitation of methylcellulose 14 from the aqueous carrier 20, and the precipitated methylcellulose polymer chains and the unbound DNA sample 16 (eg, unseeded DNA library fragments) Physically interact to form DNA-methylcellulose complex 10 within reaction chamber 28 . Any of the DNA samples 16 that have not been seeded/fixed can become entangled in the DNA-methylcellulose complex 10 . The seeded DNA library fragments are 16' hydrogen bonded to the corresponding capture primers 62, 64 and thus do not become tangled in the complex 10.

Method 300 also involves introducing an additional amount of sample fluid 12 into reaction chamber 28 of flow cell 26 . This introduction occurs as the flow-through tank 26 is heated. This introduction can occur relatively quickly, such that recombination occurs within the reaction chamber 28 and not within the fluid lines leading to the reaction chamber 28 . Additionally, the rapid introduction of additional sample fluid 12 can help to balance DNA complexing with DNA hybridization. The introduction of additional sample fluid may occur within minutes, eg, 5 minutes or less. The newly introduced methylcellulose 14 precipitates due to heat and complexes with the unbound DNA sample 16 to form additional DNA-methylcellulose complexes 10 . This concentrates the plurality of DNA-methylcellulose complexes 10 in the reaction chamber 28, as shown in Figure 5D.

The method 300 further includes cooling the flow cell 26 (or its reaction chamber 28 ) such that the temperature of the sample fluid 12 contained therein is reduced below the gelation temperature of the methylcellulose 14 . The internal or external heating assembly can be turned off to cool the flow cell 26 or the reaction chamber 28 . This involves passive cooling by allowing the sample fluid 12 to cool itself. 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.

Cooling may begin when a desired amount of sample fluid 12 has been introduced into reaction chamber 28 and/or when a desired amount of complex 10 has been concentrated in reaction chamber 28.

The temperature to which the flow cell 26 (or its reaction chamber 28 ) cools will depend upon the gelation temperature of the methylcellulose 14 in the sample fluid 12 . The flow cell 26 (or its reaction chamber 28 ) can be reduced below the gelation temperature so that the temperature of the sample fluid 12 can also be cooled below the gelation temperature.

As shown in Figure 7E, the entangled DNA-methylcellulose complex 10 is cooled in the reaction chamber 28. More specifically, cooling dissolves methylcellulose 14 in aqueous carrier 20 , which disentangles DNA-methylcellulose complex 10 and releases DNA sample 16 . The DNA sample 16 is thus concentrated in the reaction chamber 28 .

When the DNA sample 16 is released from the complex 10, seeding (eg, immobilization) of the DNA sample 16 can begin again, as shown at Figure 7F. With a higher concentration of DNA sample 16 in reaction chamber 28, seeding can be more efficient than the seeding event that occurs, for example, in FIG. must be fixed. As mentioned herein, inoculation can be performed at a temperature suitable for hybridization of the DNA sample 16 and capture primers 62,64.

Method 300 may further include introducing a wash solution to remove any remaining unbound DNA sample 16 (eg, unseeded DNA library fragments) and sample fluid 12; and then beginning a sequencing cycle. The following examples describe the sequencing by synthesis (SBS) cycle.

The SBS cycle can start 16' using cluster generation to amplify the inoculated DNA library fragments. In one example of cluster generation, DNA library fragments 16' are seeded by 3' extension from hybridizing primers 62, 64 using a high fidelity DNA polymerase. The original seeded DNA library fragments were denatured 16' and the duplicates were immobilized on capture primers 62,64. Isothermal bridge amplification or some other form of amplification can be used to amplify fixed replicas. For example, the replicated template cycles to hybridize to adjacent complementary primers 62, 64, and the polymerase replicates the replicated template to form a double-stranded bridge, which denatures to form two single-stranded strands. This two-stranded loops and hybridizes with adjacent complementary primers 62, 64 and extends again to form two new double-stranded loops. This process is repeated on each template replica by cycles of isothermal denaturation and amplification to generate dense clonal clusters. Degenerates clusters of individual double-stranded bridges. In one example, the reverse strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. Clustering causes several strands of template polynucleotides to form along reaction chamber 28 . An example of such clustering is bridge amplification, and is one example of an amplification that can be performed. It will be appreciated that other amplification techniques may be used, such as the Examplification Workflow (Illumina).

Sequencing primers can be introduced that hybridize to complementary sequences on the template polynucleotide strand. This sequencing primer renders the template polynucleotide strands ready for sequencing. The 3'-end of the template and any primers 62, 64 bound via the flow cell (not attached to the replica) can be blocked to prevent interference with the sequencing reaction and, in particular, to prevent undesired priming .

To begin sequencing, the incorporation mixture can be added to flow cell 26 . In one example, the incorporation mixture includes a liquid carrier, a polymerase, and a fluorescently labeled nucleotide. Fluorescently labeled nucleotides can include a 3' OH capping group. When the incorporation mixture is introduced into the flow cell 26, the fluid enters the reaction chamber 28, and in some instances the recess 58 (where the template polynucleotide strands are present).

Adds fluorescently labeled nucleotides to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner so that detection of the order and type of nucleotides added to the sequencing primer can be used to determine Sequence of templates. More specifically, one of the nucleotides is incorporated by a respective polymerase into a nascent strand that extends the sequencing primer and is complementary to the template polynucleotide strand. In other words, in at least some of the template polynucleotide strands across flow cell 26, the corresponding polymerase extends the hybridization-sequencing primer by one of the nucleotides incorporated into the mixture.

Incorporation of nucleotides can be detected via imaging events. An illumination system (not shown) may provide excitation light to reaction chamber 28 during an imaging event.

In some examples, the nucleotide may further include a reversible termination feature (eg, a 3' OH capping group) that terminates further primer extension once the nucleotide has been added to the sequencing primer. For example, a nucleotide analog with a reversible terminator moiety can be added to the sequencing primer so that subsequent extension cannot proceed until a deblocker is delivered to remove the moiety. Thus, for examples using reversible termination, the deblocking agent can be delivered to flow cell 26 after detection has occurred.

One or more washes can be performed between the various fluid delivery steps. The SBS cycle can then be repeated n times to extend the sequencing primer by n nucleotides, thereby detecting sequences of length n.

In some examples, the forward strands can be sequenced and removed, and then the reverse strands constructed and sequenced as described herein.

Although the SBS has been described in detail, it should be understood that the flow cell 26 described herein can be used with other sequencing schemes for genotyping or in other chemical and/or biological applications. In some cases, the primers 62, 64 of the flow cell 26 can be selected to enable simultaneous pair-wise end sequencing, where both forward and reverse strands are present on the polymeric hydrogel 66, allowing read Simultaneous base recognition. Sequential and simultaneous paired-end sequencing facilitates the detection of gene rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.

To further illustrate the invention, examples are given herein. It should be understood that these examples are provided for illustrative purposes and are not intended to be construed as limiting the scope of the present invention. Example

Example 1

Several sample fluids were prepared to examine the effect of salt on the gelling/precipitation temperature of methylcellulose. Each solution included 1 wt% methylcellulose (METHOCEL® E from Dow Chemical Company), 16 wt% polyethylene glycol (weight average molecular weight 1000 g/mol), various concentrations of sodium chloride (NaCl) and water. The concentration of NaCl is in the range of 0.4 M to 2 M.

The different solutions were exposed to different temperatures (5°C, 25°C, 45°C or 60°C) in an effort to achieve methylcellulose precipitation. The optical density of each solution was measured using a disc reader. Optical density is a measure of light scattering, and higher numbers indicate more precipitated methylcellulose. An optical density of less than 0.1 means that all the methylcellulose is in solution. The results are shown in FIG. 8 . At low temperature (5°C and 25°C) and lower salt concentration (1.25 M or lower), methylcellulose dissolves completely. With increasing salt concentration, methylcellulose can precipitate out of solution at low temperature. These results demonstrate that the salt concentration in the sample fluid disclosed herein can be used to adjust the gelling/precipitation temperature so that the complexes disclosed herein can be produced at the desired temperature.

Example 2

The sample fluid is prepared with a DNA ladder comprising a plurality of DNA inserts of different sizes. Sample fluids included water, methylcellulose (1% METHOCEL® E from Dow Chemical Company). , polyethylene glycol (5%, weight average molecular weight 1000 g/mol) and 1 M NaCl. The DNA insert is added to the solution to form the sample fluid. The sample fluid was then incubated with heat at 25°C for 5 minutes. The sample fluid is then centrifuged and decanted. The first supernatant was collected and run on an Agilent TapeStation.

The pellet was washed to remove unbound DNA and resuspended in saline sodium citrate buffer. The reconstituted sample fluid was heated again, this time at 60°C for 5 minutes. The reconstituted sample fluid is then centrifuged and decanted. The second supernatant was collected and removed, leaving behind a solid. The pelleted solid was suspended in buffer and cooled (to release any bound DNA insert). This solution was run on a TapeStation.

TapeStation results (sample intensity (normalized FU on Y-axis) versus size (number of base pairs on X-axis)) for the first supernatant and pellet are shown in FIG. 9 . The first supernatant had more smaller DNA inserts, while the pellet had more larger DNA inserts. These results demonstrate the ability of the methods disclosed herein to remove smaller DNA inserts by complexing larger DNA inserts with methylcellulose.

Example 3

One sample fluid (Ex. 1) and one comparative sample fluid (Comp. Ex. 2) were prepared with the PhiX library. Ex. 1 includes water and methylcellulose (1% METHOCEL® E from Dow Chemical Company) and PhiX library. Comp. ex. 2 includes water and PhiX library without any methylcellulose.

Both Ex. 1 and Comp. ex. 2 were incubated with heat at 60°C for 5 minutes. Ex. 1 and Comp. ex. 2 were then centrifuged and decanted. Individual pellets were washed to remove unbound DNA and resuspended in 1 M TRIS buffer and cooled (to release any bound DNA inserts). Each of the resuspension solutions was run on an Agilent TapeStation.

TapeStation results for Ex. 1 and Comp. ex. 2 are shown in Figure 10 (sample intensity (normalized FU on Y-axis) vs. size (number of base pairs on X-axis)). Ex. 1 presents a steeper peak than Comp. ex. 2, thus indicating that methylcellulose captures larger DNA inserts. Specifically, Comp. ex. 2 has a lower and broad peak at 500 bp, while Ex. 1 has a much higher and steeper peak. This indicates that Ex. 1 is more concentrated and potentially purified.

Suspension libraries from both Ex. 1 and Comp. ex. 2 were then run on the iSeq™ Sequencing System (Illumina, Inc.) to determine whether methylcellulose had any deleterious effects on the libraries themselves. Although not reproduced herein, the sequencing measures for Ex. 1 and Comp. ex. 2 were found to be similar and thus confirm that methylcellulose does not have any deleterious effect on the library itself.

Example 4

Two other sample fluids (Ex. 3 and Ex. 4) and one additional comparative sample fluid (Comp. Ex. 5) were prepared with the PhiX library. Ex. 3 includes water, methylcellulose (1% METHOCEL® from The Dow Chemical Company), and a PhiX library. Ex. 4 includes water, methylcellulose (5% METHOCEL® from The Dow Chemical Company), and a PhiX library. Comp. ex. 5 includes water and PhiX library without any methylcellulose.

Ex. 3, Ex. 4 and Comp. ex. 5 were incubated at 60°C with heating for 5 minutes. Ex. 3, Ex. 4 and Comp. ex. 5 were then centrifuged and decanted. The corresponding pellet was washed to remove unbound DNA and resuspended in 1 M TRIS buffer and cooled (to release any bound DNA insert). Each of the resuspension solutions was run on an Agilent TapeStation.

TapeStation results for Ex. 3, Ex. 4 and Comp. ex. 5 are shown in Figure 11 (sample intensity (normalized FU on Y-axis) vs. size (number of base pairs on X-axis)). The peaks around 25 bp, 75 bp and 170 bp became higher and steeper as the methylcellulose concentration increased (from Comp. ex. 5 to Ex. 3 to Ex. 4). The results indicate that high concentrations of methylcellulose do not adversely affect the capture efficiency of the polymer.

Other considerations

It should be understood that all combinations of the foregoing concepts and additional concepts discussed in greater detail below, with the proviso that such concepts are not mutually incompatible, are intended to be part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the conclusion of this disclosure are encompassed as part of the inventive subject matter disclosed herein. It should also be understood that terms expressly employed herein that may also appear in any disclosure incorporated by reference are to be accorded a meaning largely consistent with the specific concepts disclosed herein.

References throughout this specification to "one example," "another example," "an example," etc. mean specific elements described in connection with the example (eg, features, structures, and/or characteristics) are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that elements described with respect to any example may be combined in any suitable manner in various examples unless the context clearly dictates otherwise.

It is to be understood that ranges provided herein include the stated range and any value or sub-range within the stated range, as if a value or sub-range within the stated range was explicitly recited. For example, the range of about 400 nm to about 1 µm (1000 nm) should be interpreted 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 sub-ranges such as about 425 nm to about 825 nm, about 550 nm to about 940 nm, and the like. Additionally, when "about" and/or "substantially" are used to describe a value, it is meant to encompass minor variations (up to +/- 10%) of the stated value.

While several examples have been described in detail, it should be understood that the disclosed examples may be modified. Accordingly, the preceding description should be considered non-limiting.

Features of examples of the invention will become apparent with reference to the following description and drawings, in which like reference numerals correspond to similar but perhaps not identical components. For the sake of brevity, reference numbers or features having previously described functions may or may not be described in conjunction with other figures in which such reference numbers or features appear.

[ FIG. 1 ] is a flowchart illustrating an example of a method of deoxyribonucleic acid (DNA) capture;

[FIG. 2] is a schematic representation of the method of FIG. 1;

[FIG. 3] is a schematic representation of another example involving the method of FIG. 1 and the method of purification;

[ FIG. 4 ] is a flow chart illustrating an example of a method for DNA concentration;

[FIG. 5A] is a schematic representation of the flow tank assembly;

[FIG. 5B] is a cross-sectional view taken along line 5B-5B of FIG. 5A, illustrating sample fluid flow and complex formation in a temperature-controlled flow channel as temperature increases;

[FIG. 5C] is a cross-sectional view taken along line 5C-5C of FIG. 5A, illustrating the filter in the temperature-controlled flow channel and the concentration of DNA-methylcellulose complex at the filter;

[FIG. 5D] depicts the temperature-controlled flow channel of FIG. 5C as the temperature decreases;

[FIG. 5E] is an enlarged perspective view of an example of a reaction chamber having a patterned surface;

[Fig. 6] is a flow chart illustrating an example of a method of improving DNA seeding on the surface of a flow cell;

[FIG. 7A to FIG. 7F] schematically illustrate the method of FIG. 6;

[Figure 8] is a graph depicting turbidity (Y-axis, optical density) versus salt concentration (X-axis, M) for aqueous solutions including 1 wt% methylcellulose and 16 wt% polyethylene glycol when exposed to different temperatures 's diagram;

[FIG. 9] is a graph depicting sample intensity (Y-axis, normalized fluorescence units) versus DNA insert size (X-axis, base pairs) for sample fluids exposed to examples of the purification methods disclosed herein at different temperatures picture;

[FIG. 10] is a graph depicting sample intensity (Y-axis, normalized fluorescence units) versus DNA insert size (X-axis, base pairs) for control fluids and sample fluids exposed to examples of capture methods disclosed herein ;and

[FIG. 11] 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 examples of capture methods disclosed herein map.

100: Method

102: Steps

104: Steps

Claims (21)

A kit comprising: Flow tank assembly, which includes: A reaction chamber, which has: depressions separated by gap areas; and a capture primer linked within each of the recesses; a temperature-controlled flow channel in selective fluid communication with the reaction chamber inlet; and A filter disposed in the temperature-controlled flow channel, the filter i) for blocking the concentrated deoxyribonucleic acid (DNA)-methylcellulose produced in the temperature-controlled flow channel at a first temperature complexes, and ii) for passing concentrated DNA and methylcellulose released from the complexes in the temperature-controlled flow channel at a second temperature. The kit of claim 1, further comprising a sample fluid, the sample fluid comprising: aqueous carrier; DNA samples; Methylcellulose; Polymers that are chemically inert to DNA hybridization; and Salt. The kit of claim 2, wherein the polymer chemically inert to DNA hybridization is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidine having a weight average molecular weight in the range of about 500 to less than about 200,000 Ketones, polyvinyl alcohol, and combinations thereof. As in the set of claim 2, wherein: the DNA sample is present in the sample fluid at a first molar concentration in the range of 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 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 in the range of greater than 0 M to about 2 M. The kit of claim 1, wherein the flow cell assembly further comprises: a bypass line in fluid communication with the temperature-controlled flow channel inlet and with the temperature-controlled flow channel outlet; a first bypass valve for controlling the flow of sample fluid to the inlet of the temperature-controlled flow channel; and The second bypass valve is used to control the flow of the concentrated DNA and the methylcellulose to the reaction chamber. The kit of claim 1, wherein at least one surface of the temperature-controlled flow channel includes a heating plate. A method that includes: The DNA sample is combined with a solution to form a sample fluid consisting of: aqueous carrier; Methylcellulose; Polymers that are chemically inert to DNA hybridization; and Salt, and the solution is at a temperature in the range of about 5°C to about 30°C; and The sample fluid is heated to at least the gelling temperature of the methylcellulose, thereby forming a DNA-methylcellulose complex in the aqueous carrier. The method of claim 7, further comprising cooling the sample fluid below the gelling temperature of the methylcellulose, thereby disentangling the DNA-methylcellulose complexes to release the DNA sample and the methylcellulose. The method of claim 7, further comprising increasing the concentration of the salt in the solution, thereby reducing the gelling temperature of the methylcellulose. The method of claim 7, wherein the DNA sample comprises cell-free DNA, library DNA, whole genome amplified DNA, or a combination thereof. As in the method of claim 7, wherein: The DNA sample includes a plurality of DNA inserts of different sizes, the inserts including small DNA inserts and large DNA inserts; At least some of the large DNA inserts are entangled in the DNA-methylcellulose complexes; At least some of the small DNA inserts are not entangled in the DNA-methylcellulose complexes; and The method further comprises performing a purification process to separate at least some of the small DNA inserts from the DNA-methylcellulose complexes after heating. The method of claim 11, wherein the purification process involves filtration, centrifugation, decantation, or a combination thereof. The method of claim 11, further comprising cooling the DNA-methylcellulose complexes below the gelling temperature of the methylcellulose, thereby decomposing the DNA-methylcellulose complexes entangle to release at least some of the large DNA inserts and the methylcellulose. A method that includes: A sample fluid including: aqueous carrier; DNA samples; Methylcellulose; Polymers that are chemically inert to DNA hybridization; and Salt; Heating the temperature-controlled flow channel upon introduction of the sample fluid causes the temperature of the sample fluid contained therein to increase to at least the gelation temperature of the methylcellulose, thereby forming DNA-methyl groups in the temperature-controlled flow channel Cellulose complex; Continuing to flow the sample fluid through the temperature-controlled flow channel while heating the temperature-controlled flow channel, thereby concentrating a plurality of the DNA-methylcellulose complexes at the filter in the temperature-controlled flow channel; and cooling the temperature-controlled flow channel such that the temperature of the sample fluid contained therein is lowered below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complexes to The DNA sample and the methylcellulose are released, whereby the DNA sample and the methylcellulose can pass through the filter. The method of claim 14, further comprising selecting a heating temperature of the temperature-controlled flow channel based on the concentration of the salt in the sample fluid. The method of claim 14, further comprising transporting the DNA sample and the methylcellulose from the temperature-controlled flow channel to a flow cell. A method that includes: Introduce a sample fluid into the flow cell, which includes: aqueous carrier; DNA samples; Methylcellulose; Polymers that are chemically inert to DNA hybridization; and Salt; And the flow cell includes a reaction chamber having the following: depressions separated by gap areas; and a capture primer linked within each of the recesses; inoculating and hybridizing at least some of the DNA samples in at least some of the wells; heating the flow cell such that the temperature of the sample fluid contained therein increases 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 the sample fluid into the flow cell; cooling the flow cell so that the temperature of the sample fluid contained therein is lowered below the gelling temperature of the methylcellulose, thereby disentangling the concentrated DNA-methylcellulose complexes to release the DNA sample and the methylcellulose; and Inoculation and hybridization of at least some of the released DNA samples in the wells and at least some of the DNA samples from the additional sample fluid are initiated. The method of claim 17, further comprising selecting the heating temperature of the flow cell based on the concentration of the salt in the sample fluid. A kit comprising: A sample fluid, which consists of: aqueous carrier; Deoxyribonucleic acid (DNA) samples; Methylcellulose; Polymers that are chemically inert to DNA hybridization; and A salt or solvent to adjust the gelling temperature of the methylcellulose. As in the kit of claim 19, wherein: the DNA sample is present in the sample fluid at a first molar concentration in the range of 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 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 includes the salt, and the salt is present in the sample fluid at a second molar concentration in the range of greater than 0 M to about 2 M. The kit of claim 19, further comprising: Flow tank assembly, which includes: a temperature-controlled flow channel for receiving the sample fluid; A reaction chamber, which has: depressions separated by gap areas; a capture primer linked within each of the recesses; an inlet in selective fluid communication with the temperature-controlled flow channel; and a filter disposed in the temperature-controlled flow channel, the filter i) for blocking concentrated DNA sample-methylcellulose complexes generated in the temperature-controlled flow channel when the sample fluid is exposed to a first temperature, and ii) to pass the concentrated DNA sample and methylcellulose released from the complexes in the temperature-controlled flow channel at the second temperature.

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