WO2018057938A1

WO2018057938A1 - Comprehensive system for collecting, processing, processing and analyzing cell free nucleic acids in urine

Info

Publication number: WO2018057938A1
Application number: PCT/US2017/053031
Authority: WO
Inventors: Nicholas Nelson; Vlada MELNIKOVA; Andrea BACCONI; Barbara EATON; Karsten Schmidt; Janel Dockter; Walter BALFOUR; Adam CRAFT; Zachary FANNING
Original assignee: Trovagene, Inc.
Priority date: 2016-09-23
Filing date: 2017-09-22
Publication date: 2018-03-29

Abstract

Provided is a collection container for collecting a sample of a fluid. The collection container comprises the following components: a cup for collecting the sample, the cup comprising an open top, and a closed bottom; a lid that engages the open top and seals the cup; and an additive. In these embodiments, when the lid engages the open top, the additive is released into the sample. Also provided is a method of collecting a fluid sample. The method comprises inserting the sample into the above collection container and engaging the lid onto the open top, releasing the additive into the sample. Additionally provided is an adaptor for suspending a column above the bottom of a test tube. Also provided is a method of accelerating the passage of a fluid through a chromatography column. Further provided are methods of isolating cell-free nucleic acids from a urine sample from a subject. Additionally provided is a method of verifying the identity of a subject as being a source of a sample of a bodily fluid. Kits for practicing any of the above methods are also provided.

Description

COMPREHENSIVE SYSTEM FOR COLLECTING, PROCESSING AND ANALYZING

CELL FREE NUCLEIC ACIDS ΓΝ URINE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/398,630, filed September 23, 2016, U.S. Provisional Application No. 62/453,455, filed February 1, 2017, and U.S. Provisional Application No. 62/413,297, filed October 26, 2016, all of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present application generally relates to systems for collecting, preserving, processing and analyzing nucleic acids in urine. More specifically, this application provides integrated devices and methods for collecting, preserving, processing and analyzing nucleic acids in urine.

(2) Description of the related art

Various nucleic acids are present extracellularly in biological fluids such as blood, placental fluid and urine. These nucleic acids are from, e.g., genomic DNA from tissue and blood cells (including DNA having mutations associated with cancer, called circulating tumor DNA [ctDNA]), DNA from infectious agents, transplants, fetal DNA in maternal fluids (collectively, cell free DNA (cfDNA)), and RNA including miRNA (US Patent 8,486,626). The half-life of systemic ctDNA is 115 - 140 minutes (Diehl et al, 2008; Lo et al, 2003).

Circulating DNA is cleared from the plasma into the glomerular filtrate and remains in the urine. About 10% of the systemic ctDNA is believed to be cleared via the transrenal route (Tsui et al., 2012).

Urinary transrenal DNA (trDNA) and miRNA provide a systemic nucleic acid footprint and can be analyzed to determine status of a patient by analyzing non-host urinary nucleic acids deriving from, e.g., a transplant, a fetus, or a pathogen (US Patent RE39920E1), miRNA (US Patent 8,486,626; US Patent Publication 2012/0225925; PCT Publication WO 2012/089630) or mutations in host proteins relating to cancer (US Patent 8,501,924; US Patent Publications 2016/0115556 and 2013/0164746) or other diseases (US Patent Publication 2015/0132256). Total urinary DNA is driven by total urinary tract (UT) cell count; there is no direct correlation of total urinary DNA with systemic DNA. However, high UT cell counts can negatively influence assay sensitivity due to presence of apoptotic and non-apoptotic DNA from those cells. Generally, cell count in urine correlates with whether it is sampled from first-void (high) or mid-stream (low). See Manoni et al. 2012.

The ability to isolate, detect and characterize cf nucleic acids in bodily fluids are powerful innovations in the field of cancer and other gene-associated diseases, and particularly useful as indicators for the detection and monitoring of diseases and abnormal conditions which may be present in areas of the body other than where the fluid sample was obtained (Al-Yatama et al. 2001 ; Utting et al., 2002).

Cell-free nucleic acids in bodily fluids are often present at very low concentrations and can be fragmented to an average size of 150-300 bp, as shown in FIG. 9. In plasma, cell-free DNA has a bimodal distribution, with peaks at about 167 and 340 bp, corresponding to the length of DNA around mono- and dinucleosomes (Chandrananda et al., 2015). Cell-free DNA in urine appears to be smaller than plasma cfDNA, with a reported size range of around 35-350 bp (FIG. 9)·

Because of the small sizes and low concentrations, methods for cf nucleic acid isolation from bodily fluids is more difficult than other nucleic acid isolation procedures and generally require the use of hazardous chemicals, or extra drying steps, are difficult to automate, require a large minimal quantity of starting sample for sufficient detection sensitivity, and/or achieve low yield and preferentially isolates high molecular weight DNA and RNA. Such high molecular weight yields interfere with testing for genomic DNA sequences not originating in the bodily fluid (e.g., blood cell DNA and RNA in plasma, or DNA and RNA from urinary tract cells in urine). This is particularly true with urine, where all genomic DNA (except from the urinary tract) must pass the glomerular size barrier, and so are necessarily small.

In the first step toward analyzing DNA in a urine sample, the collection of the sample should be optimized by the immediate addition of a preservative to prevent nuclease degradation of the cell free nucleic acids. To that end, it is useful to have the additive as part of the sample collecting device configured in such a way that the additive is automatically added to the sample when a cover is put on the sample container. See, e.g., US Patents 4,473,530; 4,927,605; 5,496,736; 6,138,821; 6,935,493; 8,221,381; 8,642,323; 8,714,808; and 9,079,181; and US Patent Application Publications 2004/0170536; 2008/0293156; 2009/0155838; 2013/0164738; 2013/0006146; 2014/0120531; and 20150056716. The present invention provides, inter alia, designs for containers that add an additive to the sample when the container is closed. These designs are improvements over previously known containers, and are part of the integrated, comprehensive system for analyzing nucleic acids in urine provided herewith.

Anion exchange columns are useful for isolating cf nucleic acids. See US Patent

9,163,229. That patent describes binding of the urine sample onto Q Sepharose® in columns, with elution of the cf nucleic acids in a 2.0 M NaCl solution, and the collection of fractions of the eluate, and retention of the fractions that have the transrenal nucleic acids.

There is a need for additional methods whereby a greater sensitivity and or enrichment of target sequences can be achieved with efficiency and ease and which are integrated with the collection device. The present invention addresses this need by providing a method for isolating assay-ready nucleic acids directly from whole or concentrated urine.

BRIEF SUMMARY OF THE INVENTION

Provided is a collection container for collecting a sample of a fluid. The collection container comprises the following components:

a cup for collecting the sample, the cup comprising an open top, and a closed bottom; a lid that engages the open top and seals the cup; and

an additive. In these embodiments, when the lid engages the open top, the additive is released into the sample.

Also provided is a method of collecting a fluid sample. The method comprises inserting the sample into the above collection container and engaging the lid onto the open top, releasing the additive into the sample.

Additionally provided herewith are methods for isolating and purifying cf nucleic acids from bodily fluids such as urine and plasma that is much simpler and easier to automate than previous methods. Thus, in some embodiments, a method of isolating cell-free (cf) nucleic acids from a urine sample from a subject is provided. The method comprises

(a) adding a preservative to the urine sample, wherein the preservative prevents degradation of the cf nucleic acids;

(b) adding a first magnetic particle to the urine sample under conditions where the cf nucleic acids in the sample bind to the first magnetic particle, wherein the first magnetic particle comprises an anion exchange moiety; (c) separating the first magnetic particle from the rest of the urine sample using a magnet; and

(d) eluting the cf nucleic acids from the first magnetic particle using a salt solution.

An adaptor for suspending a column above a bottom of a test tube is also provided herewith. With this adaptor, the column comprises an open entry and an open exit, with the entry wider than the exit, and the test tube has an open entrance and closed end.

The adaptor comprises a cylindrical tube having an open top, an open bottom comprising an opening, and a side between the top and the bottom, the side having a diameter at the top and the bottom that is less than the diameter of the test tube and wider than the diameter of the open exit of the column, and an outwardly directed top extension at the open top, wherein the width of the adaptor with the top extension is greater than the diameter of the test tube.

When the column is placed into the open top of the adaptor and the adaptor is placed into the open top of the test tube, the adaptor is suspended from the top of the test tube by the extension and the column is suspended from the top of the adaptor such that the bottom of the column is suspended above the bottom of the test tube.

Additionally provided is a method of accelerating the passage of a fluid through a chromatography column, where the chromatography column comprises an open entry and an open exit, with the entry wider than the exit. The method comprises

insert the chromatography column into the above-described adaptor,

insert the adaptor-chromatography column into the test tube,

add the fluid to the chromatography column, and

centrifuge the test tube-adaptor-chromatography column, thus accelerating passage of the fluid through the chromatography column.

Further provided is a method for isolating cell-free nucleic acids from a urine sample. The method comprises

(a) obtain the urine sample;

(b) centrifuge the sample until cells in the sample are pelleted;

(c) separate supernatant of the sample from the cells;

(d) add an anion exchange resin to the supernatant and incubate the supernatant-resin to allow binding of nucleic acids to the resin;

(e) separate the supernatant from the anion exchange resin;

(f) wash the anion exchange resin with a salt solution that is less than 500 mM; (g) elute nucleic acids from the anion exchange resin with a salt solution that is 500 mM or greater, using the adaptor of claim 58 to suspend a chromatography column comprising the anion exchange resin above the closed end of a test tube, then centrifuging the test tube- chromatography column-adaptor to accelerate elution of the nucleic acid from the anion exchange resin.

Additionally provided is a method of verifying the identity of a subject as being a source of a sample of a bodily fluid. The method comprises:

obtaining a reference sample from the subject, where the reference sample comprises DNA from the subject;

obtaining the sample of the bodily fluid;

analyzing DNA in the reference sample to determine a genetic fingerprint of the subject; analyzing DNA in the sample of the bodily fluid to determine a genetic fingerprint of the sample of the bodily fluid; and

comparing the genetic fingerprint of the reference sample with the genetic fingerprint of the sample of the bodily fluid. In these embodiments, if the genetic fingerprint of the reference sample is the same as the genetic fingerprint of the sample of the bodily fluid, then the subject is a source of the sample of the bodily fluid, and if the genetic fingerprint of the reference sample is not the same as the genetic fingerprint of the sample of the bodily fluid, then the subject is not a source of the sample of the bodily fluid.

Further provided are methods for isolating and purifying cf nucleic acids from bodily fluids such as urine and plasma that is much simpler and easier to automate than previous methods. Thus, in some embodiments, a method of isolating cell-free (cf) nucleic acids from a urine sample from a subject is provided. The method comprises

(b) adding a first magnetic particle to the urine sample under conditions where the cf nucleic acids in the sample bind to the first magnetic particle, wherein the first magnetic particle comprises an anion exchange moiety;

(c) separating the first magnetic particle from the rest of the urine sample using a magnet; and

(d) eluting the cf nucleic acids from the first magnetic particle using a salt solution. In other embodiments, a kit comprising any ingredient and/or component utilized in any of the above methods is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A provides an illustration of an embodiment of the invention collection container.

FIG. IB provides illustrations of an embodiment of the invention collection container.

FIG. 1C provides illustrations of an embodiment of the invention collection container.

FIG. ID provides an illustration of a lid of an embodiment of the invention collection container.

FIG. 2 A provides an illustration of an embodiment of the invention collection container with photographs of centrifuge components useful therewith.

FIG. 2B provides illustrations of an embodiment of the invention collection container and centrifuge components useful therewith.

FIG. 3A provides illustrations of an embodiment of the invention collection container. FIG. 3B provides an illustration of an embodiment of the invention collection container with photographs of centrifuge components useful therewith.

FIG. 3C provides an illustration of a cup component of an embodiment of the invention collection container.

FIG. 3D provides illustration of a portion of a cup and a lid of an embodiment of the invention collection container.

FIG. 4 provides illustrations of an embodiment of the invention collection container. FIG. 5A provides illustrations of a lid and a preservative subassembly of an embodiment of the invention collection container.

FIG. 5B provides illustrations of an embodiment of the invention collection container. FIG. 6 provides illustrations of components of an embodiment of the invention collection container.

FIG. 7A provides illustrations of an embodiment of the invention collection container. FIG. 7B provides illustrations of a fluid container of an embodiment of the invention collection container.

FIG. 7C provides exploded views of an embodiment of the invention collection container. FIG. 8A provides exploded views of an embodiment of the invention collection container.

FIG. 8B provides illustrations of an embodiment of the invention collection container.

FIG. 9 is a graph showing comparisons of urinary and plasma DNA size.

FIG. 10 is a graph showing comparing relative recoveries of four sizes of DNA fragments among three different molarities of NaCl concentration.

FIG. 11 is a graph showing sizes of DNA in urine eluted from magnetic particles using 1.8 M NaCl or 5 M NaCl.

FIG. 12 is a graph showing recovery of a 103 bp DNA fragment spiked into urine of five donors. The urine was then concentrated, and cf nucleic acids were isolated and purified from the concentrated urine by the methods described in Examples 1-3.

FIG. 13 is a graph showing recovery of a 103 bp DNA fragment spiked into concentrated urine from six donors. Cell-free nucleic acids were then isolated and purified from the concentrated urine by the methods described in Examples 2 and 3.

FIG. 14 is a graph showing the recovery of cancer mutations in urine of cancer patients.

DNA isolated from urine using low salt (1.8 M) ("Low") or high salt (5 M) ("High") to elute the DNA from magnetic particles. The various patients, numbered along the bottom, provided 1, 2 or 3 samples ("Rep").

FIG. 15 is a graph showing the effect on recovery of a 103 bp fragment spiked into urine containing three concentrations of tris(2-carboxyethyl)phosphine (TCEP) before concentrating urine and isolating and purifying cf nucleic acids from the concentrated urine.

FIG. 16 is a graph showing recovery of a 103 bp fragment spiked into urine, which was not concentrated before the cf nucleic acids were isolated and purified therefrom.

FIG. 17A is a side view of an embodiment of an adaptor of the present invention.

FIG. 17B is a top view of an embodiment of an adaptor of the present invention.

FIG. 18A is a side view of devices used in embodiments of methods of the present invention.

FIG. 18B is a side view of devices used in embodiments of methods of the present invention.

FIG. 18C is a side view of devices used in embodiments of methods of the present invention. FIG. 19 is a flow chart of an embodiment of the invention cf nucleic acid collection and purification method.

FIG. 20 is a graph showing recoveries of the invention transrenal nucleic acid collection and purification method.

FIG. 21 is graphs comparing the V2 method vs the VI method as to percent SRY and total SRY detected in pregnancy samples. The median total DNA and SRY copies from DNA extracted from 28 samples of urine from women bearing a male fetus is shown.

FIG. 22 is graphs showing Fragment Analyzer profiles of pregnancy samples. Representative Fragment Analyzer profiles of four samples of urine from women bearing a male fetus. Profiles show that the V2 wash did not recover any DNA fragments, the V2 EB-S elution recovered small fragments < 400 bp, and the LF elution recovered large fragments between 20- 20,000 bp. The VI (PNI) samples look similar to the LF elution in that they are recovering large fragments between 20-20,000 bp.

FIG. 23 is graphs showing a comparison between the V2 method and the VI method, in percent mutant and total mutant copies in cancer samples. Extracted DNA from 2 tissue-positive cancer samples: a KRAS-positive sample and an Exl9del-positive sample. The EB-S Elution represents a combination of elutions SI (500 mM NaCl) and S2 (600 mM NaCl) for TROV-044- 052, and SI A (500 mM NaCl), SIB (550 mM NaCl), S2A (600 mM NaCl) and S2B (650 mM NaCl) for TROV-044-1001.

FIG. 24 is graphs showing Fragment Analyzer profiles of a cancer sample. Fragment

Analyzer profile of a KRAS-positive cancer sample, shows that the V2 wash recovered small DNA fragments, the V2 EB-S elution recovered small fragments (EB-S 1 A Elution is 500 mM NaCl, EB-S IB is 550 mM NaCl, EB-S2A is 600 mM NaCl, and EB-S2B is 650 mM NaCl) < 400 bp, and the EB-L elution recovered larger fragments between 20-20,000 bp. The VI (PNI) samples look similar to the EB-L elution (without a previous EB-S elution) in that they recovered large fragments between 20-20,000 bp.

FIG. 25 is graphs showing a comparison between the V2 method and various commercial kits for extracting DNA from urine, measuring extracted DNA from 10 samples of urine from women bearing a male fetus.

FIG. 26 is graphs showing Fragment Analyzer profiles of pregnancy samples.

Representative Fragment Analyzer profiles of samples from pregnant females bearing a male fetus. Profiles show that the V2 EB-S elution recovered small fragments <400 bp and the three competitor kits recovered large fragments between 20 - 40,000 bp.

FIG. 27 is graphs showing a comparison between the V2 method and various commercial kits for extracting DNA from urine, measuring extracted DNA from a KRAS-positive cancer sample. Percent mutant was calculated by total MT copies divided by total Quant-iT™ copies + total MT copies. A two-step EB-S elution was performed with fractions at 500 and 600 mM NaCl.

FIG. 28 is graphs showing total DNA and the SRY signal from three samples from urine from two women bearing a male fetus.

FIG. 29 is graphs showing extracted DNA (EB-S elution) from a pool of pregnant donors.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, the use of "or" is intended to include "and/or", unless the context clearly indicates otherwise.

The present invention provides a comprehensive, integrated system for collecting, preserving, extracting and analyzing cell-free nucleic acids, e.g., nucleic acids in urine (including transrenal nucleic acids), to provide for very efficient and sensitive analysis of those nucleic acids.

Collection containers

In one aspect, provided are liquid sample collection containers that release an additive upon enclosing the container with a lid after the sample is taken. In these collection containers, there are no exposed sharp edges or points, used to cut open an additive container in other designs. See, e.g., US Patent 8,221,381 and US Patent Publication 2015/0251176.

The collection containers provided herein comprise the following components:

a cup for collecting the sample, where the cup comprises an open top, and a closed bottom;

a lid that engages the open top and seals the cup; and

an additive that is released into the sample in the cup when the lid engages the open top.

Any cup, of any shape suitable for a given sample can be used. In some embodiments the cup has round sides and a flat bottom. Additionally, the cup can hold any suitable volume, e.g., more than 1 ml of sample, more than 2 ml, more than 5 ml, more than 10 ml, more than 25 ml, more than 50 ml, or any volume in between.

Any suitable additive can be used in these containers. Nonlimiting examples of additives include an additive that

- prevents bacterial and/or viral growth;

- induces lysis of cells present in the fluid;

- prevents and/or delays lysis of cells present in the fluid;

- comprises magnetic particles with an affinity to the specific sample component;

- stabilizes cell membranes and/or surface antigens on the surface of cells present in the fluid;

- prevents aggregation of particles and/or cells present in fluid;

- prevents lysis of nucleated cells present in the fluid;

- prevents adsorption of an analyte to the container walls;

- is a preservative that prevents degradation of a molecule (e.g., a small molecule or a macromolecule) in the fluid.

or any combination thereof.

In various embodiments, the preservative prevents degradation of a macromolecule in the fluid. The macromolecule can be, e.g., a protein, a nucleic acid (for example, DNA, RNA, mRNA, microRNA, etc.), a carbohydrate, a polyphenol, a lipid, a glycolipid, a glycoprotein, a proteoglycan, a phospholipid, a synthetic polymer or any combinations. In some of these embodiments, the macromolecule is a nucleic acid, for example a nucleic acid in urine. Where a molecule is intended to be identified, it is preferred that at least portions of components that contact the fluid comprise a material to which the molecule does not significantly adsorb.

The fluid can be any fluid to which sampling is desired. In some embodiments, the fluid is an environmental fluid, such as waste water, municipal water, sea water, rain water, petroleum products, a spill from a leaking storage container, etc. In other embodiments, the fluid is a biological fluid. Nonlimiting examples of such a bodily fluid is urine, breast milk, saliva, sputum, blood, serum, plasma, lymph, amniotic fluid, cerebrospinal fluid, interstitial fluid, bile, peritoneal fluid, sweat, tears, ejaculates, ascites, or any wound fluid. In various embodiments, the biological fluid is urine.

The container can be made of any suitable material, including any type of plastic. The skilled artisan could select a suitable material for any application without undue experimentation. In some embodiments, at least a portion of the container is transparent, in order to observe the contents inside while the sample is being deposited into the container and afterward. In some of these embodiments, the transparent portion is tinted to prevent degradation of light sensitive substances in the collected fluid.

The additive may be stored in the container in a number of ways, for example as shown in the figures and described below. In some embodiments, the additive is sealed in a fluid container in an underside of the lid by a breakable seal. See, e.g., FIGS. 1, 5, 6, 7 and 8, and the discussion below, providing nonlimiting examples of those embodiments. In other embodiments, the additive is sealed in a fluid container inside the cup which prevents overfilling of the cup when the additive is added by positive displacement of the collected fluid. See, e.g., FIG. 4.

The breakable seal can be any material suitable for the particular design incorporating it. In some embodiments, the breakable seal comprises a foil (e.g., aluminum foil), either reinforced or not reinforced, and with or without a liner (e.g., a plastic liner to reinforce the foil and/or to prevent interaction of the foil with the additive). In other embodiments, the breakable seal is a rigid or flexible synthetic polymer or plastic film, which, in some embodiments, is transparent. In further embodiments, the breakable seal is a natural polymer such as cellulose, chitin, chitosan, lignin etc. The breakable seal can also be a fabric or a composite material, e.g., plastic or foil with reinforcing fibers of another material, for example cellulose, plastic, or metal, or a blend of synthetic and natural polymers).

In some embodiments, the fluid container further comprises at least one tower directed from the lid to the breakable seal that supports the breakable seal, preventing warping or premature breaking of the breakable seal before the lid is engaged with the open top, e.g., during storage and transport. FIG. ID illustrates a lid below the breakable seal, having six such towers 32.

In various embodiments, the cup near the open top and the lid have complementary screw threads such that the lid engages the open top by screwing onto the open top. See, e.g., FIGS. 1, 4, 5, 7, and 8. The screw threads can be right-handed or left-handed. Alternatively, a lever (as in FIG. 6), a latch, or any other means known in the art can be used to engage and secure the lid to the open top.

The collection container can be manufactured by any suitable method known in the art. In some embodiments, the collection container is subjected to a terminal sterilization process (e.g., autoclaving with saturated steam under pressure, or radiation), and enclosed in a sterile environment, such as a plastic bag. In other embodiments, the collection container is manufactured under aseptic processing conditions, where components, materials and equipment are handled in such a manner that foreign microbial and endotoxin contaminants that exceed pre- determined acceptable levels are not introduced to the product stream. In some of these embodiments, the aseptic manufacturing process comprises blow-fill-seal (BFS) aseptic processing, where the container is molded from plastic, aseptically filled and hermetically sealed in one continuous, integrated and automatic operation, without human manipulation.

In some embodiments, exemplified in FIGS. 1-3, of the invention collection container 10 comprises a cup 11 with an open top 13 and a lid 30 with complementary screw threads 14, 31 such that the lid 30 engages the open top 13 by screwing onto the open top 13. Here, the additive is sealed in a fluid container 40 in the underside of the lid 30 by a breakable seal 42.

In these illustrated embodiments, the cup 11 further comprises an upper section 12 comprising the open top 13 and a lower section 15 below the upper section 12, wherein the upper section 12 is wider than the lower section 15. The cup 11 also comprises at least one upwardly directed push bar 17 that engages the breakable seal 42, breaking open the seal and releasing the additive into the cup 11 when the lid 30 is screwed unto the open top 13, wherein the push bar 17 does not comprise a sharp edge or point.

FIG. 1A shows an example of this collection container 10 in profile, with the lid 30 on. FIG. IB shows an example of the collection container 10 with the lid 30 off, and three push bars 17. As shown in FIG. 1C, top illustration (cross section of lid 30), the push bar 17 pushes into the breakable seal 42 of the fluid container 40 when the lid 30 is screwed on.

These collection containers 10 have an upper section 12 of the cup 11 that is wider than the lower section 15. In some embodiments, the lower section 15 is of a size that fits into a centrifuge rotor bucket or adapter. In such embodiments, the wider upper section 12 allows a larger volume of sample to be taken and centrifuged. For example, the exemplified collection container illustrated in FIG. 2 and FIG. 3B are designed to fit into Adapter 022638921 52 for Rotor A-8-81 50 of Eppendorf centrifuges 5804, 5804 R, 5810, and 5810 R (see Centrifuge 5804/5804 R/5810/5810 R Operating manual, 2012, Eppendorf AG, Hamburg). As shown in FIG. 2B, the wider upper section 12 adds volume to allow the cup 11 to hold a sample of up to 300 ml even though the adapter 52 has a volume of about 100 ml. FIG. 2B also shows the specific dimensional requirements for the segmented adapter 022638921 52. The exemplified design uses three stacked 62mm diameter adapter segments 54 (total bore height 60mm) and then expands the diameter in order to accommodate high fluid volumes. Any of the collection containers provided herein can be designed to fit any centrifuge rotor or adapter of any size without undue experimentation.

FIG. 1C illustrates an embodiment where the underside of the lid 30 further comprises at least one spike 44 sealed in the fluid container 40 wherein, when the lid 30 is screwed onto the top 13, the push bar 17 pushes the breakable seal 42 into the spike 44, which breaks open the seal 42. When one or multiple spikes 44 are present, they can take on any shape (e.g., sharp point, edge or blade) that will break the seal 42 when the push bar 17 pushes the seal 42 onto the spike(s) 44.

In those embodiments, the push bar 17 pushes the breakable seal 42 in a circular motion since the push bar 17 contacts the breakable seal 42 while the lid 30 is being screwed on. With multiple spikes 44 and/or multiple push bars 17 (as illustrated in FIG. 1C, showing three push bars 17, and FIG. 3 A, showing a lid 30 having 12 spikes 44), the push bar(s) 17 will slide around the lid, pushing the breakable seal 42 into multiple spikes 44, assuring rapid and complete release of the additive sealed in the fluid container 40.

Since the push bar 17 does not comprise a sharp edge or point, and, in the embodiment illustrated in FIG. 1C, the sharp edge or point (spike 44) is not exposed but is inside the fluid container 40, there is no sharp edge or point that can lead to injury to the person providing or obtaining the sample, or the person that processes the sample.

In some embodiments, there is no spike in the cup 11, but the push bar pushes the breakable seal 42 sufficiently to break it open. In those embodiments, the breakable seal 42 must be made of a material that can be broken by the push bar 17 pushing onto the seal 42 in a circular motion, while the lid 30 is being screwed on.

In some embodiments, the fluid container 40 is integrated directly into the lid 30 and sealed with the breakable seal 42. In other embodiments, the fluid container 40 is molded (e.g., injection molded) and sealed separately then affixed to the underside of the lid 30.

The breakable seal 42 can be of any material that can be penetrated by the push bar 17 (when a spike 44 in the fluid container 40 is not relied on), or pushed up by the push bar 17 into a spike 44 in the fluid container. In certain embodiments, the fluid container comprises at least one tower protruding outward from the lid, to support the breakable seal, e.g., by preventing warping or premature breaking of the breakable seal before the lid is screwed on to the open top. FIG. ID illustrates a lid 30 below the breakable seal, having six such towers 32. The tower can be of any shape, including, but not limited to, a pole shape as illustrated, a semi-circle, a square, or a circle.

In various embodiments, exemplified in FIG. 3C, the cup 11 comprises a conical insert 18 forming a closed bottom 16. The conical bottom 18 can be used to accumulate solid material in the sample. The sample solid material can accumulate in the conical bottom 18 by any means, e.g., natural gravity or by centrifugation. In some embodiments, particles with an affinity to a specific sample component (e.g., non-magnetic or magnetic particles with antibodies for binding specific proteins to the particles or nucleic acids complementary to specific DNA sequences to bind those DNA sequences to the particles). Such non-magnetic particles can be centnfuged into the conical bottom or allowed to settle to the conical bottom by gravity. Magnetic particles can be pulled to the inner wall of the conical insert by applying a magnetic force.

In some embodiments, exemplified in FIG. 3C, the collection container comprises a graduated scale 19 readable on the outer surface of the cup, allowing determination of the quantity of fluid in the cup.

In other embodiments, the collection container comprises a premature piercing prevention mechanism preventing screwing of the lid onto the open top before the urine sample is put into the cup. A nonlimiting example of such a mechanism is a rotational lock preventing clockwise rotation of the lid while screwed on, unless the lid is first completely unscrewed.

In additional embodiments, exemplified in FIGS. 3 A and 3D, the collection container comprises at least one lobe 21 and/or 33 on a screw thread 14 and/or 31 that engages with the complementary screw thread 31 and/or 14, creating a haptic and/or tactile and/or visible feedback when the lid 30 is fully and tightly screwed on to the cup 11 such that leakage and/or contamination of the contents is prevented during storage and transport. As shown in FIG. 3D, where the interacting click lobe 21 on the cup 11 perimeter at the bottom of the screw thread 14 and the inner surface of the lid 30 interact to create a click. Any other haptic, tactile or visible feedback mechanism now known or later discovered can be used to indicate a tight closure.

In some of those embodiments, the lid can be unscrewed from the top after the feedback mechanism is actuated. In other embodiments, for example when taking samples for drug testing, the lid cannot be unscrewed from the top after the feedback mechanism is actuated. This can be accomplished by any means known in the art, for example by replacing one of the lobes (21 or 33) shown in FIG. 3D with lobes that do not allow reversal.

In various embodiments, the lid has ergonomic features that allows the user to provide increased torque in applying rotational force when screwing the lid onto the top of the cup. Such features are particularly useful for frail or elderly users with a weak grip. Nonlimiting examples are the protruding lobes 34 and grip lines 35 shown in the lids in FIG. 3A.

These collection containers can also comprise an area on the upper section of the cup with markings indicating a desired fill level (e.g., 22 in FIG. 3C) and where a finger or thumb should be placed while collecting the fluid sample, such that a change in temperature sensed by the finger or thumb (e.g., warmth from urine) indicates the desired fill level has been achieved.

In further embodiments, the collection container comprises a skirt below the bottom supporting a firm stand of the container thereon. Such a skirt 23 is exemplified in FIG. 3C. The supporting skirt can take any shape, for example creating a cylinder extending the perimeter of the lower section, as shown in FIG. 3C, or straight perpendicular sections below the bottom of the cup, either parallel or intersecting, e.g., forming an X. The skirt can also extend outward beyond the perimeter of the lower section, to create added stability.

Any feature described above for the design shown in FIGS. 1-3, for example, wider upper section, use in a centrifuge, the construction of the fluid container, the conical insert, the feedback mechanism, the skirt, etc., is also conceived for the designs shown in FIGS. 4-8 if appropriate for those designs.

As exemplified in FIG. 4, other embodiments of the invention collection container 100 comprises a lid 130 that engages an open top 112 by screwing onto the open top 112;

the additive is in a vessel 140 located inside the cup extending from above the open top 112, wherein the elongate vessel 140 has a top 142 and a pierceable bottom 144, thereby providing a positive displacement mechanism that prevents overfilling of the cup upon release of the additive. Below the vessel is a piercing and draining mechanism 114. The lid 130 comprises an underside with a curved ramp 132. In this design, screwing the lid 130 onto the open top slides the top of the vessel 142 along the curved ramp 132, pushing the pierceable bottom 144 of the vessel 140 into the piercing and draining mechanism 114, causing the pierceable bottom 144 to be pierced and the additive to escape the vessel 140 and mix with fluid in the cup.

The vessel can be of any shape, and it can engage the piercing and draining mechanism 144 at the bottom of the cup 110 (as exemplified in FIG. 4) or at any level below the top of the cup 112. The pierceable bottom 144 is analogous to the breakable seal 42 discussed above for the design exemplified in FIGS. 1-3.

The piercing and draining mechanism can be of any design. As shown, the piercing and draining mechanism 144 has four posts 116 that engage the pierceable bottom 144 when the additive container 140 is pushed into them.

In some of these embodiments, the vessel is elongate, extending to near the bottom of the cup.

In some embodiments of this design, the vessel comprises a mechanism to prevent accidental pushing of the vessel. In the exemplified container shown in FIG. 4, an elastic spacer 118 is inserted into the piercing and draining mechanism, preventing the vessel 140 from accidently engaging the piercing and draining mechanism 114. In other embodiments, a spring, either extended up from outside the vessel, or joining the vessel to the inner wall of the cup, provides resistance to pushing of the vessel into the piercing and draining mechanism.

In further embodiments of the invention collection container, exemplified in FIG 5, the lid 230 comprises an underside 232 and engages the open top by screwing onto the open top, and the additive is enclosed in a subassembly 240. The subassembly 240 comprises a receptacle 242 overlapped by an outer cover 243, and the receptacle 242 is affixed to the underside of the lid 232, with the outer cover 243 is distal thereto. The receptacle 242 screws into the outer cover 243 using threads 244 that are reversed from threads used to screw the lid 230 onto the open top 212 of the cup 210.

In these embodiments, the outer cover 243 further comprises at least one outwardly extended tab 245; and at least one slot 246 at a portion of the outer cover 243 that is closest to the bottom of the cup 212 when the lid 230 is screwed onto the open top 212. In these embodiments, the slot 246 is internally covered by the receptacle 242 when the receptacle 242 and outer cover 243 are screwed together.

When the lid 230 is screwed onto the open top 212, the tab 245 engages with the cup 210 preventing rotation when the lid 230 is screwed onto the cup 210, and causing the receptacle 242 and to unscrew from the outer cover 243, uncovering the slot 246 opening a gap 247 to allow additive to drain into the cup 210.

FIG. 5A shows the subassembly 240, including the outer cover 243 and receptacle 242.

The subassembly 240 is affixed to the underside of the lid 230 (top right). When assembled and positioned to screw onto the top of the cup 212 (FIG. 5B), the outer cover 243 is facing downward (bottom right of FIG. 5A), where the slot(s) 246 in the lid are at the bottom of the subassembly 241. In this position, the receptacle 242 is extended to the bottom of the inside of the lid 234, covering the slot(s) 246 and preventing additive from leaking out of the slot 246. When the lid 230 is screwed onto the top of the cup 212, the tab(s) 245 in the outer cover 243 engage the cup 210 and prevent the outer cover 243 from turning with the top of the cup 212. Since the receptacle 242, attached to the underside of the lid 232, is turning but the outer cover 243 is not, the receptacle 242 unscrews from the outer cover 243, opening a gap 247 between the receptacle 242 and outer cover 243, exposing the slot(s) 246 to the additive, which leaks through the gap 247 into the cup 210.

In additional embodiments of the invention collection container, exemplified in FIG. 6, the additive is sealed in a fluid container 340 in an underside of the lid 330 by a breakable seal 342, and the lid 330 further comprises a lever 332 operably linked to at least one piercing pin 334 in the fluid container 340, whereby, after the lid 330 engages the open top of the cup (not shown) and the lever 332 is actuated, the piercing pin 334 pierces the seal 342, releasing the additive from the fluid container 340 into the cup.

The lever 332 can operate to pierce the breakable seal 342 by any mechanism known in the art. In some embodiments, the lever 332 is operably linked to the piercing pin 334 by a cam 336 inside the fluid container 340 and joined to the lever 332, the cam 336 pushing the piercing pin 334 through the seal 342 when the lever 332 is actuated.

In some of these embodiments, the lid 332 engages the open top of the cup by screwing onto the open top. In alternative embodiments, actuating the lever 332 also closes the lid 332 onto the open top, preventing leakage.

In other embodiments of the invention collection container, exemplified in FIG. 7, the lid 430 engages the open top 412 by screwing onto the open top. The additive is sealed in a fluid container 440 in the underside of the lid 432, and the fluid container comprises at least one protrusion 442 directed away from the underside of the lid 432.

In these embodiments, the collection container further comprises a circular blade assembly 450 surrounding the fluid container 440. The blade assembly 450 comprises at least one blade 452 adjacent to the protrusion 442 and directed thereto, and at least one tab 454 that fits inside a guide 414 in the cup 410, preventing rotation of the blade assembly 450 when the lid 430 is screwed onto the open top 412. Screwing the lid 430 onto the open top of the cup 412 causes the blade 452 to cut the protrusion 442, releasing the preservative from the fluid container 440 into the cup 410.

FIG. 7A, right, shows the fluid container 440 and the circular blade assembly 450 in the underside of the lid 432. The fluid container 440 is affixed to the lid 430, and, when the lid 430 is placed on the top of the cup 412, at least one tab 454 engages the cup 410 in a guide 414, preventing the blade assembly 450 from turning when the lid 430 is screwed onto the top of the cup 412. Since the fluid container 440 turns with the lid 430 and the blade assembly 450 is stationary, the blade(s) 452 slice into the protrusions 442 of the fluid container 440, releasing the additive into the cup 410. FIG. 7B shows the fluid container 440.

In some embodiments, for example as shown in FIG. 7C, the container further comprises a screen 460 adjacent to the blade assembly 450 and distal thereto, that will stop the cut protrusions 442 from falling into the fluid sample in the cup 410.

In further embodiments of the invention collection container, exemplified in FIG. 8, the lid 530 engages the open top 512 by screwing onto the open top 512 and the additive is sealed in a fluid container 540 affixed to the underside of the lid by a breakable seal. The fluid container may be affixed to the underside of the lid 530 by holders 532, as shown in FIG. 8A, bottom. The collection container further comprises a puncture ring 550 adjacent to the breakable seal 542.

The puncture ring 550 comprises at least one puncture point 552 directed toward the breakable seal 542, and at least one tab 554 that fits inside a guide 514 in the cup 510, preventing rotation of the puncture ring 550 when the lid 530 is screwed onto the open top 512. In these embodiments, screwing the lid 530 onto the open top 512 causes the puncture point 552 to pierce, then rotationally slice through the breakable seal 542, releasing the preservative from the fluid container 540 into the cup 510.

FIG. 8A is exploded views of the entire container (top) and the lid assembly, comprising the lid 530, the fluid container 540, and the puncture ring 550. FIG. 8B shows the tabs 554 on the puncture ring 550 engaged with the open top of the cup 512 (bottom) and the puncture of the fluid container 540 when the lid 530 is screwed onto the top 512.

The present invention is also directed a method of collecting a fluid sample. The method comprises inserting the sample into any of the above-described collection containers, and engaging the lid onto the open top, releasing the additive into the sample. In some embodiments, the fluid is urine, breast milk, saliva, sputum, blood, serum, plasma, lymph, amniotic fluid, cerebrospinal fluid, interstitial fluid, bile, peritoneal fluid, sweat, tears, ejaculates, ascites, or any wound fluid. In certain of these embodiments, the fluid is urine.

In other embodiments, the additive is a preservative that prevents degradation of a small molecule or a macromolecule, e.g. a lipid, a carbohydrate, a protein or a nucleic acid.

The invention is also directed to a method of collecting a urine sample. The method comprises inserting the sample into the collection container exemplified in FIGS. 1-3, then screwing the lid onto the open top, releasing the additive into the urine in the cup. In various embodiments, the additive the additive is a preservative that prevents degradation of a small molecule or a macromolecule.

In some embodiments, these methods further comprise centrifuging the collection container after the lid is screwed onto the open top, then separating a resulting liquid portion from a resulting pelleted portion present in the container.

In other embodiments, these methods further comprise adding magnetic particles with an affinity to a specific sample component to the urine, allowing the specific sample component to bind to the magnetic particles, then pulling the magnetic particles to the inner wall of the conical insert by applying a magnetic force.

Also provided is a method of isolating cells from urine. The method comprises collecting the urine in a collection container, e.g., as exemplified in FIGS. 1-3; screwing the lid onto the open top releasing the additive into the urine; centrifuging the collection container sufficiently to pellet the cells in a conical insert; removing the liquid, and isolating the cells from the conical insert.

Nucleic acid isolation and purification

(a) Magnetic particle

Additionally provided herewith are methods for isolating and purifying cf nucleic acids from bodily fluids such as urine and plasma. These methods are much simpler and easier to automate than previous methods. While the present methods are exemplified with cfDNA in urine, the methods would also be expected to be effective in isolating and purifying any nucleic acid species including but not limited to DNA from cells disrupted in a sample, mRNA, or miRNA, from any bodily fluid, e.g., urine, blood, plasma, serum, saliva, pancreatic juice, semen, stool, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid or the like. Thus, in some embodiments, a method of isolating cell-free (cf) nucleic acids from a urine sample from a subject is provided. The method comprises

Any cf nucleic acid can be isolated by these methods, including any DNA or RNA, or any mixture thereof. In some embodiments, the cf nucleic acids are cfDNA. In some of those embodiments, the cfDNA is transrenal cfDNA (trDNA).

The cf nucleic acids can be eluted from the first magnetic particle using any salt solution. Non-limiting examples include chaotropic agents (e.g., guanidinium salts, urea, lithium perchlorate), Good's buffers (e.g., MES, PIPES, MOPS, tris, HEPES, tricine, etc.), or an amino acid salt (see, e.g., Vandeventer et al., 2013). In some embodiments, as exemplified in the Examples below, the salt is sodium chloride (NaCl).

In some embodiments, the salt solution has a molarity of less than 2.0 M. In other embodiments, the salt has a molarity of 2.0 M. In additional embodiments, the salt solution has a molarity greater than 2.0 M. See, e.g., Examples 2 and 4, using 5.0 M. However, higher molarity salt solutions will elute nucleic acids of all sizes, so for applications where small (such as transrenal nucleic acids that are less than 500 basepairs/nucleotides) and not larger nucleic acid fragments are desired, elution using a salt that is less than 2.0 M is preferred since very little larger nucleic acids will be eluted (see FIG. 11), so interference from a large amounts of larger nucleic acids can be avoided.

Thus, the salt molarity can be greater than 5.0, between 3.0 and 5.0 inclusive, between 2.0 and 3.0 inclusive, 1.95 M, 1.9 M, 1.8 M, 1.7 M, 1.6 M, or molarity in between, or any molarity below 1.6 M. As shown in FIG. 10, 1.8 M NaCl was superior to 1.7 M and 1.6 M for eluting a range of nucleic acids between 103 bp and 414 bp. Also, FIG. 11 shows that very little longer (greater than 1000 bp) nucleic acids eluted when 1.8 M NaCl was used. Thus, 1.8 M salt is a preferred molarity for isolating transrenal nucleic acids from urine without isolating many longer pieces.

Any preservative now known or later discovered to preserve nucleic acids in urine may be used in these methods. Nonlimiting examples include chelating agents such as ethylenediaminetetraacetic acid (EDTA). EDTA added to the urine sample to a concentration of 0.25 - 1.0 M is effective in preventing degradation of urine DNA. In some embodiments, the EDTA in the sample is 0.5 M.

In some embodiments, the urine sample is greater than 90 ml, with fully hydrated urine. As is known, urine concentrations can vary up to 10-fold during the day. More concentrated urine should have more cf nucleic acids.

The cell count in urine can also affect the amount of longer nucleic acid pieces that have not been filtered by the glomeruli. Since there is greater numbers of cells in the first void, the urine may be collected mid-stream to avoid high cell counts.

These methods are not narrowly limited to the use of any particular anion exchange moiety on the first magnetic particle. In some embodiments, the anion exchange moiety is a quaternary ammonium moiety, e.g., a 2-hydroxypropyl trimethylammoniumchlorid moiety (see Example 2). The first magnetic particle can be superparamagnetic or ferromagnetic. In some embodiments, the first magnetic particle is superparamagnetic.

Any magnetic core may be used in the first magnetic particle, e.g., magnetite (Fe₂0₄) or maghemite (gamma Fe₂0₃). In some embodiments, the first magnetic particle has a maghemite core.

The first magnetic particle can be any size and have any dimensions known in the art. In

1 9

some embodiments, the first magnetic particle is 1.0 μιη size with about 1.8 x 10 particles/g, and a surface area of about 50 m /g.

As described in Example 1 and in PCT Patent Publication WO 2015/164435, the urine sample may be concentrated after adding the preservative and before adding the first magnetic particle.

The urine sample may be unconcentrated, or concentrated by any means known in the art. In some embodiments, the urine sample is concentrated by ultrafiltration.

When concentrated, the sample may be concentrated to any volume, e.g., from 0.2 ml to

20 ml. In some embodiments, the sample is concentrated to a volume of less than 5 ml. The skilled artisan, without undue experimentation, can select a pore size for the filter used for ultrafiltration. In some embodiments, the filter has a pore size of 4000-6000 Dalton, e.g., 5000 Dalton.

These methods are not narrowly limited to the use of any particular binding buffer to bind the urine cf nucleic acids to the first magnetic particle. In some embodiments, the buffer comprises 2-propanol, tris-HCl and EDTA, e.g., as described in Example 2.

A reducing agent can also be added to the urine sample to prevent proteins in the urine, e.g., uromodulin, from preventing recovery of some of the cf nucleic acids. Any reducing agent may be used in these embodiments. Nonlimiting examples of the reducing agent are dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). See Example 5.

The cf nucleic acids eluted from the first magnetic particle can be further purified by any means known in the art. In some embodiments, the purifying comprises

(e) adding a second magnetic particle to the eluted cf nucleic acids under conditions where the cf nucleic acids bind to the second magnetic particle;

(f) separating the second magnetic particle from the rest of the eluted cf nucleic acids using a magnet; and

(g) eluting purified cf nucleic acids from the second magnetic particle.

The second magnetic particle can comprise any moiety known to bind to nucleic acids. In some embodiments, the nucleic acid-binding moiety is a silane, a silanol, silicon carbide, iron oxide, or a carboxyl moiety.

The second magnetic particle can be superparamagnetic or ferromagnetic. In some embodiments, the second magnetic particle is superparamagnetic.

Any magnetic core may be used in the first magnetic particle, e.g., magnetite (Fe₂0₄) or magnemite (gamma Fe₂0₃). In some embodiments, the second magnetic particle has a maghemite core.

The second magnetic particle can be any size and have any dimensions known in the art.

1

In some embodiments, the second magnetic particle is 1.0 μιη size with about 1.8 x 10 particles/g, and a surface area of about 50 m /g.

These methods are not narrowly limited to the use of any particular binding buffer to bind the cf nucleic acids to the second magnetic particle. In some embodiments, the buffer comprises 2-propanol, guanidinium thiocyanate and polyethylene glycol. After the cf nucleic acids are bound to the second magnetic particle, they may be washed by any means known in the art, e.g., in an ethanol solution, for example a 70% ethanol solution. In some embodiments, e.g., as in Example 3, the second magnetic particle with bound cf nucleic acids is washed (i) in 70% ethanol with guanidinium thiocyanate then (ii) 70% ethanol.

The purified cf nucleic acids may be eluted from the second magnetic particle with any elution buffer known in the art. In some embodiments, e.g., those described in Example 3, the elution buffer is 10 mM Tris-HCl.

These methods are easily automated with any suitable laboratory automation system, for example the KingFisher™ Flex System, as in Examples 1-3.

The isolated and purified cf nucleic acids obtained using these methods are suitable for assaying for the presence of a nucleic acid sequence that does not originate from endogenous nucleic acid sequences of the subject, e.g., cf nucleic acids that originate from a pathogen, transplant tissue, or, where the subject is pregnant, a fetus, e.g., as described in US Patent RE39920E1. The cf nucleic acids resulting from these methods are also suitable for assaying for the presence of a nucleic acid sequence that originates from endogenous nucleic acid sequences of the subject, for example miRNA (US Patent 8,486,626; US Patent Publication 2012/0225925; PCT Publication WO 2012/089630) or genomic DNA of the subject, e.g., a mutation associated with a cancer (US Patent 8,501,924; US Patent Publications 2016/0115556 and 2013/0164746) or other diseases (US Patent Publication 2015/0132256).

Also provided is a kit comprising any ingredients utilized in the above-described methods. Such kits may include, e.g., a magnetic particle having one or more anion exchanger groups such as, for example, a quaternary ammonium group; buffers and solutions for elution; written protocol sheet; and or positive and negative control samples. Kits may also include materials for safe sampling from a subject (e.g., gloves, alcohol swabs, container for storage of samples having address of destination lab, sample identification number, patient code, etc.). (b) Anion exchange chromatography

The present invention also includes methods of purifying cell free nucleic acids from urine using anion exchange chromatography that is an improvement on the methods provided in US Patent 9,163,229. The improvements include adaptations that ease flow-through and allow for easy size fractionation of the nucleic acids.

In some embodiments, the improved methods utilize an adaptor that suspends a column (e.g., a chromatography column) above a bottom of a test tube. In these embodiments, the column comprises an open entry and an open exit, with the entry wider than the exit, and the test tube has an open entrance and closed end.

The adaptor comprises a cylindrical tube having an open top, an open bottom comprising an opening, and a side between the top and the bottom, the side having a diameter at the top and the bottom that is less than the diameter of the test tube and wider than the diameter of the open exit of the column. The adaptor also comprises an outwardly directed top extension at the open top, wherein the width of the adaptor with the top extension is greater than the diameter of the test tube. In these embodiments, when the column is placed into the open top of the adaptor and the adaptor is placed into the open top of the test tube, the adaptor is suspended from the top of the test tube by the extension and the column is suspended from the top of the adaptor such that the bottom of the column is suspended above the bottom of the test tube.

An example of this adaptor is illustrated in FIG. 17A and 17B. These illustrations show the adaptor 600, the open top 610, the open bottom 620, the side 630, and the top extension 612, shown therein as an outward circumferential flange. In this example, the adaptor also comprises at least one wing 632 extending outwardly from the side.

In some embodiments, the open bottom of the adaptor further comprises an inwardly directed bottom extension that narrows the opening. That bottom extension is illustrated in FIG. 17B as an inward circumferential flange 622.

In various embodiments, the column is a chromatography column, for example the column illustrated in FIG. 18A (left side)(700), which has an open entry 710, an open exit 720 (when the snap-off tip 740 on the bottom is removed) and a shoulder 730. In these embodiments, the shoulder 730 sits on the bottom extension 622 when the chromatography column is inserted into the adaptor 600.

In some embodiments, the adaptor, chromatography column and test tube form a chromatography assembly, illustrated in FIG. 18A (right side), where the chromatography column (open entry 710 shown) is inserted into the adaptor (top extension 612 and side 630 shown, the latter through the side of the test tube 800), and the column + adaptor is inserted into the test tube 800 such that the open exit 720 is suspended above the closed end 820 of the test tube 800.

The adaptor, column and test tube can be any size. It should be understood that the diameter of the adaptor open top, the top extension and the bottom extension must be such that the chromatography column will fit within the adaptor and be held by the adaptor, and that the top extension rests on the open entrance of the test tube. In certain embodiments, the test tube is a standard 50 ml tube, where the diameter of the adaptor open top is between 2.0 cm and 2.5 cm, e.g., between 2.2 cm and 2.4 cm, and the diameter of the test tube open entrance is between 2.5 cm and 3.0 cm, e.g., between 2.7 cm and 2.9 cm. Of course, the adaptor top extension must have a greater diameter than the test tube open entrance, since that top extension must rest on top of the open entrance to hold up the adaptor and column. In some of these embodiments, the column open entry is between 1.7 and 2.2 cm.

The present invention is also directed to a method of accelerating the passage of a fluid through a chromatography column, where the chromatography column comprises an open entry and an open exit, with the entry wider than the exit. The method comprises

insert the chromatography column into the adaptor described above,

insert the adaptor-chromatography column into the test tube,

add the fluid to the chromatography column, and

centrifuge the test tube-adaptor-chromatography column.

In some of these embodiments, the width of the open top is between 2.2 cm and 2.4 cm, the test tube open entrance is between 2.5 cm and 3.0 cm and the chromatography column open entry is between 1.7 and 2.2 cm. These embodiments are exemplified with the chromatography assembly illustrated on the right side of FIG. 18A.

In various embodiments of these methods, the chromatography column further comprises a chromatography resin. Any chromatography resin now known or later discovered could be used in the chromatography resin, including a silica-based resin, a reversed phase resin, or an ion-exchange resin, e.g., a cation exchange resin or an anion exchange resin such as Q Sepharose® (see Example 5).

The fluid in these embodiments is not narrowly limited to any particular fluid, and can include an environmental sample such as waste water, or a bodily fluid, for example , e.g., urine, blood, plasma, serum, saliva, pancreatic juice, semen, stool, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid or the like.

The present invention is also directed to another method for isolating cell-free nucleic acids from a urine sample. As discussed above, this method is an improvement on the methods provided in US Patent 9,163,229. An example of the use of these methods is provided in Example 7 below.

This method comprises (a) obtain the urine sample;

(b) centrifuge the sample until cells in the sample are pelleted;

(c) separate supernatant of the sample from the cells;

(e) separate the supernatant from the anion exchange resin;

(f) wash the anion exchange resin with a salt solution that is less than 500 mM; and

(g) elute nucleic acids from the anion exchange resin with a salt solution that is 500 mM or greater, using the above-described adaptor to suspend a chromatography column comprising the anion exchange resin above the closed end of a test tube, then centrifuging the test tube- chromatography column-adaptor to accelerate elution of the nucleic acid from the anion exchange resin.

The anion exchange resin for these methods can be any type of anion exchange resin, e.g., as described in US Patent 9,163,229. In some embodiments, the anion exchange resin is Q Sepharose®.

To wash the anion exchange resin in step (f), any salt solution that is less than 500 mM may be used. In some embodiments, this salt solution is 400 mM NaCl. That step is greatly accelerated by using the above-described adaptor to centrifuge the chromatography column in the chromatography assembly as described above, to accelerate the elution of the wash solution through the column.

The elution of nucleic acids from the ion exchange resin can be tailored to isolate nucleic acids of any size range. As shown in FIG. 20, 500 mM NaCl elutes nucleic acids less than about 200 bp; 600 mM NaCl elutes nucleic acids less than about 400 bp (which includes essentially all transrenal nucleic acids); and an elution with 700 mM NaCl elutes longer nucleic acids. As exemplified in Example 7 with combining a stepwise elution with 500 mM NaCl and 600 mM NaCl almost exclusively nucleic acids of 400 bp or less, providing a greatly purified fraction of transrenal nucleic acids, and almost complete depletion of DNA fragments greater than 400 bp. Those longer nucleic acids were retained in the subsequent 700 mM NaCl elution.

In some embodiments, the nucleic acids are further purified by

(h) add sodium acetate to the eluted nucleic acids to a concentration of less than 5%, mix, then add a chaotropic salt and alcohol solution that allows binding of the nucleic acids to a silica medium; (i) apply the sodium acetate-nucleic acid-guanidine-isopropanol solution to a silica medium such that the nucleic acids bind to the silica medium;

(j) wash the silica medium with a wash buffer that removes impurities from the silica medium, while the silica medium retains the nucleic acids;

(k) elute the nucleic acids from the silica medium with an elution medium.

In step (h), any chaotropic salt/alcohol solution effective in allowing binding of the nucleic acids to the silica medium may be used. In some embodiments, the chaotropic salt is guanidine hydrochloride and the alcohol is isopropanol.

Any silica medium that can reversibly bind to nucleic acids can be used in these steps. In some embodiments, a QIAquick spin column (Qiagen) is utilized.

The wash buffer of step (j) can be any buffer solution that will not cause the desired nucleic acids to elute from the column. In some embodiments, the wash buffer comprises an alcohol, e.g., ethanol.

The elution medium of step (k) can be any medium that elutes desired nucleic acids from the silica medium. In some embodiments, the elution medium 10 mM Tris-Cl.

Analysis of nucleic acids

The quality and purity of the nucleic acids using any of the above methods is suitable for any downstream analysis, for example those described in US Patents RE39920E1; 8,486,626; and 8,501,924; US Patent Publications 2012/0225925; 2013/0164746; 2015/0132256 and 2016/0115556; and PCT Publication WO 2012/089630.

In additional embodiments, a method of verifying the identity of a subject as being a source of a sample of a bodily fluid is provided. By comparing a genetic fingerprint obtained from a reference sample known to contain DNA from the subject with a genetic fingerprint from the sample of bodily fluid, the bodily fluid sample can be verified as coming from the subject. This method is particularly useful for determining whether a subject that provides a sample of a bodily fluid such as urine or sputum is the source of the bodily fluid, for, e.g., testing for a banned drug for employment or athletic events. This method is also useful where the identification records of a medical sample is lost, where the method could be used to confirm the source of the sample. However, the method is not limited to these uses, and multiple other uses are contemplated.

The method comprises obtaining a reference sample from the subject, where the reference sample comprises DNA from the subject;

obtaining the sample of the bodily fluid;

The subject for these embodiments can be any vertebrate animal. In some embodiments, the subject is a mammal. In some of those embodiments, the subject is a human.

In some of these embodiments, the sample of the bodily fluid is collected in any of the above-described collection containers. The collection container exemplified in FIGS. 1-3 having the conical bottom for collecting centrifuged cells is particularly useful for these embodiments, particularly when cells from the bodily fluid is used to obtain the DNA to be analyzed. However, any of the other collection containers disclosed herein can also comprise a conical bottom to collect cells or other particulate material.

This method can be utilized with a sample of any bodily fluid, for example urine, breast milk, saliva, sputum, blood, serum, plasma, lymph, amniotic fluid, cerebrospinal fluid, interstitial fluid, bile, peritoneal fluid, sweat, tears, ejaculate, ascites, or a wound fluid. In some embodiments, the bodily fluid is urine.

The reference sample can be any DNA-containing sample known to be from the subject.

In some embodiments, the reference sample is a bodily fluid from the subject. Any bodily fluid known to be from the subject and containing DNA can be utilized. Examples include urine, serum, sputum, stool, sweat, plasma, or a wound fluid. Alternatively, the reference sample can be any tissue sample from the subject, for example a surgical or biopsied tissue, a buccal swab, a hair sample, skin tissue, toenail tissue or fingernail tissue.

In some embodiments, the reference sample is a forensic sample from the subject. As used herein, a forensic sample is a sample used to detect or investigate a crime or infraction. In some of these embodiments, the forensic sample is a witnessed sample taken to verify the identity of the sample of the bodily fluid. In some of these embodiments, the sample of the bodily fluid is provided for testing for a banned substance therein. Non-limiting examples of a banned substance for these methods is a performance-enhancing drug, e.g., in an athlete or a racehorse, or a psychoactive drug, e.g., to determine whether an employee, candidate employee, or student is taking illegal drugs.

The reference sample can be taken from the subject at any time before or after the sample of the bodily fluid is obtained. In some embodiments, the reference sample is taken from the subject the same day that the sample of the bodily fluid from the subject is obtained. In other embodiments, the reference sample is taken after the day that the sample of the bodily fluid from the subject is obtained. In additional embodiments, the reference sample is taken prior to the day that the sample of the bodily fluid from the subject is obtained.

These embodiments are not narrowly limited to any particular means of confirming that the reference sample comprises DNA from the subject. In some embodiments, the reference sample is obtained from the subject while the subject is witnessed in the presence of an authorized person or a camera that visualizes the sample being obtained. A nonlimiting example of this is a reference sample that is a blood or urine sample obtained from the subject by a medical professional during a visit to a doctor's office, blood donation center, clinic, or emergency room by the subject. In other embodiments, the reference sample is established as from the subject by comparing the genetic fingerprint of the reference sample with a genetic fingerprint of a third sample obtained from the subject while the subject is witnessed in the presence of an authorized person or a camera that visualizes the third sample being obtained.

In some embodiments, the sample of the bodily fluid undergoes analysis in addition to the determination of the genetic fingerprint of the sample. A nonlimiting example of such an analysis is an analysis for a foreign substance such as a drug (e.g., performance enhancing drug or psychoactive drug, a toxic substance, or a pathogen.

Any DNA in the reference sample and sample of the bodily fluid can be analyzed to determine the genetic fingerprint. In some embodiments, the DNA analyzed from the sample of the bodily fluid is from cells in the sample. In other embodiments, the DNA analyzed from the sample of the bodily fluid is cell free DNA in the sample. In certain embodiments, the sample of the bodily fluid is urine and the cell free DNA is transrenal genomic DNA. Genetic fingerprints of any DNA that is expected to be unchanged in the subject between the taking of the reference sample and the provision of the sample of the bodily fluid can be analyzed to determine the genetic fingerprint of the subject (from the reference sample) or of the sample of the bodily fluid. In some embodiments, the DNA analyzed is DNA that is expected to be unchanged through the life of the subject. In various embodiments, the analyzed DNA is mitochondrial DNA. In other embodiments, the analyzed DNA is chromosomal DNA.

Any DNA that varies among individuals can be used to determine the genetic fingerprint of the sample of the bodily fluid and the reference sample. Nonlimiting examples of such methods are short tandem repeat (STR) markers, single-nucleotide polymorphism (SNP) markers, copy number polymorphisms, small insertion and deletion polymorphisms, and chromosomal abnormalities or translocations, e.g., as described in Zietjiewicz et al., 2012.

Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.

Example 1. Concentration of urine

This method allows quick, safe, and efficient isolation of nucleic acids and other components of whole urine from a large starting volume (i.e. greater sample volume). See also

PCT Publication WO 2015/164435 incorporated by reference, particularly disclosure therein related to the various methods described for concentrating urine.

Urine volumes of about 90 - 110 can be applied to a filtration container, allowing separation of salt and fluids from other components of urine (for example, nucleic acids, and proteins).

In one example, a preservative of 10 ml of 0.5 M EDTA was added to 90-110 ml whole urine from a patient. The preservative prevents degradation of the cfDNA in the urine by nucleases. The preservative-urine mixture was dispensed into the upper chamber of a Sartorius VIVACELL 100 container. The VIVACELL 100 includes a filter pore having greater than 90% retention of molecules greater than 5000 Dalton in size wherein retention is based on protein, where the typical retention cut-off for dsDNA is 25 bp and greater. Negative pressure was applied until the original urine volume was reduced to less than 2 ml (concentrated urine) and removed to a microcentrifuge tube.

The upper chamber of the V ACELL 100 was rinsed with 3 ml binding buffer (Tris- HC1, EDTA, 2-propanol) to collect any remaining urine components, the rinse was combined with the above 2 ml concentrated urine, resulting in about 5 ml volume of concentrated urine with binding buffer. The concentrated urine may then be stored for later studies or immediately subjected to further assays or processes for nucleic acid analysis such as, for example, detection of specific ctDNA and/or cfDNA sequences/mutant alleles. Example 2. Isolation of nucleic acids using magnetic particles

Preferential isolation of small (< 500 bp) nucleic acids from concentrated urine can be achieved using magnetic particles having anion exchange moieties.

Concentrated urine with binding buffer, prepared as described in Example 1, was spiked with synthetic DNA targets consisting of plasmid pUC fragments of 103 bp and 414 bp and fragments of 200 bp and 300 bp of the EGFR gene encoding an L858R mutation. The spiked urine was combined with SiMAG-Q (Chemicell GmbH) anion exchange particles, 1.0 μιη size. SiMag-Q particles have a maghemite (Fe₃0₄) superparamagnetic core and a 1-hydroxypropyl trimethylammoniumchlorid (-Si-R-CH₂-CHOH-CH₂-N⁺(CH₃)₃ CI^") quaternary ammonium group, with strong anion exchange properties. The mixture was agitated to allow binding of sample nucleic acids to the functional groups.

The particles were separated from the rest of the mixture with a magnet and the nucleic acids were eluted using an NaCl solution of 1.6 M, 1.7 M or 1.8 M. The particles were then subjected to a 5.0 M NaCl solution, eluting larger nucleic acids.

The aspects of these methods using magnetic particles, along with the purification methods below, were automated using the Kingfisher™ Flex System (i FF)(https://tools.thermofisher.com/content/sfs/manuals/D01475~.pdf) using the following protocol:

2.0 ml or less of concentrated urine was added to a well of a deep well plate (24 wells). Binding buffer ("ΤΒΒ-Τ/ΓΡΑ") was added to the urine to a combined ratio of 1:1 urine (e.g., to 2.0 ml concentrated, 1.0 ml TBB-T and 1.0 ml 100% isopropyl alcohol) (TBB-T Buffer: 100 mM EDTA/50 mM Tris-HCl, pH 8). SiMag-Q particles (25 μΐ) were then added to the well. The magnetic beads were collected on KFF tip combs. To a separate 24 deep well plate, 250 μΐ of a "Low Salt" solution, 1.6, 1.7 or 1.8 M NaCl, was dispensed into each well. To an additional deep well plate "High Salt", 250 μΐ of 5 M NaCl, was dispensed into each well. The appropriate protocol was selected on the instrument; the plates were loaded into the processor and the run proceeded as follows. Run time was approximately 40 minutes.

Complete binding of all DNA to the beads was ensured by allowing the processor to slowly mix the urine/particle/binding buffer using its tip comb for 30 minutes. The beads were collected with the magnetic head for 30 seconds, three times, to ensure adequate separation of the particles from the binding solution. The bound particles were transferred to the low salt elution plate and mixed aggressively every 30 seconds for a total of 5 minutes at 70 °C. The beads were transferred to the 5 M NaCl plate where they underwent 30 seconds of mixing followed by a 30 second pause, for a total of 5 minutes at 70 °C.

Fragment recovery was determined using droplet digital PCR (ddPCR) for the pUC (103 bp and 414 bp) fragments and Next Generation Sequencing (NGS) for the EGFR L858R (200 bp and 300 bp) fragments.

The results are depicted in FIG. 10. For each fragment DNA species (103 bp, 200 bp, 300 bp, and 414 bp), the greatest relative recovery was obtained with 1.8M NaCl elution. The 1.6 M NaCl solution also effectively eluted 103 bp species, but was ineffective at recovering the 200 bp, 300 bp and 414 bp fragments. All three salt concentrations resulted in good relative recovery of the 103 bp fragment; the 200 bp fragment was effectively isolated at 1.7 M and 1.8 M NaCl solution, with the 300 bp fragment having about 50% less mean relative recovery (as compared to 200 bp) at 1.7 M but a somewhat greater recovery with the 1.8 M NaCl solution. NaCl at 1.8 M was also effective at isolating the 414 bp fragment.

To determine the size profile of the recovered cfDNA from urine using the above method, concentrated urine samples were subjected to the method and eluted with 1.8 M NaCl, then 5 M NaCl, after spiking the sample with a 550 bp marker and a 3500 bp marker. The samples were analyzed using gel electrophoresis imaging using Ranger Technology from Coastal Genomics. The results are shown in FIG. 11.

The 1.8 M solution eluted DNA fragments of 500 bp or less, along with small amounts of large fragment DNA. There is a clear delineation between the small, presumably transrenal, DNA from the larger pieces. Example 3. Purification of eluted nucleic acids

The nucleic acids eluted with 1.8 M NaCl were purified as follows, using the KingFisher™ Flex System. Into the well containing the 1.8 M NaCl eluate (250 μΐ) described in Example 2 above, was added 500 μΐ silica binding solution (ECBB)(33% 6 M guanidinium thiocyanate, 67% isopropyl alcohol), 20 μΐ of P75 (75% w/v PEG8000), and 20 μΐ of SiMag- Silanol Magnetic particles (maghemite core, 1.0 μιη, having silanol coating). The mixture was mixed for 10 minutes at room temperature. The particles were separated and collected for 30 seconds, three times on tip combs, the particles were washed twice in separate wells, the first wash was in 750 μΐ of 70% ethanol with guanidinium thiocyanate; the second wash was in 250 μΐ 70% ethanol. The particles were then removed from the second wash solution and let dry for 5 minutes above the splash plate to allow the residual ethanol to evaporate. The particles were then released into the silanol elution buffer (10 mM tris-HCl, pH 8.5) and mixed aggressively to ensure all DNA is released into the buffer at 65-70 °C for 5 minutes. The particles are collected for 30 seconds 3X and then removed and left in the 2nd "sample" plate. This process yields approximately 135-140 μΐ cfDNA ready for storage or immediate downstream processing.

Example 4. Recovery studies

Synthetic 103 bp DNA target (pUC) was spiked into urine samples from 5 donors. Each urine sample was processed through concentration, isolation and purification as described in Examples 1-3, where the elution from the anion exchange particles (Example 2) was with 1.8 M NaCl. Fragment recovery was determined using droplet digital PCR.

Results are shown in FIG. 12 and Table 1. Overall mean recovery was 67%, with variations between donors.

Table 1.

Mean Std Dev %cv

Donor 1 59.5 0.075 11.8

Donor 2 62.7 0.056 8.9

Donor 4 63.0 0.076 12.1

Donor 5 67.6 0.089 13.2

Donor 6 84.0 6.9 8.2 To determine the effect of the concentration step on the recovery of cfDNA in concentrated urine, urine from 6 donor samples were first concentrated as in Example 1, then the 103 bp pUC fragment was spiked into the already concentrated urine, and processed through the isolation and purification protocols described in Examples 2 and 3, respectively (1.8 M NaCl elution). Results are shown in FIG. 13 and Table 2. Considerably greater recovery was achieved with the urine spiked after concentration than before concentration.

Table 2.

Recovery of cancer mutations from urine of cancer patients was next evaluated. Urine samples were obtained from seven cancer patients, each with a BRAF mutation. The concentration, isolation and purification procedures of Examples 1, 2 and 3 above were followed with the patient urine, with a 1.8 "low salt" elution and a 5.0 "high salt" elution recovered and purified. Results are shown in FIG. 14. BRAF mutation detection was: 1.8 M elution - 10/14 reps; 5 M elution - 9/14 reps. Five of the reps (arrows) showed a greater percentage of the mutation/wild-type in the 5 M eluate than in the 1.8 M eluate. Similar results were seen in urine from 11 disease patients with KRAS mutations.

Example 5. Evaluation of entrapment of cf nucleic acids in urine as reducing recovery

The presence of Tamm-Horsfall protein (THP), a.k.a. uromodulin, in urine was evaluated to determine its effect on the recovery of cf nucleic acids using the methods described herein. THP is a glycoprotein secreted into urine by specific sections of Henle's loop. THP, constituting about one-third of total urinary protein, forms a mucous meshwork of fibrils that traps cells, cell fragments, and potentially DNA in urine. Dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) treatment can depolymerize THP by reduction of disulfide bonds (Fernandez-Llama et al, 2010). To determine the effect of TCEP on recovery of cf nucleic acids from urine, a synthetic 103 bp DNA fragment (pUC) was spiked into whole urine. Each urine sample was split and TCEP was added at 0, 5 mM, 25 mM or 50 mM and mixed at RT overnight. The samples were then concentrated, isolated and purified as in Examples 1-3, with the cf nucleic acids eluted from the anion exchange particles with 1.8 M NaCl.

Results are shown in FIG. 15. Addition of TCEP to whole urine led to a significant improvement in recovery.

Example 6. Isolation of urine nucleic acids from whole, unconcentrated urine

A synthetic 103 bp DNA fragment (pUC) was spiked into urine samples obtained from 4 normal donors. Each urine sample was divided among 50 ml conical tubes at 40 ml urine per tube. Binding buffer with magnetic beads (10 ml) was added to each conical tube and rotated at room temperature for 1 hr. Specimens were centrifuged to isolate pellet and the pellet then used for further isolation and purification steps. Fragment DNA recovery was determined and confirmed using ddPCR. The results are shown in FIG. 16.

The Overall Mean recovery was 81.1% and the Overall %CV was 14.1%. This demonstrates that similar or better recovery can be achieved using whole, unconcentrated urine.

Example 7. Collection and Purification of Cell Free Nucleic Acids from Urine using Anion Exchange Chromatography

The method described here, and components thereof, are illustrated in FIGS. 17-19, results are shown in FIG. 20. In this method, cell-free, transrenal DNA is isolated from a high volume (200 ml) urine specimen without concentrating the urine. The method utilizes a novel adaptor to speed the workflow when compared to other methods; without automation, the method allows the extraction of 12 samples in parallel using inexpensive lab equipment in less than 8 hr.

Urine samples (10) were collected in the collection container illustrated in FIGS. 1-3. Up to 200 ml urine + preservative (0.5 M EDTA) was centrifuged at 3000g for 15 minutes. Avoiding the pellet, the urine was transferred into a 225 ml Falcon PP Centrifuge Tubes (Corning Cat# 352075). Q Sepharose® (2.8 ml) (GE-Healthcare; Cat# 17-0510-01) to each 225 ml tube. The 225 ml tubes were then rotated at room temperature for at least 60 minutes, using a medium speed setting on a Glas-Col laboratory rotator (Cat# 099 A RD4512). After rotating, the 225 ml tubes were centrifuged at 3000xg for 5 minutes. The supernatant was then vacuum aspirated, avoiding the pellet. Wash buffer (4 ml; 400 mM NaCl + EDTA/EGTA) was added to the 225 ml tube to resuspend the Q Sepharose® pellet; the wash- buffer with suspended Q Sepharose® was transferred to a Poly-Prep Chromatography column (Bio-Rad; Cat# 731-1553; FIG. 18A, left side) in a chromatography assembly, comprising the Poly-Prep column inserted into the adaptor illustrated in FIG. 17, and inserting that combination into a sterile 50 ml conical test tube (Corning; Cat# 430791 and 430828)(FIG. 18A, right side). This assembly suspends and secures the Poly-Prep column above the bottom of the 50 ml test tube, allowing the Poly-Prep column to be centrifuged in the 50 ml test tube, accelerating the flow of solutions through the Poly-Prep column.

After incubation of the Poly-Prep column for 5 minutes, the assembly was centrifuged, and the flow-through was discarded. The adaptor and Poly-Prep column were then transferred to a new 50 ml test tube and 4 ml of EB-S1 Buffer (500 mM NaCl) was add to the Poly-Prep column and mixed with the pellet Q Sepharose®. After an incubation of 5 minutes at room temperature, the chromatography assembly was centrifuged at 3000 rpm for 3 minutes. Four ml of EB-S2 Buffer (600 mM NaCl) was then added and mixed with the pellet. After another incubation for 5 minutes, the chromatography assembly was again centrifuged as above. The two EB-S Buffer eluates represent the bulk of DNA fragments less than 400 bp; it is almost completely depleted of fragments greater than 400 bp (FIG. 20).

After again transferring the adaptor and Poly-Prep column to a new 50 ml test tube, 4 ml of EB-L Buffer (700 mM NaCl) was added to the Poly-Prep column. After a 5 minute incubation as above, the chromatography assembly was centrifuged as above. That eluate represents primarily fragments larger than 400 bp (FIG. 20).

Sodium acetate (2 M, pH 4.2) was added to each eluate to 3% (v/v)(240 μΐ to 8 ml from the SF buffers; 120 μΐ to 4 ml from the LF buffer). Ten volumes of PNI buffer (40% mixture of guanidine hydrochloride in isopropanol; 60% 2-propanol) was added to each elution.

The DNA in the two solutions (pooled eluate from the two EB-S buffers and eluate from the EB-L buffer) was purified by the following procedure.

The samples were each run through a QIAquick spin column (Qiagen; Cat# 28306) with a 20 ml tube extender (Qiagen; Cat# 1029526)(FIG. 18B, 900, 910) under vacuum. The tube extender was removed and 500 μΐ of PE buffer (Qiagen; Cat# 19065) was run through each spin column twice under vacuum. The QIAquick column was then placed on a collection tube (FIG. 18B, 920) and centrifuged at 14,000 rpm for 3 minutes. The spin column with the tube extender and/or with the collection tube can be conveniently placed inside a 50 ml test tube, as illustrated in FIG. 18C).

After centrifuging the QIAquick column, the collection tube was discarded and the QIAquick column was placed into a 1.5 ml Eppendorf tube. 50 μΐ of EB buffer (10 mM Tris-Cl, pH 8.5) was added to the column and incubated at room temperature for 3 minutes. The QIAquick column in the Eppendorf tube was then centrifuged at 14,000 rpm for 2 minutes. The column was disposed and the eluted DNA was retained. Example 8. Comparison of DNA Extraction Methods I

The method described in Example 7 ("V2 method") was compared with a method ("VI method") similar to that taught in PCT Patent Publication WO 2015/164435.

The VI method is comprised of a whole urine concentration step using a Vivacell-100 which is limited to a maximum urine volume of 100 ml. This method adds a $30 cost to each sample extraction and requires pressure filtration that can take up to 6-8 hours for some clinical samples.

The VI method was carried out as follows: 100 ml of each donor urine pool was concentrated to 4-5 mL using the Vivacell-100. 700 μΐ of Q Sepharose® was then added and rotated at room temperature for 60 minutes. Samples were spun at 3000 xg for 5 minutes and the supernatant removed. The Q Sepharose® pellet was resuspended with 500 μΐ Buffer PNI, loaded onto an empty Bio-Rad spin column and incubated for 5 minutes. Each column was then spun for 3 minutes at 3000 rpm. This elution step was repeated with the same elution buffer and the two eluates combined to yield ~1.2 ml. The pH was adjusted by adding 3% (v/v) of 2M NaOAc, pH 4.2 to each elution (e.g. 30 μΐ NaOac to 1 ml elution). 750 μΐ of eluate was added to each QIAquick column and spun at 14,000 rpm. The remaining eluate was added to the column and spun again. After washing the column twice with 500 μΐ Buffer PE, DNA was eluted with 50 μΐ Buffer EB.

The V2 method was developed to (a) reduce extraction cost and processing time, and (b) to increase the volume of extracted urine to above 100 ml, to increase DNA yield.

Twenty-eight urine samples from pregnant women bearing a male fetus were collected over 1-5 consecutive days. After being stored at 4 °C for 1 week to 6 months, the samples were separated into 50 mL conical tubes and spun to remove the pellet containing urinary tract cells and stored at either -20 °C or -80 °C for 1-2 months. One EGFR Exl9del-positive cancer patient sample was collected over 2 days. After being stored at 4 °C for 3 months, the samples were separated into 50 ml conical tubes and spun to remove the cells. One fresh KRAS-positive cancer patient sample was collected over 2 days then stored at 4 °C for 1 week. A minimum of 300 ml of urine from each donor or patient was thawed and pooled for extraction.

Post Isolation: The Quant-iT™ dsDNA Assay Kit (Invitrogen) was used to quantitate the total amount of DNA recovered. An RNaseP qPCR assay (WO 2015/164435) was used to quantitate total amplifiable DNA recovered. The High Sensitivity Large Fragment Analysis Kit (Advanced Analytical Technologies, Inc.) was used to determine range of fragment lengths in each elution. To test how much SRY was recovered from the male fetal pregnancy samples, an SRY-1 qPCR assay was used (US Patent Publication 2015/0329920, Example 4). To test how many copies of EGFR exl9del or KRAS G12X were recovered from the tissue-positive cancer samples, Next Generation Sequencing-based Exl9del (US Patent Publication 2016/0273022, Example 1) or KRAS (US Patent Publication 2016/0273022, Example 3) Trovera tests were run on the samples respectively.

Results

Maternal urine tests for SRY: %SRY ranged from 0.0% - 56.6% and total SRY recovery ranged from 0 - 42,012 cps for the samples extracted using the V2 method. For the VI extracted samples, %SRY ranged from 0.0% - 28.6% and total SRY recovery ranged from 0 - 36,615 cps (Table 3). The median %SRY was 3.6% for V2 samples vs 1.0% VI samples, a 3.6- fold difference, and the median total SRY recovery was 1003 copies for V2 samples vs 930 copies VI samples (FIG. 21). When examining the Fragment Analyzer plots, there was a distinct reduction in the amount of high molecular weight DNA recovered in the V2 EB-S elutions vs. the VI extracted samples (FIG. 22).

V2 200 mL EB-S Elution 34.1% 44426 6466 3344

TROV-Y_075

VI 100 mL PNI 6.6% 46241 20734 1455

V2 200 mL EB-S Elution 8.6% 91301 25070 2366

TROV-Y_076

VI 100 mL PNI 2.5% 164140 116312 3016

V2 200 mL EB-S Elution 3.7% 8522 4019 156

TROV-Y_077

VI 100 mL PNI 0.0% 48913 18851 0

V2 200 mL EB-S Elution 17.0% 42769 16546 3384

TROV-Y_078

VI 100 mL PNI 2.7% 111905 1 3103

V2 200 mL EB-S Elution 1.5% 57689 47202 741

TROV-Y_081

VI 100 mL PNI 0.4% 164282 2 635

V2 200 mL EB-S Elution 28.8% 51416 13509 5476

TROV-Y_083

VI 100 mL PNI 9.6% 92450 65569 6953

V2 200 mL EB-S Elution 16.8% 64415 55679 11264

TROV-Y_086

VI 100 mL PNI 18.5% 93938 98170 22263

V2 200 mL EB-S Elution 48.7% 16255 14225 13529

TROV-Y_089

VI 100 mL PNI 13.5% 145265 156097 24279

V2 200 mL EB-S Elution 5.3% 28550 37871 2139

TROV-Y_094

VI 100 mL PNI 4.4% 146720 119629 5532

V2 200 mL EB-S Elution 19.9% 184524 169550 42012

TROV-Y_095

VI 100 mL PNI 28.6% 135334 91349 36615

V2 200 mL EB-S Elution 0.1% 40690 46174 41

TROV-Y_098

VI 100 mL PNI 0.0% 376645 492281 39

Quant-it RNaseP SRY qPCR

TROV Donor Extraction Specimen

tiuiion o/bcSpKvT l oiai qr K l Olal l oiai

ID Method Volume

Copies Copies Copies

V2 200 mL EB-S Elution 2.5% 61244 27284 710

TROV-Y_102

VI 100 mL PNI 1.3% 95266 78140 1057

V2 200 mL EB-S Elution 2.6% 42412 26860 709

TROV-Y_107

VI 100 mL PNI 0.2% 497770 312246 670

V2 200 mL EB-S Elution 3.1% 269909 86180 2768

TROV-Y_lll

VI 100 mL PNI 0.5% 298465 264209 1232

V2 200 mL EB-S Elution 0.2% 242151 277929 603

TROV-Y_114

VI 100 mL PNI 0.0% 696278 1300714 11

V2 200 mL EB-S Elution 2.4% 169817 42021 1021

TROV-Y_116

VI 100 mL PNI 1.0% 178297 179380 1852

V2 200 mL EB-S Elution 0.8% 49440 32007 260

TROV-Y_117

VI 100 mL PNI 0.2% 230538 138061 209

V2 200 mL EB-S Elution 3.4% 16383 8142 287

TROV-Y_119

VI 100 mL PNI 0.1% 16532 13050 12

TROV-Y_120 V2 200 mL EB-S Elution 5.3% 21555 7004 1215

Table 3. V2 vs VI: percent SRY and total SRY in pregnancy samples. Total DNA yield and

SRY copies detected in 28 male fetal pregnancy samples. Percent SRY was calculated as total SRY copies divided by total RNaseP copies + total SRY copies. In the case where the RNaseP value was 0, the Quant-iT™ value was used.

Tissue-positive cancer samples: Two cancer samples, TROV-044-0052 and TROV- 044-1001, were processed using both the VI and V2 extraction methods. Step-wise EB-S elutions were performed with fractions between 500-650 mM NaCl. For the V2 extracted samples, %MT for the combined EB-S elutions was 1.2% and 0.1% and total MT recovery was 3485 and 25 cps for 044-0052 and 044-1001 respectively. For the VI extracted samples, %MT was 0.5% and 0.0% and total MT recovery was 2711 and 0 cps for 044-0052 and 044-1001 respectively (Table 4). The median %MT was 0.7% for V2 samples vs 0.3% VI samples. The median total SRY recovery was 1755 copies for V2 samples vs 1356 copies VI samples (FIG. 23). Overall, an equivalent amount of total mutant copies were recovered but there was a 2.3 fold higher %MT (0.7% vs 0.3%) using the V2 vs VI extraction methods (FIG. 23). When examining the Fragment Analyzer plots, there was a distinct reduction in the amount of high molecular weight DNA recovered in the V2 EB-S elutions (FIG. 24). This correlates with a reduction in total DNA seen in V2/ EB-S compared to Vl/PNI as determined using Quant-iT™ and RNaseP (Table 4, FIG. 23). RNaseP

TROV Quant-it RNaseP Mutant

Extraction Specimen Quant-it qPCR

Donor Elution %MT Total qPCR Total Method Volume Total ng Total ID Copies Total ng Copies

Copies

Wash 0.0% 159 48120 6 1752 2

EB-S1

3.6% 55 16658 5 1590 621 Elution

TROV

V2 200 mL EB-S2

-044- 1.0% 897 271914 288 87189 2865

Elution

0052

EB-L

0.1% 2772 840081 2227 674947 919 Elution

VI 100 mL PNI 0.5% 1918 581247 706 214070 2711

Wash FAIL 92 27982 1 202 —

EB-S1A

FAIL 2 749 5 1440 — Elution

EB-S1B

1.2% 7 2139 26 7872 25 Elution

TROV

V2 200 mL EB-S2A

-044- 0.0% 24 7149 110 33408 0

Elution

1001

EB-S2B

0.0% 60 18311 238 72000 0 Elution

EB-L

0.0% 1935 586411 1346 408000 0 Elution

VI 100 mL PNI 0.0% 317 96159 488 147840 0

Table 4. V2 vs VI : percent mutant and total mutant copies in cancer samples. Extracted DNA from 2 tissue-positive cancer samples - a KRAS-positive and an EGFR Exl9del-positive sample. Percent mutant was calculated by total MT copies divided by total Quant-iT™ copies + total MT copies.

Conclusions

V2 shows superior enrichment of transrenal DNA (%SRY or %MT) and an equivalent total transrenal DNA recovery over the VI method. V2 shows a preferential isolation of the low MW transrenal DNA in the SF elution when compared to the VI method. DNA fragment profiles are not necessarily indicative of transrenal DNA (SRY or MT) recovery likely due to contamination with post-renal DNA fragments. Example 9. Comparison of DNA Extraction Methods II

The V2 method as described in Example 7 was compared with three commercial DNA extraction kits.

Methods

Ten urine samples from pregnant women bearing a male fetus were collected over 1-5 consecutive days. After being stored at 4 °C for 1 week to 6 months, the samples were spun to remove the pellet containing urinary tract cells and stored at either -20 °C or -80 °C for 1-2 months. One fresh KRAS-positive cancer patient sample was collected over 2 days then stored at 4 °C for 1 week. A minimum of 300 ml of urine from each donor or patient was thawed and pooled for extraction. The following competitor kits were used: Norgen RNA/DNA/Protein Purification Kit, Zymo Quick-DNA Urine Kit and QIAamp Circulating Nucleic Acid Kit. 1.75 ml, 40 ml and 4 ml of urine was extracted with the Norgen, Zymo and Qiagen kits, respectively, and the extraction protocols were carried out according to the vendor manuals.

Results

Fetal pregnancy samples: Quant-iT™ and the RNaseP qPCR assay (described in Example 8) were used to quantify DNA yield and the SRY qPCR assay (Example 8) was used to quantify the SRY signal. For the V2 extracted samples, %SRY ranged from 0.1% - 17.1% and total SRY recovery ranged from 41 - 2768 cps. For the samples extracted with the three competitor kits, %SRY ranged from 0.0% - 9.9% and total SRY recovery ranged from 0 - 285 cps (Table 5). The median total SRY recovery was 657 copies for V2 samples vs 0 - 115 copies for samples extracted with other kits (FIG. 25). The median %SRY was 2.8% for V2 samples vs 0.0% - 1.3% for samples extracted with three competitor kits (FIG. 25). Overall, there was 2.2 - 2.8 fold higher %SRY and 5.7 - 657 fold more total SRY recovery using the V2 method vs competitor kits. When examining the Fragment Analyzer plots, there was a distinct reduction in the amount of high molecular weight DNA recovered in the V2 EB-S elutions (FIG. 26).

Norgen SF 1.75 mL 0.0% 29098 48517 2

Norgen LF 1.75 mL 0.0% 15257 30342 0

Zymo 40 mL 0.0% 852945 2185797 0

Qiagen 4 mL 0.0% 326022 467152 18

V2 EB-S El ution 200 mL 2.5% 61244 27284 710

Norgen SF 1.75 mL 0.0% 2692 5163 0

TROV-Y_102 Norgen LF 1.75 mL 0.4% 1793 2977 13

Zymo 40 mL 0.0% 241113 260133 0

Qiagen 4 mL 0.4% 60810 51740 221

V2 EB-S El ution 200 mL 3.1% 269909 86180 2768

Norgen SF 1.75 mL 1.2% 3586 13405 168

TROV-Y_lll Norgen LF 1.75 mL 0.0% 2146 6928 0

Zymo 40 mL 0.0% 254281 448904 0

Qiagen 4 mL 0.0% 124723 152150 0

V2 EB-S El ution 200 mL 0.2% 242151 277929 603

Norgen SF 1.75 mL 0.0% 6185 18477 0

TROV-Y_114 Norgen LF 1.75 mL 0.6% 3041 6016 35

Zymo 40 mL 0.0% 798351 1332321 0

Qiagen 4 mL 0.0% 261738 274857 3

V2 EB-S El ution 200 mL 2.4% 169817 42021 1021

Norgen SF 1.75 mL 5.3% 2838 5057 285

TROV-Y_116 Norgen LF 1.75 mL 3.0% 1762 2668 82

Zymo 40 mL 0.0% 122927 147382 0

Qiagen 4 mL 0.0% 58253 55482 10

TROV Donor Extraction Specimen Quant-it RNaseP qPCR SRY qPCR

ID Method Volume Total Copies Total Copies Total Copies

V2 EB-S El ution 200 mL 0.8% 49440 32007 260

Norgen SF 1.75 mL 3.1% 2574 3815 122

TROV-Y_117 Norgen LF 1.75 mL 9.9% 1488 1615 178

Zymo 40 mL 0.0% 157811 161625 0

Qiagen 4 mL 0.0% 37538 42011 0

V2 EB-S El ution 200 mL 3.4% 16383 8142 287

Norgen SF 1.75 mL 7.0% 1352 1437 108

TROV-Y_119 Norgen LF 1.75 mL 7.1% 817 687 52

Zymo 40 mL 0.0% 40774 29489 5

Qiagen 4 mL 0.4% 17786 18671 84

V2 EB-S El ution 200 mL 14.8% 21555 7004 1215

Norgen SF 1.75 mL 0.5% 2469 20288 92

TROV-Y_120

Norgen LF 1.75 mL 1.9% 1884 2758 52

Zymo 40 mL 0.0% 277536 285885 0 Qiagen 4 mL 0.4% 31009 30291 129

V2 EB-S Elution 200 mL 17.1% 22222 8858 1833

Norgen SF 1.75 mL 1.4% 4091 8820 124

TROV-Y_121 Norgen LF 1.75 mL 1.3% 3017 5560 76

Zymo 40 mL 0.0% 343321 428307 28

Qiagen 4 mL 0.0% 41378 53708 0

V2 SF Elution 200 mL 9.9% 68962 2723 298

Norgen SF 1.75 mL 9.2% 11728 1420 144

TROV-Y_122 Norgen LF 1.75 mL 0.0% 6241 613 0

Zymo 40 mL 0.0% 320517 39160 1

Qiagen 4 mL 0.1% 127503 22793 15

Table 5. V2 vs competitor kits: percent SRY and total SRY in pregnancy samples. Extracted DNA from 10 samples from pregnant women bearing a male fetus. Percent SRY was calculated by total SRY copies divided by total RNaseP copies + total SRY copies.

KRAS-positive cancer sample: Quant-iT™ and the RNaseP qPCR assay were used to quantify DNA yield and the KRAS assay was used to quantify mutant signal. For the V2 extracted sample, %MT was 1.2% for the combined EB-S elutions vs 0.1% - 0.4% for the sample extracted with other kits. Total MT recovery was 3486 cps for the combined V2 EB-S elutions vs 5 - 808 cps for the sample extracted with other kits. Overall, there was 9.5 - 38 fold higher %MT and 4.3 - 697 fold more total MT using the V2 method vs competitor kits (FIG. 27).

Conclusions

The V2 assay showed superior enrichment of transrenal DNA (%SRY or %MT) and total transrenal DNA recovery over all of the competitor urine extraction kits tested. Additionally, the V2 assay showed a greater preferential isolation of the low MW transrenal DNA in the EB-S elution over all of the competitor urine extraction kits tested. DNA fragment profiles were not necessarily representative of transrenal (SRY or MT) recovery.

Example 10. Reproducibility Studies I

The consistency of transrenal DNA isolation (e.g. copies/ml urine) from urine samples collected at different times from the same donor was evaluated.

Methods

Samples from two pregnant women bearing a male fetus, designated TROV-Y 077 and TROV-Y 081, were collected over 2 consecutive days. After being stored at 4 °C for 3 months, the samples were spun to remove the pellet containing urinary tract cells and stored at -20 °C for 1 month. Three time points with a minimum of 200 ml each were selected for each donor and thawed. The samples were extracted using the V2 protocol as described in Example 7.

Post Isolation: The RNaseP and SRY qPCR assays described in Example 8 were used to quantitate total amplifiable DNA and transrenal SRY respectively.

Results

Samples from subject TROV-Y 077 had SRY signals ranging from 0.0 - 6.0 cps/μΐ (avg. 3.6) with a 72% CV. RNaseP ranged from 0.7 - 3.4 ng/μΐ (avg. 1.6) with a 83% CV. Samples from subject TROV-Y 081 had SRY signals ranging from 7.4 - 128 cps/ μΐ (avg. 87.4) with a 65% CV. RNaseP ranged from 2.0 - 4.2 ng/ μΐ (avg. 3.0) with a 29% CV. Thus the DNA yield and SRY signal were not equivalent across three collection times for the same urine sample (Table 6, FIG. 28).

Conclusions

Reproducibility was not achieved across separate time points for the same urine donor within 2 collection days. Importantly, a sample that is negative for SRY at one time point does not necessarily mean there will consistently be a lack of transrenal DNA at other time points for that particular donor. Collection of urine over multiple time points is recommended.

Example 11. Reproducibility Studies II

The consistency of using NaCl to elute DNA from Q Sepharose® followed by binding/elution from a silica column was evaluated.

Methods

Four urine samples from pregnant women bearing a male fetus were collected over 2-5 consecutive days. After being stored at 4 °C for up to 4 months, the samples were spun to remove the pellet urinary tract cells and stored at -20 °C for 2 months. 200 ml from each donor was pooled into one batch of 800 ml and split equally into four 225 ml Falcon tubes. 2.8 ml of Q Sepharose® was added to each of four replicates of 200 ml urine and then rotated at room temperature for 60 minutes. The samples were spun at 3000xg then the supernatant was removed. Each Q Sepharose® pellet was resuspended with 4 mL of wash buffer (Example 7) and pooled into one slurry. The slurry was thoroughly resuspended and aliquoted evenly into four separate Poly-Prep chromatography columns. The V2 downstream extraction steps were continued as described in Example 7.

Post Isolation: The RNaseP qPCR assay described in Example 8 was used to quantitate total amplifiable DNA recovered. To test how much SRY was recovered, the SRY-1 qPCR assay (Example 8) was used.

Results

The four reps had an SRY signal ranging from 58 - 111 cps/μΐ (avg. 94) with a 23% CV. RNaseP ranged from 1.3 - 1.5 ng/μΐ (avg. 1.4) with a 6% CV. Therefore both the DNA yield and SRY signal were equivalent across four reps for the same pooled urine sample (Table 7, FIG.

Table 7. Consistency of V2 elution and downstream extraction steps.

Conclusions

EB-S elution reproducibility was achieved across four replicates for the same pooled urine sample when using the V2 method downstream of Q Sepharose® binding. The LF elution shows variability of SRY signal across replicates due to one outlier with a higher signal.

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In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.

As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

All references cited in this specification are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

Claims

What is claimed is:

1. A collection container for collecting a sample of a fluid, the collection container comprising the following components:

an additive,

wherein, when the lid engages the open top, the additive is released into the sample.

2. The collection container of claim 1, wherein the cup near the open top and the lid have complementary screw threads such that the lid engages the open top by screwing onto the open top.

3. The collection container of claim 1, wherein the additive is sealed in a fluid container in an underside of the lid by a breakable seal.

4. The collection container of claim 1, wherein the additive is a preservative that prevents degradation of a molecule present in the fluid.

5. The collection container of claim 4, wherein the molecule is a biological macromolecule.

6. The collection container of claim 5, wherein the biological macromolecule is a nucleic acid.

7. The collection container of claim 1, wherein the fluid is urine.

8. The collection container of claim 1, wherein

the cup near the open top and the lid have complementary screw threads such that the lid engages the open top by screwing onto the open top;

the additive is sealed in a fluid container in an underside of the lid by a breakable seal; the cup further comprises an upper section comprising the open top and a lower section below the upper section, wherein the upper section is wider than the lower section, and

at least one upwardly directed push bar that engages the breakable seal, breaking open the seal and releasing the additive into the cup when the lid is screwed unto the open top, wherein the push bar does not comprise a sharp edge or point.

9. The collection container of claim 8, wherein the underside of the lid further comprises at least one spike sealed in the fluid container wherein, when the lid is screwed onto the top, the push bar pushes the breakable seal into the spike, which breaks open the seal.

10. The collection container of claim 8, wherein the underside of the lid further comprises at least one tower inside the fluid container protruding outward from the lid.

11. The collection container of claim 8, further comprising a conical insert on a bottom surface inside the cup, wherein solid material in the sample can collect in the insert.

12. The collection container of claim 8, wherein the fluid container is integrated directly into the lid and sealed with the breakable seal.

13. The collection container of claim 8, wherein the fluid container is molded and sealed separately then affixed to the underside of the lid.

14. The collection container of claim 8, further comprising a skirt below the bottom supporting a firm stand of the container thereon.

15. The collection container of claim 8, wherein the fluid is urine.

16. The collection container of claim 15, wherein the additive is a preservative that prevents degradation of a nucleic acid present in the urine.

17. The collection container of claim 1, wherein

the lid engages the open top by screwing onto the open top; the additive is in a vessel in the cup extending from above the open top to near the bottom, wherein the vessel has a top and a pierceable bottom;

below the vessel is a piercing and draining mechanism; and

the lid comprises an underside with a curved ramp, wherein

screwing the lid onto the open top slides the top of the vessel along the curved ramp, pushing the pierceable bottom of the vessel into the piercing and draining mechanism, causing the pierceable bottom to be pierced and the additive to escape the vessel and mix with fluid in the cup.

18. The collection container of claim 1, wherein

the lid comprises an underside and engages the open top by screwing onto the open top; and

the additive is enclosed in a subassembly, wherein

the subassembly comprises a receptacle overlapped by an outer cover, wherein the receptacle is affixed to the underside of the lid, and the outer cover is distal thereto;

the receptacle screws into the outer cover using threads that are reversed from threads used to screw the lid onto the open top;

the outer cover further comprises

at least one outwardly extended tab; and

at least one slot at a portion of the outer cover that is closest to the bottom of the cup when the lid is screwed onto the open top, wherein the slot is internally covered by the receptacle when the receptacle and outer cover are screwed together,

wherein when the lid is screwed onto the open top, the tab engages with the cup preventing rotation when the lid is screwed onto the cup, and causing the receptacle and outer cover to unscrew, uncovering the slot and allowing additive to drain into the cup.

19. The collection container of claim 1, wherein

the additive is sealed in a fluid container in an underside of the lid by a breakable seal; and the lid further comprises a lever operably linked to at least one piercing pin in the fluid container, whereby, after the lid is screwed into the open top and the lever is actuated, the piercing pin pierces the seal, releasing the additive from the fluid container into the cup.

20. The collection container of claim 1, wherein

the lid engages the open top by screwing onto the open top;

the additive is sealed in a fluid container in an underside of the lid, wherein the fluid container further comprises at least one protrusion directed away from the underside of the lid; wherein the collection container further comprises a circular blade assembly surrounding the fluid container, the blade assembly comprising

at least one blade adjacent to the protrusion and directed thereto, and at least one tab that fits inside a guide in the cup, preventing rotation of the blade assembly when the lid is screwed onto the open top;

wherein screwing the lid onto the open top causes the blade to cut the protrusion, releasing the additive from the fluid container into the cup.

21. The collection container of claim 1, wherein

the lid engages the open top by screwing onto the open top;

the additive is sealed in a fluid container in an underside of the lid by a breakable seal; wherein the collection container further comprises

a puncture ring adjacent to the breakable seal, the puncture ring comprising

at least one puncture point directed toward the breakable seal, and at least one tab that fits inside a guide in the cup, preventing rotation of the puncture ring when the lid is screwed onto the open top;

wherein screwing the lid onto the open top causes he puncture point to pierce, then rotationally slice through the breakable seal, releasing the additive from the fluid container into the cup.

22. The collection container of claim 1, wherein the fluid sample has a volume of at least

50 ml.

23. A method of collecting a fluid sample, the method comprising inserting the sample into the collection container claim 1 and engaging the lid onto the open top, releasing the additive into the sample.

24. The method of claim 23, wherein the fluid is urine, breast milk, saliva, sputum, blood, serum, plasma, lymph, amniotic fluid, cerebrospinal fluid, interstitial fluid, bile, peritoneal fluid, sweat, tears, ejaculate, ascites, or any wound fluid.

25. The method of claim 23, wherein the fluid is urine.

26. The method claim 23, wherein the additive is a preservative that prevents degradation of a small molecule or a macromolecule.

27. The method of claim 26, wherein the preservative prevents degradation of a nucleic acid.

28. The method of claim 23, wherein the fluid sample has a volume of at least 50 ml.

29. The method of claim 25, wherein the fluid sample has a volume of at least 90 ml.

30. A method of isolating cell-free (cf) nucleic acids from a urine sample from a subject, the method comprising

31. The method of claim 30, wherein the cf nucleic acids are cfDNA.

32. The method of claim 31, wherein the cfDNA is transrenal cfDNA.

33. The method of claim 30, wherein the cf nucleic acids are eluted from the first magnetic particle using a salt solution that is less than 2.0 M.

34. The method of claim 30, wherein the cf nucleic acids are eluted from the first magnetic particle using a 1.8 M salt solution.

35. The method of claim 30, wherein the cf nucleic acids are eluted from the first magnetic particle using a 1.7 M salt solution.

36. The method of claim 30, wherein the cf nucleic acids are eluted from the first magnetic particle using a 1.6 M salt solution.

37. The method of claim 30, wherein the salt solution is NaCl.

38. The method of claim 30, wherein the preservative comprises ethylenediaminetetraacetic acid (EDTA).

39. The method of claim 38, wherein the EDTA is at a concentration of 0.5 M after added to the sample.

40. The method of claim 30, wherein the urine sample is greater than 90 ml.

41. The method of claim 30, wherein the anion exchange moiety is a quaternary ammonium moiety.

42. The method of claim 41, wherein the quaternary ammonium moiety is a 2- hydroxypropyl trimethylammoniumchlorid moiety.

43. The method of claim 30, wherein the first magnetic particle is superparamagnetic.

44. The method of claim 43, wherein the first magnetic particle has a maghemite core.

45. The method of claim 30, wherein the urine sample is concentrated after adding the preservative and before adding the first magnetic particle.

46. The method of claim 45, wherein the urine sample is concentrated by ultrafiltration to a volume of less than 5 ml.

47. The method of claim 46, wherein the ultrafiltration uses a filter with a pore size of 4000-6000 Dalton

48. The method of claim 30, wherein the cf nucleic acids bind to the first magnetic particle in a buffer comprising 2-propanol, tris-HCl and EDTA.

49. The method of claim 30, further comprising adding a reducing agent to the urine sample.

50. The method of claim 49, wherein the reducing agent is dithiothreitol (DTT) or tris(2- carboxyethyl)phosphine (TCEP).

51. The method of claim 30, further comprising further purifying the eluted cf nucleic acids.

52. The method of claim 51, wherein the purifying comprises

(g) eluting purified cf nucleic acids from the second magnetic particle.

53. The method of claim 52, wherein the second magnetic particle comprises a silanol moiety.

54. The method of claim 52, wherein the cf nucleic acids binds to the second magnetic particle in a buffer comprising 2-propanol, guanidinium thiocyanate and polyethylene glycol.

55. The method of claim 54, wherein the second magnetic particle with bound cf nucleic acids are washed in an ethanol solution.

56. The method of claim 55, wherein the second magnetic particle with bound cf nucleic acids is washed (i) in 70% ethanol with guanidinium thiocyanate then (ii) 70% ethanol.

57. The method of claim 52, wherein the purified cf nucleic acids are eluted from the second magnetic particle in 10 mM Tris-HCl.

58. An adaptor for suspending a column above a bottom of a test tube,

wherein the column comprises an open entry and an open exit, with the entry wider than the exit, and the test tube has an open entrance and closed end,

the adaptor comprising

a cylindrical tube having an open top, an open bottom comprising an opening, and a side between the top and the bottom, the side having a diameter at the top and the bottom that is less than the diameter of the test tube and wider than the diameter of the open exit of the column, and

an outwardly directed top extension at the open top, wherein the width of the adaptor with the top extension is greater than the diameter of the test tube,

wherein, when the column is placed into the open top of the adaptor and the adaptor is placed into the open top of the test tube, the adaptor is suspended from the top of the test tube by the extension and the column is suspended from the top of the adaptor such that the bottom of the column is suspended above the bottom of the test tube.

59. The adaptor of claim 58, wherein the top extension is an outward circumferential flange.

60. The adaptor of claim 58, further comprising at least one wing extending outwardly from the side.

61. The adaptor of claim 58, wherein the open bottom further comprises an inwardly directed bottom extension that narrows the opening.

62. The adaptor of claim 61, wherein the bottom extension is an inward circumferential flange.

63. The adaptor of claim 58, wherein the column is a chromatography column.

64. The adaptor of claim 61, wherein the column further comprises a shoulder that sits on the bottom extension when the chromatography column is inserted into the adaptor.

65. The adaptor of claim 58, wherein the width of the open top is between 2.0 cm and 2.5 cm.

66. The adaptor of claim 58, wherein the width of the open top is between 2.2 cm and 2.4 cm.

67. The adaptor of claim 58, wherein the test tube open entrance is between 2.5 cm and

3.0 cm.

68. The adaptor of claim 58, wherein the test tube open entrance is between 2.7 cm and

2.9 cm.

69. The adaptor of claim 58, wherein the column open entry is between 1.7 and 2.2 cm.

70. A method of accelerating the passage of a fluid through a chromatography column, wherein the chromatography column comprises an open entry and an open exit, with the entry wider than the exit, the method comprising insert the chromatography column into the adaptor of claim 58,

insert the adaptor-chromatography column into the test tube,

add the fluid to the chromatography column, and

centrifuge the test tube-adaptor-chromatography column,

thus accelerating passage of the fluid through the chromatography column.

71. The method of claim 70, wherein the width of the open top is between 2.2 cm and

2.4 cm.

72. The method of claim 70, wherein the test tube open entrance is between 2.5 cm and

3.0 cm.

73. The method of claim 70, wherein the chromatography column open entry is between 1.7 and 2.2 cm.

74. The method of claim 70, wherein the chromatography column further comprises a chromatography resin.

75. The method of claim 74, wherein the chromatography resin is an ion-exchange resin.

76. The method of claim 74, wherein the chromatography resin is an anion-exchange resin.

77. The method of claim 70, wherein the fluid comprises a bodily fluid.

78. The method of claim 77, wherein the bodily fluid is urine.

79. A method for isolating cell-free nucleic acids from a urine sample, the method comprising

(a) obtain the urine sample;

(b) centrifuge the sample until cells in the sample are pelleted;

(c) separate supernatant of the sample from the cells; (d) add an anion exchange resin to the supernatant and incubate the supernatant-resin to allow binding of nucleic acids to the resin;

(e) separate the supernatant from the anion exchange resin;

(g) elute nucleic acids from the anion exchange resin with a salt solution that is 500 mM or greater, using the adaptor of claim 58 to suspend a chromatography column comprising the anion exchange resin above the closed end of a test tube, then centrifuging the test tube- chromatography column-adaptor to accelerate elution of the nucleic acid from the anion exchange resin.

80. The method of claim 79, further comprising

(h) add sodium acetate to the eluted nucleic acids to a concentration of less than 5%, mix, then add a chaotropic salt and alcohol solution that allows binding of the nucleic acids to a silica medium;

(i) apply the sodium acetate-nucleic acid-guanidine-isopropanol solution to a silica medium such that the nucleic acids bind to the silica medium;

(k) elute the nucleic acids from the silica medium with an elution medium.

81. The method of claim 79, wherein the anion exchange resin is a quaternary ammonium resin.

82. The method of claim 81, wherein the quaternary ammonium resin is Q Sepharose®.

83. The method of claim 79, wherein the wash of the anion exchange resin of step (f) uses the adaptor of claim 58.

84. The method of claim 80, wherein the chaotropic salt and alcohol of step (h) are guanidine hydrochloride and isopropanol.

85. The method of claim 80, wherein the wash buffer of step (j) comprises an alcohol.

86. The method of claim 80, wherein the elution medium of step (k) comprises 10 mM Tris-Cl.

87. The method of claim 79, wherein the elution of nucleic acids of step (g) is by stepwise application of salt solutions of increasing molarity.

88. The method of claim 87, wherein the stepwise application of salt solutions includes stepwise elution with salt solutions of about 500 mM, about 600 mM, and about 700 mM.

89. The method of claim 79, wherein the washing of step (f) is with a salt solution of about 400 mM.

90. The method of claim 79, wherein the width of the open top is between 2.2 cm and

2.4 cm.

91. The method of claim 79, wherein the test tube open entrance is between 2.5 cm and

3.0 cm.

92. The method of claim 79, wherein the chromatography column open entry is between 1.7 and 2.2 cm.

93. The method of any one of claims 30-57 or 79-92, further comprising assaying the cf nucleic acids for the presence of a nucleic acid sequence that does not originate from endogenous nucleic acid sequences of the subject.

94. The method of claim 93, wherein the cf nucleic acids originate from a pathogen, a transplant, or, where the subject is pregnant, a fetus.

95. The method of any one of claims 30-57 or 79-90, further comprising assaying the cf nucleic acids for the presence of a nucleic acid sequence that originates from endogenous nucleic acid sequences of the subject.

96. The method of claim 95, wherein the nucleic acid sequence is of a miRNA.

97. The method of claim 95, wherein the nucleic acid sequence is in genomic DNA of the subject.

98. The method of claim 97, wherein the genomic DNA comprises a mutation associated with a cancer.

99. A method of verifying the identity of a subject as being a source of a sample of a bodily fluid, the method comprising

obtaining a reference sample from the subject, wherein the reference sample comprises DNA from the subject;

obtaining the sample of the bodily fluid;

comparing the genetic fingerprint of the reference sample with the genetic fingerprint of the sample of the bodily fluid;

wherein

if the genetic fingerprint of the reference sample is the same as the genetic fingerprint of the sample of the bodily fluid, then the subject is a source of the sample of the bodily fluid, and if the genetic fingerprint of the reference sample is not the same as the genetic fingerprint of the sample of the bodily fluid, then the subject is not a source of the sample of the bodily fluid.

100. The method of claim 99, wherein the sample of the bodily fluid is collected in the collection container of claim 1.

101. The method of claim 100, wherein the bodily fluid is urine.

102. The method of claim 101, wherein the sample is urine and the DNA in the sample is isolated by the method of any one of claims 30-57 or 79-92.

103. A kit comprising any ingredients utilized in the method of any one of claims 30-57, 79-92 or 99-102.