Devices and methods related to collection of cell-free DNA

Abstract

The present disclosure provides methods, compositions, and systems related to the collection of DNA. The method is especially advantageous for collecting cell-free DNA (cfDNA) separately from cell associated DNA including gDNA and other DNA inside cells. In particular, the present disclosure provides methods, compositions, and systems related to the collection of cfDNA from a sample for either commercial or clinical purposes in a manner that facilitates consistent, convenient, and reproducible collection of stable cfDNA from large sample volumes. Additionally, the method is designed to facilitate preparation of a sample enriched in trans-renal DNA representing internal physiology and pathophysiology of a subject.

Description

AIGN-43658.601DEVICES AND METHODS RELATED TO COLLECTION OF CELL-FREE DNARELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 690,142 filed September 3, 2024, and U.S. Provisional Patent Application No. 63 / 711,799 filed October 25, 2024, both of which are incorporated herein by reference in their entireties and for all purposes.FIELD

[0002] The present disclosure provides methods, compositions, and systems related to the collection of cell-free DNA (cfDNA). In particular, the present disclosure provides methods, compositions, and systems related to the collection of cfDNA from a sample for both commercial and clinical purposes in a manner that facilitates consistent, convenient, and reproducible collection of stable cfDNA from large sample volumes or diluted samples. Additionally, the methods disclosed herein are useful for separately collecting trans-renal DNA (trDNA) or other non-nucleosomal DNA, genomic DNA (gDNA) and nucleosomal DNA. The methods facilitate immediate, on-site collection and stabilization of the DNA by a patient or a not previously trained layperson before transport to a lab.BACKGROUND

[0003] Cell-free DNA (cfDNA) arises from apoptotic or necrotic cells, and from the active secretion of DNA fragments into the bloodstream or body fluids from normal or abnormal, native or transplanted cells. Additionally, cfDNA can arise from pathogenic sources such as tumors, viruses, bacteria, mycoplasma, and other microorganisms present within the body. Generally speaking, cfDNA is obtained from blood plasma, which has been considered the most common type of diagnostic sample from which to obtain cfDNA. cfDNA is also passed from the blood through the kidneys into the urine, providing another source of cfDNA for diagnostics. It is also present in all other body fluids tested to date. Blood samples ty pically are collected by venipuncture and immediately centrifuged for plasma separation, typically requiring sequential spins, skilled technicians, and artful separation of plasma. Plasma is then immediately processed or more ty pically frozen and transported for purification. In contrast, collection of cfDNA from other body fluids, such as urine, saliva, and semen, can be performed at home without trained healthcare personnel, without centrifugation or use of other instruments, and is completely non-invasive, which offers a more convenient and comfortable way to collect cfDNA.AIGN-43658.601

[0004] Currently, if subjects need cfDNA analysis performed, plasma samples (generally frozen or preserved immediately) or urine samples (also frozen immediately or conditioned with DNA stabilizing reagents) are shipped directly to a lab for testing. Shipping frozen plasma or urine samples both require overnight shipping on dry7ice (approx. -70°C or lower) or expensive preservative tubes. When used with DNA stabilizers, at least a portion of the DNA in the blood or urine remains stable at room temperature for up to seven days, allowing for limited travel and shipping time without the need for temperature-controlled shipping. However, cfDNA is composed of two fractions. The largest fraction is stabilized by nucleosomes, this fraction comes from gDNA that is not being actively transcribed, and a separate fraction from actively transcribed gDNA, such as oncogenes, which is loosely bound by transcription factors and not protected by nucleosomes. The stability of the latter cfDNA, which is especially important for understanding pathophysiology, has not been established and cannot be estimated from the stability' of nucleosomal DNA. Methods of preserving, transporting and isolation of the latter are especially important, but not available.|0005] Once the standard liquid samples are shipped to a lab for processing, cfDNA extraction and purification may be performed via commercially available kits (e g., Qiagen MinElute ccfDNA). These kits have a maximum sample processing volume of 4-40mL for urine kits and 0.24-10mL for plasma kits. Some may allow' 20 mis. However, there is about 0. 12 to 60.24 ng / mL cfDNA in urine compared to 65-877ng / mL cfDNA in plasma, so for the same amount of cfDNA, typically more urine volume is needed. Urine averages approximately 200-400mL per void, with a median void of 220mL, making the current commercially available urine cfDNA extraction kits (max 40mL) insufficient for the average volume of a single void. To process a full void with commercially available kits, one w ould need to use multiple preps, thereby increasing cost of materials and labor.

[0006] Additionally, currently available methods typically involve immediately disrupting nucleosomes, and binding all DNA, including nucleosomal cfDNA, to silica in the presence of chaotropic salts that are toxic and require specialized, expensive, disposal. Release and collection of the cfDNA requires multiple separation steps using centrifugation and / or magnetic particle capture, they take 90 -120 minutes. Some can be automated on expensive machines. They are optimized for the ~166bp nucleosomal DNA and many methods have only a small fraction of, or may miss entirely, the smaller non-nucleosomal DNA from the actively transcribed regions.AIGN-43658.601SUMMARY

[0007] Embodiments of the present disclosure include a method for obtaining cell-free DNA (cfDNA) and especially trans-renal cell free DNA (trDNA) and cell enclosed DNA (genomic or gDNA) from a sample. In accordance with these embodiments, passing the sample through a filter cartridge comprising at least one filter, wherein the sample comprises a pH < 5, and wherein cfDNA present in the sample binds the at least one filter. In some embodiments, the method includes adjusting the pH of the sample obtained from a subject using a DNA binding composition and passing the pH-adjusted sample through at least one filter cartridge comprising at least one filter per cartridge. In some embodiments, the cfDNA from the sample binds the at least one filter, and the sample flowthrough is removed or discarded. In other embodiments the flowthrough immediately, or subsequently, passes through a second, or additional downstream, filter cartridge(s). In some embodiments, the filter's physical properties and / or chemical properties trap or bind specific components allowing stabilization, transport, and / or purification.

[0008] In some embodiments, the method comprises adjusting the pH of the sample to a pH of < 5 prior to passing the sample through the filter cartridge. In some embodiments, the method comprises adjusting the pH of the sample to a pH of < 2 prior to passing the sample through the filter cartridge. In some embodiments, the pH of the sample is adjusted using a DNA binding composition. In some embodiments, the DNA binding composition is formulated to adjust the pH of the sample to a pH of < 5. In some embodiments, the DNA binding composition comprises one or more of acidic forms of ascorbate, acetate, citrate, borate, formate, maleate, tartrate, lactate, glycine, potassium hydrogen phthalate buffer and / or MES buffer. In some embodiments, the DNA binding composition comprises a DNA stabilization agent. In some embodiments, the DNA stabilization agent comprises EDTA. In some embodiments, the DNA binding composition is formulated as a powder or a solution.|0009] In some embodiments, the sample comprises one or more bodily fluids from the subject selected from a set including urine, whole blood, blood plasma, serum, ascites, saliva, lymph, pleural effusion, semen, sweat, tears, and cerebral spinal fluid. In some embodiments, the sample comprises water from the natural environment, or water or other liquid from a manmade or industrial water source.

[0010] In some embodiments, the sample comprises a volume from about 1-10 mL, in other embodiments, the sample comprises 20mL to about lOOOmL. In some embodiments, the pH- adjusted sample is passed through the filter cartridge at a single time point or separately overAIGN-43658.601 multiple time points. In some embodiments, the sample comprises a volume from about 20 mL to about 1000 mL, and the sample is passed through the filter cartridge immediately after the sample is obtained and / or pH adjusted. In some embodiments, the filter cartridge or set of cartridges is reused for additional samples.10011] In some embodiments, the cfDNA comprises circulating tumor DNA (ctDNA), trans-renal DNA (trDNA), cell-free fetal DNA (cffDNA), cell-free mitochondrial DNA (ccf mtDNA). DNA from a micro-organism or infectious organism, donor-derived cell-free DNA (dd-cfDNA), and / or other trans-renal DNA (trDNA). In some embodiments, the cfDNA and / or trDNA obtained using the methods and systems of the present disclosure comprises DNA from normal cells (e.g., non-pathologic cells) and / or DNA that is not associated with a pathologic disease or condition (e.g., non-mutated DNA). including but not limited to, DNA from normal cells that is present in altered concentrations and / or ratios as compared to a control or reference sample (e.g., DNA from a healthy or control subject, or a prior sample from the same patient). In some embodiments, the DNA is from diseased cells, or cells causing disease, including normal body components, altered components, such as cancer or damaged cells, transplanted organs, or infectious agents.

[0012] In some embodiments, the DNA binding composition is formulated to adjust the pH of the sample to a pH of < 5. In some embodiments, the DNA binding composition is formulated to adjust the pH of the sample to a pH of < 2. In some embodiments, the DNA binding composition comprises an acidifying agent to adjust the pH of the sample to a pH of < 5. In some embodiments, an acidifying agent is added to the DNA binding composition prior to adding the sample. In some embodiments, an acidifying agent is added to the DNA binding composition after adding the sample. In some embodiments, the DNA binding composition comprises an acidifying agent. In some embodiments, the DNA binding composition comprises acid or an acidic buffer with one or more of the acidic forms of ascorbate, acetate, citrate, borate, formate, maleate, tartrate, lactate, glycine, potassium hydrogen phthalate buffer and / or MES buffer.

[0013] In embodiments, seeking to isolate only trans-renal DNA (trDNA), urine is buffered to a pH of about 3 to about 6.5. and in some embodiments, to a pH of about 4 to about 5. In some embodiments, when seeking to disrupt nucleosomes, a lower pH is used; in this situation bladder cfDNA is also freed to be able to bind to the capture material.

[0014] In some embodiments, the DNA binding composition comprises a DNA stabilization agent. In some embodiments, the DNA stabilization agent comprises EDTA.AIGN-43658.601

[0015] In some embodiments, the DNA binding composition is formulated as a powder or a solution. In some embodiments, the DNA binding composition is encased in a material that rapidly dissolves in the biologic sample.

[0016] In some embodiments, the at least one fdter in the fdter cartridge comprises a DNA- binding fdter. In some embodiments, the at least one fdter in the fdter cartridge comprises a positively charged polyelectrolyte. In some embodiments, the positively charged polyelectrolyte comprises chitosan, or a variant or derivative thereof. In some embodiments, the at least one fdter comprises chitosan with a DNA binding capacity ranging from about 1 ng to about 1000 mg. In some embodiments, the at least one fdter comprises a DNA binding capacity ranging from about 1 ug to about 100 ug. In some embodiments, the at least one fdter comprises a pore size ranging from about 0. 1 pm to about 10.0pm. In some embodiments, the pore size is from 0.25 to 10pm. In some embodiments, the at least one fdter comprises polyacrylonitrile, glass fiber, and / or poly ethersulfone.

[0017] In some embodiments, the at least one fdter comprises a pre-filter. In some embodiments, the pre-filter comprises a pore size ranging from about 0.2pm to about 10pm. In some embodiments, the pre-filter comprises polyacrylonitrile, glass fiber, and / or poly ethersulfone.

[0018] In some embodiments, the at least one fdter comprises a DNA-binding fdter and a pre-filter. In some embodiments, the DNA-binding fdter and the pre-filter are stacked inside a single cartridge. In some embodiments, the flowthrough from a first fdter cartridge immediately passes through a second. In some embodiments, the second has a smaller area. In some embodiments, the second cartridge is easily removed so DNA can be retrieved, from either cartridge, without disassembly of the cartridge. In some embodiments, the DNA-binding fdter is contained in one cartridge and the pre-filter contained in a separate cartridge. In some embodiments, the DNA-binding fdter cartridge and the pre-filter cartridge are stacked. In some embodiments, the sample is passed through a pre-filter before being passed through the at least one fdter in the fdter cartridge.

[0019] In some embodiments, the at least one fdter in the one of the fdter cartridges comprises a digestive enzyme. In some embodiments, the digestive enzyme is a protease. In some embodiments, it is proteinase K or papain.

[0020] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the sample is obtained from the human, and wherein the sample comprises urine from one or more voids. In some embodiments, the one or more voidsAIGN-43658.601 are passed through the filter cartridge by the human subsequent to urination. In some embodiments, the at least one filter in the filter cartridge comprises a positively charged polyelectrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 1 minute to about 5 minutes from the time the subject voids. In some embodiments, the human is experiencing or has experienced one or more of the following: cancer, transplanted organ, pregnancy, physical exercise training, heart attacks or other cardiovascular conditions, stroke, neurologic disease, Alzheimer's Disease, psychiatric conditions, autoimmune disease, sepsis, inflammatory disease and / or urologic disease or is at risk for one of these diseases or is being tested to rule in or rule out the presence of one of these diseases.

[0021] In some embodiments, the human sends the filter cartridge to a lab for processing. In some embodiments, the human passes a wash buffer and / or a stabilization buffer (e.g., or water-based solution or alcohol-based solution) through the at least one filter in the filter cartridge before sending the cartridge to a lab for processing. In some embodiments, the filter cartridge is stored for no more than about 7 days prior to processing. In some embodiments, the filter cartridge is transported / stored for about 8 hours, about 16 hours, about 32 hours, about 64 hours, about 128 hours, or about 1 week prior to processing. In some embodiments, the filter is dried and stored before all or part of it is processed.

[0022] In some embodiments, the method further comprises processing the filter cartridge to obtain the cfDNA. In some embodiments, the processing comprises passing a wash buffer or a stabilization buffer through the at least one filter in the filter cartridge. In some embodiments, the wash buffer and / or the stabilization buffer comprises a pH of about 7 or below. In some embodiments, the wash buffer and / or the stabilization buffer comprises a pH of about 6 or below. In some embodiments, the wash buffer contains physiologic or hypertonic salt and / or detergents.

[0023] In some embodiments, the method further comprises processing the filter cartridge to obtain the cfDNA. In some embodiments, the processing comprises passing a DNA release buffer (e.g., an elution buffer) or a stabilization buffer through the at least one filter in the filter cartridge. In some embodiments, the processing comprises treating the one or more filters with a protease.

[0024] In some embodiments, the processing comprises eluting the cfDNA from the one or more filters. In some embodiments, the processing comprises eluting the cfDNA from the one or more filters using a buffer having a pH from about 6 to about 11. In some embodiments, the cfDNA is further purified and / or concentrated.AIGN-43658.601

[0025] In some embodiments, the processing is performed at a lab. In some embodiments, the method further comprises assessing the cfDNA for one or more biomarker sequences or types of sequencing.

[0026] In some embodiments, the filters are stacked in a cartridge. In some embodiments, the filter stack, in the filter cartridge, contains multiple filters, including filters designed to bind unwanted component (e.g., cellular debris and / or contaminants), filters designed to bind cells that are a source of gDNA (e.g., gDNA from the urogenital tract), proteases, or other enzymes, and as well as a filter to bind cfDNA. In some embodiments, these filters are housed in separate filter cartridges.

[0027] In some embodiments, the cell-binding filter is processed in a filter cartridge with flow generated by a hand-held or mechanical syringe or pump, or by standard lab techniques, to obtain gDNA (e.g., prevent or reduce gDNA contamination).|0028] In some embodiments, the filter cartridge is opened to remove one or more filters, and / or to add reagents such as a protease, or a filter containing protease, before the filter cartridge is closed again and the filters are further processed with a syringe.

[0029] In some embodiments, all processing and separation steps are done in the filter cartridge with simple hand pumping of the needed reagents.

[0030] In some embodiments, filter cartridges are placed in a manifold for simultaneous processing by manual or automatic pumping mechanisms.

[0031] In some embodiments, one or more of the filters are processed with standard lab techniques and methods, including but not limited to, adding reagents, centrifugation, or air pressure with devices designed to separate liquids from the filter.

[0032] In some embodiments, one or more voids of urine are sequentially passed through the same filter cartridge and filter(s).

[0033] In some embodiments, the flowthrough comprises histone-bound DNA. In some embodiments, histone-bound DNA in the flowthrough is further processed and / or assessed for one or more nucleosomal cfDNA or other biomarkers. In some embodiments, both materials from the further processed fl owth rough and that captured on the prefilter as well as the cfDNA binding filter are preserved and tested.

[0034] In some embodiments, the flowthrough from the first cartridge is adjusted, by change in pH, salt, detergent or other buffer properties before flowing through the second filter.

[0035] Embodiments of the present disclosure also include methods for selectively obtaining urothelial cell-free DNA (cfDNA) from a urine sample from a subject. In accordance with these embodiments, the method includes buffering the urine sample to a pH at or betweenAIGN-43658.601 a pH of 0.5 and a pH of 3, and passing the buffered urine sample through a filter cartridge comprising at least one filter, thereby selectively binding the cfDNA present in the urine sample to the at least one filter.

[0036] Embodiments of the present disclosure also include methods for selectively obtaining trans -renal DNA (trDNA) from a urine sample from a subject. In accordance with these embodiments, the method includes buffering the urine sample to a pH at or between a pH of 3 and a pH of 6. and passing the buffered urine sample through a filter cartridge comprising at least one filter, thereby selectively binding the trDNA present in the urine sample to the at least one filter.

[0037] Other embodiments and embodiments of the disclosure will be apparent in light of the following detailed description and related figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIGS. 1A-1C: Representative schematic diagram of affinity' capture of DNA (FIGS. 1A-1B). FIG. 1A illustrates that blood nucleosomal cfDNA does not penetrate into the urine and any nucleosomes in the urine are from bladder or elsewhere in the urogenital tract. IB illustrates mixing of buffer and pH adjustment occurs during the voiding process and as urine is poured into 250 ml syringe. Pushing urine through the filter stack comprising in this case, a prefilter to capture cells and their gDNA followed by a chitosan coated filter takes about 1 minute of easily applied mild pressure. At this point, a person would seal the used filter holder, typically in a plastic bag and mail it to a lab for processing. The methods of the present disclosure are particularly useful for isolating trans-renal DNA from urine (FIG. 1A &1B). Since this trans-renal DNA is the DNA representative of physiologic and / or disease processes in the body, enriching for it is critical for interrogation of internal body processes. In contrast, as shown in FIG. 1A & IB, the nucleosomal DNA in the blood is excluded from the ureter. In the urine any' nucleosomal DNA, or any DNA >1 OObp, comes from the bladder. The glomerular filtration barrier is about 5nm so even nucleosomal DNA, as well as complete nucleosomes (l lnm) do not pass.

[0039] Representative data from DNA capture experiments (FIG. 1C). Unmodified membranes show no effective capture of DNA. The chitosan modified membranes show DNA capture of 73.2%. A person at a distant site might open the cartridge and send just one or more of filters to the lab for processing. In the embodiments using multiple cartridges only the second, typically’ smaller cartridge containing the cfDNA might be sent. In a particular embodiment, the entire complex of filter cartridges is sent to a lab for processing. At the lab,AIGN-43658.601 the prefilter can be used to obtain bladder cells and their gDNA. while the polyelectrolyte modified cfDNA capture filter is used to obtain a sample enriched in trans-renal DNA. If bladder cfDNA is desired, one of many methods, but typically very low pH, is used to dissociate the nucleosomes and allow capture of the nucleosomal DNA.10040] FIGS. 2A-2B: Effect of pore size on maximum urine volume (FIG. 2 A) and DNA yield (FIG. 2B) from various filter combinations. Urine was filtered through the pre-filter and the chitosan filter in succession (filters were not physically stacked). Bars (Mean+SD) are for the filter combinations with replicates. DNA was quantified by AccuBlue HS dsDNA kit.

[0041] The optimal combination yielded 11.5 ug of cfDNA from 330 mis of urine from a female volunteer. Specimens from women have more cells and thus are harder to process.|0042] FIG. 3: Total DNA yield from stacked filter combinations with either 2.7pm or 5pm prefilter and 2.7pm or 5pm filter chitosan-modified filter. The same batch of urine (frozen) from healthy human female was filtered through each filter stack until filter became clogged and no more urine could be pushed through. DNA was quantified by AccuBlue HS dsDNA kit. Two elutions of 2.5 mis were required for efficient recover}' of all the DNA from these 47mm diameter filters.

[0043] FIG. 4: Recovery rate of DNA using chitosan extraction method using Nanodrop. 50-mer oligos were spiked into 20mL of 50mM MES pH 5 buffer (input DNA = 2ng / pL; 40pg total DNA) and eluted with 2.5mL of 50mM Tris pH9 buffer (theoretical yield = 40pg / 2.5mL = 16ng / pL). Subtracting the background (0 ng / pL eluent value) from the eluent from 2ng / pL spiked sample = 13.663ng / pL actual yield. Capture efficiency = 13.663 / 16 = 85%. Values below 2 are below the accurate lower limit of quantification.|0044] FIG. 5: Total DNA recovered from filters that were modified with either chitosan supernatant (completely dissolved portion of chitosan solution) or complete chitosan solution (with undissolved chitosan particulates / hydrogels). 2-sample t-test p-value > 0.05 = ns (not significant). F-test to test for equal variance p-value > 0.05 (ns). Statistical analysis was performed in GraphPad Prism.

[0045] FIGS. 6A-7D: Weeklong stability study showing DNA yield and protein contamination measured by AccuBlue and NanoOrange assays, respectively. Slopes for (FIG. 6A) DNA yield over time and (FIG. 6B) protein contamination over time were tested to see if they were significantly different from 0 (p-values listed in Table 5). Overall (FIG. 6C) DNA yield and (FIG. 6D) protein contamination were subjected to ANOVA analysis in GraphPad Prism. Blue window in FIG. 6B shows the (mean DNA yield ± SD) of immediately washedAIGN-43658.601 and eluted filters for comparison. P-values were adjusted via Tukey’s multiple comparisons. P-value: >0.05 = ns, <0.05 = *, <0.01 = **, <0.001 = ***, <0.0001 = **** Over the first 4 days allowing the filters to remain in the filter housing (’‘dried, washed, eluted”) resulted in no change in the DNA yield, while DNA yield at 7 days decreased. The size of the eluted DNA after the Dried / Washed / Eluted process was analyzed by CE as shown in 6E.

[0046] FIG. 7: Representative electropherogram from elution of 180mL urine using chitosan membrane filter. Fluorescent units (FU) on the y-axis represent the fluorescence detected from 1 pL of a 1 :5 dilution of chitosan filter eluate. The graph indicates the presence of DNA fragments at approximately 80 bp (x-axis) with a concentration of 89.74 pg / pL within the eluate, which w as extracted from 180 mL of urine.DETAILED DESCRIPTION

[0047] Cell-free DNA (cfDNA) has been appreciated for its utility in clinical applications such as monitoring organ health in organ transplant patients, identifying various infections, screening for genetic abnormalities during fetal development, determining if cancer is present, determining what therapy a cancer patient should have, and monitoring cancer response to determine response or progression.

[0048] It is important to note that genomic DNA (gDNA); large fragment size DNA comes from normal cells. It contaminates cfDNA preparation and decreases sensitivity in these applications. Because current methods for cfDNA extraction and processing are labor-intensive and / or use high-tech laboratory equipment, cfDNA testing is expensive, limiting its use. Most importantly, obtaining the specimen typically requires a visit to a clinic for venipuncture, this involves exposure to patients and attendant infection risk, as well as expense and time for travel, loss of work or leisure hours, which may be critical for terminal patients, and the physical and especially psychologic trauma of a blood draw. Both long-term repeat monitoring and screening compliance are dramatically lower when venipuncture visits are required. As described further herein, embodiments of the present disclosure include the development of a novel device that can collect cfDNA from urine at-home, or any setting, successfully processing up to 420mL of urine at once and if used repeatedly in either separate devices or the same one, can sequentially process a full day’s, or more, worth of urine.

[0049] In addition to being more convenient, having less risk, reducing skilled labor needed, reducing cost, and increasing speed, the new process yields at least an order of magnitude more DNA. As current sequencing and digital PCR systems can detect a sequence if present in the sample, the key limiting factor is the presence in a sample. The typical small samples previousAIGN-43658.601 methods resulted in thus is a limiting factor in sensitivity. High sensitivity is often required, especially for the early detection of cancer or the sensitive and early detection of its recurrence.

[0050] A system has been recently developed that uses chitosan printed onto a lateral flow filter device to directly extract cfDNA from blood plasma via pH-based capture and elution methods. Chitosan is a biopolymer that is obtained from marine waste and exhibits different characteristics depending on pH. Chitosan’s isoelectric point is approximately at the pH of 6.5, meaning that in solution with pH <6.5. chitosan is positively charged. The amino groups in chitosan become protonated by the hydrogen ions in acidic solutions to become positively charged protonated amino groups which then bind to the negatively charged DNA fragments in solution. By increasing the pH of the solution, the amino groups become deprotonated and are no longer positively charged, thereby releasing the negatively charged DNA molecules from the chitosan-DNA complex. It has also been shown that when DNA is bound to chitosan, DNA degradation by nucleases is inhibited by 99.9% (+ / - 0.1) due to the chitosan having a higher binding efficiency to DNA than nucleases, thereby protecting the bound DNA.

[0051] While this lateral-flow filter method does not require electricity and can be used in low-resource settings to isolate cfDNA from blood plasma, the maximum sample size for this system is only about 50pL of blood. For most cfDNA applications, at least 2-4 mL of plasma is required to obtain adequate material needed for accurate diagnostics, making this lateralflow system insufficient for many high sensitivity diagnostic needs. Modem methods require even more DNA to take advantage of their enhanced depth of sequencing.

[0052] This lateral flow filter system was also not tested with urine. It was hypothesized that, depending on the methods used to carefully control pH, the mechanism behind cfDNA binding to chitosan could be applied to urine. Current clinical cfDNA isolation methods do not utilize pH dependent binding and release, rather they depend on the binding of cfDNA to silica in the presence of chaotropic salts. Some proprietary kits use binding to magnetic or paramagnetic particles, and a magnet to hold the bound DNA during a series of sequential steps. Other companies require centrifugation of silica particles. Both require lab equipment. The magnetic systems work well for small, but not large volumes.

[0053] As described further herein, embodiments of the present disclosure include systems and methods for extracting cfDNA from a sample. In some embodiments, the systems and methods of the present disclosure include an affinity-based (e.g., poly cationic) system for the extraction, collection, stabilization, transport and purification (in a scaled-up manner) of cfDNA from urine. It is also instrument free, requiring only reusable / recyclable industrialAIGN-43658.601 syringes and filter cartridges. This required trans / cross flow of the urine rather than lateral flow. As trans flow means the length of filter being traversed is small, it was uncertain if conditions sufficient for efficient capture could be found, especially when extraction of large volumes was targeted. The critical time of contact per volume, defining if binding can occur, is orders of magnitude lower.

[0054] Also, because a much larger volume of urine is used, and because the ratio of urine to the length of filter being traversed is orders of magnitude different, the results were very uncertain. A cfDNA extraction kit, using any method, for capturing DNA from an entire void in a single prep, or even reasonably easy to combine multiple preps, is currently not available. This is critical as current data indicates that the amount of cfDNA harvested, and thus the amount of cancer DNA or pathogen DNA or other DNA of interest, is limiting. Thus, experiments were performed to develop a method using a syringe setup, that requires no machinery, that can be used to collect urine cell-free DNA (ucfDNA) from a full bladder void. This novel system specifically captures cfDNA by using a syringe to push urine through a glass microfiber pre-filter that filters out epithelial cells and other debris, and a chitosan-modified glass microfiber filter to capture ucfDNA. The method often includes in the collection vessel a method to assure pH of the urine is adequate to bind chitosan, and DNA preservatives, allowing virtually instantly inhibiting DNAses.

[0055] Urine contains cfDNA derived from the urogenital track. This is the classic nucleosomal sized (~ 166bp) and this cfDNA is protected by a complex of histones. This is the only type of cfDNA for which kits have been constructed. Urine also contains DNA from the blood stream, trans-renal DNA (trDNA), while this is potentially the most interesting and informative DNA, no commercial method selective for its isolating exists. Some are co-purified in the kits for urine cfDNA. Some research methods have first steps that precede standard cfDNA purification involving many, many, steps that may enrich for trDNA.|0056] As described further herein, embodiments of the present disclosure include a novel syringe filter-based cfDNA extraction device for capturing ucfDNA, and especially trDNA in any setting, including a home setting, to be transported to a laboratory facility for processing based on elegantly simple pH change without the need for instrumentation or skilled labor. It requires 1 / 10 the lab time of any current method, concomitant with a dramatic reduction in steps. This device has been shown to capture cfDNA from up to 420mL of urine, which is 10.5 times more processing volume than the maximum processing volume of currently commercially available urine cfDNA extraction kits, the latter requiring laboratory trained personnel and often over $100,000 of equipment. The devices and systems of the presentAIGN-43658.601 disclosure also demonstrated preferential binding of cfDNA while excluding gDNA that can cause decreased sensitivity and inconsistent results. Embodiments of the present disclosure demonstrate that cfDNA remains stable on the filter for at least 4 days after collection, giving sufficient time for shipment to the laboratory facility.10057] In accordance with the above, the compositions and methods of the present disclosure are particularly useful for obtaining ucfDNA and trDNA from a urine sample(s) using the above-described trans flow method in which initial collection and biochemical binding are performed in any, including at-home, settings. The trDNA specific system utilizes mild acidification, often pH 4.5, which does not disrupt the histone DNA binding of urothelial nucleopsomes. This is in marked conceptual conflict with currently available methods and systems that specify initial steps that remove the positively charged histones from the negatively charged DNA. These often use proteases, salts, and / or especially chaotropic agents, which means that the bladder nucleosomes are dissociated, releasing the bladder nucleosomal DNA so that it is mixed with, and contaminates, the desired trDNA. In order to obtain a sample enriched for trDNA. the systems and methods of the present disclosure selectively isolate trDNA from DNA associated with positively charged histones by capturing based on charge. The histone charge neutralizes the DNA charge, so the complex does not bind. A distinctive, convenient trans-flow syringe-based methodology' was developed, in which the sample flows through a filter cartridge based on pressure from a syringe. In contrast, current magnetic and centrifugation based methods involve moving silica particles; however, the systems and methods of the present disclosure not only obviate the need for this additional purification step, but are specifically designed to ensure the histone-bound DNA does not bind the trDNA collection filter, which is typically positively charged. Thus, the systems and methods of the present disclosure facilitate obtaining samples enriched in trDNA from urine, which represents cfDNA from blood. Such trDNA potentially includes DNA from cancers, tumors, internal infections, sepsis causing organisms, organisms growing on the heart valves, tuberculosis bacilli, DNA from transplanted organisms, DNA from a fetus, or DNA from any physiologic or pathologic process in the body. When all urine cfDNA is desired, the methods herein utilize much lower pH to dissociate the histones in the bladder nucleosomes. Thus, in some embodiments, these methods mimic the cfDNA collected by current methods, but it is especially important in generating a preparation of cfDNA enriched in actively transcribed trDNA.|0058] As described further herein, a preparation enriched in trans-renal cfDNA from the blood could be obtained using conditions that did not dissociate the positively charged histonesAIGN-43658.601 on the bladder cfDNA. Electrostatic interactions were exploited to separate the naked trDNA from histone-bound bladder DNA, as the former is highly negatively charged, while the negative charge on the DNA of the latter is neutralized by the positively charged histones. Only the highly negatively charged trDNA is bound to the positively charged capture material in the filters, whereas histone-bound DNA does not bind.

[0059] As described further herein, when the cfDNA is released from the capture material, a preparation enriched in trans-renal cfDNA is obtained. The capture matenal may be on a filter directly, on beads, on nanoparticles, and / or on other particles that may be trapped on a filter. It may be on solid or porous surfaces, in a column, again fixed in relationship to a Plowing liquid. Such particles may be temporarily free in solution but able to be captured later in a column or on a solid or porous surface. Any of these can be used in a system in which the liquid is moved without movement of the cfDNA being purified. Particles with positive surfaces may be magnetic or paramagnetic, and may be of any size. The capture may be coupled to a solid surface such as a microtiter plate or in a capillary, microfluidic, nanofluidic device or microfluidic device or column.

[0060] As would be appreciated by one of ordinary skill in the art based on the present disclosure, histone-bound DNA that is typically discarded via the flowthrough methods of the present disclosure can, in certain embodiments, be saved and used as a sample that is enriched in bladder cfDNA (and depleted in trDNA). Samples enriched for bladder cfDNA can be especially beneficial for assessing, for example, bladder and renal cancer or the health of a transplanted kidney.

[0061] Thus, embodiments of the present disclosure include systems and methods that separate, purify, and / or isolate histone-bound DNA from histone-free DNA. While useful for urine, these systems and methods can be applied to other bodily fluids (e.g.. blood, serum, plasma, etc.) to obtain a preparation enriched in histone-bound DNA and / or a preparation enriched in histone-free DNA (or both). This may be especially beneficial as the histone-bound DNA is that which was relatively inactive when in the cell, while the non-histone bound DNA, some of which may been less strongly bound to transcription factors, is the more active portion of the DNA. This applies even to the DNA in blood. Thus, while described herein as trDNA it applies to all DNA that is not strongly bound to histone proteins.

[0062] Section headings as used in this section, and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.AIGN-43658.6011. Definitions

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art based on the present disclosure. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0064] The terms “comprise(s),” “include(s),” ‘'having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0065] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.10066] The term “derived from” as used herein refers to cells or a biological sample (e.g., blood, tissue, bodily fluids, plants, etc.) and indicates that the cells or the biological sample were obtained from the stated source at some point in time. The term includes directly obtained from, isolated and cultured, or obtained, frozen, and thawed. The term “derived from” may also refer to a component or fragment of a cell obtained from a tissue or cell, including, but not limited to, a protein, a nucleic acid, a membrane or fragment of a membrane, a vesicle, and the like.

[0067] ‘ ‘Controls” as used herein generally refers to a reagent whose purpose is to evaluate the performance of a measurement system in order to assure that it continues to produce results within permissible boundaries (e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end). To accomplish this, a control should be indicative ofAIGN-43658.601 patient results and optionally should somehow assess the impact of error on the measurement (e.g., error due to reagent stability, calibrator variability, instrument variability, and the like).

[0068] As used herein, the term ‘'sample” is used in its broadest sense. In one sense, it is meant to include a specimen obtained from any source, including biological samples. Biological samples may be obtained from animals (including humans) or plants and encompass fluids, solids, tissues, and gases. Such examples are not, however, to be construed as limiting the sample types. Preferably, a sample is a fluid sample such as a liquid sample or solid sample that is then dispersed in or solubilized in a fluid. Examples of liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, pleural effusions, ascites, thin needle aspirates and spinal fluid). Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. For example, throat or genital swabs may be suspended in a liquid solution to make a sample. Samples can comprise biological materials, such as cells, microbes, organelles, and biochemical complexes. Liquid samples can be made from solid, semisolid, or highly viscous materials, such as fecal matter, tissues, organs, biological fluids, or other samples that are not fluid in nature. For example, solid or semisolid samples can be mixed with an appropriate solution, such as a buffer, a diluent, and / or extraction buffer. The sample can be macerated, frozen and thawed, or otherwise extracted to form a fluid sample. Residual particulates may be removed or reduced using conventional methods, such as filtration or centrifugation.

[0069] “Sample,” '‘test sample,” “sample from a subject,” “biological sample,” and “patient sample” as used interchangeably herein may be a sample of blood, such as whole blood (including for example, capillary7blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, amniotic fluid, an anal sample (such as an anal swab specimen), lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. In other embodiments, the sample is a non-biological sample (i.e., a sample not obtained directly from a biological sample from subj ect or patient), including but not limited to, water from a natural environment, or water from a manmade or industrial water source, whether initially in liquid or frozen form.AIGN-43658.601

[0070] A variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluids may include sections of tissues such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, an anal sample (such as an anal swab specimen), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration. A tissue or cell type may be provided by removing a sample of cells from a human and a non-human animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and / or purification may not be necessary.|0071] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species kept as pets, or livestock or used as “model systems” for research purposes, such a mouse models, prokaryotic models (e.g., bacteria), archaea, and single-celled eukaryotes(e.g., yeast). Likewise, subjects may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., humans and non-humans) that may benefit from the uses of compositions and methods contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the subject is a human.

[0072] As used herein, “cell-free DNA” (cfDNA) generally refers to DNA that is released from cells and is present in the blood or other bodily fluid. The majority of cfDNA in the blood comes from hematopoietic cells, but all cells in the body, even brain cells, contribute to cfDNA. Tissue-specific methylation signatures can identify the organ that released a particular cfDNA molecule. cfDNA is degraded quickly and can be excreted in urine, but only after it is separated from its protective histones.

[0073] As used herein, “circulating tumor DNA” (ctDNA) generally refers to DNA fragments that are associated with cancer that can be found in the blood and are commonly detected in liquid biopsies. Liquid biopsies are non-invasive or minimally invasive ways toAIGN-43658.601 sample bodily fluids for cancer biomarkers. ctDNA can be used for cancer detection, personalized therapy, and monitoring treatment response or recurrence.

[0074] As used herein, '‘trans-renal DNA” (trDNA) generally refers to cell-free DNA that originated from the blood and passed through to kidney so that is obtained from a urine sample(s) . TrDNA is generally made up of fragments of circulating cell free DNA (which may include ctDNA, fetal DNA, infectious DNA. donor derived DNA from transplanted organisms, or other cfDNA) that pass from the bloodstream into the urine through the kidney. These are believed to be small enough to penetrate the glomerular fdtration system. In some embodiments, trDNA is associated with cancer, and can be referred to as “trans-renal cell-free tumor DNA"’ (TR-ctDNA), and any variations thereof. TR-ctDNA can be indicative of the presence of cancer; it is circulating ctDNA that has passed from the plasma into the urine.

[0075] Urine nucleosome DNA or urine cellular DNA is not trDNA, it is useful for the analysis of kidney, urothelium and bladder condition and pathophysiology; it may also be enriched in DNA from nearby tissues such as the prostate, vagina, cervix, fallopian tube and ovary.

[0076] Preferred methods and materials are descnbed below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.2. Methods and Systems

[0077] Liquid biopsies (LBs) have proven critical for understanding basic pathophysiology and translating this into medical advances, blood and urine are both standard clinical specimens. Translational clinical trials of cell free DNA (cfDNA) for heart attacks and other cardiovascular conditions, organ transplantation, infectious disease, cancer, maternal health including eclampsia, fetal health including 25 mutations and aneuploidy, Alzheimer’s and even psychiatry show great promise. More basic studies using cfDNA to understand and diagnose autoimmune disease, and neurological diseases (including ALS. MS, TB1. and stroke) are in progress. Blood cfDNA (bcfDNA) is the most common sample and it is rarely obtained more than once daily. bcfDNA has a short half-life (~2hrs) and is subject to changes throughout the day. Urine sampling is easier, less expensive, less invasive and it can be done in any setting, including at home. It can capture changes that occur during the day, or be used to monitor daily throughout the week after a change in therapy, without undue stress on the subject.AIGN-43658.601

[0078] As described further herein, the methods and systems of the present disclosure include urine collection devices that facilitate cfDNA collection, be it a one-time event and especially from multiple samples / day, waking day timed collections and / or 24hr collection. The method for 24-hour samples does not use the typical collection in a single vessel of multiple voids and then process them all at once; this allows degradation in the collection vessel. Instead, this disclosure describes methods that prevent degradation by immediate processing and binding onto a filter. In addition, preservatives are preloaded in the collection vessel, so they instantly contact the voided urine. Unlike a one-point blood test, urine cell free DNA (ucfDNA), especially using the methods disclosed herein, makes 12 or 24-hour integration of cfDNA production feasible. Given the known variation during the day this allows greater accuracy. Of critical importance for high sensitivity testing, the methods and systems of the present disclosure can also provide lOx to lOOx more cfDNA. As described further herein, the methods and systems of the present disclosure limit the cfDNA degradation that begins immediately. Simple, immediate capture, at-home, ucfDNA sample collection systems and methods are not currently available. In addition to collection at home, the methods and systems of the present disclosure facilitate immediate DNA processing at ambient temperature, use of routine shipping, and for the first time institute a biochemical step that results in at least partial purification in the home. It allows immediate separation as well as collection of both cells, including their gDNA, and cfDNA. It is also unique in allowing totally instrument free purification as well as collection. The product would facilitate basic science, translational work, clinical applications, novel cfDNA based screening tests, and serve the large and growing existing commercial market for FDA approved tests, for Laboratory Developed tests and for a variety of research projects.

[0079] For example, patients in clinical trials might be monitored more frequently, with higher compliance, lower cost and less risk of immunosuppressed patients acquiring infections while at, or traveling to, blood draw sites. Blood samples require immediate trained personnel processing or stabilization in expensive tubes to prevent cfDNA degradation. Transporting blood or plasma is biohazardous, expensive, and may require a cold chain. Centrifugation must be followed immediately by careful harvesting of the plasma, a biohazardous procedure, and a second spin to obtain clean plasma. Then there is multistep isolation of cfDNA requiring expensive reagents, often using expensive equipment, and requiring hours. Sensitivity is often limited by the volume processed and Poisson distribution, meaning the small sample simply lacks the diagnostic sequence. UcfDNA is more fragmented, however existing computational methods can compensate for this. Current commercial urine kits require highly trained medicalAIGN-43658.601 technicians and research-level skills and generally handle only 10s of mis per sample. Therefore, there is an unmet commercial and clinical need for a reliable, inexpensive, easy method to collect, stabilize and allow easy shipping of ucfDNA from large volumes (100’s of mis) of urine collected at home. Across a wide variety of diseases, this will be translational, it will facilitate clinical decisions based on basic science and make available abundant excess clinical cfDNA for basic science research.

[0080] In accordance with the above, and as described further herein, embodiments of the present disclosure include methods and systems for cfDNA collection and partial purification from large volume samples using polyelectrolyte-modified filter membranes. In some embodiments, the pre-filter used in the methods and systems of the present disclosure is configured to have low DNA-binding so that the DNA present in a biological sample is able to move past the prefilter and bind to the chitosan on the DNA-binding filter. For example, because the median volume of urine in a bladder void is about 220mL, one prefilter that allowed for that volume of urine flowthrough was the 5pm glass fiber (G / F) filter. In other embodiments, to ensure that enough particulates were filtered out to prevent clogging of the eventual secondary filter, a 2.7pm G / F filter can also be used.

[0081] Although the [2.7pm prefilter -> 1.6pm chitosan] and [2.7pm prefilter2.7pm chitosan] filter combinations had approximately the same urine flowthrough, the total DNA yield was 1617% higher in the filter combination with the 2.7 m chitosan filter compared to the filter combination with the 1.6pm chitosan filter (FIG. 2; Table 1). Likewise, despite the [5pm prefilter 2.7pm chitosan] filter combination having 21.4% less urine flowthrough than the [5pm prefilter -> 5pm chitosan] filter combination, the 2.7pm chitosan filter captured 4996% more DNA than the 5pm chitosan filter (FIG. 2; Table 1). This shows that the chitosan- modified 2.7pm G / F filter had the highest DNA binding capacity, likely related to its greater surface area and chitosan binding, and that the 5 pm prefilter is sufficient for allowing the 2.7 pm chitosan filter to handle the full void volume.

[0082] Table 1: Pre-filter (non-modified) and chitosan filter combinations (in succession, not physically stacked) performance for maximum urine volume and total DNA yield. All filters are glass fiber (G / F). Average urine volume indicates the average of the maximum urine volume from a female that could be pushed through until either the filter got clogged or there was no urine left. DNA was quantified using AccuBlue HS dsDNA kit.AIGN-43658.601

[0083] With the 5 pm prefilter allowing for the most urine flowthrough and the 2.7pm chitosan filter capturing the most DNA, it was concluded that the best filter combination going forward was the 5pm G / F prefilter and the 2.7pm G / F chitosan-modified filter. Once the filters were stacked in the same filter housing, results were consistent with the findings from FIG. 2, showing similar urine flowthrough volume and DNA recovery even when stacked.

[0084] As described further herein. cfDNA was shown to have no significant decrease for at least 4 days (slopes of DNA yield for all filter treatments were not significantly different from zero to over a week, with p-values ranging from 0.1314 to 0.7958 for all treatments; FIG. 6A; Table 2). While cfDNA was stable on the filter, it was evident that the 70% and 91% isopropanol rinses resulted in significantly less cfDNA yield than the samples that were immediately processed (p-values = 0.0057 and 0.0293, respectively), while drying without isopropanol rinses did not have significantly different cfDNA yield (p = 0. 177; FIG. 6C; Table 3). This demonstrated that if a patient w ere to push their urine through the filter and ship the filter to the lab after drying, cfDNA yield would not be significantly affected for up to one week.

[0085] Table 2: Comparison of simple linear regressions betw een different filter treatment groups (immediately washed and eluted, stored-washed-eluted, 70% isopropanol-stored- w ashed- eluted, and 91% isopropanol -stored-washed-eluted).AIGN-43658.601

[0086] Table 3: ANOVA results for overall DNA yield and protein contamination in weeklong stability study between different filter treatment groups (immediately washed and eluted, stored-washed-eluted, 70% isopropanol-stored-washed-eluted, and 91% isopropanol- stored-washed-eluted).AIGN-43658.601

[0087] Experiments were also conducted to determine which filter treatment resulted in the eluate with the least amount of protein contamination. It has been established that isopropanol / water mixtures denature and coagulate proteins; therefore, it was envisioned that 70% or 91% isopropanol would lead to decreased protein contamination in the final eluate. It was found that the act of drying and / or isopropanol rinses did not result in a statistically significant decrease in protein contamination in the final sample, in fact it appeared to trap DNA on the filter and lower yield of DNA. (FIG. 6D; Table 3), however there is an apparent, though NS, decrease in protein in 70% ETOH.

[0088] Urine cfDNA fragment size has been shown to mostly range from 40 bp to 120 bp with a modal size of approximately 81 bp. Given that chitosan extraction collected fragments between 35 and 150bp, the low molecular weight chitosan that was used captured only low fragment size DNA consistent with smaller cfDNA fragments found in urine. While initial experiments produced an eluate with smaller mean fragment size, (FIG. 6E), procedure optimization resulted in the ideal 80 bp mean peak (FIG. 7).|0089] In accordance with the above embodiments and the experimental results described herein, embodiments of the present disclosure include a method for obtaining cfDNA from aAIGN-43658.601 sample. In some embodiments, the method includes the subject ensuring that the pH of the sample is acidic and at an optimum pH by use of a DNA binding composition and passing the pH-adjusted sample through a filter cartridge comprising at least one filter. In some embodiments, the cfDNA from the sample binds the at least one filter, and the sample flowthrough is removed, discarded, or otherwise separated from the filter or filter cartridge. In some embodiments, the flowthrough immediately passes through a second filter cartridge. In some embodiments, the first filter cartridge has a larger area and contains the prefilter and the flowthrough immediately proceeds to a second cartridge containing a small filter with the DNA capture material; this conformation allows easy separation and in cartridge further purification of the samples in each cartridge separately. In some embodiments, the smaller second cartridge facilitates effective washing and elution of the DNA in a smaller volume.

[0090] In some embodiments, the sample comprises one or more bodily fluids from the subj ect selected from urine, whole blood, blood plasma, serum, ascites, saliva, lymph, pleural effusion, semen, and cerebral spinal fluid. In some embodiments, the sample comprises water from the natural environment, or water from a manmade or industrial water source. Other sample types can also be used, as would be recognized by one of ordinary skill in the art based on the present disclosure. For example, the sample can also comprise plant material, which can be extracted in larger volumes as compared to currently available systems and methods. In some embodiments, the systems and methods of the present disclosure can be used to obtain DNA from plant material immediately subsequent to obtaining the material in the field. In other embodiments, the systems and methods of the present disclosure can be used to obtain DNA from plant material after storage (e.g., frozen samples).

[0091] In some embodiments, the sample comprises a volume from about 20mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 50mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 75mL to about lOOOmL. In some embodiments, the sample comprises a volume from about lOOmL to about lOOOmL. In some embodiments, the sample comprises a volume from about 200mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 300mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 400mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 500mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 600mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 700mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 800mL to about lOOOmL. In some embodiments, the sample comprises a volume from about 900mL to aboutAIGN-43658.601 lOOOmL. In some embodiments, the sample comprises a volume from about 20mL to about 900mL. In some embodiments, the sample comprises a volume from about 20mL to about800mL. In some embodiments, the sample comprises a volume from about 20mL to about700mL. In some embodiments, the sample comprises a volume from about 20mL to about600mL. In some embodiments, the sample comprises a volume from about 20mL to about500mL. In some embodiments, the sample comprises a volume from about 20mL to about400mL. In some embodiments, the sample comprises a volume from about 20mL to about300mL. In some embodiments, the sample comprises a volume from about 20mL to about200mL. In some embodiments, the sample comprises a volume from about 20mL to about lOOmL. In some embodiments, the sample comprises a volume from about 20mL to about75mL. In some embodiments, the sample comprises a volume from about 20mL to about 50mL. In some embodiments, the sample comprises a volume from about 200mL to about 800mL. In some embodiments, the sample comprises a volume from about 450mL to about 650mL. In some embodiments, the sample comprises a volume from about 150mL to about 500mL.

[0092] In some embodiments, multiple samples are passed through the same filter resulting in the accumulation of ucfDNA from up to a liter of sample. In some embodiments, multiple samples are passed through the same filter resulting in the accumulation of ucfDNA from up to 2 liters of sample. In some embodiments, the pH-adjusted sample is passed through the filter cartridge and then the flowthrough is further pH adjusted and passed through an additional filter cartridge(s). In some embodiments, a sample is divided into multiple fractions, each pH adjusted to a different value and passed through different filter cartridge(s).

[0093] In some embodiments, the methods and systems of the present disclosure can be used and / or optimized for collecting, isolating, and / or purifying any type of cfDNA from a sample (e.g., a biological sample or a non-biological sample). In some embodiments, the sample comprises circulating tumor DNA (ctDNA), trans-renal DNA (trDNA), cell-free fetal DNA (cffDNA), cell-free mitochondrial DNA (ccf mtDNA), DNA from a micro-organism or infectious organism, donor-derived cell-free DNA (dd-cfDNA), and / or other trans-renal DNA (trDNA). In some embodiments, the sample can include other types of DNA not yet identified or characterized.

[0094] In some embodiments, the cfDNA and / or trDNA obtained using the methods and systems of the present disclosure comprises DNA from normal cells (e.g., non-pathologic cells) and / or DNA that is not associated with a pathologic disease or condition (e.g., non-mutated DNA), including but not limited to, DNA from normal cells that is present in altered concentrations and / or ratios as compared to a control, previous sample, or reference sampleAIGN-43658.601(e.g., DNA from a healthy or control subject). For example, normal DNA or DNA from normal calls can be important indicators of a disease or condition, including but not limited to normal neuronal cfDNA as a marker for Alzheimer’s Disease (e.g., increased amounts can indicate whether moderate cognitive difficulty will progress to AD); DNA released from normal WBCs and endothelial cells (e.g., enables / enhances diagnosis of infections, cancers, autoimmune and inflammatory’ diseases, and the like); release of normal skeletal muscle DNA can be an indication of over-exercise and may be used to optimize training / prevent overtraining (e.g., beneficial exercise levels increase endothelial and WBC DNA, but counterproductive muscle damaging exercise causes muscle cfDNA to increase); and normal, but increased, abnormal amounts of cardiac muscle cfDNA can be an indication of heart disease or other cardiovascular issues.

[0095] In some embodiments, the methods and systems of the present disclosure include the use of a DNA binding composition to facilitate the binding of the DNA to one or more filters in the filter cartridge (e.g., to increase affinity of DNA for the one or more filters in the filter cartridge). As shown in FIG. 1, the DNA binding composition can be added to the sample prior to passing the sample through the filter cartridge. In one embodiment, the DNA binding composition comprises an acidifying agent, and in another embodiment, a preservative is included in the collection device prior to the subj ect voiding. In some embodiments, the subj ect voids directly into the syringe used to push the sample through the filter. In some embodiments, a stopcock can be used as a connector between a syringe and a filter cartridge.

[0096] In some embodiments, the DNA binding composition is formulated such that a user (e.g., human subject) can adjust the pH of the sample to a pH of < 5, which can ensure binding of the DNA to the filter (e.g., a pH-adjusted sample). In some embodiments, the DNA binding composition comprises an acidifying agent to adjust the pH of the sample to a pH of < 5. In some embodiments, an acidifying agent is added to the DNA binding composition prior to adding the sample. In some embodiments, an acidify ing agent is added to the DNA binding composition after adding the sample. In some embodiments, the DNA binding composition comprises an acidifying agent. In some embodiments, the DNA binding composition comprises acid or an acidic buffer with one or more of the acidic forms of ascorbate, acetate, citrate, borate, formate, maleate, tartrate, lactate, glycine, potassium hydrogen phthalate buffer and / or MES buffer. In some embodiments, the pH is adjusted to under pH 2.

[0097] In accordance with the above, and as described further herein, embodiments of the present disclosure include methods for selectively obtaining urothelial cell-free DNA (cfDNA) from a urine sample from a subject. In some embodiments, the method includes buffering theAIGN-43658.601 urine sample to a pH at or between a pH of 0.5 and a pH of 3, and passing the buffered urine sample through a filter cartridge comprising at least one filter, thereby selectively binding the cfDNA present in the urine sample to the at least one filter. Additionally, and as described further herein, embodiments of the present disclosure include methods for selectively obtaining trans-renal DNA (trDNA) from a urine sample from a subject. In some embodiments, the method includes buffering the urine sample to a pH at or between a pH of 3 and a pH of 6, and passing the buffered urine sample through a filter cartridge comprising at least one filter, thereby selectively binding the trDNA present in the urine sample to the at least one filter.

[0098] In some embodiments, the DNA binding composition comprises other components to aid in the DNA binding process, and / or to stabilize the DNA in the sample. In some embodiments, the DNA binding composition further includes a DNA stabilization agent. In some embodiments, the DNA stabilization agent comprises EDTA. In other embodiments, the DNA binding composition comprises a preservative, a buffer, or other component to aid in the passing of the sample through the filter cartridge. In accordance with these embodiments, and as described further herein, the present disclosure also includes a kit in which the DNA binding composition is formulated as a powder and contained in a packet that can be included in a sample collection container. In some embodiments, the DNA binding composition comprises an acidifying agent, a buffer or buffering agent, a stabilization agent, and / or a preservative, all or any of which can be formulated as a powder and included in the packet. In some embodiments, the collection cup, DNA binding composition, syringe, filter cartridges(s) preloaded with the filter(s) are provided in a kit with instmctions for a lay user. In some embodiments, the syringe barrel and filter cartridges, but not syringe plunger, are assembled as a unit for ease of use.

[0099] As would be recognized by one of ordinary skill in the art based on the present disclosure, the DNA binding composition used to facilitate the binding of DNA in the sample to the filter in the filter cartridge (e.g., to increase affinity of DNA for the one or more filters in the filter cartridge) can be formulated as a powder, such that a user adds the powdered formulation to the sample prior to passing the sample through the filter cartridge, or in one embodiment, to the collection cup prior to voiding. In other embodiments, the DNA binding composition is formulated as a solution and added to the sample.

[0100] In some embodiments, the at least one filter in the filter cartridge comprises a DNA- binding filter. In some embodiments, the at least one filter in the filter cartridge comprises a positively charged polyelectrolyte. In some embodiments, the positively charged polyelectrolyte comprises chitosan, or a variant or derivative thereof. In some embodiments.AIGN-43658.601 the at least one filter is made of polyacrylonitrile, glass fiber, poly ethersulfone, or any combinations thereof. As would be recognized by one of ordinary’ skill in the art based on the present disclosure, the at least one filter and / or filter cartridge can include any suitable filtration material known in the art, including but not limited to, membranes, columns, and / or beads. In accordance with this, the at least one filter and / or filter cartridge of the present disclosure can include any DNA-adsorbent material known in the art, including but not limited to, silica, magnetic iron oxide particles, glass particles, diatomaceous earth, anion-exchange carriers, and / or modified polyurethane material.

[0101] In some embodiments, the at least one filter used in the systems and methods of the present disclosure comprises a chitosan capacity ranging from about 1 pg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 10 pg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 100 pg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 mg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 10 mg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 100 mg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 250 mg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 500 mg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 pg to about 1000 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 pg to about 500 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 pg to about 100 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 pg to about 10 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 pg to about 1 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 1 mg to about 500 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 75 mg to about 500 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 250 mg to about 500 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 10 mg to about 250 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 50 mg to about 500 mg. In some embodiments, the at least one filter comprises a chitosan capacity ranging from about 100 mg to about 300 mg.AIGN-43658.601

[0102] In some embodiments, the at least one filter comprises a pore size ranging from about 1.0pm to about 5.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 1.0pm to about 4.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 1.0pm to about 3.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 1.0pm to about 2.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 2.0pm to about 5.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 3.0pm to about 5.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 4.0pm to about 5.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 2.0pm to about 4.0pm. In some embodiments, the at least one filter comprises a pore size ranging from about 1.5pm to about 3.4pm. In some embodiments, the at least one filter comprises a pore size ranging from about 3.0pm to about 4.0pm.

[0103] In some embodiments, and as described further herein, the methods and systems of the present disclosure include the use of a pre-filter to remove various components of the sample (e.g., cellular debris) and / or contaminants. In some embodiments, the sample is passed through a pre-filter before being passed through the at least one filter in the filter cartridge. In some embodiments, the DNA binding filter includes an integrated pre-filter. In some embodiments, the DNA-binding filter and the pre-filter are stacked. In some embodiments, the DNA-binding filter and the pre-filter are not stacked, but used in succession. In some embodiments, the at least one filter is made of polyacrylonitrile, glass fiber, polyethersulfone, or any combinations thereof.|0104] In some embodiments, the pre-filter comprises a pore size ranging from about 1.0pm to about 5.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 1 0pm to about 4.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 1.0pm to about 3.0pm. In some embodiments, the pre-filter comprises apore size ranging from about 1.0pm to about 2.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 2.0pm to about 5.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 3.0pm to about 5.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 4.0pm to about 5.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 2.0pm to about 4.0pm. In some embodiments, the pre-filter comprises a pore size ranging from about 1.5pm to about 3.4pm.AIGN-43658.601In some embodiments, the pre-filter comprises a pore size ranging from about 3.0pm to about 4.0pm.|0105] In some embodiments, the DNA binding filter and / or a pre-filter (if present) can be formulated to include various other agents to aid in the binding of the DNA from the sample to the filter, to help stabilize the DNA on the filter, and / or to aid in the removal of various components of the sample, and any contaminants. For example, in some embodiments, the filter or filter cartridge can include one or more digestive enzymes. In some embodiments, the digestive enzyme is a protease. In some embodiments, the digestive enzy me is a proteinase K or papain or try psin.

[0106] In accordance with the above embodiments, the methods and systems of the present disclosure can be used to obtain cfDNA from a biological sample from a human subject. In some embodiments, the sample is obtained from one or more biological fluids from the human subject. In some embodiments, the sample includes urine from one or more voids. In some embodiments, the urine is frozen prior to processing. In other embodiments, the urine is obtained shortly after a subject voids. In some embodiments, the sample includes urine from multiple voids. In some embodiments, the one or more voids are passed through the filter cartridge by the human subject or an assistant. In some embodiments, the one or more voids are passed through the filter cartridge by the human subject directly after (or immediately subsequent to) urination. In some embodiments, the filter in the filter cartridge comprises a positively charged polyelectrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 30 seconds to about 10 minutes following the completion of the void. In some embodiments, the filter in the filter cartridge comprises a positively charged polyelectrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 1 minute to about 8 minutes. In some embodiments, the filter in the filter cartridge comprises a positively charged polyelectrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 1 minute to about 5 minutes. In some embodiments, the filter in the filter cartridge comprises a positively charged poly electrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 1 minute to about 4 minutes. In some embodiments, the filter in the filter cartridge comprises a positively charged polyelectrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 2 minutes to about 8 minutes. In some embodiments, the filter in the filter cartridge comprises a positively charged polyelectrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 4 minutes to about 8 minutes. In some embodiments, the filter in the filter cartridge comprises a positively charged polyelectrolyteAIGN-43658.601 formulated to bind and / or stabilize the cfDNA from the urine within about 3 minutes to about 5 minutes.

[0107] In some embodiments, the methods and systems of the present disclosure can be used to collect cfDNA from a human subject who is suspected of, or at risk of, having a disease or condition, and the collected cfDNA can be used to assess the human subject for that disease or condition. In some embodiments, the human subject is experiencing or has experienced one or more of the following: cancer, transplanted organ, pregnancy, physical exercise training, heart attacks or other cardiovascular conditions, stroke, neurologic disease, Alzheimer’s Disease, psychiatric conditions, autoimmune disease, sepsis, and / or urologic disease.

[0108] As would be recognized by one of ordinary skill in the art based on the present disclosure, any size filter cartridge can be used in accordance with the systems and methods described herein. For example, in some embodiments, the filter cartridge is from about 1 cm to about 10 cm in diameter. In some embodiments, the filter cartridge is from about 1 cm to about 8 cm in diameter. In some embodiments, the filter cartridge is from about 1 cm to about 5 cm in diameter. In some embodiments, the filter cartridge is from about 1 cm to about 3 cm in diameter. In some embodiments, the filter cartridge is from about 2 cm to about 8 cm in diameter. In some embodiments, the filter cartridge is from about 2 cm to about 5 cm in diameter. In some embodiments, the filter cartridge is from about 5 cm to about 25 cm in diameter. In some embodiments, a filter cartridge is configured so as to minimize space around the filter in a filter cartridge. In some embodiments, the filter cartridge is configured to minimize space in and around the filter, so as to reduce the final volume of an eluate, which can be advantageous for downstream processing. In some embodiments, a stopcock can be used as a connector between a syringe and a filter cartridge.

[0109] In some embodiments, the human subject sends the filter cartridge (comprising the bound DNA from the biological sample) to a lab for processing. In some embodiments, the human subject passes a wash buffer and / or a stabilization buffer through the at least one filter in the filter cartridge before sending the cartridge to a lab for processing. In some embodiments, the filter cartridge is stored for no more than about 7 days prior to processing. In some embodiments, the filter cartridge is stored for about 8 hours, about 16 hours, about 32 hours, about 64 hours, about 128 hours, or about 1 week prior to processing. In some embodiments, the methods and systems of the present disclosure can be used to isolate cfDNA from a sample from a subject and facilitate its storage on the filter in a substantially stable form for up to 1 week without significant degradation or significantly compromised yield or purity. As described further herein, the methods and systems of the present disclosure can be adjusted inAIGN-43658.601 order to optimize one or more parameters important for downstream processing. For example, the methods and systems of the present disclosure can be adjusted to optimize cfDNA yield and / or cfDNA purity, depending on the particular requirements of a downstream assay or further purification process.10110] In some embodiments, the method further comprises processing the filter cartridge to purify the cfDNA. In some embodiments, the processing comprises passing a wash buffer or a stabilization buffer through the at least one filter in the filter cartridge. In some embodiments, the wash buffer and / or the stabilization buffer comprises a pH of 7 or below. In some embodiments, the wash buffer and / or the stabilization buffer comprises a pH of 6 or below. In some embodiments, a wash buffer of about 7 is used. In some embodiments, the processing comprises treating the one or more filters with a protease. In some embodiments, the processing comprises eluting the cfDNA from the one or more filters. In some embodiments, the cfDNA is further purified and / or concentrated. In some embodiments, the processing is performed at a lab by a laboratory technician.

[0111] In some embodiments, the method further comprises processing the filter cartridge to release the cfDNA using an elution buffer. In some embodiments, the elution buffer has a pH that is from about 5 to about 11. In some embodiments, the elution buffer has a pH that is from about 6 to about 11. In some embodiments, the elution buffer has a pH that is from about 6 to about 7. In some embodiments, the elution buffer has a pH that is from about 7 to about 8. In some embodiments, the elution buffer has a pH that is from about 8 to about 9. In some embodiments, the elution buffer has a pH that is from about 9 to about 11. In some embodiments, the elution buffer contains salts, or detergents, especially ionic detergents or zwitterionic detergents, or other methods known in the art to decrease electrostatic binding to facilitate release of the DNA.

[0112] In some embodiments, the methods of the present disclosure can include assessing the cfDNA by performing quantitative or qualitative analysis, including but not limited to, nucleic acid sequencing, methylation analysis, PCR, qPCR, array analysis ddPCR, nanopore sequencing, and the like. In some embodiments, the method includes assessing the cfDNA obtained from the filter cartridge for one or more biomarkers, such as one or more biomarkers indicative of a disease or condition.

[0113] In some embodiments, the systems and methods of the present disclosure can also be used to obtain genomic DNA (gDNA). For example, gDNA can be collected and purified from one of more of the pre-filer cartridges described herein. In some embodiments, a cartridge contains a filter to capture cells. Following the capture of the cells, with or without washing toAIGN-43658.601 remove debris, a downstream cartridge can be used immediately under the cell capture cartridge, and cell lysis components (e.g., detergents) can be added to release the gDNA, which is captured on a downstream DNA capture filter. In some embodiments, the detergent is a nonionic detergent such as Tween or NP-40. In some embodiments, such as those in which the DNA binding filters action is not inhibited by an ionic detergent, the detergent can be ionic such as SDS.

[0114] In some embodiments, upon receipt in the lab, a pH indicator, such as a dye or dipstick, is applied to the filter or filter cartridge contents to QC the collection and binding to make sure that the proper pH was obtained. In some embodiments, the indicator contains further testing means to determine if the subj ect has a kidney, metabolic, or urinary' abnormality that may alter the DNA binding or may be important for assessing their health. For example, an indicator can contain one or more test strips to measure, without limitation, leukocytes, nitrite, urobilinogen, protein, pH, kidney function, specific gravity', ketone, bilirubin, free radicals, calcium, vitamin C (as corbate / as corbie acid), pH, glucose, microalbumin, creatinine, blood, specific gravity. In some embodiments, the results from the test strips are used to exclude some samples as unsuitable. In some embodiments, the results from the test strips confirm proper pH adjustment. In some embodiments, the results from the test strips are used to normalize the DNA content.

[0115] In some embodiments, a kit for laboratory use containing urine tester strips, the wash buffer, the elution buffer and instructions may be supplied. In some embodiments, this kit may also contain syringe(s) and collection tubes. In some embodiments, the user kit and laboratory kit are distributed together, in others they are directed to the appropriate address.

[0116] The methods and systems of the present disclosure can also include disposable / consumable components that are utilized for sample analysis and / or sample preparation. The system or kit may further contain additional containers or devices for use with the methods disclosed herein. Also within the scope of the present disclosure are kits that include any or all of the components of the systems described here. For example, in some embodiments, the kits include a pH measurement component, a control or calibrator component, a sample isolation component, a sample reservoir component, a sample purification component, and / or a sample dispensing device. In some embodiments, the kits also include calibration and / or control samples. Individual member components of the kits may be physically packaged together or separately. The components of the kits may be provided in bulk packages (e.g., multi-use packages) or single-use packages. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars.AIGN-43658.601 flexible packaging, cardboard, plastics and the like. In one embodiment of the present disclosure, the kit includes a DNA binding composition formulated as a powder and contained in a packet, which is included in a sample collection container. In some embodiments, the DNA binding composition comprises an acidifying agent, a buffer or buffering agent, a stabilization agent, and / or a preservative, all or any of which can be formulated as a powder and included in the packet.

[0117] In some embodiments, the lab portion of the kit includes liquid wash and elution buffers. In some embodiments, the lab portion includes powder packets, capsules, or tablets used to make wash and elution buffers. In some embodiments, the lab kit includes syringes and tubes for washing and sample collection. In some embodiments, the collection kit is designed for shipment to the subject and contains materials the subject needs including return shipment packaging and logs. In some embodiments, the lab kit is separate and contains materials comprising buffers to wash the filters and elute the DNA. In some embodiments, these may include protease on filters, or in solutions. In some embodiments, the lab kits are designed to handle 10, 25, 50 or 100 subject sample kits.

[0118] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kits. The materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols, which may include diagrams and pictures, for using the compositions, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kits or as a separate member component, either as a paper form or an electronic form which may be supplied on a computer readable memory' device or downloaded from an internet website, or as recorded presentation.

[0119] It is understood that the disclosed kits can be employed in connection with the disclosed methods.3. Materials and Methods

[0120] Quantification methods. Accublue High Sensitivity (HS) DNA kit (Biotium; Cat#: 31006) was used according to the manufacturer’s protocol. All chitosan extractions were run on Bioanalyzer (Bioanalyzer High Sensitivity (HS) DNA kit (Agilent; Cat# 5067-4626)) at a 1:5 dilution to allow for tolerable levels of Tris EDTA. Nanodrop was used according to the manufacturer’s instructions. NanoOrange Protein Assay (Invitrogen™; Cat# N6666) was used according to manufacturer’s protocol for 96-well plate.AIGN-43658.601

[0121] Chitosan Filter Preparation. Membrane filter was soaked in 1% w / v chitosan (Sigma Aldrich; Cat# 448869) solution in 50mM MES pH 5 buffer (Thermo Scientific; Cat# 28390) until completely saturated by gently swirling the filter in solution for approximately one minute. Chitosan-modified filter was placed on a mesh / grid surface and left to dry overnight at room temperature. Filters were rinsed five times with fresh 50mM MES pH 5 buffer and left to dry overnight at room temperature. Filters were subsequently removed from the grid surface carefully so that the filters did not stick to the mesh surface and tear. Chitosan- modified filters were stored in their original container until use.

[0122] Chitosan Extractions. For all chitosan-filter extractions (unless specified otherwise), the urine sample was brought to pH 5 using ascorbic acid (NOW; Cat# B001F0QVXY), then the full volume was pushed through the specified filter membrane in 150-250mL increments using a 200 mL or 250 ml syringes with an adapter to connect the syringe to the filter housing in a leakproof manner. Various experiments were done to optimize syringe volume as the diameter defines the force that can be conveniently applied by home users. While smaller syringes could supply more pressure, they required more refilling; in contrast, larger volume syringes were generally of too large a diameter to conveniently supply the force / sq area needed. The optimum syringe barrel size is about 45mm. The range may vary by user from 30 mm to 65 mm. Women, who shed more cells, may need 45 mm or smaller barrels, for some, 50 mm requires excessive force.

[0123] The prefilter was removed to take away any epithelial cells or other debris, and the filter holder reassembled with the chitosan-modified filter inside. In some embodiments, the prefilter is in a different cartridge and separation does not require opening the cartridge. The cfDNA loaded chitosan filter was washed by pushing 20mL 50mM MES pH 5 buffer through the filter by using a syringe. The DNA that remained on the filter was eluted by pushing 2.5mL 50mM Tris ImM EDTA pH9 buffer (Tris Base = Promega, Cat#: H5133; EDTA = Thermo Scientific, Cat#: J15694-AE) through the filter, directly into a 5mL conical. A schematic of this process can be seen in FIGS. 1A-1B, along with representative data from DNA capture experiments (FIG. 1C). In some embodiments, a second elution is performed and combined with the first. In some embodiments, the elutions are concentrated further. This may be done by a variety of methods known in the art, including alcohol precipitation, extraction of aqueous phase through dialysis, or other porous tubing, spin concentration or other methods known in the art. In some embodiments, the elution(s) totaling 2- 2.5 or 4-5 or up to 10 mis are pH adjusted and applied to another DNA capture filter with 1 / 10 or less area and subsequently eluted in 0.25 to 0.5 mis of elution buffer to concentrate the DNA sample.AIGN-43658.601

[0124] Choosing Filter Combination. Various 47mm membrane filters were inserted into the filter holder housing and fresh urine was pushed through with a 200mL syringe until either no more urine could be pushed through (due to a clogged filter) or there was no urine left. The membrane filters tested as a pre-filter were as follows: 1 ,6pm glass fiber (G / F) (Omicron; Cat#: 130047), 2.7pm G / F (Omicron; Cat#: 129047), 5pm G / F (ValuSep; Cat#: 26547), 5pm polyethersulfone(PES) (Sterlitech; Cat# PES5047100), 8pm PES (Sterlitech; Cat#: PES8047100), 0.2pm polyacrylonitrile(PAN) (Sterlitech; PAN0247100).

[0125] For certain experiments, including some on optimizing yield, urine was first pushed through the prefilter, and then the pre-filtered urine was pushed through the chitosan-modified filter separately (filters were not stacked). DNA was quantified by Accublue.

[0126] After individual filters were optimized, the pre-filter and chitosan-modified filters were stacked in the same filter holder to confirm adequate performance. To confirm recovery of DNA from the filter, a second elution was also performed on the same filters with 2.5mL of elution buffer. Accublue HS dsDNA assay was performed on the eluted samples to quantify DNA.10127] Filter Holder Comparison testing. Due to leakage in filter holders when filters were stacked, various methods were used to try to prevent leakage. A syringe was connected to the output of the filter holder to pull liquid through the filter so that there would be negative pressure in the filter housing rather than positive pressure induced by pushing. Filters were also dampened with 50mM MES pH 5 buffer when loading the filter holder to decrease space between filters and create a better seal around the gasket. The filter holders were then tested with water, and leakage was categorized as none (0% leakage by volume), mild (1-5% leakage), moderate (5-10% leakage), or severe (10%+ leakage).

[0128] Recovery Efficiency. A known amount of 50-mer DNA oligos (IDT), 50-mer DNA oligo sequence in Table 5 were spiked into MES buffer and a chitosan extraction was performed on the sample. DNA concentration was measured for the input, filtrate, and elution on Nanodrop. Blanks were measured for each respective buffer in the sample (MES vs Tris).

[0129] Stability of urine DNA on Filter. The same volume of pH 5 urine (adjusted to pH 5 using ascorbic acid - determined by urine test strip) was pushed through a new filter stack and holder for each of the following treatments: a) washed and eluted immediately, b) washed immediately and stored 48hrs, then eluted, and c) stored for 48hrs, then washed and eluted. To dry, filters were taken out of the filter holders and placed in a petri dish with desiccant. DNA was quantified by Accublue.AIGN-43658.601

[0130] An additional experiment was performed to expand on different fdter treatment options. The same volume of pH 5 urine (adjusted to pH 5 using ascorbic acid - determined by urine test strip) was pushed through a new filter stack for each of the following filter treatments: a) washed and eluted immediately, b) stored for 48hrs, then washed and eluted, c) rinsed with lOmL 70% isopropanol and stored for 48hrs, then washed and eluted, and d) rinsed with lOmL 91% isopropanol and stored for 48hrs, then washed and eluted. Filters were left in the filter holders (to replicate a consumer not being able to disassemble the filter holder for shipment) and stored in a sealed bag with desiccant. DNA was quantified using Accublue assay, and protein quantification was performed by NanoOrange protein assay to see if any of the filter treatments also resulted in increased / reduced protein contamination.

[0131] Replicability Study for Chitosan Modification. A 1% chitosan solution was centrifuged at HOOx rpm and the supernatant portion without any chitosan solids was saved. Several 2.7pm 47mm G / F filters were prepared with the chitosan supernatant solution. The same amount of DNA-spiked MES buffer was pushed through the filters made with complete chitosan preparation vs. supernatant only (5 of each). DNA quantification was performed using Accublue HS dsDNA assay. Statistical testing for differences in total DNA yield between groups was performed via unpaired two-tailed 2-sample t-test, and F-test was performed to check for equal variance between groups. Statistical analyses were executed in GraphPad Prism.

[0132] Week-long Stability Study. Fresh urine from a healthy female was pre-treated to pH 5 using ascorbic acid (confirmed by urine pH strip). The same volume of pH 5 urine was pushed through each filter for the following filter treatments: a) immediately washed and eluted, b) immediately stored to dry, c) 70% isopropanol rinse then stored to dry, and d) 91% isopropanol rinse then stored to dry. After the same volume of urine was pushed through each filter stack, the prefilter was removed. For treatments with the isopropanol rinse, the chitosan filters w ere kept in the same filter housings and lOmL of the appropriate isopropanol concentration was pushed through the filter. Forty 5mm-sized punches were taken from each filter (10 punches in each quadrant) and placed in a petri dish with desiccant to dry, keeping each punchout in its respective quadrant. At day 0, 1, 2, 4, and 7 time points, two punches from each quadrant w ere pooled for each sample (eight punches total) and processed (except the immediately w ashed and eluted filter - all punches for that filter treatment were processed immediately as five replicates - 8 punches per replicate). Each sample was processed by soaking the eight punches in a 6cm petri dish with 7mL 50mM MES pH 5 buffer for one minute with gentle swirling.AIGN-43658.601Filter punches were placed into a 1.5mL microcentrifuge tube and gently squeezed between the forceps and the side of the tube, and the excess MES buffer was removed via pipette and discarded. The tube was centrifuged for one minute at 2,000 x g and any further excess MES buffer was removed and discarded. 5()LLL IX TE pH8 buffer was added to the filter punches in the tube. Tubes were quickly spun down to get all filters into contact with the TE buffer at the bottom of the tube and samples were incubated for 2 minutes at room temperature. Filters were squeezed between a pipette tip and the sides of the tube to “wring” out as much elution buffer as possible. The sample elution was transferred to a clean microcentrifuge tube and was centrifuged for 2 minutes at 10,000 x g. Without disturbing the filter paper pellet, the supernatant was transferred to a new tube and stored at 4°C. DNA was quantified by Accublue HS dsDNA assay and protein contamination was quantified by NanoOrange HS Protein Assay. ANOVA was used to analyze differences in total weekly DNA yield and overall protein contamination between different treatments with Tukey’s adjustment for multiple comparisons. The rate of change in DNA yield over time (slope) for each filter treatment was analyzed via simple linear regression in GraphPad Prism to determine if any treatments resulted in significant DNA degradation over time.

[0133] All information on materials and reagents with manufacturer and catalog number is listed in the supplementary materials (Table 6).

[0134] UltraPrep Extraction. Below is an exemplary protocol for performing UltraPrep extraction. Buffers were made before starting the procedure (Table 4).

[0135] Prep frozen samples. For 1.5mL tubes: place frozen at RT for 30 minutes. For 15mL tubes or larger: place in a RT water bath, replacing water every 20-30 minutes until thawed (to decrease time it takes to thaw). Spin down tubes at 500 x g for 5 minutes. Transfer supernatant to a clean tube and avoid pellet / solids.

[0136] Table 4: Exemplary buffer compositions.AIGN-43658.601

[0137] Bring up the volume of sample to the nearest mL with NucleaseFree-H2O (NF- H2O). From this point on, use the nearest mL volume as the sample volume: For ImL sample, use 5mL conical. For2-3mL sample, use 15mL conical. For 4-10mL sample, use 50mL conical. Add lOuL Proteinase K per mL of starting sample. Add 650uL Digestion Buffer per mL of starting sample. Vortex. Incubate samples at 56°C for 60 minutes, vortexing every' 30 minutes. Add 3.3mL Binding Buffer per mL of starting sample. Add 40uL of 400nm magnetic silica beads to digested sample per mL of starting sample. Vortex. Incubate beads for 10 minutes at room temperature. Place sample tubes on magnetic rack until solution is clear, typically about 1-5 minutes depending on tube size, and discard the supernatant. Take tubes off magnetic rack and resuspend beads in 500uL Wash Buffer # 1 per mL of starting sample and transfer to clean 1.5mL tubes (for 4-10mL starting, stay in current tube). Vortex, spin down, place on magnetic rack, and discard supernatant. Take tubes off magnetic rack and resuspend beads in 500uL Wash Buffer #2 per mL of starting sample. Vortex, spin down, place on magnetic rack, and discard supernatant. Take tubes off magnetic rack, resuspend beads in lOOuL of 100% EtOH per starting mL of sample (now transfer to new 1.5mL tube if you started with 4- 1 OmL sample). Vortex, spin down, place on magnetic rack, and discard EtOH supernatant. Incubate samples at 37°C for 2-10 min with caps open until all ethanol in samples is evaporated. Add 15uL of lx TE Buffer per 1 mL of starting sample to each tube and resuspend beads by vortexing (but no less than 30 uL TE) but do not pipette up / down, as beads will get stuck in the pipette tip in this step. Place tubes on magnetic rack. Save the supernatant (DNA) in new tube labeled with sample ID number and “UP” for Ultraprep.

[0138] Solution Preparations - GITC solution preparation (for use in buffers). About 8M GITC Procedure (for 32 mL of 8M GITC for Digestion buffer): Measure out 30.36g of GITC in weigh boat. Measure 32mL of MilliQ H2O in graduated cylinder then pour into 250mL beaker with stir bar in bottom. Mark on the beaker where the water level hits (with stir bar in beaker) then pour the water back into the graduated cylinder. Making sure the stir bar is in beaker, add about half the powder into the beaker - careful not to go over the water level mark and leaving some room. Add water from graduated cylinder until you are still a little under 32mL water level mark. Put beaker on stirring hot plate with stirring on and temperature set between 30-100. Add in rest of GITC powder. Add in any more water needed to reach 32mL water level mark. Let solution sit stirring on heated plate until it is completely clear.|0139] Digestion buffer: 31.25 mL 8 M GITC; 12.5 mL Tween 20; 500 uL Tris buffer 1 M, pH 8; 2.5 mL 0.5 M EDTA; 3.25 mL NF-H2O.AIGN-43658.601

[0140] Binding buffer: 43.75 mL 8 M GITC; 45 mL 100% Isopropanol; 2.5 mL Tween 20;1 mL Tris buffer 1 M, pH 8; 200 uL 0.5 M EDTA; 7.55 mL NF-H2O.

[0141] Wash buffer 1: 18.75 mL GITC 8 M; 15 mL 100% Isopropanol; 2.5 mL Tween 20;10 mL Bis-Tris buffer 0.2 M, pH 6; 200 uL 0.5 M EDTA; 3.55 mL NF-H2O.|0142] Wash buffer 2: 2.5 mL Tris buffer 1 M, pH 8; 50 uL 0.5 M EDTA; 40 mL 100% Ethanol; 7.45 mL NF-H2O.

[0143] TE buffer: 150 uL Tris buffer 1 M, pH 8; 3 uL 0.5 M EDTA; 14.85 mL NF-H2O.

[0144] Table 5. Oligo DNA information and subsequent hybridization protocol.

[0145] DNA Oligos were purchased from IDT. Stock tubes were reconstituted at 500uM in low TE buffer (lOmM Tris, 0.1 mM EDTA).

[0146] Oligo Hybridization. Prepare hybridization mixture as follows: lOOuL primary oligo at 500 (uM); lOOuL complementary oligo at 500 (uM); lOOuL Tris buffer (100 mM Tris, 150 mM NaCl) autoclaved; mix gently by flicking and spin down. Incubate at 95 C for 5 minutes. Samples were slowly brought back down to 25C at -1C per minute (~70 minutes). Once at 25 C, tubes were stored in 4C overnight. Add 200uL volume of low TE buffer (lOmM Tris, 0. ImM EDTA) for a final 100 (uM) stock of hybridized oligo.[0147| Table 6. Reagents and materials information, including manufacturer and catalog number.AIGN-43658.601

[0148] Table 7. Thickness, weight, and chitosan capacity of 1.6um, 2.7um, and 5um G / F filter membranes. N=20 for each filter. The chitosan capacity is determined by weighing dried filters before and after chitosan binding.4. Examples

[0149] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0150] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.Example 1

[0151] Filter Stack Optimization. To develop a filter stack that would be capable of handling a full volume of a bladder void, including urine that has epithelial cells and other potential precipitates, numerous experiments were required. In this example, urine from a healthy human female was used to optimize the filter stack as urine from males has much few er cells that mayAIGN-43658.601 block the filters. First, the top two prefilters were selected from various filters with different pore sizes and materials based on urine flowthrough volume. The two prefilters with the highest average flowthrough volumes were the 2.7pm G / F filter (87mL) and the 5pm G / F filter (400mL) (Table 8).

[0152] Table 8. Various pre-filter options tested with urine from healthy human female. Different materials include PAN, G / F, and PES. Average urine volume indicates the average of the maximum urine volume that could be pushed through until either the filter got clogged or there was no urine left.

[0153] Combinations of pre-filters and chitosan-modified filters were then used in succession (filters w ere not stacked) to determine which pre-filter / chitosan-filter combination resulted in the highest DNA yield while being able to maintain a flowthrough volume below the median void volume of 220mL. The top filter combination based on both flowthrough and DNA yield was the 5pm G / F pre-filter with 2.7pm G / F chitosan-modified filter with 330mL of urine flowthrough and a yield of 1.86ng of total DNA (Table 9; FIGS. 2A-2B).

[0154] Table 9. Pre-filter (non-modified) and chitosan filter combinations (in succession, not physically stacked) performance for maximum urine volume and total DNA yield. All filters are G / F. Average urine volume indicates the average of the maximum urine volume that could be pushed through until either the filter got clogged or there was no urine left. DNA was quantified using AccuBlue HS dsDNA kit.|0155] The top two performing filter combinations from FIG. 2 were then stacked in the same filter holder and used to process urine to determine if it still performed well, and a secondAIGN-43658.601 elution step was performed on the filter stack immediately after the first elution. This increased total DNA yield was 660% higher with the increased urine volume allowed by the 5um G / F prefilter (FIG. 3). It was also suggested that 2.5mL of elution buffer might need to be increased to elute all the DNA that was captured on the filter (FIG. 3).10156] However, after filters were physically stacked on top of each other in the filter holder, the filter holders leaked urine. To eliminate leakage, three different filter holders were obtained, and the two following methods were tested: a) pulling the output from the bottom of the filter holder to create negative pressure, and b) loading filters dampened with 50mM pH 5 MES buffer into the filter holder to create a better seal around gasket. The filter holders were tested with water, and leakage was categorized as none (0% leakage by volume), mild (1-5% leakage), moderate (5-10% leakage), or severe (10%+ leakage). It was determined that the best method for alleviating leaking was to load the filters into the filter holder while damp, as there was no longer any leakage (Table 10). The clear filter holder can have smooth or ridged edges and can be used to stack filters without leakage. Tightening methods have been developed that allow alternative cartridges to be used without leakage.

[0157] Table 10: Filter holder leakage with stacked membrane filters and various methods used to try to prevent leakage. The filter holders were tested with water, and leakage was categorized as none (0% leakage by volume), mild (1-5% leakage), moderate (5-10% leakage), or severe (10%+ leakage). Methods to improve leakage include pulling the output from the bottom of the filter holder instead of pushing into the top of the filter holder or loading dampened filters to screw filter holder tighter.Example 2

[0158] Recovery Rate of Chitosan System. Because urine has contaminants including proteins and other salts that may affect DNA quantification readings, a known amount of 50- mer oligos were spiked into MES buffer and processed, and DNA concentration was measured for the input, filtrate, and elution on Nanodrop to determine % recovery of the chitosan system. This chitosan filter system had an 85% recover}' (FIG. 4).AIGN-43658.601Example 3

[0159] DNA Stability on Filter. Chitosan has previously been shown to have limited solubility in MES buffer. To examine if the undissolved hydrogel and particle were needed to obtain good ucfDNA binding, the supernatant portion was separated out from the chitosan solids, and the supernatant was used to prepare chitosan fdters as described above. The same amount of spiked 50-mer oligos in MES buffer was processed through chitosan-supernatant filters and complete-chitosan solution modified filters (n=5 for each). DNA quantification of the eluates was performed using the AccuBlue HS DNA kit. There was no significant difference between DNA recovery between chitosan-supernatant filters and complete-chitosan solution modified filters based on unpaired two-tailed 2-sample t-test assuming unequal variances (p-value = 0.3411), and there was no significant difference in the variability between filters of the same type based on F-test for equal variance (p = 0.1124; FIG. 5).

[0160] Due to the differing trends in the yield between '‘immediately washed and eluted” and the “stored-washed-eluted” filters in the first two stability experiments, an additional experiment was performed over one week to reflect possible shipping times for the filter to reach the lab. The same volume of pH 5 urine was pushed through each filter for the following filter treatments: a) immediately washed and eluted, b) immediately stored to dry, c) 70% isopropanol rinse then stored to dry, and d) 91% isopropanol rinse then stored to dry. To eliminate variation between filters from the same treatment over time in the third stability experiment, ten 5mm punches were taken from each quadrant of each filter so that two randomly selected punches from each quadrant could be pooled together for each time point of 0, 1, 2, 4, and 7 days for an accurate representation of all quadrants on the same filter. According to the linear regression comparison, none of the filters resulted in significantly different DNA yield or protein contamination over time (FIGS. 6A-6B; slopes and p-values listed in Table 2). Filters that were rinsed with 70% and 91% isopropanol had significantly lower yield overall (p = 0.0057 and 0.0293, respectively; FIG. 6C; Table 2). To test whether the higher yield of the '‘stored-washed-eluted” filter compared to the isopropanol rinsed filters was due to increased protein contamination, protein content was assayed with NanoOrange. No significant difference in protein contamination was found between the groups according to ANOVA with Tukey’s multiple comparisons (FIG. 6D; Table 3).

[0161] Table 4. Comparison of simple linear regressions between different filter treatment groups (immediately washed and eluted, stored-washed-eluted, 70% isopropanol-stored- washed-eluted, and 91 % isopropanol-stored- washed-eluted).AIGN-43658.601

[0162] Table 5: ANOVA results for overall DNA yield and protein contamination in weeklong stability study between different filter treatment groups (immediately washed and eluted, stored-washed-eluted, 70% isopropanol-stored-washed-eluted, and 91% isopropanol- stored-washed-eluted).AIGN-43658.601AIGN-43658.601Example 4

[0163] Approximately 180mL of urine was passed through the membrane filter, which consists of a 5-micron prefilter sitting on top of a 2.7-micron chitosan-treated filter. Urine flowthrough was discarded and the prefilter was removed. The cartridge was reassembled, and the urine-soaked chitosan filter was washed using an acidic buffer (20 mL 50mM MES / lmM EDTA pH 5) and flowthrough was discarded. Lastly, a basic buffer (2.5 mL 50mM Tris / lmM EDTA pH 9) was used to elute the ucfDNA bound to the chitosan filter and collected in a 5 mL Eppendorf tube. The wash and elution steps were performed using smaller graduated syringes (sizes 60mL for the MES buffer wash and 20 mL for the elution) that were attached to the cartridge with a plastic adapter.

[0164] The 2.5mL eluates were then quantified by fluorometry using the AccuBlue® High Sensitivity dsDNA Quantitation Kit (cat: 31006) from Biotum, following the protocol provided with the kit. The total concentration of DNA within the eluate was 485.3 ng / mL, though subsequent analyses are needed to measure the amount of ucfDNA within that sample.

[0165] Following quantification, Agilent’s 2100 BioAnalyzer instrument was used along with software to analyze the size of the DNA captured and to assess whether ucfDNA was isolated. All samples were diluted 1 :5 to adjust the concentration of the 50mM Tris / lmM EDTA pH 9 buffer to 1 OmM Tris, which is required for the instrument to give a proper reading. luL of each diluted sample was loaded onto the BioAnalyzer chip using the Agilent High Sensitivity DNA Kit (cat: 5067-4626) according to the protocol provided with the kit. The electropherogram generated by the 2100 Expert software (version B.02.11.SI824) showed the calculated size and concentration of DNA within the sample (FIG. 7).

[0166] The data from the BioAnalyzer analysis showed that the diluted eluate extracted from the 180mL urine sample contained 89.74 pg / uL of 80 bp DNA fragments, which is theAIGN-43658.601 average size of ucfDNA. This calculates a total of 1.12 ug of ucfDNA within a 2.5 mL sample, before the 1 :5 dilution. These results indicate that the membrane filter is capable of recovering 6.23 ng of ucfDNA per milliliter of urine loaded onto the filter.

[0167] This DNA was used in real time PCR (rtPCR). Primers for beta actin, a “single copy” gene, as illustrated in the Table 6 (below) produced a signal present at a cycle value of <38, which was very distinct from the blank. The corresponding negative control blank was >40. Further, when run on a Bioanalyzer capillary electrophoresis beta actin amplification was seen to produce the expected 41 bp fragment, further documenting successful PCR.

[0168] Table 6: Results of PCT testing.

[0169] This eluted cfDNA was sent to the University of Colorado sequencing core, where QC tests were performed, including TapeStation, Qubit and qPCR, and Ovation® Ultralow System V2 was used to make the library followed by Illumina NovaSeq X NGS (50 million reads). All QC measurements were satisfied. Library preparation and sequencing was then performed. As shown in Table 7 (below), the cfDNA isolated using the methods of the present disclosure performed comparably to that from blood. Some decrease was expected due to the shorter size; however, the standard software used compensated for this and accurate sequence data was obtained. Since the blood DNA is larger than the urine DNA, due to the glomerular filtration limit, the similarity of the Q30 and MQS between the blood and urine is excellent. The 90% Q30 is also excellent; Illumina criteria is >70-80%.

[0170] Table 7: Results of NGS sequencing.Example 5

[0171] Approximately 250ml of urine was passed through the membrane filter, which consists of a 5-micron prefilter sitting on top of a 2.7-micron chitosan-treated filter. In thisAIGN-43658.601 example, the chitosan had been bound to a Whatman Fusion 5 filter paper. This is a variant of glass fiber filter and the methods described in this example may be used on the standard Omicron filters used above. Is this example, filters were first washed on a rocker in 100 ml 0.2 M NaOH for 8 minutes, washed 3 times with 200 ml deionized water and dried under vacuum at 45°C. The filters were soaked in 0.25% w / v chitosan dissolved in 0.1 M acetic acid for 16 hours at room temperature on a rotary shaker. The membranes were washed in deionized water 5 times and dried under vacuum at 45°C.

[0172] Urine flowthrough was discarded and the prefilter was removed. The cartridge was reassembled, and the urine-soaked chitosan filter was washed using an acidic buffer (20 mL 50mM MES / lmM EDTA pH 5) and flowthrough was discarded. Lastly, a basic buffer (2.5 mL 50mM Tris / lmM EDTA pH 9) was used to elute the ucfDNA bound to the chitosan filter and collected in a 5 mL Eppendorf tube. The wash and elution steps were performed using smaller graduated syringes (sizes 60mL for the MES buffer wash and 20 mL for the elution) that were attached to the cartridge with a plastic adapter.

[0173] The 2.5mL eluates were then quantified by fluorometiy using the AccuBlue® High Sensitivity dsDNA Quantitation Kit (cat: 31006) from Biotum, following the protocol provided with the kit. As above, larger (e g., pg) amounts of trans-renal DNA were captured and eluted. Again, they were successfully used for PCR and sequencing.Example 6

[0174] In an at-home setting, approximately 250ml of urine was passed through the membrane filter, which consists of a 5-micron prefilter sitting on top of a 2.7-micron chitosan filter. The chitosan w as bound to silica beads and the beads w ere trapped on the surface of, or embedded in, a glass fiber or cellulose filter. Chitosan-coated silica beads were fabricated as described previously. Briefly. 30 pm silica beads (0.1 g) were suspended in 1 mL NaOH, vortexed, and sonicated for 2 min. The suspension was then mixed on a LabQuake® rotator (Bamstead Thermolyne Corporation Dubuque, IA) for 30 min, centrifuged, the supernatant removed, and the beads washed with water until at a neutral pH was reached. The beads were then rinsed with ethanol and acetone and placed in a 60-70 °C oven for 5-10 min to dry. The beads were cleaned with a piranha solution (1 : 1 H2SO4:H2O2) for 30 min, the piranha solution removed and the beads were again w ashed with w ater, until a neutral pH was reached, then washed with ethanol and acetone, and dried in an oven (60-70 °C) for 1-2 hrs or until dry. A solution (500 pL in water) containing 20 mg of chitosan oligosaccharide and 2 pL of (3-(3- Glycidyloxypropyl)trimethoxy silane (GPTMS) was vortexed, sonicated for 2 min. and mixedAIGN-43658.601 using the LabQuake® rotator for 2 hrs. This solution was then added to the 30 gm silica beads and the suspension mixed for 18 hours using the LabQuake® rotator. The suspension was centrifuged, the supernatant removed, and the beads rinsed with 0.1 M HC1. The suspension was centrifuged and the supernatant of HC1 removed and the beads dried in an oven (60-70 °C) for 2 hrs or until dry. Lastly, the beads were rinsed with water, ethanol, and acetone and placed in the oven for 30 min or until neutral pH reached.

[0175] Urine flowthrough was discarded. The urine-soaked chitosan filter cartridge was sent to the lab. Subsequently the prefilter was removed. The cartridge was reassembled, and then was washed using an acidic buffer (20 mL 50mM MES / lmM EDTA pH 5) and flowthrough was discarded. Lastly, a basic buffer (2.5 mL 50mM Tris / lmM EDTA pH 9) was used to elute the ucfDNA bound to the chitosan filter and collected in a 5 mL Eppendorf tube. The wash and elution steps were performed using smaller graduated syringes (sizes 60mL for the MES buffer wash and 20 mL for the elution) that were attached to the cartridge with a plastic adapter.

[0176] The 2.5mL eluates were then quantified by fluorometry using the AccuBlue® High Sensitivity dsDNA Quantitation Kit (cat: 31006) from Biotum, following the protocol provided with the kit. As above larger amounts (e.g., pg) of trans-renal DNA were captured and eluted. Again, they were successfully used for PCR and sequencing.Example 7

[0177] Approximately 250ml of urine was passed through the membrane filter, which consists of a 5-micron prefilter sitting on top of a 2.7-micron chitosan-treated filter. In this example, the chitosan had been bound to the filter using the method described above except that instead of activating the filter with the toxic Piranha acid solution the filter was activated with ozone. The ozone was created by aJHH-3Y030-A ozone generator and the filters were placed on wire racks to allow gas penetration for 2 hours. From 10 min to 10 hours can be used.

[0178] Urine flowthrough was discarded and the prefilter was removed. The cartridge was reassembled, and the urine-soaked chitosan filter w as washed using an acidic buffer (20 mL 50mM MES / lmM EDTA pH 5) and flowthrough was discarded. Lastly, a basic buffer (2.5 mL 50mM Tris / lmM EDTA pH 9) was used to elute the ucfDNA bound to the chitosan filter and collected in a 5 mL Eppendorf tube. The wash and elution steps were performed using smaller graduated syringes (sizes 60mL for the MES buffer wash and 20 mL for the elution) that w ere attached to the cartridge with a plastic adapter.|0179] The 2.5mL eluates were then quantified by fluorometiy using the AccuBlue® High Sensitivity dsDNA Quantitation Kit (cat: 31006) from Biotum, following the protocol providedAIGN-43658.601 with the kit. As above, larger amounts of trans-renal DNA were captured and eluted. Again, they were successfully used for PCR and sequencing.Example 8|0180] Approximately 250ml of urine was passed through the membrane filter, which consists of a 5-micron prefilter sitting on top of a column of chitosan bound silica particles, the chitosan having been bound by the methods described by the GTMPS method above. The column was attached to the outlet of the prefilter cartridge, and can be from 1 mm to 50 mm, with additional ranges of 3 mm to 30 mm, and / or 5 mm to 25 mm. The column has an inert support to hold the silica in place. The beads may be from nanometer to millimeter size. Paramagnetic particles may be used in some applications, and typically, they are 0.5 to 10 micrometers in size while non-magnetic particles can be from 150-600 micrometers in size. Non-magnetic beads of 500 microns size were used in this example. Urine flowthrough was discarded and the prefilter was removed. The urine-soaked chitosan column was sent to the lab and then washed using an acidic buffer (20 mL 50mM MES / lmM EDTA pH 5) and flowthrough was discarded. Lastly, a basic buffer (1 mL 50mM Tris / lmM EDTA pH 9) was used to elute the ucfDNA bound to the chitosan filter and collected in a 5 mL Eppendorf tube. The wash and elution steps were performed using smaller graduated syringes (sizes 60mL for the MES buffer wash and 20 mL for the elution) that were attached to the cartridge with a plastic adapter.

[0181] The 2.5mL eluates were then quantified by fluorometry using the AccuBlue® High Sensitivity dsDNA Quantitation Kit (cat: 31006) from Biotum, following the protocol provided with the kit. As above larger amounts of trans-renal DNA were captured and eluted. Again, they were successfully used for PCR and sequencing.Example 9|0182| Approximately 250ml of urine was passed through the membrane filter, which consists of a 5-micron prefilter sitting on top of a column of positively charged cationic material. There are numerous suitable materials that can be used as charged cationic material, including but not limited to, strong anion exchangers such as Q Sepharose (GE Healthcare), DEAE Sepharose, Mono Q, Source Q, and Q Sephadex; weak anion exchangers such as DEAE cellulose, DEAE Sephadex, and ANX Sepharose. Quaternary Ammonium (Q) based resins can also be used, such as Dowex, Amberlite, and Bio-Rex. Polyethyleneimine (PEI) based materials can also be used, such as PEI-functionalized magnetic beads, and PEI-cellulose.AIGN-43658.601Other commercial resins that can be used include Capto Q (GE Healthcare), Fractogel EMD (Merck), Toyopearl, and Macro-Prep (Bio-Rad).

[0183] In this example, Q-Sepharose was used; following binding of the DNA, the resin was washed with 10 rnL low-salt buffer in this case 0.3 mol / L LiCl and 10 mmol / L NaOAc, pH 5.5, but a variety of buffers can be used. DNA was eluted with from 0.5 to 1.5 ml high-salt buffer, in this case 2 mol / L LiCl and 10 mmol / L NaOAc. pH 5.5, but a variety of buffers can be used. This eluate was washed and concentrated in Qiagen mini -spin column. In some cases, further purification may be used, for example the eluate may be mixed with 2-5 mL 95% ethanol and applied incrementally to a QIAquick column (Qiagen; 800 x g?30 seconds). The column was washed twice with 0.5 mL 2 mol / L LiCl in 70% ethanol and twice with 0.5 mL 75 mmol / L KOAc, pH 5.5, in 80% ethanol (800 x g?30 seconds). The column was dried (20,000 x g, 3 minutes) and DNA was eluted (20,000xg, 2 minutes) in 106 pL elution buffer (Qiagen). |0184] The eluates were then quantified by fluorometry using the AccuBlue® High Sensitivity dsDNA Quantitation Kit (cat: 31006) from Biotum, following the protocol provided with the kit. As above larger amounts of trans-renal DNA were captured and eluted. Again, they were successfully used for PCR and sequencing.

[0185] The tables below contain examples of materials with different charges at different pHs. Given the above examples and description, one skilled in the art may use these materials in an analogous manner to purify DNA in a trans-flow manner described in this invention.

[0186] Table 8: Exemplary resin materials.

[0187] Table 9: Exemplary resin materials.AIGN-43658.601

Claims

AIGN-43658.601CLAIMSWhat is claimed is:

1. A method for obtaining cell-free DNA (cfDNA) from a sample from a sub ect comprising passing the sample through a filter cartridge comprising at least one filter, wherein the sample comprises a pH < 5, and wherein cfDNA present in the sample binds the at least one filter.

2. The method of claim 1, wherein flowthrough from the sample is removed.

3. The method of claim 1 or claim 2, wherein the method comprises adjusting the pH of the sample to a pH of < 5 prior to passing the sample through the filter cartridge.

4. The method of any one of claims 1 to 3, wherein the method comprises adjusting the pH of the sample to a pH of < 2 prior to passing the sample through the filter cartridge.

5. The method of claim 3 or claim 4, wherein the pH of the sample is adjusted using a DNA binding composition.

6. The method of any one of claims 1 to 5, wherein the DNA binding composition is formulated to adjust the pH of the sample to a pH of < 5.

7. The method of claim 6, wherein the DNA binding composition comprises one or more of acidic forms of ascorbate, acetate, citrate, borate, formate, maleate, tartrate, lactate, glycine, potassium hydrogen phthalate buffer and / or MES buffer.

8. The method of claim 6 or claim 7, wherein the DNA binding composition comprises a DNA stabilization agent.

9. The method of claim 8, wherein the DNA stabilization agent comprises EDTA.

10. The method of any one of claims 1 to 9, wherein the DNA binding composition is formulated as a powder or a solution.AIGN-43658.60111. The method of any one of claims 1 to 10, wherein the sample comprises one or more bodily fluids from the subject selected from urine, whole blood, blood plasma, serum, ascites, saliva, lymph, pleural effusion, semen, and cerebral spinal fluid.

12. The method of any one of claims 1 to 11, wherein the sample comprises water from the natural environment, or water from a manmade or industrial water source.

13. The method of any one of claims 1 to 12, wherein the sample comprises a volume from about 20 mL to about 1000 mL, and wherein the sample is passed through the filter cartridge immediately after the sample is obtained and / or pH adjusted.

14. The method of any one of claims 1 to 13, wherein the cfDNA comprises circulating tumor DNA (ctDNA), trans-renal DNA (trDNA), cell-free fetal DNA (cffDNA), cell-free mitochondrial DNA (ccf mtDNA). DNA from a micro-organism or infectious organism, donor-derived cell-free DNA (dd-cfDNA), and / or other trans-renal DNA (trDNA).

15. The method of any one of claims 1 to 14, wherein the at least one filter in the filter cartridge comprises a DNA-binding filter.

16. The method of any one of claims 1 to 15, wherein the at least one filter in the filter cartridge comprises a polyelectrolyte comprising a positive charge at the pH to which the sample has been adjusted.

17. The method of claim 16, wherein the positively charged poly electrolyte comprises chitosan, or a variant or derivative thereof.

18. The method of claim 17, wherein the at least one filter comprises a chitosan capacity ranging from about 10 mg to about 100 mg.

19. The method of any one of claims 1 to 18, wherein the at least one filter comprises a pore size ranging from about 1.0pm to about 5.0pm.AIGN-43658.60120. The method of any one of claims 1 to 19, wherein the at least one filter comprises polyacrylonitrile, glass fiber, and / or polyethersulfone.

21. The method of any one of claims 1 to 20, wherein the at least one filter comprises a pre-filter.

22. The method of claim 21, wherein the pre-filter comprises a pore size ranging from about 0.2pm to about 10pm.

23. The method of claim 21 or claim 22. wherein the pre-filter comprises polyacrylonitrile, glass fiber, and / or poly ethersulfone.

24. The method of any one of claims 1 to 23, wherein the at least one filter comprises a DNA-binding filter and a pre-filter.

25. The method of claim 24, wherein the DNA-binding filter and the pre-filter are stacked.

26. The method of claim 24, wherein the DNA-binding filter is contained in one cartridge and the pre-filter contained in a separate cartridge.

27. The method of claim 26, wherein the DNA-binding filter cartridge and the pre-filter cartridge are stacked.

28. The method of any one of claims 1 to 27, wherein the sample is passed through a prefilter before being passed through the at least one filter in the filter cartridge.

29. The method of any one of claims 1 to 28, wherein the at least one filter in the filter cartridge comprises a digestive enzyme.

30. The method of claim 29, wherein the digestive enzy me is a protease.

31. The method of any one of claims 1 to 30, wherein the subject is a human.AIGN-43658.60132. The method of claim 31 , wherein the sample is obtained from the human, and wherein the sample comprises urine from one or more voids.

33. The method of claim 32, wherein the one or more voids are passed through the filter cartridge by the human subsequent to urination.

34. The method of claim 33, wherein the at least one filter in the filter cartridge comprises a positively charged poly electrolyte formulated to bind and / or stabilize the cfDNA from the urine within about 0.5 minutes to about 10 minutes.

35. The method of any one of claims 31 to 34, wherein the human is experiencing, has experienced, or is at risk of experiencing, one or more of the following: cancer, transplanted organ, pregnancy, physical exercise training, heart attacks or other cardiovascular conditions, stroke, neurologic disease, Alzheimer's Disease, psychiatric conditions, autoimmune disease, sepsis, and / or urologic disease.

36. The method of any one of claims 31 to 35, wherein the human sends the filter cartridge to a lab for processing after passing the sample through it and discarding what is not retained.

37. The method of any one of claims 31 to 36, wherein the human passes a wash buffer and / or a stabilization buffer through the at least one filter in the filter cartridge before sending the cartridge to a lab for processing.

38. The method of any one of claims 31 to 37, wherein the filter cartridge is stored or in transit for no more than about 7 days prior to processing.

39. The method of any one of claims 31 to 38. wherein the filter cartridge is stored or in transit for about 8 hours, about 16 hours, about 32 hours, about 64 hours, about 128 hours, or about 1 week prior to processing.

40. The method of any one of claims 1 to 39, wherein the method further comprises processing the filter cartridge to obtain the cfDNA, wherein the cfDNA comprises circulatingAIGN-43658.601 tumor DNA (ctDNA), trans-renal DNA (trDNA), cell-free fetal DNA (cffDNA), cell-free mitochondrial DNA (ccf mtDNA). DNA from a micro-organism or infectious organism, donor-derived cell-free DNA (dd-cfDNA), and / or other trans-renal DNA (trDNA).

41. The method of claim 40, wherein the processing comprises passing a wash buffer or a stabilization buffer through the at least one fdter in the fdter cartridge.

42. The method of claim 41 , wherein the wash buffer and / or the stabilization buffer comprises a pH of < 6.

43. The method of any one of claims 40 to 42. wherein the processing comprises treating the one or more filters with a protease.

44. The method of any one of claims 40 to 43, wherein the processing comprises eluting the cfDNA from the one or more filters.

45. The method of any one of claims 40 to 44, wherein the processing comprises eluting the cfDNA from the one or more filters using a buffer having a pH from about 6 to about 11.

46. The method of claim 44 or claim 45. wherein the cfDNA is further purified and / or concentrated.

47. The method of any one of claims 40 to 46, wherein the processing is performed at a lab.

48. The method of claim 46, wherein the method further comprises assessing the cfDNA for one or more biomarkers.

49. The method of any one of claims 1 to 48, wherein the flowthrough comprises histone- bound DNA.

50. The method of claim 49, wherein the histone-bound DNA in the flowthrough is further processed and / or assessed for one or more biomarkers.AIGN-43658.60151. The method of claim 49 of claim 50, wherein the histone-bound DNA in the flowthrough is further processed by dissociating the histones from the DNA and assessing the histone-free DNA for one or more biomarkers.

52. A method for selectively obtaining urothelial cell-free DNA (cfDNA) from a urine sample from a subject comprising buffering the urine sample to a pH at or between a pH of 0.5 and a pH of 3, and passing the buffered urine sample through a filter cartridge comprising at least one filter, thereby selectively binding the cfDNA present in the urine sample to the at least one filter.

53. A method for selectively obtaining trans-renal DNA (trDNA) from a urine sample from a subject comprising buffering the urine sample to a pH at or between a pH of 3 and a pH of 6, and passing the buffered urine sample through a filter cartridge comprising at least one filter, thereby selectively binding the trDNA present in the urine sample to the at least one filter.

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