DEVICES, METHODS AND SYSTEMS FOR EXTRACTION OF GENETIC DNA FROM CELL SAMPLES USING TUNABLE SIZE SELECTION - Patent application

Abstract

A microfluidic flow-based device for extracting genomic DNA fragments of a selected size range from cells or cell nuclei is disclosed. The device includes a microfluidic channel and an array of micropillars arranged in the microfluidic channel in a defined configuration. A system including such a device and a fluid control module is also disclosed. A method for selecting parameters for extracting genomic DNA fragments with a desired size metric from cells or cell nuclei and a method for isolating genomic DNA fragments with a selected size metric from cells or cell nuclei are further disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 242,216, filed September 9, 2021; and U.S. Provisional Application No. 63 / 320,389, filed March 16, 2022, the entire contents of which are incorporated by reference herein.

[0002] Description of rights This invention was made with government support under Grant No. 1940395 awarded by the National Science Foundation. The Government has certain rights in this invention. [Background technology]

[0003] background Extraction of DNA, such as genomic DNA, from samples containing cells is necessary for a variety of applications. For example, the extracted DNA may be required for pulsed-field gel electrophoresis, amplification, and detection of specific loci, or sequencing, among many other applications. These applications may in turn be useful in understanding important phenomena such as cancer, infectious diseases, and many other diseases.

[0004] The use of microfluidic devices is one of the options for extracting DNA from cells. For example, a micropillar array can be used in the device to immobilize genomic DNA (gDNA) from lysed cells and then extract the gDNA from the array. Despite having many advantages, these methods often result in a mixture of gDNA fragments of various sizes, and often limit the range of samples that can be processed.

[0005] Therefore, there is a need in the art to further develop methods for extracting gDNA from cell samples. Summary of the Invention [Means for solving the problem]

[0006] Abstract The present invention, in some aspects, relates to a method for extracting and isolating genomic DNA (gDNA) of selectable fragment sizes from samples containing cells or cell nuclei. The extracted DNA can be analyzed by various techniques, such as fragment size sorting, selective amplification, or sequencing. This capability is useful in biological research and medical applications, such as diagnosing disease and prescribing treatment. By selecting an optimal gDNA fragment size for a particular sequencing technique, the efficiency of the process is improved. The disclosed approach also reduces the number of steps required for sample preparation and should be efficient for small sample sizes.

[0007] To extract gDNA from selected cells or cell nuclei at selected fragment sizes, enzymatic approaches are often used to shear the DNA to the desired size and release shorter DNA fragments from pillar-like obstacles in a microfluidic device on which the pillar-like obstacles are immobilized. The gDNA can then be removed from the device or subjected to further processing such as buffer exchange or concentration in a second stage of the device. The gDNA is then compatible with subsequent analytical processes such as selective amplification, detection, or sequencing.

[0008] Methods for making microfluidic devices use standard lithography and etching approaches to create molds that can be replicated in polydimethylsiloxane, (PDMS), or other polymers using molding, embossing, or injection molding. One example of a method for making a device is described in Benitez et al. The molded PDMS can be easily bonded to a glass plate to complete the structure, or devices can be constructed with a combination of different polymer materials.

[0009] Alternatively, microfluidic devices can be fabricated using other materials, such as glass, plastic, metal, silicon, or any combination thereof, in addition to polymers (e.g., PDMS).

[0010] The combination of channel dimensions, pillar size, and pillar array organization can be varied for a desired sample type or volume, for example, a smaller pillar spacing would be used to capture smaller cells or cell nuclei, while a larger spacing would be used to select larger cells from a mixture containing smaller entities in the sample.

[0011] In some aspects, a microfluidic flow-based device for extracting fragments of genomic DNA of a selected size range from cells or cell nuclei comprises a microfluidic channel having an inlet and an outlet to allow flow in a flow direction from the inlet to the outlet. In a preferred embodiment, the device comprises an array of micropillars disposed within the microfluidic channel.

[0012] In some embodiments, the diameter of the micropillars is preferably between at least about 3 micrometers and about 15 micrometers, but may range from at least about 1 micrometer to about 100 micrometers. 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 micrometers; or any range therebetween.

[0013] In some embodiments, the preferred height of the micropillars is at least about 15 to about 25 micrometers, but can range from at least about 1 to about 200 micrometers. In some embodiments, the height is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 , 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 1 62, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 micrometers; or any range therebetween.

[0014] In some embodiments, the micropillars are separated from one another by a distance preferably between at least about 2 micrometers and about 150 micrometers, but may range from 0.1 micrometers to more than 300 micrometers. In some embodiments, the distance is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 micrometers; or any range therebetween.

[0015] In some embodiments, each array of micropillars is bounded within an area of ​​25 square microns or more. In some embodiments, each array of micropillars is bounded within an area of ​​1 square millimeter or more. In some embodiments, the area is at least about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 ,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8 .5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20 0, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 square millimeters; or any range between these values. In some embodiments, each array of micropillars is bounded within an area of ​​less than about 200 square millimeters. In some embodiments, each array of micropillars is bounded within an area of ​​less than about 100 square millimeters. In a preferred embodiment, each array of micropillars is bounded within an area of ​​less than about 1600 square centimeters.

[0016] In some embodiments, the micropillars are arranged in an array with defined configurations (eg, as shown in Figures 9 and 10).

[0017] In some embodiments, the micropillars comprise a polymer (e.g., the micropillars are made of a polymer), a plastic, a metal, a silicone, a glass, or a combination of any two or more of the foregoing. In some embodiments, the micropillars are made using a lithographic process. In some embodiments, the device further comprises one or more canals extending into the area of ​​the array of micropillars. In some embodiments, the one or more canals are aligned along the flow direction. In some embodiments, the diameter, the spacing, the area, the shape, or a combination thereof is selected based on the type of the cell or cell nucleus. In some embodiments, the cell is a white blood cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a T cell. In some embodiments, the nucleus is a plant cell nucleus (or a cell nucleus from another organism). In some embodiments, the shape is narrow near the inlet, such that the shape is capable of extracting fragments of genomic DNA from a single cell. In some embodiments, the shape is capable of extracting fragments of genomic DNA from multiple cells or cell nuclei. In some embodiments, the device further comprises an agent that binds to the cell or nucleus. In some embodiments, the agent is an antibody, an antigen-binding fragment thereof, or an aptamer. In some embodiments, the device further comprises a secondary channel perpendicular to the microfluidic channel. Alternatively, the secondary channel intersecting the first channel can be at any angle. In some embodiments, the secondary channel allows for lysing the cells after they bind to the agent. In some embodiments, the micropillar comprises polydimethylsiloxane.

[0018] In some aspects, a system including any embodiment of these devices further includes a fluid control module suitable for connection to the inlet.

[0019] In some embodiments, the fluid control module includes a pipette or syringe. In some embodiments, the fluid control module includes a pressurized air source (e.g., compressed air), a controlled pressure source, a controlled air pressure source, a pressure-driven pump, a syringe pump, a vacuum pump, or a peristaltic pump. In some embodiments, the system further includes one or more collection reservoirs suitable for connection to an outlet. In some embodiments, the one or more collection reservoirs include a sample collection reservoir and a waste collection reservoir. In some embodiments, the system further includes a controlled voltage source for electrophoretically driving the DNA in the microfluidic channel. In some embodiments, the system includes an array of a plurality of micropillars in a plurality of microfluidic channels. In some embodiments, the array is a staggered configuration in the device. In some embodiments, the array is an ordered configuration that is aligned with the multiwell plate configuration. In some embodiments, the aforementioned multiwell plate is a 6-well, 12-well, 24-well, 48-well, 96-well, 384-well plate, 1536-well, or other similar format. In some embodiments, the fluidic control module is capable of delivering cells, a lysis buffer, a digestion buffer comprising one or more enzymes, and a wash buffer into the microfluidic channel. In some embodiments, the system further comprises a digestion buffer as described above, where the one or more enzymes comprise one or more restriction enzymes. In some embodiments, the system further comprises a digestion buffer as described above, where the one or more enzymes comprise one or more nucleases (such as endonucleases).

[0020] In some embodiments, a method of selecting parameters for extracting fragments of genomic DNA having a desired size metric from a cell or cell nucleus includes the steps of processing a sample comprising at least one cell or cell nucleus with a system of the disclosure at least once using parameters including: a concentration of each of said one or more enzymes; a number of said one or more enzymes; a buffer composition of said digestion buffer; a digestion time; and a digestion temperature; determining a fragment size metric of the collected sample; and, based on said fragment size metric, selecting said parameters to be used if said fragment size metric corresponds to said desired size metric; selecting said parameters to be used if said fragment size metric is less than said desired size metric. decreasing said concentration, decreasing said number, decreasing said digestion time, changing said digestion temperature to decrease enzyme efficiency, changing said buffer composition to decrease enzyme efficiency, or a combination thereof, until said fragment size metric is no less than said desired size metric; and if said fragment size metric is greater than said desired size metric, increasing said concentration, increasing said number, increasing said digestion time, changing said digestion temperature to increase enzyme efficiency, changing said buffer composition to increase enzyme efficiency, or a combination thereof, until said fragment size metric is no greater than said desired size metric.

[0021] In some embodiments, the fragment size metric is the average fragment size or the median fragment size. In some embodiments, the processing step comprises loading the sample into the device and washing at least one cell or cell nucleus in the device using a washing buffer. In some embodiments, the method further comprises inactivating one or more enzymes. In some embodiments, the inactivation comprises heat inactivation.

[0022] In some embodiments, a method for isolating fragments of genomic DNA having a selected size metric from a cell or cell nucleus comprises processing a sample comprising at least one cell or cell nucleus with a system of the present disclosure using parameters selected by the methods described herein; and collecting the fragments of genomic DNA isolated by said processing.

[0023] In some embodiments, the method further comprises recovering the collected fragments. In some embodiments, said recovering comprises removing the fragments from the collection reservoir by manual pipetting or through tubing. In some embodiments, said recovering comprises removing the fragments from the collection reservoir via an electrophoresis channel attached to the collection reservoir. [Brief description of the drawings]

[0024] [Figure 1] Overview of the Workflow Using Exemplary Embodiments of the Disclosed System for Sample Preparation for Genomic Analysis Here, we outline the overall workflow using the disclosed platform technology for cell sample preparation in four key steps, which can be generally described as: (1) obtaining a biological sample (e.g., whole blood or cell sample solution) containing at least one cell, (2) loading said sample into the disclosed device (which would then be loaded into the platform instrument responsible for fluidic control and user interface), (3) removing the processed / conditioned sample from the platform and then analyzing the sample, and (4) analyzing the data and drawing conclusions from step 3.

[0025] [Diagram 2]Figure 2A-D. Overview of an exemplary workflow of micropillar array technology used for the described method of adjustment of gDNA size fragments collected from cellular gDNA extraction. Here, the four-step genomic DNA (gDNA) extraction process performed by micropillar array technology begins with (A) fixing the cells within the sample, (B) chemically lysing the cells with a lysis buffer (such as a surfactant or high salt buffer), (C) allowing the gDNA to become physically entangled within adjacent pillars, then processing the gDNA while it remains entangled on the micropillar array and / or fluorescently staining the gDNA for imaging, and finally, (D) shearing the gDNA into smaller fragments for release from the pillar array.

[0026] [Diagram 3] Micrograph of genomic DNA extracted using micropillar array technology. The genomic DNA has been stained with a fluorescent intercalating dye and will be visualized by fluorescence microscopy. Figure 3 shows a device containing a hexagonal lattice (staggered orientation) in which the "step" gradient of interpillar spacing is altered to densely increase the number of pillars (decreasing micropillar spacing) in the direction toward the outlet.

[0027] [Figure 4] Illustration of the workflow of target cell capture and subsequent gDNA extraction. Non-target cells (red) are able to flow unimpeded through the larger structural array and are not lysed along with the target cells (purple). Target cells that become arrested on the surface of the larger pillars (at the intersections) due to their surface binding properties can then be lysed vertically and subjected to gDNA extraction as described in Figures 2 and 3. Although a vertical secondary channel is shown as an example, the secondary channel intersecting the first channel can be at any angle relative to the first channel.

[0028] [Diagram 5]FIG. 5A-C. Illustration of an exemplary single-cell workflow. The general steps shown here are identical to those in FIG. 2, with the main difference being the channel structure. A solid wall structure surrounds the micropillar array, while single-cell-sized openings at the tip of the array allow only single cells to be loaded into each micropillar array region, while additional cells are funneled into further downstream pillar arrays via side "groove-like" channels or discarded, depending on the required application. The two right panels are micrographs showing a human cancer single cell loaded at the tip of the micropillar array, and the gDNA of the single cell imaged after lysis and staining.

[0029] [Figure 6] Various pillar diameters of the micropillars within the array portion of the microfluidic chip. (Dimensions shown in microns.) Shown here are four separate examples of pillar diameter sizes ranging from about 4 μm to about 6.4 μm.

[0030] [Figure 7] Various inter-pillar spacings within the pillar array portion of the microfluidic chip. (Dimensions shown in microns). Inter-pillar spacings of 2.8 μm to 105 μm are shown here. These can be freely adjusted as needed depending on cell size for a particular application.

[0031] [Figure 8] Sizes of different micropillar regions within the microfluidic chip. (Measurements shown in microns.) The length and width of the micropillar array regions can be varied to accommodate different cell input sizes ranging from single cell applications to applications requiring hundreds of thousands of cells per sample.

[0032] [Figure 9]Various chip configurations, microchannel distributions, and overall layouts of microfluidic channels. The orientation and size of each channel is freely adjustable, and separate channels can be fabricated separately from other channels on the same silicon wafer master.

[0033] [Figure 10] 96-well "microplate" configuration and microfluidic channel layout. If the channels are spaced such that the input and output ports match the spacing of the separate wells in a 96-well microplate, a configuration with a total of 48 channels per microplate is possible. This allows the disclosed technology to be operated in a standardized microplate / 96-well format while retaining the functionality of the disclosed micropillar technology. Each of the 48 channels can be considered to comprise a single micropillar array channel or set, which can then be operated with a fluidic system that can control all 48 channels simultaneously or address each channel individually. The degree of freedom and flexibility of operation can depend on the application.

[0034] [Figure 11] Photograph of a typical microfluidic chip device with tubing attached to the inlets.

[0035] [Figure 12] Schematic of the pressure-driven flow system: A pressurized air source is connected to the device through tubing, forcing solution through the chip and out to an exit port for collection.

[0036] [Figure 13] Schematic diagram of a fluidic control system for introducing and withdrawing fluids from a microfluidic chip.

[0037] [Figure 14] FIG. 13 is a schematic diagram of fluid control using interleaved fluid control showing that different types of pump and valve configurations can be used.

[0038] [Figure 15] Schematic diagram of a fluidic control system that applies an electric potential to drive charged molecules such as DNA or RNA in combination with a conventional pump to drive the fluid.

[0039] [Figure 16] Schematic diagram of a fluid control system capable of directing different fluid discharge components to different collection volumes.

[0040] [Figure 17] 1 is a flow chart showing a method for obtaining DNA fragments of a selected size. Using the disclosed method, DNA fragment size can be tuned by varying conditions on the disclosed device.

[0041] [Figure 18] Pulsed-field capillary electrophoresis analysis using the Femto Pulse system reveals the size distribution of fragmented gDNA generated from mammalian cells using the disclosed technology. The size range obtained for the specific conditions used for this sample is narrow, with a major peak at 8 kb.

[0042] [Figure 19] Pulsed-field capillary electrophoresis analysis using the Femto Pulse system reveals the size distribution of fragmented gDNA generated from mammalian cells using the disclosed technology. The size range obtained for the specific conditions used for this sample is narrow, with a major peak at 10 kb.

[0043] [Figure 20] Pulsed-field capillary electrophoresis analysis using the Femto Pulse system reveals the size distribution of fragmented gDNA generated from mammalian cells using the disclosed technology. The size range obtained for the specific conditions used for this sample is narrow, with a major peak at 14 kb.

[0044] [Figure 21] Pulsed-field capillary electrophoresis analysis using the Femto Pulse system reveals the size distribution of fragmented gDNA generated from mammalian cells using the disclosed technology. The size range obtained for the specific conditions used for this sample is narrow, with a major peak at 25 kb.

[0045] [Figure 22] Pulsed-field capillary electrophoresis analysis using the Femto Pulse system reveals the size distribution of fragmented gDNA generated from mammalian cells using the disclosed technology. The size range obtained for the specific conditions used for this sample is broad, with a major peak at 200 kb.

[0046] [Figure 23] FIG. 23A-B. Integrated "canal" construction to facilitate further / deeper loading of cells into the micropillar array region. (A) Shows a typical micropillar array region where the micropillars are evenly spaced in the column such that the spacing becomes denser the further down the canal in the flow direction. In this scenario, the loaded cells may localize in similar regions in the pillar array and therefore the flow may be blocked or clogged. In (B), a canal is constructed in the pillar array region to allow direct passage through the initial part of the pillar array to allow the final cell loading to spread out. This facilitates both cell lysis and final DNA processing by preventing the extracted gDNA from bundling into a denser "rope-like".

[0047] [Figure 24] Staggered micropillar array region in a microfluidic device. This image shows one possible such configuration of micropillar arrays in a device, where multiple pillar arrays are staggered to improve cell loading but spreading the cells over a larger area, rather than the cells all becoming confined and localized in a small area.

[0048] [Diagram 25] 25A-B. An exemplary microfluidic device. (A) A cutaway image of two parallel microfluidic channels out of a total of eight channels for this device design is shown with the flow direction being up and down. (B) shows the micropillars arranged in what is termed a "square" lattice arrangement herein, whereby the pillars are arranged in such a way that they have distinct rows and columns spaced apart at specified distances from each other. Thus, a "smooth" gradient of pillars of various spacing in a square lattice configuration defines the micropillar array region. The micropillar channels are identical to each other, and the eight parallel channels are also meant to serve as an alternative to creating one giant channel.

[0049] [Figure 26] 26A-C. An exemplary microfluidic device. (A) Similar to FIG. 25, this device design shows two parallel microfluidic channels as cutaway images of a total of eight channels, with the flow direction being up and down. (B) and (C) show that the square lattice micropillar array has different spacing, which is intentionally designed and can be changed to any other desired spacing or dimensions. Unlike FIG. 25, this device has a "stepped" gradient rather than a "smooth" gradient in the channels, and rather than the spacing transition being smooth from the top to the bottom of the micropillar array, the spacing is held uniformly at one period and then changes to the next period. Also, the micropillar channels are identical to each other, meaning that the eight parallel channels serve as an alternative to creating one giant channel.

[0050] [Figure 27]FIG. 27A-C. An exemplary microfluidic device. (A) Similar to FIG. 25 and FIG. 26, two parallel microfluidic channels out of a total of eight channels for this device design are shown as cutaway images, with flow direction up and down. (B) and (C) show that the "hexagonal" lattice micropillar array has different spacing, which is intentionally designed and can be changed to any other desired spacing or dimensions. Similar to FIG. 25 and FIG. 26, the gradient in pillar spacing can be either smooth or stepwise. Also similarly, the two micropillar channels are identical to each other compared to the other six out of a total of eight parallel channels, also serving as an alternative to creating one giant channel.

[0051] [Figure 28] FIG. 28A-F. An exemplary microfluidic device. (A) The flow direction is up and down, showing all eight parallel microfluidic channels zoomed out. (B) Also zoomed in on two channels from panel (A). (C) shows that this design intermixes square and hexagonal lattice regions within the channels to determine if this design improves sample (cells or nuclei) loading over only square or only hexagonal micropillar arrays. (D), (E), and (F) show cropped images zoomed in even further to give a closer look at the alternating square and hexagonal lattice regions.

[0052] [Figure 29]FIG. 29A-B. An exemplary microfluidic device. (A) The flow direction is up and down, showing all eight parallel microfluidic channels zoomed out. (B) This design is intended to truncate / shorten the entire micropillar array region, so that the micropillar array is shorter in length than other designs shown herein (e.g., FIG. 25 or FIG. 26), and the overall length can be adjusted as desired, similar to how any other dimension of the channel (width, height) or micropillar (height, diameter, shape, spacing, etc.) can be freely adjusted as desired. The larger, flared pillars downstream of the micropillar array region (near the outlet; bottom half of panel (B)) are support pillars that are simply there to maintain structure, however, they can be modified to other feature designs as desired.

[0053] [Diagram 30] FIG. 30A-D. An exemplary microfluidic device. (A) Flow direction is up and down, showing all eight parallel microfluidic channels zoomed out. (B) This design is intended to have repeating micropillar array regions separated by a distance in a support structure, where the micropillar array can be either a square lattice, a hexagonal lattice, or a combination thereof, any gradient of smooth or step spacing is possible, and the repeating micropillar regions can be identical, but also completely different. (C) Zoomed in on a single channel with alternating micropillar and blank / support structure regions. (D) A further zoomed in and enlarged example of two micropillar array regions separated by a blank / support structure region. In this design, as with all other designs of all microfluidic channels shown herein, the support structures can vary in spacing, size, shape, and periodicity.

[0054] [Diagram 31]FIG. 31A-C. (A) The flow direction is up and down, showing all eight parallel microfluidic channels zoomed out. In this figure, we have designed repeating regions of micropillar arrays as in FIG. 30, but now there are three micropillar array regions rather than two. Also, each micropillar array region is separated by a spacing of blank / support structure regions. (B) and (C) are further zoomed-in screenshots of the CAD drawing to better show the different regions. It is important to note that the spacing for the three micropillar regions in (B) can be the same or different (e.g., increased micropillar density / reduced distance between pillars, or any other desired variation or alternative spacing). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Detailed Description The present disclosure relates to systems, methods, and devices for extracting genomic DNA from cell samples with tunable size selection. Each of the following patents and publications is incorporated by reference in its entirety herein: U.S. Pat. No. 10,947,528, Craighead et al. Microfluidic device for extracting, isolating, and analyzing DNA from cells; U.S. Pat. No. 9,926,552, Craighead et al. Microfluidic device for extracting, isolating, and analyzing DNA from cells; U.S. Pat. No. 9,856,513, Cerf et al. Methods and arrays for controlled manipulation of DNA and chromatin fragments for genetic and epigenetic analysis; Benitez, JJ, Topolancik, J., Tian, ​​HC, Wallin, CB, Latulippe, DR, Szeto, K., Murphy, PJ, Cipriany, BR, Levy, SL, Soloway, PD, et al. (2012). Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells. Lab on a Chip 72,4848;Tian,HC,Benitez,JJ,and Craighead,HG(2018).Single cell on-chip whole genome amplification via micropillar arrays for reduced amplification bias.PLOS ONE 13,e0191520;Reinholt,S.&Craighead,HG(2018)Microfluidic Device for Aptamer-Based Cancer Cell Capture and Genetic Mutation Detection.Anal Chem.90 2601-2608.doi:10.1021 / acs.analchem.7b04120; and HWHou et al.Microfluidic Devices for Blood Fractionation.Micromachines 2011,2(3),319-343;https: / / doi.org / 10.3390 / mi2030319. .

[0056] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0057] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art (e.g., within 2 standard deviations of the mean).About can be understood to be within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.Unless otherwise clear from the context, all numerical values ​​provided herein are modified with the term about.

[0058] All numerical ranges provided herein are understood to be shorthand for all decimal and fractional values ​​within the range. For example, the range of 1 to 50 may be any number from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. , combinations of numbers, or subranges, as well as all intervening decimal values ​​between the aforementioned integers (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9, etc.) and all intervening fractional values ​​between the aforementioned integers (e.g., 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 8, and 1 / 9, etc.), and all multiples of the aforementioned values. With respect to subranges, "nested subranges" from either end of the aforementioned ranges are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0059] As used herein, the term "sample" will be understood to encompass any fluid, solution, or mixture isolated or detected as a component of a more complex mixture, or synthesized from precursor species. The term "sample" encompasses any biologically derived sample or biological sample, including, but not limited to, blood, plasma, serum, lymph, saliva, tears, cerebrospinal fluid, urine, sweat, plant or edible plant extracts, semen, in vitro cell cultures, tissue homogenates (e.g., animal tissue homogenates, plant tissue homogenates, etc.), solutions containing dissociated cells or cell nuclei, and ascites fluid. A sample may be native or non-native. For example, a sample may include acellular species (e.g., disrupted cells, nuclei). Additionally, a sample may be fixed or otherwise processed. In some embodiments, a sample or biological sample includes at least one cell, at least one cell nucleus, and / or at least a portion of gDNA (partially isolated or completely isolated).

[0060] The device of the present disclosure can vary in size.The isolation technology of gDNA or its fragments is not limited by the size of the device.Therefore, the dimensions provided herein are for practical purposes only, and the device dimensions can be larger or smaller than those indicated.

[0061] In some embodiments, the length or width of the device may be about 450 millimeters or less.

[0062] In some embodiments, the height of the device or the height of an internal channel within the device can range from 1 μm to 200 μm. In some embodiments, the height can be at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μm. In some embodiments, the height can range from 15 μm to 35 μm.

[0063] The present invention is based on a method for extracting and isolating genomic DNA (gDNA) from a sample containing cells. Figure 1 shows an exemplary workflow of the disclosed system.

[0064] As shown in Figure 2, an exemplary method begins with the introduction of a sample (e.g., whole blood, or a cell suspension) into a microfluidic device that includes one or more microfluidic channels. This can be done manually by pipetting or any other means utilizing a syringe pump, a peristaltic pump, or other means that controllably drive fluids.

[0065] When a sample is introduced into a microfluidic channel, a slight pressure applied to the fluid forces the cell solution through the channel to an output reservoir. During this process, the cell solution passes through a region of the channel that contains an array of micropillars, altering the flow of the fluid in the channel.

[0066] These micropillars can have any shape or form. For example, the micropillar heads and / or the micropillar tips can include shapes that are circular (thereby forming cylindrical micropillars), elliptical, square, triangular, rectangular, cross, hexagonal, diamond, polygonal, dome-shaped (three-dimensional), pyramidal, or any combination(s) thereof.

[0067] The micropillars can be arranged with variable spacing dimensions depending on the size of the target cells of interest. Most animal cells (e.g., mammalian cells) have a diameter between 10 μm and 100 μm. HeLa cells are usually 10-40 μm in diameter depending on the culture conditions. Red blood cells (one of the smallest human cells) have a diameter of less than about 8 μm. On the other hand, muscle fiber cells and neurons can be extremely long. The diameter of a mammalian cell nucleus (e.g., human cell nucleus) can be about 10 μm. Plant cells tend to be larger than animal cells, but can still be 10 μm to 100 μm in diameter. The diameter of a fungal cell can be between 2 μm to 10 μm. The diameter or length of a protist can be between 1 μm to 3 millimeters. The diameter of the budding yeast Saccharomyces cerevisiae is about 4 μm. The average diameter of a coccus is 0.5 μm to 2.0 μm. For rod-shaped bacteria or filamentous bacteria, the length is 1 μm to 10 μm, and the diameter is 0.25 μm to 1 μm. The size of the cell and the size of the nucleus are well known in the art. The cell or cell nucleus can be derived from any organism, including animals (mouse, dog, cat, human, cow, etc.), fungi, protozoa, bacteria, or plants.

[0068] The next step in the process is to perform a short wash step with a suitable buffer (e.g., phosphate buffered saline), which acts to remove any undesired material from the buffer and helps to wash away any loose particles that may be stuck in the channel. The genomic DNA (gDNA) of the captured cells is then released by flowing a lysis buffer into or through the device. Upon cell lysis, the genomic DNA is released from the nucleus and the relatively long gDNA molecules become captured and are held in the micropillars, while all other cellular components are flowed into an output reservoir, thereby isolating the gDNA from all other cellular components. Another wash step is then performed to remove the lysis buffer from the channel. This is shown in Figure 3.

[0069] As the cells traverse the array, the target cells become trapped in the array due to the constraint of the pillar spacing, while all particles in the solution with a size smaller than the restrictive pillar spacing will pass freely towards the output reservoir. For example, in a whole blood sample, larger cells such as white blood cells will become trapped between the micropillars due to the restrictive micropillar spacing, while smaller cells (e.g., red blood cells, platelets, bacteria) and other acellular material (e.g., viral particles, proteins, lipids, RNA) will pass freely through the micropillar array to reach the output reservoir.

[0070] Alternatively, when an affinity selection approach is employed, the channel can be pretreated with an antibody or antibodies (or other molecules that confer specific affinity, such as aptamers) to functionalize the channel, and pillar spacing can be less restrictive (e.g., larger than required to capture cells) so that cells of similar size expressing different surface markers can be selected from. Using this approach, only cells expressing a certain marker or set of markers will be captured in the pillar array, while other cells will pass through the array. Also, a combination of size selection and affinity selection can be used. Such a combination can be in any arrangement or configuration. In some embodiments, the array of micropillars for affinity capture can be placed closer or further from the inlet compared to the array of micropillars for size selection. In other embodiments, a mixture of micropillars for affinity capture and micropillars for size selection can be placed in the same area in the channel. In still other embodiments, different arrays of micropillars (e.g., for affinity capture vs. for size selection) can be placed in different channels (e.g., orthogonal or branching channels below, see e.g., FIG. 4).

[0071] This channel surface functionalization strategy can be utilized to capture cells of interest from within a sample of mixed biological components. An example application would be to utilize channel functionalization in a larger structure at the intersection of two orthogonal microfluidic channels, as shown in FIG. 4. Although an orthogonal secondary channel is shown as an example in FIG. 4, the secondary channel intersecting the first channel can be at any angle relative to the first channel. The larger structure can be functionalized with antibodies, DNA / RNA aptamers, or any other molecule that confers the ability to selectively target and restrain a specific cell type while allowing all other cells to pass unimpeded. The captured target cells can then be lysed toward the pillar array region in the orthogonal channel. This allows selective capture and extraction of DNA from the cells of interest, and then collection and analysis of the genetic material of the target cells, while other cells or debris can be separated from this collection.

[0072] To recover gDNA, in the following system, the entangled DNA is cut into smaller fragments by using an enzyme that cuts double-stranded DNA. The smaller fragments of DNA escape the pillars more easily and flow towards the drainage reservoir. The gDNA can then be removed from the device for downstream processing or subjected to further processing such as heat inactivation of the enzyme in the second stage of the device, buffer exchange, sample enrichment, or library preparation for a sequencing platform. The gDNA is compatible with any analytical process such as subsequent amplification, gene identification, or sequencing. The present disclosure encompasses various means for cutting the entangled DNA into smaller fragments (e.g., endonuclease digestion (e.g., restriction enzymes), sonication, high intensity light, alteration of flow conditions, any other method of cutting gDNA into smaller fragments known in the art, or any combination of these (see below for further discussion).

[0073] The scalability of the device can be scaled down to single cell processing while retaining the functionality and performance of the micropillar array technology. Figure 5 shows the single cell workflow, which is very similar to the multi-cell workflow described in Figure 2. Here, an integral wall surrounding the micropillar array area allows only single cells to be captured at the tip of the micropillar array. Once a cell is captured, access to the micropillar array is prevented for further cells, and any further cells entering the channel are redirected via a "grooved" branch channel to a downstream waste channel or connected to a further single cell channel. Upon cell lysis, only single cell gDNA is collected in each micropillar array, allowing the DNA to be sheared and further analyzed for the single cell at the time of collection.

[0074] Figures 2 to 5 show a microfluidic device consisting of an array of pillars fabricated in PDMS by a lithographic process. The PDMS structure is capped with a glass plate, thereby forming a fluidic channel incorporating microstructures for trapping selected cells and subsequently trapping and selecting DNA of selected fragment lengths. The micropillar array can be constructed in all dimensions as required, and it can be specifically tailored to small or large samples. Figures 6 to 11 show the different device designs.

[0075] The functionality of the microfluidic system can be maintained in a wide range of channel geometries and dimensions. Within the microchannel, this configurability includes the size, shape, density, and spacing of the microstructures responsible for cell capture and extraction and retention of genomic DNA during cell lysis. The overall chip dimensions can be altered in channel height, width, and length while maintaining the functionality of the system. This includes scaling the overall size and capacity of the chip as well as configuring the chip into various formats such as 96-well or microwell plate formats suitable for single cell processing, suitable for processing large numbers of cells, and allowing for additional levels of integration into other sample preparation and / or analysis systems that operate within the microwell plate format. These alternative microwell plate systems include, but are not limited to, other robotic sample preparation platforms, flow cytometer platforms, plate readers, and the like.

[0076] The present disclosure herein demonstrates and contemplates several systems, devices, and methods for preparing and collecting fragments of DNA of defined sizes from selected cells, which improve DNA preparation for sequencing and other analyses.

[0077] system As shown in FIG. 11, the disclosed system includes a microfluidic "chip" device for processing cells and DNA and a system for delivering samples to the microfluidic chip device and controlling the sequential flow of different reagents through the microfluidic chip.

[0078] One popular fluidic system is one in which volumes of fluid samples and reagents from about 10 microliters to about 50 mL are conveniently pipetted into the inlets. In some embodiments, the volumes are at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 7 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mL; or any range between these values, and is incorporated into the PDMS portion of the fluidic chip. Figure 6 shows an image of a typical microfluidic device fitted with inlet and outlet ports. Fluid is driven through the microfluidic chip device by application of several PSI of air pressure from compressed air. Air pressure is applied for the period required to drive the desired volume of fluid by inserting a tube connected to a controlled pressure source into the inlet of the device. After flow occurs, the tube is removed and the next liquid or reagent required for the process is pipetted into the inlet, followed by another pressure-driven step.

[0079] The present disclosure encompasses other systems that do not require the attachment and detachment of tubing to apply pressure to drive the required fluids. Figure 8 shows a schematic of a system in which the fluids can be driven by any pump type (such as a pressurized air source (e.g., compressed air), a controlled pressure source, a controlled air pressure source, a pressure driven pump, a syringe pump, a vacuum pump, or a peristaltic pump) with a selection valve to select the fluid required for the required process step. Flow rates and flow rates can be measured and controlled by standard processes.

[0080] method The method for extraction of size-selected DNA fragments involves using a specific combination of a cocktail of DNA cleavage enzymes, applied flow pressure, incubation time, and duration to produce gDNA fragments of specific fragment lengths.

[0081] Any sample or solution (including but not limited to digestion buffer, wash buffer, elution buffer, lysis buffer, etc.) can be loaded into the microfluidic device using applied pressure (e.g., measured in positive psi or flow rate) or vacuum pressure (e.g., measured in negative psi or flow rate).

[0082] In some embodiments, the applied pressure or vacuum pressure is at least about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152 , 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 psi; or any range therebetween. As used herein, psi with respect to applied pressure refers to positive psi, whereas psi with respect to vacuum pressure refers to negative psi.

[0083] In some embodiments, the flow rate is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 20 0, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、696、697、698、699、700、701、702、703、704、705、706、707、708、709、710、711、712、713、714、715、716、717、718、719、720、721、722、723、724、725、726、727、728、729、730、731、732、733、734、735、736、737、738、739、740、741、742、743、744、745、746、747、748、749、750、751、752、753、754、755、756、757、758、759、760、761、762、763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 8 23, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 88 3, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943 , 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 μl / min.In some embodiments, the flow rate ranges from about 0.1 μl / min to about 500 μl / min.

[0084] Loading of cell-containing samples into microfluidic devices A volume ranging from at least about 10 microliters to about 50 mL (e.g., at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, A sample (e.g., dissociated animal cells (e.g., mammalian cells), whole blood) containing cells in a suspension of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mL; or any range between these values) is loaded into a microfluidic device containing one or more channels. Each channel consists of an inlet, an array of micropillars, an output reservoir, and a second stage for further processing including heating the sample, exchanging buffers, concentrating the sample, and preparing libraries.

[0085] Loading of the sample onto the device is performed either by manually pipetting the solution into an instrument that interfaces with a microfluidic channel capable of holding the sample volume, or by a pressurized air source (e.g., compressed air), a controlled pressure source, a pressure-driven pump, a syringe pump, a vacuum pump, or a peristaltic pump, or similar device. The sample, including the cells, is then forced through the channel using applied pressure (0.001-50 psi) on the instrument or driven through a pump system at a defined speed.

[0086] The sample is then flowed through the device until the desired volume is driven through the device (from 1 second to several hours). During this time, the sample passes through an array of micropillars. The size of the pillars, the spacing, and the length of the array can be customized to create pillar arrays tailored to specific cell types with specific spacing between pillars (in certain embodiments, as small as 1 micrometer). When a cell encounters the array, any cell larger than the pillar spacing will become physically trapped and will not be able to continue through the array and channel. Any cell or other particle smaller than the pillar spacing can flow unrestricted through the channel toward the drainage reservoir.

[0087] For example, for a sample containing whole blood, an array with a gradient of pillar spacing ranging from 100 to 8 micrometers will capture white blood cells or circulating tumor cells in the array while allowing all other components of whole blood (e.g., erythrocytes, thrombocytes, albumin, bacteria, viruses, etc.) to pass unrestricted through the pillars due to their smaller size. These smaller components will continue to flow through the channels and into a pool in the exhaust reservoir where they can be removed as waste or used for further off-device analysis.

[0088] If affinity selection is desired, the channels and pillars can be functionalized with specific antibodies (or aptamers or any affinity-conferring proteins, solutions, molecules, or nucleic acids). For example, if it is desired to isolate mature T cells from whole blood, the channels and arrays can be treated to be functionalized with anti-CD3 (a specific marker for mature T cells), and the pillars can be defined so that all white blood cells physically cross the pillars. Thus, CD3- cells will pass through, while CD3+ will be captured by affinity. Antibodies that bind to various cell surface markers are commercially available. For example, antibodies that bind to CD3 can be purchased from vendors such as R&D systems (representative catalog numbers MAB100, MAB100R, FAB100A, etc.), Abeam (representative catalog numbers abll089, ab5690, ab52959, etc.), ThermoFisher Scientific (representative catalog numbers 14-0037-82, 16-0037-81, 48-0037-42, 16-0038-81, 13-0037-82, etc.), and BioLegend (representative catalog numbers 317319, 317301, 344801, etc.). Also, labeled antibodies (e.g., biotin-labeled antibodies) are available from vendors and can then be readily attached to streptavidin-coated micropillars.Finally, the channel containing the functionalized (e.g., with an antibody or aptamer) micropillars is exposed to a solution containing at least one cell or cell nucleus at a pressure (about 0.001 to about 50 psi; or at least about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9, 0.1, 0.4, 0.5, 0.6, 0.9, 0.8, 0.9, 0.1, 0.5, 0.7, 0.8, 0.9, 0.1, 0.6, 0.7, 0.8, 0.9, 0.1, 0.8, 0.9, 0.1, 0.9 ... , 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 psi; or any range between these values) or at a prescribed flow rate for one minute to several hours or days. In some embodiments, the total volume of solution flowing through the device can range from at least about 10 μl to about 100 ml (or range between these values). In some embodiments, it may be possible to stop the flow and incubate the cells with the functionalized micropillars to maximize binding. Similar to cell binding, the captured / bound cells can be washed by exchanging the solution with a wash buffer (e.g., phosphate buffered saline, pH 7-7.9) using the same pressure, flow rate, or volume of buffer as indicated above.

[0089] Lysis of captured cells and trapping of gDNA To release the gDNA contents from the cells trapped between the pillars, a lysis buffer suitable for lysis of the nuclear membrane of the cells (e.g., 1% SDS in PBS (pH 7-8), or 4 M guanidinium isothiocyanate) is added to the device and pressure (about 0.001 to about 50 psi, e.g., 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59 , 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 psi) was flowed over the cells for about 0.001 to about 120 minutes. The composition of the lysis buffer can be altered to retain native histone complexes upon lysis. Thus, the amount of protein binding to the recovered genomic DNA can be controlled and tuned by the type of lysis buffer and / or wash buffer (e.g., containing harsher or milder denaturants, etc.). During lysis, all cellular contents are released from the cell or cells, but while the genomic DNA becomes entangled and captured on the micropillars, all other contents (lipids, proteins, RNA, etc.) flow past the pillars toward an exhaust reservoir, where the aforementioned other contents can be removed from the device as waste or for further analysis. The pillars retain nearly 100% of the genomic DNA expected to be contained in the cell or cells. Phosphate buffered saline is then added to the device to flush out the lysis buffer under pressures of about 0.001 to about 50 psi for about 0.1 seconds to about 120 minutes.In some embodiments, the pressure is at least about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 psi. Under these conditions, the captured gDNA is retained on the pillars for several days.

[0090] Tunable shearing and release of gDNA To release and recover gDNA from the micro-pillars, the following exemplary method may be used: 1. Enzymatic cleavage 2. Changing flow conditions 3. Sonication 4. Light-based methods (e.g. high-intensity light) 5. Spraying 6. Mechanical methods / melting or collapsing of pillars 7. Any combination of 1 to 5 In addition to the methods described above, one of skill in the art can combine any method of cleavage / shearing gDNA known in the art with the devices, systems, and / or methods of the present disclosure.

[0091] enzymatic method Using different enzymatic processes and conditions, the size of the recovered gDNA fragments can be tailored to a specific size range to meet optimal conditions for the desired downstream analytical application. Any enzyme capable of nuclease cleavage of double-stranded DNA can be used to accomplish this.

[0092] Such examples include, but are not limited to, restriction enzymes (e.g., type I restriction enzymes (restriction and modification enzymes that cut DNA away from the enzyme's recognition sequence), type II restriction enzymes (blunt-end or overhanging-end cutters), type III restriction enzymes (cut outside their recognition sequence and require two such sequences in inverted orientation within the same DNA molecule for cleavage), type IV restriction enzymes (enzymes that recognize / cleave modified (e.g., methylated) DNA), homing endonucleases, transposases, type II CRISPR-Cas9 proteins, or other commercially available enzymes that cleave dsDNA (e.g., dsDNA Fragmentase from New England Biolabs (representative catalog numbers: M0348S, M0348L)). The size of the gDNA recovered from the device can be selectively adjusted based on several conditions, such as: type and number of enzymes used, concentration of the enzyme or enzymes, buffer composition, incubation temperature, incubation time, and flow conditions (psi and time at a particular psi).

[0093] Overview of enzymatic methods using restriction endonucleases To recover gDNA fragments within a specific fragment size range, prepare a reaction mixture containing: One or more type II restriction enzymes and / or homing endonucleases (final 1 × 10 -4 ~5U / microliter) The buffer solution contains: Tris-HCl (0-500 mM) NaCl (0-500 mM) MgCl2 (0-100 mM) Potassium acetate (0-500mM) Tris acetate (0-500 mM) DTT (0-10 mM)

[0094] Introduce buffer into the microfluidic channel by instrumentation (by pipetting or syringe pump). Set the device at a controlled reaction temperature (10-70 °C). Allow the reaction to proceed under flow pressure of 0.01-50 psi, preferably for 1-60 min, then 0.5-50 psi for 1-30 min. Notably, in certain circumstances, the higher the flow rate during DNA release (compared to the flow rate used during enzyme incubation), the higher the recovery of gDNA fragments. In some embodiments, a higher flow rate ("blast") may be applied during DNA release using a pressure of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 psi. In some embodiments, a blast may be applied at a flow rate ranging from at least about 0.1 μl / min to about 1000 μl / min (or any range between these values). In some embodiments, the blast may be applied for a duration of approximately a fraction of a second (e.g., in the millisecond range). In some embodiments, the blast may be applied for a duration of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 seconds. In some embodiments, the blast may be applied for a duration of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes. In preferred embodiments, the blast is applied for a duration ranging from at least about 0.1 seconds to 10 minutes.

[0095] In general, the following principles allow the user to adjust the recovered fragment size: The modifications can be used separately or in various combinations to obtain different fragment sizes. 1. When the fragment size is large 1. Decrease in total enzyme concentration 2. Reduction in the number of different enzymes 3. Changing the buffer to reduce the efficiency of the enzyme 4. Decreased digestion time 5. Lowering digestive temperature 6. Changing flow conditions 2. When the fragment size is small 1. Increase in total enzyme concentration 2. Increase in the number of different enzymes 3. Changing the buffer to increase enzyme efficiency 4. Increased digestion time 5. Increase in digestive temperature 6. Changing flow conditions

[0096] Other enzymatic and nonenzymatic approaches for DNA fragmentation Alternatively, other enzymatic approaches may be used to shear gDNA to a defined size and release it from the pillars. Type II CRISPR-Cas9 Transposase Fragmentase Tagmentation approaches, such as the bacterial transposase Tn5, which in addition to fragmenting DNA, leave the DNA altered (e.g., barcoding the ends of the fragmented DNA with nucleotides or other adapters used in downstream methods) Commercially available enzymes that fragment double-stranded DNA

[0097] Physical forces can also be used to fragment DNA as an alternative to enzymatic cleavage. For example, increasing the flow rate of the liquid will generate hydrodynamic forces on the immobilized gDNA, thereby fragmenting the DNA. Ultrasonic agitation of the fluid (e.g., sonication) is also known to cleave DNA and can be applied to fragment the immobilized gDNA. Ultraviolet light can be irradiated onto the device, alone or in combination with other processes mentioned above, to fragment / release the gDNA.

[0098] Thus, the devices, systems, and / or methods of the present disclosure may further comprise at least one agent and / or at least one instrument that facilitates (a) cleavage of gDNA into fragments and / or (b) modification of the gDNA fragments or proteins associated with said fragments.

[0099] In some embodiments, the at least one agent comprises at least one enzyme. In some embodiments, the at least one enzyme is a DNA cleaving enzyme, a DNA modifying enzyme (e.g., a DNA methyltransferase, a terminal transferase capable of labeling DNA fragments, a T4 DNA ligase capable of ligating a PCR primer to a DNA fragment for amplification, a DNA polymerase (e.g., for PCR amplification; e.g., Taq polymerase, Pfu polymerase, etc.), and / or an enzyme that modifies a DNA associated protein (e.g., histones, transcription factors, etc.) (e.g., histone methyltransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, ubiquitin ligase, deubiquitinase, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), E3 These may include SUMO ligase, SUMO-specific protease, protease, Poly-ADP ribose polymerase, (Adp-ribosyl)hydrolase ARH1 and ARH3, protein arginine deiminase 4 (PAD4), Fpr4 (proline isomerase).

[0100] In some embodiments, at least one instrument comprises an ultrasonic liquid handler (eg, a sonicator).

[0101] In some embodiments, at least one instrument comprises a light source for high intensity light to fragment gDNA. In some embodiments, the light source for high intensity light emits / generates UV light, X-rays, gamma rays, and / or ionizing radiation. In a preferred embodiment, the light source emits UV light.

[0102] In some embodiments, at least one device comprises a nebulizer. It is recognized in the art that nebulization fragments DNA (see, e.g., Lentz et al. (2005) J Aerosol Sci 36:973-990; Sambrook and Russell (2006) CSH Protoc pdb.prot4536, each of which is incorporated herein by reference).

[0103] In some embodiments, at least one device is a device that generates hydrodynamic forces capable of shearing gDNA (e.g., a Point-sink Shearer (PtS), a recirculating point-sink flow system, a bubbling system, and / or a sieving system such as those described in, for example, Yew and Davison (1968); Thorstenson et al. (1998) Genome Res 8:848-855; Shui et al. (2013) Rsc Adv 3:13115-13118; Oefner c / a / . (1996) Nucleic Acids Res 24:3879-3886; Nesterova et al. (2012) Lab Chip 12:1044-1047; Joneja and Huang (2009) Biotechniques 46:553-556; Shui et al. (2011) Nanotechnology 22:494013; Li et al. (2012) Nanotechnology 22:494013; Li et al. (2012) Nanotechnology 22:494013; Li et al. (2012) Nanotechnology 22:494013; Li et al. (2012) Nanotechnology 22:494013). (2017) Scientific Reports 7:40745, each of which is incorporated herein by reference).

[0104] Light Method In certain embodiments, non-enzymatic approaches / methods are used to cleave and release gDNA from the micropillars. Such methods may include the use of light (which may be high intensity) directed directly or focused on the area of ​​the chip cartridge (device) where gDNA is captured / extracted. In this approach, one or more light sources can be applied directly to the top or bottom of the channel (or scanned across the area) at an angle or series of angles defined by coordinates. The light alone or in conjunction with chemicals (including but not limited to DNA binding agents or fluorescent dyes) can generate double-stranded breaks; alternatively, the light should be of a minimum intensity strong enough to weaken the physical / mechanical properties of the collective gDNA and then create multiple single-stranded DNA breaks that can more easily release or release the gDNA from the anchoring pillars in the micropillar array. This can be combined with an auxiliary increase in shear force by increasing fluid flow or changing the internal pressure of the channel, or enzymatic cleavage, or any other cleavage method described.

[0105] Additionally, the same principles utilized in optical methods can be extended to wavelengths beyond the visible spectrum of the human eye (including x-rays, ultraviolet light) or to other wavelengths (such as, but not limited to, radioactive wavelengths).

[0106] Sonication In certain embodiments, the non-enzymatic approach to shearing and releasing gDNA from the micropillars includes the use of sonication, which can shear the extracted gDNA and thereby release it from the micropillars, and sonication can be performed with or without a constant or intermittent flow of fluid through the microfluidic channel. In this application, one or more sonication sources can be embedded directly into the channel, directly sealed above or below the panel, or the entire chip can be housed or placed in a sonication chamber or sonication solution / bath. When sonication forces are applied intermittently or constantly, the gDNA will be sheared. When a flow of fluid is subsequently or simultaneously applied to the microfluidic channel, the sheared / sonicated DNA is believed to be freed from the physical / mechanical entanglement forces that initially tethered the gDNA during extraction / cell or nucleus lysis, and therefore can be subsequently flowed / recovered / removed / collected.

[0107] How to change the flow The flow rate can be manipulated in several ways that affect the force and method used to release the gDNA or gDNA fragments from the pillars. For example, while using an enzyme to cleave the gDNA, the flow rate can be decreased to allow the enzyme to interact and process with the DNA more frequently or faster, thereby cleaving more DNA. Conversely, the flow rate can be accelerated to minimize enzyme-DNA interaction events. In another application, after weakening the physical / mechanical entanglement forces between the DNA and the micropillars by enzymatic cleavage, increasing the flow rate can induce higher shear forces on the DNA molecules, which will further unravel or shear the DNA from the micropillars, thereby acting to "clean / wash" the channel, helping to improve / maximize the recovery / yield of the DNA collected from the original cell or nuclear sample loaded into the chip cartridge(s).

[0108] Mechanical methods / pillars for melting or collapsing To improve the release and recovery / yield of extracted gDNA physically entangled within the micropillars without using any biochemical / enzymatic treatment, some methods may include the use of micropillars that can be mechanically bent (no longer perpendicular to the channel surface), mechanically stored (in one wall or cap layer of the chip cartridge / microfluidic channel), mechanically removed by peeling of a layer attached to the micropillar, or chemically dissolved in whole or in part to allow the gDNA to slide or flow down from the micropillar in the fluid flow, and the unfragmented or minimally fragmented collected in an outlet port. The micropillars can be constructed from soft polymers (such as PDMS) containing paramagnetic particles, and then a single magnetic body located at the top of the channel can be used to manipulate or actuate the micropillars to tilt in one direction. Similarly, to release the gDNA by dissolving the micropillars for collection, a solution can be flowed through the channel, dissolving some or all of the micropillar material while being inert or minimally damaging to the gDNA in the channel.

[0109] gDNA fragment size The disclosed devices, systems, and methods can recover intact genomic DNA, including entire chromosomes. Alternatively, the disclosed devices, systems, and methods can generate gDNA fragments of various sizes. For example, the size of the gDNA fragments can be at least about between 1 kb and 500 kb; or any range between these values. In some embodiments, the size of the gDNA fragments can be at least about between 1 kb and 500 kb; or any range between these values. In some embodiments, the size of the gDNA fragments is at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500 kb; or any range between these values.

[0110] The term "size of gDNA fragments" does not mean that all fragments have the same size (e.g., length, molecular weight). Rather, it refers to a representative or average size of the gDNA fragments in the recovered solution. For example, in some embodiments, when the size of the gDNA fragments is at least about 5 kb, at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, %, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% have an average size of at least about 5 kb.

[0111] In some embodiments, the size of the gDNA fragments represents an average size of at least about 50% of the gDNA fragments in the recovered solution.

[0112] In some embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the total genomic DNA in the channel after cell / nuclear lysis contains the desired fragment size + / - 10 kb.

[0113] In some embodiments, at least about 50% of the total genomic DNA in the channel after cell / nuclear lysis contains the desired fragment size + / - 10 kb.

[0114] Sample recovery from the device As the gDNA is sheared during the shearing reaction, it will be released from the pillars and flow toward the output reservoir. Upon reaction completion, the gDNA can then be processed in one of two ways.

[0115] (1) The first method involves removing the sheared gDNA from the output reservoir by manual pipetting or through tubing, whereby the sample is pumped out of the device into a collection tube, and the total output volume containing the size-selected gDNA is at least about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.40, 0.42, 0.46, 0.48, 0.49, 0.50, 0.52, 0.56, 0.60, 0.70, 0.80, 0.90, 0.12, 0.14, 0.16, 0.18, 0.22, 0.24, 0.26, 0.28, 0.32, 0.34, 0.36, 0.44, 0.50, 0.60, 0.70, 0.80, 0.90, 0.12, 0.14, 0.16, 0.18, 0.22, 0.24, 0.26, 0.28, 0.32, 0.34, 0.36, 0.40, 0.52, In a preferred embodiment, the total drained volume containing the size-selected gDNA can range from 1 microliter to 5 mL; or any range between these values. If an enzyme is used, the sample is incubated at the particular temperature indicated for the particular enzyme used (eg, 80° C. for 15 minutes) for the particular time period to inactivate the enzyme present in the sample.

[0116] (2) The second approach involves a sequential step of a second channel: upon completion of the shear reaction, a valve downstream of the drain reservoir is opened to expose the sample to the electrophoresis channel.

[0117] After enzymatic inactivation, the gDNA samples are ready for downstream applications such as pulsed-field gel electrophoresis, amplification and detection of specific loci, or sequencing.

[0118] In some embodiments, any sample or buffer (e.g., lysis buffer, wash buffer, elution buffer) may further include a blocking agent (e.g., BSA, ionic detergent, or another charged agent) and / or a lubricant (e.g., polymer) to maximize the yield of recovered gDNA fragments or the recovery of intact chromosomes or gDNA. Such blocking and / or lubricants may reduce entanglement of gDNA to the micropillar or channel surfaces and avoid shearing of intact or large gDNA by physical traction. See below for further discussion.

[0119] Mechanical and electrical pertubation of micropillars Mechanical perturbation of the micropillars can be performed in various ways on one or more of the micropillars in the micropillar array, either simultaneously, sequentially, or at other intervals. Pertubation can include mechanical tilting or collapse of the micropillars, mechanical or physical retraction of the micropillars, or removal of the entire micropillar by peeling off the layer / wall of the channel to which the micropillars are attached. These micropillars can also be embedded with particles that can be mechanically or electrically acted upon to change shape or other mechanical or physical properties with the goal of being able to release the extracted genomic DNA without the need for DNA shear or fragmentation. Electrical perturbation can include application of an electric charge or electric field to force or induce a change in the shape of the entire micropillar by a power-receiving component (wire, metallic anchor, etc.) in the micropillar.

[0120] Chemical pertubation of micropillars Chemical perturbation can include partial or complete dissolution or decomposition of the micropillar structure material via a solution flowed in the microfluidic channel. Chemical perturbation can also include damaging or decomposing specific portions of the micropillars in such a manner as to allow release of the micropillars from their fixed or attached portions in the channel. This perturbation can also be done by targeting specific subsets of micropillars in any number of micropillar arrays on the chip cartridge / in the channel for perturbation, and can be selected by choosing the material of the fabricated micropillars (not limited to such structures) for DNA extraction and subsequent perturbation, conceptually can be applied to support structures, walls, or other designed structures in the channel.

[0121] Coating micropillars for DNA sliding One or more micropillars within the micropillar array or even the entire channel can be coated with a chemical or material that would allow the DNA extracted and entangled within the micropillar array to slide off the micropillars and thus be recoverable / collected without the need for fragmentation. The incubation time within the channel as well as the molecules (such as PEG or PEO polymers, or even BSA or other biomolecules) can be tailored and will correlate with the coverage of the total interior surface of the channel or micropillar and / or with the thickness of the coating on the surface of such structures themselves.

[0122] Other Device Configuration Variations for the Disclosed Microfluidic Channels In addition to the basic micropillar array layout, where there are variations in pillar shape, pillar dimensions, and inter-pillar spacing, tertiary structures can also be designed into the micropillar array to improve functionality during cell loading. Exemplary variations are described below.

[0123] Additional canals in the micropillar array For example, "canals" (shown in Figures 23 and 24) can be created in the micropillar array by removing a piece of the pillar in a manner that would allow for further loading of cells into the array. This would allow the cells to be more spread throughout the array area rather than all stagnating around a localized area. Not only would this reduce the possibility of clogging or flow disturbances within the pillar array area, but it would also help prevent DNA from roping in localized areas with densely loaded cells. Similarly, this would facilitate not only DNA extraction from cells, but also removal and off-chip collection of the extracted DNA during enzymatic digestion.

[0124] Therefore, constructing such a canal structure within our micropillar array would facilitate cell loading, increase the cell volume within the device, help prevent clogging of localized cell clusters, and improve the on-chip DNA extraction capacity.

[0125] Multiple Channels In certain aspects, the disclosed device or system may include at least one channel. In some embodiments, the disclosed device or system may include multiple channels (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more channels). In some embodiments, such channels may be arranged horizontally. In some embodiments, multiple channels may be arranged parallel to each other. In other embodiments, multiple channels may be arranged in a staggered configuration (e.g., FIG. 24).

[0126] Stacked devices (two or more devices stacked together, either with or without adhesive) The micropillar array regions themselves can be strategically positioned in a stacked fashion to facilitate even better spreading of the cell sample during the cell loading step.

[0127] For example, the disclosed devices and systems may include an arrangement of separate or connected / glued channels or chip cartridges stacked vertically (in a vertical plane) on top of each other. These stacked chip cartridges or channels may be of the same or different designs, both in the macroscale and in the microfluidic channel itself. This may allow for a higher degree of processing of the channel volume / micropillar area without the need to enlarge the horizontal footprint of the channel, which the inventors anticipate will be useful for increasing or enhancing the total recovery of gDNA, reducing the horizontal extent of the overall chip cartridge footprint, and / or improving the throughput of the chip cartridge during processing of biological (cell or nuclear) samples.

[0128] Other variations Other variations include those shown in Figures 25 to 31. Such designs are also described in the Examples section below.

[0129] 29-31 include shorter / shortened micropillar array regions. In some embodiments, the length of the shorter / shortened micropillar array region (measured in the direction of fluid flow) is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 1 %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or less.

[0130] In some embodiments, the length of the shorter / shortened micropillar array region is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the length of the channel.

[0131] In other embodiments, the length of the shorter / shortened micropillar array region is less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the length of the channel.

[0132] In some embodiments, the length of the shorter / shortened micropillar array region may range from 5 μm to 450 μm. In some embodiments, the length is between 20 μm and 50 μm. In some embodiments, the length is less than 50 millimeters.

[0133] In some embodiments, the length of the shorter / reduced micropillar array region is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, or 450 μm.

[0134] Additional Equipment The devices, systems, or methods of the present disclosure may further include instruments, agents, and / or methods for (a) dissociating tissue into single cells, (b) analyzing cells that enter the microfluidic device, (c) analyzing recovered gDNA or gDNA fragments, or (d) any combination thereof.

[0135] In some embodiments, the device, agent, and / or method may dissociate the tissue into single cells. Numerous devices and agents are commercially available. For example, a sonicator, a homogenizer (e.g., TissueLyserII (Qiagen), TissueRuptorII (Qiagen), Dounce homogenizer, gentleMACS™ Octo Dissociator with heater (Miltenyi Biotec, catalog number 130-096-427), gentleMACS™ Dissociator (Miltenyi Biotec, catalog number 130-093-235), Singulator100 (S2 Genomics)) and / or a tissue grinder may be used to break the tissue into single cells. Further devices and methods include those described in WO2021 / 236666A1, WO2018 / 102471A1, and WO2019 / 232504A2, each of which is incorporated by reference herein.

[0136] In some embodiments, the device, agent, and / or method may analyze cells that enter the microfluidic device. For example, cells or cell nuclei may be sorted by size, morphology, stiffness, mechanical properties of cells, or the presence of certain cell surface markers. Thus, in some embodiments, the device, system, or method of the present disclosure may further include a cell fractionator, a flow cytometer, a fluorescent activated single cell sorter (FACS), a microscope (such as an optical microscope, a fluorescent microscope, etc.), or a combination thereof.

[0137] In some embodiments, the device, agent, and / or method may analyze the recovered gDNA or gDNA fragments. In some embodiments, the recovered gDNA or gDNA fragments may be analyzed by pulsed-field gel electrophoresis, hybridization, microarray, amplification, mass spectrometry (e.g., LC-MS), Southern blotting, sequencing, or a combination thereof. Thus, the device, system, or method of the present disclosure may further include an instrument capable of performing the aforementioned analyses (e.g., gel electrophoresis, PCR, real-time PCR, mass spectrometry (e.g., LC-MS, LC-MS / MS), sequencer (e.g., next-generation sequencing, third-generation sequencing), microarray, or a combination thereof).

[0138] Sequencing Any of a variety of sequencing reactions known in the art can be used to directly sequence the gDNA fragments recovered. Examples of sequencing reactions include those based on the technology developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Set. USA 74:560 or Sanger (1977) Proc. Natl. Acad. Set. USA 74:5463. It is also contemplated that any of a variety of automated sequencing procedures (Naeve (1995) Biotechniques 19:448-53) can be utilized, including sequencing by mass spectrometry (see, for example, PCT International Publication No. WO94 / 16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159). In particular, mass spectrometry (e.g., LC-MS, LC-MS / MS) can be used to sequence DNA (see Chowdhury and Guengerich (2013) Curr Protoc Nucleic Acid Chem 7:Unit-7.1611).

[0139] In certain embodiments, detection of gDNA fragments is accomplished using techniques such as sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), wobble sequencing (PCT / US05 / 27695), multiplex sequencing (U.S. Patent Application Serial No. 12 / 027,039, filed Feb. 6, 2008; Porreca et al. (2007) Nat. Methods). 4:931), polymerizing colony (POLONY) sequencing (U.S. Patent Nos. 6,432,360, 6,485,944, and 6,511,803, and PCT / US05 / 06425); and nanogrid rolling circle sequencing (ROLONY) (U.S. Patent Application No. 12 / 120,541, filed May 14, 2008). High throughput sequencing methods can also be utilized, such as by cyclic array sequencing using platforms such as Roche 454, Illumina Solexa or MiSeq or HiSeq, AB-SOLiD, Helicos, and Polonator platforms. High throughput sequencing methods are described in U.S. Patent Application No. 61 / 162,913, filed March 24, 2009. A variety of light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmacogenom. 1:95-100; and Shi (2001) Clin. Chem. 47:164-172) (see, e.g., U.S. Patent Application Nos. 2013 / 0274117, 2013 / 0137587, and 2011 / 0039304).

[0140] Next-generation sequencing (NGS) is a technology for determining DNA sequences to study genetic variations associated with disease or other biological phenomena. When it was introduced for commercial use in 2005, the method was initially called "massively parallel sequencing" because it allows multiple DNA strands to be sequenced simultaneously, instead of one DNA strand at a time, such as traditional Sanger sequencing by capillary electrophoresis (CE).

[0141] Due to the speed, throughput, and accuracy of NGS, NGS can interrogate hundreds of thousands of gDNA fragments in multiple samples at once, and can discover and analyze different types of genomic features, from single nucleotide variations (SNVs) to copy number and structural variants, and even DNA fusions, in a single sequencing run. NGS offers an ideal throughput per run, allowing research to be performed quickly and cost-effectively. Additional advantages of NGS include lower sample input requirements than Sanger sequencing, higher accuracy, and the ability to detect variants at lower allele frequencies.

[0142] Analysis of the whole genome using next generation sequencing (NGS) provides a base-by-base representation of all genomic alterations, including single nucleotide variants (SNVs), insertions and deletions, copy number changes, and structural variations. Paired-end whole genome sequencing involves sequencing both ends of a DNA fragment, which increases the likelihood of alignment with a reference and facilitates detection of genomic arrangements, repetitive sequences, and gene fusions.

[0143] In some embodiments, the Illumina "phased sequencing" platform can be used, which uses a combination of long and short paired ends. In some embodiments, the Illumina "long read assay" can obtain continuous reads of DNA fragments of about 10 kb in length. In other embodiments, third generation single molecule sequencing technology (e.g., ONT) can obtain much longer reads of DNA sequences.

[0144] In some embodiments, "deep sequencing" or a high-recovery version of Illumina NGS can be used. Deep sequencing refers to the sequencing of a single sample multiple times, sometimes hundreds or even thousands of times. Deep sequencing can detect miRNAs, rare clonotypes, cells, or microorganisms that may be present in as little as 1% of the original sample. Illumina's NovaSeq performs such whole genome sequencing efficiently and cost-effectively, with scalable output generating up to 6 Tb and 20 billion reads in dual flow cell mode with a simple, streamlined, automated workflow.

[0145] Microarrays In certain embodiments, gDNA fragments can be analyzed using microarrays. High-throughput microarrays have been developed to identify and detect the presence of certain loci or DNA abnormalities (e.g., mutations) (e.g., substitutions, deletions, insertions, duplications, DNA fusions, chromosome fusions, etc.) in various samples (e.g., tissue and cell types).

[0146] In some embodiments, covalent attachment of fluorophores can be used to directly label gDNA molecules for use in microarray analysis, for example, using commercially available kits for labeling DNA.

[0147] In certain embodiments, the recovered gDNA (e.g., chromatin) in its native form containing DNA-binding proteins (e.g., transcription factors) can be used in a microarray in combination with chromatin immunoprecipitation (also called a "ChIP chip").

[0148] Exemplary embodiments 1. A microfluidic flow-based device for extracting fragments of genomic DNA of a selected size range from cells or cell nuclei, comprising: at least one microfluidic channel having at least one inlet and at least one outlet that allows flow in a flow direction from the inlet to the outlet; and At least one array of micropillars disposed within said microfluidic channel. where The diameter of said micropillars is between 3 micrometers and 50 micrometers; said micropillars being separated from one another by a spacing between 2 micrometers and 150 micrometers; said array being bounded within an area of ​​between 1 square millimeter and 1600 square centimeters; the height of said micropillars is between 1 and 200 micrometers; and A device, wherein the micropillars are arranged in the array in a defined configuration.

[0149] 2. The device of 1, wherein the shape of said micropillars is circular, elliptical, square, triangular, rectangular, cross-shaped, hexagonal, diamond, polygonal, dome-shaped, pyramidal, or any combination of two or more of the foregoing.

[0150] 3. The aforementioned channel, (a) Random orientation; (b) square lattice; (b) a hexagonal lattice; or (c) any combination of two or more of the foregoing. 3. The device according to claim 1 or 2, comprising micropillars.

[0151] 4. The device of 3, wherein the channel comprises alternating regions of square lattice micropillars, randomly oriented micropillars, and hexagonal lattice micropillars.

[0152] 5. The device of claim 4, wherein the alternating regions of square and hexagonal lattice micropillars are (a) contiguous or (b) discontinuous with the regions being separated by structural regions.

[0153] 6. A device according to any one of 1 to 5, wherein the micropillars are arranged in a gradient of variable spacing.

[0154] 7. The device of 6, wherein the variable interval gradient comprises: (a) a smooth gradient; (b) a step gradient; or (c) a combination of (a) and (b).

[0155] 8. A device according to any one of 1 to 7, wherein the channel comprises at least one shorter micropillar array region (e.g., FIG. 29).

[0156] 9. The device of 8, wherein said channel comprises at least two or at least three shorter micropillar array regions, optionally wherein said shorter micropillar array regions are separated by structural regions.

[0157] 10. The device of 8 or 9, wherein the length of the shorter micropillar array region is less than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the length of the channel.

[0158] 11. A device according to any one of 1 to 10, wherein said device further comprises one or more canals extending into said micropillar array region.

[0159] 12. The device of claim 11, wherein said one or more canals are aligned along said flow direction, and optionally said one or more canals are straight, branched, curved or intersecting.

[0160] 13. The device of 11 or 12, wherein the device includes at least two canals of different lengths.

[0161] 14. The device of any one of 1 to 13, wherein the diameter, spacing, area, shape, or combination thereof is selected based on the type of cell or cell nucleus.

[0162] 15. The device according to any one of 1 to 14, wherein said cell or cell nucleus is an animal, fungal, protist or plant cell or cell nucleus, optionally a human cell or cell nucleus.

[0163] 16. The device according to any one of 1 to 15, wherein the cell or cell nucleus is a cell or cell nucleus of a white blood cell or a T cell.

[0164] 17. A device according to any one of 1 to 16, wherein the device comprises (a) a single-cell sized opening at the tip of the array, and / or (b) a narrower feature (e.g., micropillar spacing) near the inlet capable of extracting fragments of genomic DNA from single cells.

[0165] 18. The device of any one of 1 to 17, further comprising a "groove-like" branch channel.

[0166] 19. A device according to any one of 1 to 16 and 18, wherein the device comprises (a) a multi-cell sized opening at the tip of the array and / or (b) wider features (e.g., micropillar spacing) near the inlet, thereby enabling extraction of fragments of genomic DNA from multiple cells.

[0167] 20. A device according to any one of 1 to 19, further comprising an agent that binds to said cell or cell nucleus.

[0168] 21. The device according to 20, wherein the agent is an antibody, an antigen-binding fragment thereof, or an aptamer.

[0169] 22. The device of 20 or 21, further comprising a secondary channel to the microfluidic channel.

[0170] 23. The device of claim 22, wherein the secondary channel (e.g., FIG. 4) enables lysis of the cells and capture of the genomic DNA after the cells bind to the agent.

[0171] 24. The device of any one of 1 to 23, wherein the micropillars comprise a polymer, plastic, metal, silicon, glass, or any combination of two or more of the foregoing.

[0172] 25. The device of any one of 1 to 24, wherein the micropillars comprise polydimethylsiloxane (PDMS).

[0173] 26. A device according to any one of 1 to 25, wherein the micropillars are separated from one another by spacings in the range of about 2 to 30 micrometers, optionally 5 micrometers or less.

[0174] 27. A device according to any one of 1 to 26, wherein the height of the micropillars is between 5 and 50 micrometers.

[0175] 28. A device according to any one of 1 to 27, wherein the device comprises at least two microfluidic channels comprising an array of micropillars.

[0176] 29. The device of any one of 1 to 28, wherein the device includes at least 6, 12, 8, 12, 24, 48, 96, 384, or 1536 channels.

[0177] 30. (a) the channels are in a staggered or side-by-side configuration in the device; and / or (b) The device of 28 or 29, wherein the channel is an ordered feature that is aligned with a multiwell plate feature.

[0178] 31. A device described in any one of 28 to 30, wherein at least two of the microfluidic channels are vertically stacked.

[0179] 32. The device of any one of 1 to 31, further comprising an instrument for isolating cells and / or cell nuclei from tissue.

[0180] 33. The device of 32, wherein the equipment comprises a homogenizer, a sonicator, and / or a tissue grinder, optionally wherein the homogenizer is selected from TissueLyser II (Qiagen), TissueRuptor II (Qiagen), a Dounce homogenizer, gentleMACS™ Octo Dissociator with heater (Miltenyi Biotec, catalog number 130-096-427), gentleMACS™ Dissociator (Miltenyi Biotec, catalog number 130-093-235), and Singulator 100 (S2 Genomics).

[0181] 34. The device of 32 or 33, wherein the tissue is unprocessed, frozen, flash frozen, formalin-fixed paraffin-embedded (FFPE), or preserved in optimal cutting temperature compound (OCT).

[0182] 35. A device according to any one of 1 to 34, further comprising an apparatus for sorting cells or cell nuclei.

[0183] 36. The device according to 35, wherein the instrument comprises a flow cytometer, a fluorescence activated cell sorting (FACS) instrument, and / or a microscope.

[0184] 37. The device of any one of 1 to 36, further comprising at least one agent that facilitates lysis of cells or cell nuclei (e.g., a lysis buffer, a denaturant, and / or a wash buffer).

[0185] 38. The device of any one of 1-37, further comprising a digestion buffer, one or more enzymes, a DNA labeling agent (e.g., biotin, a fluorescent molecule, a barcode), a hybridization agent (e.g., a primer (e.g., labeled with a detectable marker, e.g., a fluorescent molecule, biotin)), and / or a wash buffer.

[0186] 39. The device according to 38, wherein the one or more enzymes are selected from DNA cleaving enzymes, DNA modifying enzymes (e.g., DNA methyltransferase, terminal transferase, T4 DNA ligase, DNA polymerase), and enzymes that modify DNA-associated proteins.

[0187] 40. The device of 38 or 39, wherein the one or more enzymes are selected from a restriction enzyme, an endonuclease, a homing endonuclease, a transposase, a type II CRISPR-Cas9 protein, and a dsDNA fragmentase.

[0188] 41. The device of any one of 38 to 40, wherein the one or more enzymes include one or more restriction enzymes.

[0189] 42. A device according to any one of 1 to 41, further comprising at least one apparatus for facilitating DNA cleavage and / or DNA release from the channel.

[0190] 43. The device of 42, wherein the at least one instrument is selected from a sonicator, a nebulizer, a light source (e.g., emitting visible light, UV light, X-rays, gamma rays, or ionizing radiation), or any combination of two or more of the foregoing.

[0191] 44. A device according to any one of 1 to 43, wherein the micropillars are foldable, meltable, mechanically retractable, dissolvable, mechanically peelable, mechanically bendable and / or electrically bendable.

[0192] 45. A device according to any one of 1 to 44, further comprising at least one instrument for analyzing the isolated DNA.

[0193] 46. ​​The device of 45, wherein the at least one instrument is selected from a microscope, a fluorescence microscope, an optical microscope, a microarray, a mass spectrometer (e.g., LC-MS, LC-MS / MS), PCR, real-time PCR, gel electrophoresis, capillary gel electrophoresis, and a sequencer (e.g., NGS, third generation sequencer).

[0194] 47. A device according to any one of 1 to 46, further comprising a robotic sample preparation platform and / or a plate reader.

[0195] 48. A device according to any one of 1 to 47, wherein the recovered genomic DNA comprises at least one DNA-binding protein.

[0196] 49. The device of 48, wherein the at least one DNA-binding protein comprises a histone, a transcription factor, a replication protein, a DNA repair protein, a DNA-modifying enzyme (e.g., DNA methyltransferase), and / or a nuclease.

[0197] 50. A system comprising at least one device described in any one of 1 to 49, further comprising at least one fluid control module suitable for connection to at least one inlet.

[0198] 51. The system of claim 50, wherein the fluid control module includes a pipette or a syringe.

[0199] 52. The system of 50, wherein the fluid control module comprises a pressurized air source (e.g., compressed air), a controlled pressure source, a controlled air pressure source, a pressure-driven pump, a syringe pump, a vacuum pump, or a peristaltic pump.

[0200] 53. The system of any one of 50-52, further comprising one or more collection reservoirs suitable for connection to an outlet, optionally wherein said one or more collection reservoirs comprise a sample collection reservoir and a waste collection reservoir.

[0201] 54. A system described in any one of 50 to 53, further comprising a controlled voltage source for electrophoretically driving DNA within the microfluidic channel.

[0202] 55. The system of any one of 50-54, further comprising at least one temperature control module, optionally wherein (a) said at least one temperature control module is capable of heating different portions of said device at different temperatures, and / or (b) said at least one temperature control module is capable of being controlled in a time-controlled manner (e.g., capable of heating for a certain duration).

[0203] 56. The system described in 55, wherein the at least one temperature control module is capable of setting the temperature of the device in the range of approximately 2°C to 100°C.

[0204] 57. A system according to any one of 50 to 56, wherein the system comprises at least two devices, and optionally, wherein the at least two devices are vertically stacked.

[0205] 58. A method for selecting parameters for extracting fragments of genomic DNA having desired size metrics from cells or cell nuclei, comprising: A sample containing at least one cell or cell nucleus is subjected to a process using a device according to any one of 1 to 49 or a system according to any one of 50 to 57. a concentration of each of the one or more enzymes mentioned above; one or more of the enzymes listed above; the buffer composition of the digestion buffer as described above; Digestion time; and Digestion temperature; processing at least once using parameters including: determining a fragment size metric for the collected sample; and based on said fragment size metric, selecting said parameters to be used if said fragment size metric corresponds to said desired size metric; if said fragment size metric is less than said desired size metric, decreasing said concentration, decreasing said number, decreasing said digestion time, changing said digestion temperature to reduce enzyme efficiency, changing said buffer composition to reduce enzyme efficiency, or a combination thereof, until said fragment size metric is no longer less than said desired size metric; and If the fragment size metric is greater than the desired size metric, increasing the concentration, increasing the number, increasing the digestion time, changing the digestion temperature to increase enzyme efficiency, changing the buffer composition to increase enzyme efficiency, or a combination thereof, until the fragment size metric is no longer greater than the desired size metric. A method comprising:

[0206] 59. The method of claim 58, wherein said fragment size metric is an average fragment size or a median fragment size.

[0207] 60. The method of 58 or 59, wherein said processing step comprises loading said sample into said device and washing at least one cell or cell nucleus in said device using said washing buffer.

[0208] 61. The method according to any one of 58 to 60, further comprising the step of inactivating said one or more enzymes.

[0209] 62. The method of claim 61, wherein said inactivation comprises heat inactivation.

[0210] 63. A method for isolating cells or cell nuclei from a sample, comprising the steps of: flowing said sample through a device according to any one of 1 to 49 or a system according to any one of 50 to 57; and harvesting said cells or cell nuclei captured in said device. A method comprising:

[0211] 64. The method of claim 63, further comprising the step of lysing the cell membrane or cell wall while leaving the nuclear membrane intact.

[0212] 65. The method according to 63 or 64, further comprising the step of washing the captured cells or cell nuclei.

[0213] 66. The method according to any one of 63 to 65, wherein said cell or cell nucleus is an animal, fungal, protist or plant cell, optionally a plant cell or cell nucleus.

[0214] 67. The method of any one of 63 to 66, wherein the sample is selected from blood, plasma, lymph, saliva, urine, an in vitro cell culture, and a tissue homogenate, and optionally, wherein the tissue homogenate is a plant tissue homogenate.

[0215] 68. The method of any one of 63 to 67, wherein said sample further comprises a blocking agent (e.g., BSA, an ionic detergent, another charged agent, PEG, or a PEO polymer).

[0216] 69. A method for isolating large size genomic DNA (e.g., intact gDNA, entire chromosomes, or fragments of gDNA greater than 500 kb), comprising processing a sample containing at least one cell or cell nucleus with a device described in any one of 1-49 or a system described in any one of 50-57, optionally wherein said device comprises micropillars that are foldable, meltable, mechanically retractable, dissolvable, mechanically peelable, mechanically bendable, and / or electrically bendable.

[0217] 70. A method for isolating fragments of genomic DNA having a selected size metric from at least one cell or cell nucleus, comprising: (a) processing a sample containing at least one cell or cell nucleus with a device according to any one of 1 to 49 or a system according to any one of 50 to 57; and (b) collecting the fragments of genomic DNA isolated by the process. A method comprising:

[0218] 71. The method according to claim 70, wherein the gDNA fragments are produced by digestion with a DNA-cutting enzyme, sonication, nebulization, alteration of flow conditions, optical methods, mechanical methods, or a combination of two or more of these methods.

[0219] 72. The method according to claim 70 or 71, wherein the method comprises using parameters selected by a method according to any one of claims 58 to 62.

[0220] 73. The method according to any one of 70 to 72, further comprising a method for isolating a cell or cell nucleus according to any one of 63 to 68.

[0221] 74. (a) washing the captured cells or cell nuclei prior to said lysis; (b) washing said genomic DNA prior to said digestion; and / or (c) inactivating the one or more enzymes (e.g., heat inactivation) 74. The method according to any one of claims 70 to 73, further comprising:

[0222] 75. A method according to any one of 70 to 74, wherein the genomic DNA fragment is in the range of about 1 kb to 500 kb.

[0223] 76. The method of any one of claims 70 to 75, wherein the fragment of genomic DNA is in the range of 200 kb, 30 kb, 25 kb, 14 kb, 10 kb, or 8 kb.

[0224] 77. A method according to any one of 70 to 76, wherein the genomic DNA is derived from a single cell or the nucleus of a single cell.

[0225] 78. A method according to any one of 70 to 77, wherein the step of collecting the fragments of genomic DNA comprises removing the fragments from a collection reservoir by manual pipetting or through tubing.

[0226] 79. A method according to any one of 70 to 78, wherein the step of collecting fragments of the genomic DNA comprises removing the fragments from the collection reservoir via an electrophoresis channel attached to the collection reservoir.

[0227] 80. The method of any one of 70 to 79, further comprising the steps of sequencing, amplifying, hybridizing, end-labeling, and / or barcoding the genomic DNA.

[0228] 81. The method according to any one of 70 to 80, wherein said cell or cell nucleus is an animal, fungal, protist or plant, optionally a human cell or cell nucleus.

[0229] 82. The method according to any one of 70 to 81, wherein the cell or cell nucleus is a cell or cell nucleus of a white blood cell or a T cell.

[0230] 83. The method of any one of 70 to 82, wherein the sample comprises blood, plasma, lymph, saliva, urine, semen, in vitro cell cultures, and / or tissue homogenates.

[0231] 84. A method according to any one of 70 to 83, wherein the sample comprises partially purified or fully purified cells or cell nuclei.

[0232] 85. A method for detecting a mutation in a genome, comprising: (a) isolating genomic DNA or a fragment thereof according to any one of the methods described in any one of 69 to 84; and (b) analyzing the recovered DNA, thereby detecting the mutation. A method comprising:

[0233] 86. The method according to claim 85, wherein said mutation comprises a substitution, deletion, insertion, duplication, DNA fusion, and / or chromosomal fusion.

[0234] 87. The method of claim 85 or 86, wherein the DNA is analyzed using one or more selected from gel electrophoresis, capillary electrophoresis, hybridization, PCR, real-time PCR, mass spectrometry (e.g., LC-MS, LC-MS / MS), Southern blotting, sequencing (e.g., NGS, third generation sequencing), microarray, and a combination of any two or more of these techniques.

[0235] 88. The method according to any one of 85 to 87, wherein said genomic DNA is animal, fungal, protist or plant, optionally human genomic DNA.

[0236] 89. The method of any one of 69 to 88, wherein the sample or buffer (e.g., lysis buffer, wash buffer, elution buffer, digestion buffer, etc.) comprises a blocking agent (e.g., BSA, an ionic detergent, another charged agent, PEG, or a PEO polymer).

[0237] 90. The device of any one of 1-49 or the system of any one of 50-57, wherein at least one of the channels comprises a blocking agent (e.g., BSA, an ionic surfactant, another charged agent, PEG, or a PEO polymer), optionally wherein the blocking agent coats the inner surface of the channel and / or the micropillars within the channel. EXAMPLES

[0238] The present disclosure will be further illustrated with reference to the following specific examples, which are offered for illustrative purposes and are not meant to limit the following disclosure or the claims.

[0239] Example 1: Fabrication of a microfluidic device To create the master mold, Microposit S1813 photoresist (Shipley; Marlborough, MA) is spun onto a silicon-on-insulator (SOI) wafer (Ultrasil; Hayward, CA) and exposed by UV contact lithography (EVG620, EVG Group; Albany, NY). The exposed resin resist is developed with 726MIF developer (Microchemicals) and the pattern is transferred to a 20 μm thick top silicon layer by the Bosch process in an Unaxis SLR 770 deep active ion etching system (Unaxis USA Inc.; St. Petersburg, FL). A monolayer of (1H,1H,2H,2H perfluorooctyl)trichlorosilane is evaporated onto the etched wafer in an MVD100 molecular wafer deposition system (Applied Microstructures; San Jose, CA) to prevent PDMS from sticking to the mold. Sylgard 184 (Dow Coming; Midland, MI) PDMS base resin is mixed with a curing agent in a 10:1 ratio, degassed under vacuum at room temperature, poured into the master mold, and cured at 150°C for 45 minutes. The cast elastomer is then peeled from the mold, and access holes to the inlets and outlets of the microchannels are created using a 1.5 mm biopsy punch (Sklar Instruments; West Chester, PA). To complete the channel fabrication, the patterned PDMS is treated with oxygen plasma for 1 minute and bonded to a 170 μm thick quartz glass wafer (Mark Optics; Santa Ana, CA).

[0240] Example 2: Exemplary protocol for extraction of 10 kb DNA 1. Preparation of cell samples. In order to be effectively loaded onto the device, any sample must be in solution. Preparation of two sample types is detailed below. Blood: A lancet is used to prick a human finger to obtain 1-2 drops of blood (approximately 100uL). The blood is then transferred to a tube with 500uL of 1x PBS containing 5mM EDTA and mixed gently. The cell solution is then stored on ice until loading onto the device. Animal cells (e.g., mammalian cells): Immortalized or primary animal cells (e.g., mammalian cells) growing in culture can be prepared using standard methods. Briefly, media is aspirated from the plate on which the cells are growing and the cells are washed once with 1×PBS. The cells are then treated with trypsin+EDTA to release the cells from the plate. The cells are then washed with media and spun down at 2,000 RPM for 2 minutes. The supernatant is removed and the cells are resuspended in media. The resuspended cells are then filtered using a 40 micrometer cell strainer and stored on ice until ready to load onto the device. Plant cells: Removal of plant compounds or secondary metabolites from crude plant preparations When sequencing plant DNA, nuclei must first be prepared from plant tissue. A method for releasing plant nuclei is often used that releases the approximate nuclei from the cell wall, but this method also contains undesirable plant compounds, including secondary metabolites such as starch and polyphenols. These particles are difficult to remove and are undesirable because they interfere with the sequencing reaction. The crude sample is loaded into a microfluidic device with micropillars, where the micropillars capture the nuclei while the much smaller plant compounds or secondary metabolites (such as starch or polyphenols) can evade the array. Due to the small particle size of these compounds, a micropillar spacing size of about 5 micrometers is ultimately used for effective separation. Thus, the plant nuclei are isolated / captured within the array and purified from the undesired plant compounds. 2. Preparation of the microfluidic device. The microfluidic device (made of PDMS) is first prepared prior to cell loading. The device is briefly plasma cleaned for 1-3 min, then backloaded with 100% ethanol to fill the entire channel. The input ports are then connected with the pressure and solution delivery mechanisms. In this case, the instrument is plugged directly into the device, which contains a space that holds the volume of solution and is plugged with a pressure valve, regulator, and hose connected to a tank. 3. Cell loading. Optimal loading is achieved when the micropillar array is filled with enough cells to obtain the desired gDNA yield without overloading. Cells are loaded for a few seconds to a few minutes at 0.1-0.5 psi depending on cell concentration. After loading is complete, replace the solution from the instrument by pipetting 1x PBS for a few minutes or by flowing at 0.1-0.5 psi. At this point, only cells are captured in the device - all other components of the medium or any other particles not captured in the array are washed out to the exhaust port. 4. Lysis of the cells. To lyse the cells, exchange the solution in the device for a suitable lysis buffer (in this case 1% SDS in 1x PBS (any other chemical lysis buffer would work)) for 5 minutes under 0.1-0.5 psi pressure. Lysis occurs immediately, but lysis is allowed to continue for several minutes to ensure complete lysis. The buffer in the device is then flushed out and exchanged for 1x PBS, and the device is then washed in this solution for 5 minutes at 0.1-0.5 psi. 5. gDNA Fragmentation and Release. To release the gDNA captured on the micropillars with a fragment length of 10 kb, the following buffer is used: 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2 (containing 1 mM DTT). To 200 uL of this buffer, the following enzymes are added: 0.2 U EcoRV, 0.2 U EcoRI, 0.2 U HindIII, and 0.2 U SeaI. The buffer is exchanged in the instrument and run at 0.3-0.4 psi for 15 minutes at 27°C. After 15 minutes, the pressure is increased to 1.6 psi for 5-10 minutes until all the solution has passed through the device. The gDNA is then collected from the tip and heated at 80°C for 15 minutes to inactivate the enzymes, then cooled on ice and stored at 4°C. 6. Analysis of gDNA. Samples are first quantified using Qubit to determine yield and concentration. If necessary, samples are then diluted to 400ng / uL for fragment size analysis using a Femto Pulse pulsed-field capillary electrophoresis system to confirm fragmentation quality and size. Specific examples are outlined below that demonstrate how the above variables can be varied in different combinations to produce gDNA fragments of various sizes. All of the data below was generated using cultured HeLa cells and a device with the following parameters and dimensions: 1 inlet and 4 outlets for each device Array area of ​​4 mm wide x 3.6 mm long for each channel in each device Spacing between micropillars (center-to-center) within the array: continuous gradient from 33.3 micrometers to 18 micrometers Micropillar diameter: 6.3 micrometers Micropillar height: 18-20 micrometers

[0241] Example 3 (Figure 18): Fragment size adjustment using type II restriction enzymes: 8 kb range. To recover gDNA with a main peak at approximately 8 kb, a reaction mixture was prepared containing: Four type II restriction endonucleases (final 0.8-1.2U / uL) The buffer solution contains: -Tris-HCl (10 mM) -NaCl (50 mM) -MgCl2 (10 mM) -DTT (1 mM) Buffer solutions were introduced into the microfluidic channels through the instrument by pipetting or a syringe pump. The device was set to a controlled reaction temperature (27°C). The reaction was allowed to proceed under a flow pressure of 0.3 psi for 15-20 min, then at 1.3-1.8 psi for 5-15 min.

[0242] Example 4 (Figure 19): Fragment size adjustment using type II restriction enzymes: 10 kb range To recover gDNA with a main peak around 10 kb, a reaction mixture was prepared containing: Four type II restriction endonucleases (final 0.7-0.9U / uL) The buffer solution contains: -Tris-HCl (10 mM) -NaCl (50 mM) -MgCl2 (10 mM) -DTT (1 mM) Buffer solutions were introduced into the microfluidic channels through the instrument by pipetting or a syringe pump. The device was set to a controlled reaction temperature (27°C). The reaction was allowed to proceed under a flow pressure of 0.3 psi for 15-20 min, then at 1.3-1.8 psi for 5-15 min.

[0243] Example 5 (Figure 20): Fragment size adjustment using type II restriction enzymes: 14 kb range To recover gDNA with a main peak around 14 kb, a reaction mixture was prepared containing: Four type II restriction endonucleases (final 0.6-0.7U / uL) The buffer solution contains: -Tris-HCl (10 mM) -NaCl (50 mM) -MgCl2 (10 mM) -DTT (1 mM) Buffer solutions were introduced into the microfluidic channels through the instrument by pipetting or a syringe pump. The device was set to a controlled reaction temperature (27°C). The reaction was allowed to proceed under a flow pressure of 0.3 psi for 15-20 min, then at 1.3-1.8 psi for 5-15 min.

[0244] Example 6 (Figure 21): Fragment size adjustment using type II restriction enzymes: 30 kb range To recover gDNA fragments ranging from 1 to 165 kb with a main peak around 25-30 kb, a reaction mixture was prepared containing: 〇One type II restriction enzyme (final 0.8U / uL) The buffer solution contains: -Tris-HCl (10 mM) -NaCl (50 mM) -MgCl2 (10 mM) -DTT (1 mM) Buffer solutions were introduced into the microfluidic channels through the instrument by pipetting or a syringe pump. The device was set to a controlled reaction temperature (27° C.). The reaction was allowed to proceed under flow pressures of 0.35-0.4 psi for 15 min, then at 1.6 psi for 5 min.

[0245] Example 7 (Figure 22): Fragment size adjustment using type II restriction enzymes: 200 kb range To recover a gDNA fragment with a main peak around 200 kb, a reaction mixture containing the following was prepared: 〇One type II restriction enzyme (final 0.2U / uL) The buffer solution contains: -Tris-HCl (10 mM) -NaCl (50 mM) -MgCl2 (10 mM) -DTT (1 mM) Buffer solutions were introduced into the microfluidic channels through the instrument by pipetting or a syringe pump. The device was set to a controlled reaction temperature (27° C.). The reaction was allowed to proceed under a flow pressure of 0.2 psi for 15 min, then 0.3 psi for 5 min, then 1.6 psi for 15 min. A number of the more possible fragment sizes, ranging from a few hundred base pairs to several Mb base pairs, can be accommodated by adjusting the above parameters.

[0246] Example 8: Analysis of recovered DNA (real-time PCR) A typical protocol involves: the recovered gDNA is diluted 1:20 and 2pl is used in a real-time PCR with forward and reverse PCR primers hybridizing to a specific locus. The gDNA fragment is added to a master mix containing 10ul of 2x PowerSYBR Green Master Mix (Applied Biosystem), 1ul of 10uM primer mix, and 7ul of water. The reaction is incubated at 95°C for 10 minutes in an Applied Biosystems 7500 real-time PCR system, followed by 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 32 seconds. A dissociation step / melting curve analysis is then performed. When analyzed, gDNA fragments recovered using the disclosed devices, systems, and / or methods surprisingly outperformed traditionally prepared (e.g., CsCl purified) gDNA fragments in identical real-time PCR reactions.

[0247] Example 9: Amplification of gDNA We have demonstrated successful on-chip and off-chip DNA amplification. For on-chip, we demonstrated isothermal amplification using multiple displacement amplification (MDA) (see Tian et al. (2018) PLOS ONE 13(2):e0191520, incorporated herein by reference). We then successfully collected the amplified DNA and performed whole-exome sequencing on an Illumina sequencer as a demonstration of the method's coverage, also showing a smaller amplification bias compared to in-tube MDA (see also Example 10). In the off-chip case, we successfully collected purified DNA from the chip device after extraction and DNA fragmentation / cleavage. We demonstrated that we could take DNA samples, then amplify the DNA with PCR and qPCR, and then successfully sequence the samples on an Illumina sequencer. A combination of on-chip amplification (either PCR or isothermal) and off-chip amplification (either PCR or isothermal) is also possible.

[0248] Example 10: Analysis of recovered DNA (Next Generation Sequencing) The recovered gDNA fragments were sequenced using next-generation sequencing (NGS) according to the manufacturer's instructions (Illumina, San Diego, CA). The results were surprisingly and unexpectedly superior to sequencing performed using traditionally prepared (e.g., CsCl-purified) gDNA fragments.

[0249] Example 11: Exemplary Devices Exemplary devices that were fabricated are shown in the figures provided herein (e.g., Figures 3, 5, 23(A and B), 24, and 25-31). Further explanation of selected designs is provided below. One aspect / objective of the various designs is to improve the loading of samples (samples containing at least one cell or cell nucleus). Improved sample loading includes better spreading, so that cells or nuclei loaded into the channel can be constrained / trapped in the micro-pillar array far enough apart from each other to reduce / minimize the effect of clogging the fluid flow. The following devices are designed for better sample loading. While any lattice of micropillars (including a random lattice) will work for extracting gDNA or fragments thereof, square and hexagonal lattices in particular provide better sample loading. For the gradients described below (both smooth and step gradients), various numbers of gradients were tested, in particular step gradients with only two steps (two steps in total, each with a different spacing, excluding the adjacent micropillar array regions), and gradients with as many as 30 steps were tested. FIG. 25 shows a device having at least one channel with square lattice micropillars, where the pillars are arranged in such a way that they have distinct rows and columns spaced apart from each other at a specified distance. Square lattice refers to a lattice of micropillars where each gap (between pillars) is aligned with the next adjacent gap, in the case of a micropillar array region, which means that the spacing is uniform. In other words, each pillar is aligned with the next pillar. The device incorporated a smooth gradient of different pillar spacings in a square lattice configuration. The device also incorporated a smaller range of pillar spacings as a gradient. For higher molecular weight genomic DNA of interest, a gradient with reduced micropillar spacing near the outlet may be desirable, as such a gradient can better spread out the cells within the sample and away from other smaller particles in the sample. Figure 26 shows a device with a square lattice micropillar array that contains a step gradient in the micropillar spacing, where rather than the spacing transition being smooth from the top to the bottom of the micropillar array, the spacing is held uniformly for one period and then changed to the next period. Figure 27 shows a device with a hexagonal lattice micropillar array. Hexagonal lattice refers to a lattice of micropillars arranged such that each gap is crossed by a pillar. In other words, the pillars and gaps are alternated to maximize the possibility or probability that any particle flowing through the micropillar array will encounter an object (e.g., the next micropillar adjacent and / or downstream). The gradient in pillar spacing can be either smooth or stepped. FIG. 28 shows a device incorporating intermixed square and hexagonal lattice regions. FIG. 29 shows a device incorporating an array of shorter micropillars compared to the devices of FIGS. 25-28. The shorter length of the micropillar array may improve the yield of DNA / amount of DNA recovered from the microfluidic chip cartridge, and the area in the channel not occupied by the shorter micropillar array may include certain structures to prevent the chip cartridge from collapsing, which may be necessary for devices made with flexible materials such as PDMS. However, if structural integrity is not a consideration in a particular device (e.g., made with plastic or metal), such areas may be empty (i.e., lacking structures or micropillars). As used herein, the area not occupied by the shorter micropillar array is referred to as a "structural area" even if the area is empty and lacks any structures. Figure 30 shows a device incorporating mixed micropillar regions with non-micropillar / blank / support structure regions. Here, two micropillar array regions were separated by a distance of support structure. With the two micropillar arrays pulled apart, smaller cells or nuclei are further spaced apart from each other, which may improve sample loading by reducing / minimizing inhibition of fluid flow from heavily aggregated / roped DNA during sample lysis and DNA extraction steps. Figure 31 shows three micropillar array regions separated by support structures. The three mixed micropillar array regions may perform better than the two mixed micropillar array regions of Figure 30. Additionally, having more repeating micropillar array regions may result in better final DNA yields collected from the chip cartridge after DNA shearing.

[0250] While various embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like are possible without departing from the spirit of the disclosure, and therefore are deemed to be within the scope of the disclosure as defined in the following claims.

[0251] Incorporation by Reference All U.S. patents and U.S. and PCT patent application publications referred to herein are incorporated by reference in their entirety as if each individual patent or patent application publication was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application, including any definitions herein, shall control.

[0252] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A microfluidic flow-based device for extracting fragments of genomic DNA of a selected size range from cells or cell nuclei, comprising: a microfluidic channel having an inlet and an outlet that allows flow in a flow direction from the inlet to the outlet; and an array of micropillars disposed within the microfluidic channel; where: the diameter of the micropillars is between 3 and 50 micrometers; the micropillars are separated from one another by a spacing between 2 micrometers and 150 micrometers; the array is bounded within an area of ​​between 1 square millimeter and 1600 square centimeters; the height of the micropillars is between 1 and 200 micrometers; and A device wherein the micropillars are arranged in the array in a defined configuration.

2. 10. The device of claim 1, wherein the micropillars have a shape that is circular, oval, square, triangular, rectangular, cross, hexagonal, diamond, polygonal, dome-shaped, pyramidal, or any combination of two or more thereof.

3. The channel (a) random orientation; (b) square lattice; (c) a hexagonal lattice; or (d) any combination of two or more thereof. The device of claim 1 , comprising:

4. 10. The device of claim 1, wherein the micropillars are arranged in (a) a smooth gradient, (b) a step gradient, or (c) a variable spacing gradient comprising a combination of (a) and (b).

5. 10. The device of claim 1, wherein the micropillars are separated from one another by a spacing in the range of about 2 to 30 micrometers, optionally at a spacing equal to or less than 5 micrometers.

6. the device comprises at least 2, 6, 8, 12, 24, 48, 96, 384, or 1536 channels, optionally wherein: (a) the channels are in a staggered or side-by-side configuration in the device; and / or 10. The device of claim 1, wherein (b) the channel is an ordered configuration that is aligned with a multiwell plate configuration.

7. 10. The device of claim 1, further comprising a digestion buffer, one or more enzymes, a DNA labeling agent, a hybridization agent, and / or a wash buffer, wherein the one or more enzymes are selected from DNA cleaving enzymes, DNA modifying enzymes, and enzymes that modify DNA-associated proteins, restriction enzymes, endonucleases, homing endonucleases, transposases, Type II CRISPR-Cas9 proteins, and dsDNA fragmentases.

8. 10. The device of claim 1, further comprising at least one instrument that facilitates DNA cleavage and / or release of DNA from the channel, optionally wherein the at least one instrument is selected from a sonicator, a nebulizer, a visible light source, a UV light source, an X-ray source, a gamma ray source, or an ionizing radiation source, or any combination of two or more thereof.

9. 1. A method for extracting fragments of genomic DNA having desired size metrics from cells or cell nuclei, comprising: A sample containing cells or cell nuclei is subjected to a device according to any one of claims 7 to 8, the concentration of each of the one or more enzymes; the number of said one or more enzymes; the buffer composition of the digestion buffer; Digestion time; and Digestion temperature; collecting a sample containing fragments of genomic DNA by processing the sample using a set of parameters comprising: determining a fragment size metric for said collected sample; comparing the fragment size metric of the collected sample with the desired size metric; and if the fragment size metric of the collected sample does not have the desired size metric, modifying the set of parameters to modify the fragment size metric of the collected sample. A method comprising:

10. If the fragment size metric of the collected sample is smaller than the desired size metric, changing the set of parameters to: reducing said concentration; Reducing said number; reducing the digestion time; changing the digestion temperature to reduce the efficiency of the enzyme; or Altering the buffer composition to reduce enzyme efficiency 10. The method of claim 9, wherein the collected sample is modified by one or more of:

11. If the fragment size metric of the collected sample is greater than the desired size metric, then changing the set of parameters to: increasing said concentration; Increasing said number; increasing the digestion time; Varying the digestion temperature to increase enzyme efficiency, or Varying the buffer composition to increase enzyme efficiency 10. The method of claim 9, wherein the collected sample is modified by one or more of:

12. The method of claim 9 , wherein the fragment size metric is an average fragment size or a median fragment size.

13. 10. The method of claim 9, further comprising the step of inactivating the one or more enzymes.

14. 1. A method for isolating fragments of genomic DNA having selected size metrics from at least one cell or cell nucleus, comprising: (a) processing a sample containing at least one cell or cell nucleus with a device according to any one of claims 1 to 8; and (b) collecting the fragments of genomic DNA isolated by the process. A method comprising:

15. The method of claim 14, wherein the fragments of gDNA are produced by digestion with a DNA-cleaving enzyme, sonication, nebulization, alteration of flow conditions, optical methods, mechanical methods, or a combination of two or more thereof.

16. 15. The method of claim 14, wherein the method comprises using parameters selected by the method of claim 9.

17. (a) washing the captured cells or the cell nuclei prior to said lysis; (b) washing the genomic DNA prior to digestion; and / or (c) inactivating the one or more enzymes 15. The method of claim 14, further comprising:

18. The method of claim 14, wherein the fragments of genomic DNA are in the range of about 1 kb to 500 kb, optionally wherein the fragments of genomic DNA are greater than or equal to 200 kb, 30 kb, 25 kb, 14 kb, 10 kb, or 8 kb.

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