DEVICES AND METHODS FOR NUCLEIC ACID PREPARATION AND

ANALYSIS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial Nos. 61/625,743 and 61/783,601, respectively filed on April 18, 2012 and March 14, 2013, both entitled "DEVICE FOR PREPARING A SAMPLE"; U.S. Provisional Application Serial Nos. 61/625,745 and 61/784,399, respectively filed on April 18, 2012 and March 14, 2013, both entitled "DEVICE FOR STRETCHING A POLYMER IN A FLUID SAMPLE"; U.S. Provisional Application Serial Nos. 61/635,263 and 61/784,061, respectively filed on April 18, 2012 and March 14, 2013, both entitled "INTERCALATION METHODS AND DEVICES"; and U.S. Provisional Application Serial No. 61/635,221, filed on April 18, 2012, and entitled "SINGLE-MOLECULE SORTING AND MAPPING OF LONG DNA FRAGMENTS FOR SEQUENCING-BASED ANALYSIS" and U.S. Provisional Application Serial No. 61/790,721, filed on March 15, 2013, and entitled "SELECTION OF SINGLE NUCLEIC ACIDS BASED ON OPTICAL SIGNATURE", the entire contents of all of which are incorporated herein by reference.

FIELD OF INVENTION

The present invention is directed in part to a device for preparing a fluid sample, including but not limited to samples which include genomic DNA. More particularly, aspects of the present invention are directed to a device with a reaction chamber and a porous membrane. The present invention is also directed in part to a device for stretching at least one polymer in a fluid sample, where the device includes a tapered channel. The invention is also directed in part to methods for labeling nucleic acids with backbone stains during transit through one or more of the microfluidic devices of the invention. The invention is also directed in part to methods for physically separating nucleic acids from each other based on probe binding profiles. The invention is also directed in part to the analysis of nucleic acids such as nucleic acids from populations of cells of differing sources, species and/or strains, including for example bacterial populations or human cells. BACKGROUND OF INVENTION

The ability to prepare, manipulate, analyze and identify single nucleic acids such as single DNA molecules has a variety of clinical and non-clinical applications.

Manipulation of DNA may include fragmentation, labeling with one or more probes and/or a backbone stain. Non-specific backbone staining of nucleic acids with intercalating dyes is a fundamental requirement of certain nucleic acid manipulations and analyses. (Chan, Goncalves et al. 2004; Jung, Bharadwaj et al. 2006; Larson, Yantz et al. 2006; Shackman and Ross 2007; Protozanova, Zhang et al. 2010). As an example, in Direct Linear Analysis (DLA) or Genome Sequence Scanning (GSS™), backbone specific staining is used to determine presence, velocity and length of nucleic acids.

Typically, intercalation reactions are performed by adding an appropriate mass of a specific intercalating dye to a known mass of DNA, resulting in a fixed molar ratio of individual fluorophores to nucleic acid base pairs. However, variable starting cell load, genomic content, and DNA extraction efficiencies can cause the mass yields between sample preparations to be inconsistent. This can complicate analyses by requiring that nucleic acid mass be pre-quantified.

De novo sequencing is typically not required for the vast majority of applications, including for example some types of human diagnostics and pharmacogenomics. Instead, sequencing may be used to identify genetic differences between individuals, using a process called targeted re-sequencing. Similarly, analysis of a bacterial population does not need to include complete sequencing of the whole population. Instead, only parts of a bacterial genome that sufficiently identify the host genome can be selected for analysis.

SUMMARY OF INVENTION

The invention provides a variety of devices and methods useful in the preparation of a nucleic acid sample, analysis of such a sample, and physical separation of nucleic acids within the sample based on probe binding profile. The devices include but are not limited to

microfluidic devices and the methods may be carried out in microfluidic devices although they are not so limited.

According to one aspect, a device for preparing a sample is provided. The device includes a body having a chamber with an inlet and a membrane positioned in the body. The membrane has a first side and a second side, where the inlet is positioned on the first side of the membrane. The device also includes a plurality of channels optionally coupled to the bottom of the chamber, where the plurality of channels are optionally positioned on the second side of the membrane. Each of the plurality of channels extends outwardly from the membrane, the plurality of channels including at least a first channel and a second channel, where the first channel extends outwardly from a central portion of the membrane, and where the second channel extends outwardly from a peripheral portion of the membrane.

According to another aspect, a device for preparing a sample is provided. The device includes a body having a chamber with an inlet and a membrane positioned in the body. The membrane has a first side and a second side, where the inlet is positioned on the first side of the membrane. The membrane includes at least a first zone and a second zone, where the first zone is the central portion of the membrane and the second zone is the peripheral portion of the membrane and there is a barrier which separates the first zone of the membrane from the second zone of the membrane. The device also includes a plurality of channels coupled to the bottom of the chamber, where the plurality of channels are positioned on the second side of the membrane, in some embodiments.

According to another aspect, a device for stretching at least one polymer in a fluid sample is provided. The device includes an elongation structure, where said elongation structure includes a tapered channel that decreases in width from a first end to a second end. The tapered channel includes a first zone having a first tapered shape, and a second zone having a second tapered shape, where the second tapered shape is different than the first tapered shape. The at least one polymer, when present, moves along said tapered channel from said first end to said second end and is stretched. The first tapered shape may include an increasing strain rate taper, and the second tapered shape may include a constant strain rate taper.

According to another aspect, a method includes moving a polymer through a tapered channel past at least one detection station, where the tapered channel has a constant strain portion. The method also includes detecting an object-dependent impulse that conveys information about structural characteristics of the polymer, such as a nucleotide sequence or hybridization of the polymer to a sequence- specific probe, and obtaining an observed trace based on the detected impulse, where the observed trace is an intensity versus time trace. The method also includes applying an acceleration correction to the observed trace and obtaining a corrected intensity versus distance trace from the application of the acceleration correction to the observed trace. According to yet some other aspects, the invention provides various methods and related systems for uniformly labeling nucleic acids with, for example, backbone stains without knowledge of the amount (i.e., mass) and thus the concentration of nucleic acid being labeled. The invention contemplates that the methods and systems provided herein can be used to label nucleic acids with a number of agents including, but not limited to, backbone stains, and in particular mono-intercalating backbone stains, as will be discussed in greater detail herein.

In one aspect, the invention provides a method for uniformly labeling a nucleic acid with an intercalator comprising providing a capillary coupled to a microfluidic device, wherein the capillary comprises an intercalator, placing a first end of the capillary in a vessel comprising a nucleic acid, applying hydrodynamic or electrokinetic force sufficient to move the nucleic acid from the vessel through the capillary and to the microfluidic device, wherein flow of nucleic acid through the capillary is laminar.

In some embodiments, the nucleic acid is DNA.

In some important embodiments, the intercalator is a mono-intercalator. In some embodiments, the intercalator is a mono-cyanine intercalator. In some embodiments, the intercalator is positively charged at about neutral pH. In some embodiments, the intercalator is PO-PRO. In some embodiments, the intercalator is PO-PRO-1 or PO-PRO-3.

In some embodiments, the capillary comprises the intercalator in a buffered solution.

In some embodiments, the capillary has dimensions of about 15 cm in length and about 150 micron internal diameter. In some embodiments, the capillary is pre-filled with intercalator by hydrodynamic force. In some embodiments, the nucleic acid is labeled with the intercalator at a frequency of one intercalator molecule per three base pairs.

In some embodiments, the microfluidic device is coated with an electroosmotic flow (EOF) suppressor on one or more of its interior surfaces. In some embodiments, the capillary comprises an intercalator and an electroosmotic flow (EOF) suppressor in a buffered solution. In some embodiments, the electroosmotic flow (EOF) suppressor is a water soluble

methylhydroxyethyl derivative of cellulose, polyvinylalcohol, polyvinylpyrrolidone,

polyethyleneglycol or Triton X-100.

In some embodiments, an electrokinetic force is applied to move the nucleic acid and a cathode is present in a waste reservoir and an electrode is present in the vessel.

In some embodiments, the nucleic acid and intercalator move through the capillary in separate streams. In another aspect, the invention provides a system comprising a microfluidic device coupled to one end of a capillary comprising an intercalator.

In some embodiments, the system further comprises a waste reservoir coupled to a vacuum. In some embodiments, the system further comprises a waste reservoir comprising a cathode.

In some embodiments, the intercalator is a mono-cyanine. In some embodiments, the intercalator is positively charged at about neutral pH. In some embodiments, the intercalator is PO-PRO. In some embodiments, the intercalator is PO-PRO-1 or PO-PRO-3. In some embodiments, the capillary comprises the intercalator in a buffered solution.

In some embodiments, the microfluidic device is coated with an electroosmotic flow (EOF) suppressor on one or more of its internal surfaces. In some embodiments, the capillary comprises an intercalator and an electroosmotic flow (EOF) suppressor in a buffered solution.

In some embodiments, the electroosmotic flow (EOF) suppressor is a water soluble methylhydroxyethyl derivative of cellulose, polyvinylalcohol, polyvinylpyrrolidone, polyethyleneglycol or Triton X-100.

In another aspect, the invention provides a method for uniformly labeling a nucleic acid with an intercalator comprising providing a microfluidic device comprising a sample inlet, a sheath fluid inlet, an elongation region, and a waste reservoir downstream of the elongation region, introducing a nucleic acid into the microfluidic device through the sample inlet, introducing intercalator into the microfluidic device through the sheath fluid inlet, and applying hydrodynamic force sufficient to move the nucleic acid from the sample inlet and the intercalator from the sheath fluid inlet through the elongation region to the waste reservoir, wherein flow of the nucleic acid and intercalator is laminar.

In some embodiments, the intercalator is a mono-cyanine. In some embodiments, the intercalator is positively charged at about neutral pH. In some embodiments, the intercalator is PO-PRO. In some embodiments, the intercalator is PO-PRO-1 or PO-PRO-3.

In some embodiments, intercalator is flowed through and is present in the microfluidic device prior to introduction of the nucleic acid. In some embodiments, the microfluidic device comprises two sheath fluid inlets positioned on opposite sides of the sample inlet, and intercalator is introduced into the microfluidic device through both sheath fluid inlets.

In some embodiments, the intercalator is present in a buffered solution. In some embodiments, nucleic acids are exposed to intercalator individually. In some embodiments, nucleic acids are exposed to the intercalator while under tension. In some embodiments, the nucleic acids are exposed to and bind with the intercalator while in the elongation region.

In some embodiments, nucleic acids are exposed to the intercalator for a time that is controlled by (1) fluid velocity through the micro fluidic device and (2) geometry of the elongation region.

In some embodiments, the intercalator is present at a concentration ranging from about 50 nM to less than or about 10 μΜ, or from about 50 nM to about 500 nM, or from about 1 μΜ to less than or about 10 μΜ.

In some embodiments, the microfluidic device is coated on its interior surfaces.

In some embodiments, the nucleic acid and intercalator move through the elongation region in separate streams, optionally wherein there are 2-5 streams.

In another aspect, the invention provides a microfluidic device comprising a sample inlet port, a sheath inlet port, an elongation region, and a waste reservoir downstream of the elongation region, wherein the sheath inlet port and the elongation region comprise an intercalator prior to introduction of a nucleic acid sample. In some embodiments, the device comprises two sheath inlet ports positioned on opposite sides of the sample inlet port. In some important embodiments, the intercalator is a mono-cyanine. In some embodiments, the intercalator is positively charged at about neutral pH. In some embodiments, the intercalator is PO-PRO. In some embodiments, the intercalator is PO-PRO-1 or PO-PRO-3. The structure of PO-PRO-1 is provided in FIG. 27.

In another aspect, the invention provides a method for uniformly labeling a nucleic acid with an intercalator comprising providing a microfluidic chip comprising a sample inlet, an intercalator inlet, two sheath fluid inlets, an elongation region, and a waste reservoir downstream of the elongation region, wherein the intercalator inlet comprises two channels ("intercalator channels") extending therefrom and merging with a channel extending from the sample inlet ("sample channel") wherein the intercalator channels are positioned on opposite sides of the sample channel; and the two sheath fluid inlets are positioned on opposite sides of, and feed into, the elongation region, introducing a nucleic acid into the microfluidic chip through the sample inlet, introducing intercalator into the microfluidic chip through the intercalator inlet, and applying hydrodynamic force sufficient to move the nucleic acid from the sample inlet and the intercalator from the intercalator inlet through the elongation region to the waste reservoir, wherein flow of the nucleic acid and intercalator is laminar. In some important embodiments, the intercalator is a mono-cyanine. In some embodiments, the intercalator is positively charged at about neutral pH. In some embodiments, the intercalator is PO-PRO. In some embodiments, the intercalator is PO-PRO- 1 or PO-PRO-3.

In another aspect, the invention provides a microfluidic device comprising a sample inlet, an intercalator inlet, two sheath fluid inlets, an elongation region, and a waste reservoir downstream of the elongation region, wherein the intercalator inlet comprises two channels ("intercalator channels") extending therefrom and merging with, and positioned on opposite sides of, a channel extending from the sample inlet ("sample channel"), and the two sheath fluid inlets are positioned on opposite sides of, and feed into, the elongation region.

According to yet some other aspects, the methods of the invention can be used, inter alia, to isolate fragments of genomic DNA containing specific genes of interest, or manifesting specific probe hybridization profile or barcodes of interest, from multiple sources and from large genomes. The fragment sorting may be accompanied by simultaneous measurement of genomic maps that complement the information about the specific genes, profiles and/or barcodes and can help to build sequence contigs. This technology provides efficient and rapid analysis, selecting only a fraction of genomic material, relevant for an application or analysis. These methods can be used to more rapidly type and obtain sequence for nucleic acid fragments of interest.

The invention contemplates, in some aspects, the analysis of microbiomes (i.e., populations including multiple bacteria) based on sequencing their housekeeping genes, using the method and devices provided herein. The invention contemplates, in other aspects, the analysis of human genomes. In further aspects, the invention provides a device to physically select a limited amount of nucleic acid (e.g., DNA) for further analysis (e.g., targeted

resequencing) from a bulk nucleic acid population such as for example genomic DNA including genomic DNA isolated from a microbiome sample. Various aspects and embodiments of the invention are described in terms of DNA, but it is to be understood that other nucleic acids may be used also and that the invention is not limited to analysis of DNA molecules only.

Thus, in one aspect, the invention provides a method comprising (1) labeling nucleic acid fragments with a nucleic acid probe that is a specific probe, (2) labeling the fragments with a non-specific probe, wherein the specific probe and the non-specific probe are labeled with different detectable labels, and wherein the labeling with specific and non-specific probes occurs simultaneously or concurrently (in either order), (3) analyzing individual nucleic acid fragments for the presence of signal from the specific and non-specific probes, and (4) obtaining, for fragments having signal from the specific probes, a profile of signals from the specific and nonspecific probes along the length of the fragment.

In another aspect, the invention provides a method comprising (1) labeling nucleic acid fragments with a non-specific nucleic acid probe, such as a bisPNA probe, (2) analyzing individual nucleic acid fragments for the presence of signal from non-specific probe such as a bisPNA probe, and (3) obtaining, for fragments having signal from the non-specific probe such as a bisPNA probe, a profile of signals from the non-specific probe such as a bisPNA probe, and optionally including signals from more than one non-specific probe (if the nucleic acid was so labeled) along the length of the fragment.

Thus, in yet other aspects, the invention encompasses labeling a nucleic acid fragment with one or more non-specific probes, or one or more specific probes, obtaining signals from the non-specific probes and/or the specific probes, and separating nucleic acids based on the presence of signals from non-specific probes and/or specific-probes. Signals can be obtained and nucleic acids can be separated from each other using a microfluidic device. The probes may be bisPNA probes and the entire method may be carried out on the basis of signals from such probes.

More than one non-specific probe (e.g., bisPNA probe) can be used with the same label. More than one non-specific probes (e.g., bisPNA probe) can be used with different labels. Such probes, whether identically or differentially labeled, may differ in their binding specificity. As an example, one might bind to AT-rich regions and one may bind to GC-rich regions.

The non-specific probes in the context of aspects of invention are probes complementary to short nucleic acid sequences (e.g., 6-8 bases) that are present in genomes on average about once every 10 kb. Thus, the sequences are relatively frequent in a genome. In contrast, the specific probes of aspects of the invention are probes that are specific to sequences that exist uniquely in target genes (and thus can be used as a marker of a target gene). The profile of signals from the specific and/or the non-specific probes along the length of the fragment may be referred to herein as a signature.

It is to be understood that in addition to the foregoing probes, various aspects and embodiments of the invention also contemplate labeling of nucleic acids with backbone stains. Such stains bind along the entire length of the nucleic acid in a sequence-independent (or sequence non-specific) manner. These stains are to be contrasted with the specific or non- specific probes that bind to nucleic acids in a sequence-dependent manner (i.e., their binding to a nucleic acid is dependent upon its underlying nucleotide sequence).

In some embodiments, the method further comprises separating fragments having signal from the specific probes from fragments lacking signal from specific probes. In some embodiments, the method comprises separating fragments having certain signals (or patterns) from the non-specific probes from fragments lacking such signals (or patterns). This allows the analysis to continue on only those fragments of interest (e.g., those having signal from the specific probes). In some instances, the nucleic acid fragments of interest, whether they be identified on the basis of specific or non-specific probes or a combination of specific and nonspecific probes, are rare in the population of nucleic acids.

In important embodiments, the separation of fragments occurs in a microfluidic device. In some embodiments, all or any or any combination of steps (2) through (4) and the separation step occur in the same microfluidic device. The nucleic acids may be present in the same fluid that are used as sheathing fluids, or at a minimum they are present in fluids that are miscible with the sheathing fluids. The nucleic acids are not encapsulated in droplets.

In some embodiments, the method further comprises sequencing fragments, such as fragments having signal from specific probes or fragments having signal from non-specific probes such as bisPNA probes.

In some embodiments, the method further comprises fragmenting genomic DNA to produce the nucleic acid fragments. In some embodiments, the nucleic acid fragments are generated using restriction enzyme digestion. The restriction enzyme digestion may involve the use of one or more restriction enzymes. The fragments may be of various lengths, including for example 10-500 kb (kilobases), 30-500 kb, 50-500 kb, 50-400 kb, 50-300 kb, 50-200 kb, or 50- 100 kb. The fragments may be about or more than 100 kb, about or more than 200 kb, about or more than 300 kb, about or more than 400 kb, or about or more than 500 kb.

In some embodiments, the specific and/or non-specific probes are labeled with fluorophores. In some embodiments, the specific probes are labeled with multiple detectable labels and the non-specific probes are labeled with fewer detectable labels.

In some embodiments, the nucleic acid fragments are labeled under non-denaturing conditions.

In another aspect, the invention provides a method comprising (1) labeling nucleic acid fragments with three nucleic acid probes, wherein two of the probes are bisPNA probes and one of the probes is a non-bisPNA probe that is distinguishable from the bisPNA probes based on for example different brightness or intensity (e.g., it may be labeled with two or more detectable labels or with detectable labels of greater brightness or intensity while the bisPNA probes may be labeled with fewer detectable labels or with detectable labels of comparatively lesser brightness or intensity), (2) labeling the fragments with a non-specific probe, wherein the non- bisPNA probe and the non-specific probe are labeled with different detectable labels, and wherein the labeling steps of (1) and (2) may occur simultaneously or concurrently (in either order), (3) analyzing individual nucleic acid fragments for the presence of signal from the non- bisPNA and non-specific probes, and (4) obtaining, for fragments having signal from the non- bisPNA probe, a profile of signals from the non-bisPNA and non-specific probes along the length of the fragment.

In this and other embodiments, detectable labeling that is distinguishable based on intensity or brightness may be achieved using for example varieties of fluorophore-impregnated beads, quantum dots, dendrimeric probes, and the like.

In some embodiments, the method further comprises separating fragments having signal from the non-bisPNA probes from fragments lacking signal from non-bisPNA probes. In some embodiments, the separating occurs in a microfluidic device such as a microfluidic chip. In some embodiments, all or any, or some combination of steps (2) through (4) and the separating step occur in a microfluidic device. In some embodiments, the method further comprises sequencing fragments having signal from non-bisPNA probes.

In another aspect, the invention provides a method comprising (1) nicking nucleic acid fragments with an engineered restriction enzyme that possesses nicking activity in only one domain, extending a 3' end at a nicked site using a polymerase that lacks 5' -> 3' exonuclease activity to generate a displaced single strand, and labeling the displaced single strand with a specific probe that comprises at least two detectable labels or is otherwise detectably labeled in a manner that distinguishes it from other probes based on increased intensity or brightness, (2) labeling the fragments with a non-specific probe, wherein the specific probe and the nonspecific probe are labeled with different detectable labels, (3) analyzing individual nucleic acid fragments for the presence of signal from the specific and non-specific probes, and (4) obtaining, for fragments having signal from the specific probe, a profile of signals from the specific and non-specific probes along the length of the fragment. In some embodiments, the method further comprises separating fragments having signal from the specific probes from fragments lacking signal from the specific probes. In some embodiments, the separating occurs in a microfluidic device such as a microfluidic chip. In some embodiments, all or any or any combination of steps (2) through (4) and the separating step occur in a microfluidic device. In some embodiments, the method further comprises sequencing fragments having signal from specific probes.

In another aspect, the invention provides a microfluidic device comprising an inlet port coupled to a microfluidic taper comprising an elongation region and interrogation region, a cathode and anode positioned on opposite ends (i.e., of an axis) of a sorting channel that is present at or near the end of the microfluidic taper and that is oriented perpendicular to the microfluidic channel, a first DNA reservoir coupled to the sorting channel, optionally having a second elongation region and a second interrogation region located between the sorting channel and the first DNA reservoir, a second DNA reservoir positioned downstream of the sorting channel, optionally coupled to a third elongation region and a third interrogation region, and an outlet waste port downstream of the microfluidic taper, sorting channel, and second DNA reservoir, wherein a vacuum may be applied to the first and second DNA reservoirs, and the waste port.

In another aspect, the invention provides a microfluidic device comprising an inlet port coupled to a microfluidic taper comprising an elongation region and interrogation region, a cathode and anode positioned on opposite ends of a sorting channel that is present at or near the end of the microfluidic taper. The sorting channel is located beyond (or after) the microfluidic taper. In some embodiments, the device comprises two exit ports, one for discarded nucleic acids and one for selected nucleic acids. The stretching module may be separated from the sorting module by a distance that is sufficiently long to provide any delay required to perform analysis of the nucleic acid and comparison with templates in a database. In one embodiments, electrodes generate fields that are non-parallel to the direction of nucleic acid movement (and which may be perpendicular or at an angle to the direction of nucleic acid movement.

In yet other aspect, the invention provides a microfluidic device comprising an inlet port coupled to a microfluidic taper comprising an elongation region and interrogation region, a reservoir beyond the interrogation region where nucleic acids are sheathed by side fluid flows, and two exit ports, one each for discarded and selected nucleic acids. Discarded and selected nucleic acids can be directed to the corresponding ports by changing the balance of sheathing flows. In both cases, nucleic acids and sheathing flows can be driven either by positive pressure applied to the inlet and sheathing ports, or by vacuum applied to the exit ports, or by a combination thereof. The flows can be also controlled by positive pressure applied to the inlet port and the sheathing fluid ports.

The present invention further encompasses methods of making and/or using one or more of the embodiments described herein.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying Figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

BRIEF DESCRIPTION OF FIGURES

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like reference numeral. In some cases, different reference numerals may refer to identical or nearly identical components. For purposes of clarity, not every component may be labeled in every drawing. Various embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic view of a device for preparing a sample according to one embodiment;

FIGS. 2A-2D are schematic views of a plurality of sample preparation steps that may be performed with the device illustrated in FIG. 1;

FIG. 3 is a perspective view of a portion of a device for preparing a sample according to one embodiment;

FIG. 4 is another perspective view of the first portion of the device shown in FIG. 3; FIG. 5 is a detailed perspective view of the device shown in FIGS. 3 and 4;

FIG. 6 is a perspective view of a portion of the device for preparing a sample according to one embodiment;

FIG. 7 is another perspective view of the portion of the device shown in FIG. 6; FIG. 8A is a schematic representation of a microfluidic chip with an elongation structure according to one embodiment;

FIG. 8B is a schematic representation of a tapered channel with two zones according to one embodiment;

FIG. 8C is a schematic representation of a DNA sample being stretched in a tapered channel according to one embodiment;

FIG. 8D includes representative fluorescent signals generated from a single stretched DNA molecule;

FIG. 9 includes a chart which includes data from prior art tapered funnels and also data from two embodiments of the present invention;

FIG. 10A is a schematic representation of a DNA sample being stretched in a tapered channel according to one embodiment;

FIGS. 10B- 10F represent single molecule stretching morphologies;

FIGS. 1 lA-11C illustrate molecule extension in increasing fluid velocity;

FIGS. 12A-12D illustrate the effect of channel depth on single molecule morphology

FIGS. 13A-13B illustrate improved DNA stretching efficiency in constant strain rate detection funnels;

FIG. 14A- 14C illustrate the accelerated corrected site specific fluorescence traces;

FIG. 15 illustrates the acceleration correction in constant tension fluidics;

FIG. 16. The native conformation of DNA is shown here, as well as the varying positions where different types of intercalators may bind. A mono-intercalator is shown binding to the nucleic acid (see first red symbol shown at the top of the DNA helix). This is in contrast to the structure shown at the bottom which is a bis-intercalator.

FIG. 17. On-chip intercalation using PO-PRO- 1 eliminates DNA aggregation and surface sticking.

FIG. 18. On-chip intercalation of DNA spanning 32-fold serial concentration dilutions using PO-PRO- 1 in sheath buffers (250 nM) results in uniform DNA stretching (closed circle). In-tube intercalation of DNA spanning only 7 fold concentration dilution, however, exhibits significant effect on DNA stretching, using fixed intercalator concentration (1 μΜ, open triangles).

FIG. 19. Effect of intercalation of DNA stretching. (A) E. coli K12 Notl digested DNA was intercalated on-chip with increasing concentrations of PO-PRO- 1 ( A 50 nM, Δ 100 nM,■ 150 nM,□ 250 nM, · 400 nM, o 500 nM). Observed molecule length (μιη) is plotted as a function of known fragment length from sequence (in kb). (B) Stretching coefficient (μΓη/kb) for above conditions plotted as function of average molecule tension, as defined herein.

(C) Stretching profile for DNA intercalated with PO-PRO-1 (triangles) compared to POPO-1 (squares) Duplicate data sets are presented and are indicated by open and closed symbols in (C).

FIG. 20. A capillary is first connected to the 20 μιη region of a RTC microfluidic device. The sample loop is filled with an intercalator such as PO-PRO-1, then introduced to the DNA sample. Vacuum is applied at the waste, and intercalation occurs via transverse lateral diffusion. The sample loop may be more deeply etched than other channels in the microfluidic chip. As an example, it may have a depth of about 20 microns as compared to other channels downstream from it which may have depths of about 2 microns. The microfluidic devices of the invention comprise microfluidic chips. The chip is illustrated by the square in the figures such as FIG. 20. The chips may vary in size. Exemplary chips are 8 mm x 8 mm.

FIG. 21. Again, a capillary is connected to the 20 μιη region of an RTC microfluidic device, and the sample loop is filled with an intercalator such as PO-PRO-1. Electrodes are then placed in the sample and waste ports, and current is driven across the system.

FIG. 22. DNA is loaded into the sample injection port, and TE containing an intercalator such as PO-PRO-1 is loaded into the sheathing buffer ports. The system is then driven under vacuum. DNA is introduced to the intercalator as it enters the interrogation region, and intercalation occurs.

FIG. 23. DNA is partially stained over the 300 ms it spends in the presence of an intercalator such as POPO-1. However, the ends are never fully elongated and require a longer reaction time to be fully intercalated. This can be seen in the curved intensity profile of the DNA backbone. The plot is an average intensity per unit length versus length plot.

FIG. 24. An intercalator such as PO-PRO-1, DNA, and sheathing buffer such as TE are introduced to their respective loading ports. When vacuum is applied at the waste, laminar flow brings the intercalator and DNA into contact, and the short diffusion distance allows them to mix within milliseconds. Full seconds pass between mixing and the introduction of DNA to the detection region. The residence time of nucleic acids in this configuration is about 3-8 seconds. This could be increased by increasing the flow rate through the sheath inlets as doing so reduces the flow of nucleic acid and keeps the nucleic acids together with the intercalator for a longer period of time. FIG. 25. 2D COMSOL fluid velocity simulation of the mixing geometry interface, where DNA is nested between adjacent intercalator (such as PO-PRO-1) streams.

FIG. 26. 3D COMSOL fluid velocity simulation of the proposed multi-planar geometry, and projection views of the three dimensional streamlines. Laminar flow of intercalator (such as PO-PRO-1) from the bottom 2 μιη cross channel interfaces with the upper DNA injection channel, and passes through the outlet. The total diffusion distance where the two intersect is 1 μιη;

FIG. 27 provides the chemical structures of POPO-1 and PO-PRO-1;

FIG. 28. Positions (base numbers) of the conservative genomic sequences in the gene of 16S rRNA used for hybridization of forward (F) and reverse (R) primers.

FIG. 29. Schematic of Genome Sequence Scanning™ (GSS™). (I) Genomic DNA is extracted from bacteria, specifically cut with a restriction endonuclease, and tagged with a fluorescent probe. (II) Tagged DNA fragments are stretched into linear conformation by microfluidics. (Ill) Fluorescence pattern of the hybridized probes measured for every DNA molecule. (IV) The detected patterns are compared with the database for identification.

FIG. 30. Multiple fluorescent traces generated from E. coli K12 genome by digestion with SanDl enzyme and probes, recognizing GAGAAAGA (green) and GAAGAGAA (red) motifs. Positions of the corresponding DNA fragments are shown in the circular E. coli genome (center).

FIGS. 31A-31B. Scheme of the DNA sorting.

FIG. 31 A. General sorting approach based exclusively on GSS™ analysis using nonspecific probes. If the pattern (comprised of one or more signals) does not match a template, the DNA molecule proceeds to drain (tO. Only DNA fragments with matching patterns are directed to the selected nucleic acids reservoir (t ₂ ).

FIG. 3 IB. Selection using specific probes. If only probes with short cognate sequences (non-specific probes) are detected (single trace), the DNA fragment is permitted to follow the default route to drain (tO. If the DNA fragment with a target (e.g., a housekeeping) gene is detected by the presence of specific probes (spiked intensity profile), the DNA fragment is redirected to the collection reservoir for selected fragments (t ₂ ).

FIG. 32. Formation of a PD-loop. A DNA fragment (A) is hybridized to two bisPNA probes (B and C). If these probes are sufficiently close to each other, they displace a long strand of DNA, which in turn can be hybridized with an oligonucleotide (D), carrying two fluorophores. The whole process does not require denaturing conditions at any stage.

FIG.33. Suitable site for PD-loop formation at position 1215 bp in the 16S rRNA gene. One of the sites for bisPNA hybridization includes a pyrimidine; therefore, bisPNA will hybridize only with 7 bases. The sites are separated by a single base.

FIG. 34. Suitable site for PD-loop formation at position 1531 bp in the 16S rRNA gene. Each of the sites for bisPNA hybridization includes a couple of pyrimidine inserts; therefore, bisPNAs will hybridize only with 7 bases, The sites are separated by 2 bases.

FIGS. 35A-35C. Single strand flap hybridization.

FIG. 35A. One strand nick is performed by a specific nicking enzyme.

FIG. 35B. The nicked DNA strand is displaced by synthesizing the complementary sequence from the nick using Vent(exo-) polymerase.

FIG. 35C. A complementary fluorescent oligonucleotide is hybridized to the displaced DNA strand.

FIG. 36. Microfluidic chip for DNA sorting. Pictures of pre-sorting DNA reader I and DNA sorting structure II are presented in panels B and C, respectively.

FIGS. 37A-37B. Scheme of a microfluidic chip for DNA sorting using hydrodynamics. FIG. 37A shows normal flow of DNA (referred to herein as DNA, DNA fragments or DNA molecules, interchangeably) through the chip to the waste (labeled as W).

FIG. 37B shows the change of flow of selected DNA to the collection reservoir (labeled as C).

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying Figures, which are schematic and are not intended to be drawn to scale. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. DETAILED DESCRIPTION OF INVENTION

The invention broadly provides a variety of devices, systems and methods for the preparation, manipulation, and analysis of nucleic acids, and the physical separation of nucleic acids of interest from other nucleic acids.

Preparation of a Nucleic Acid Sample

According to some aspects, the invention provides devices and methods of use thereof for positioning or manipulating or concentrating agents within a fluid, including but not limited to polymers such as genomic DNA. Aspects of the invention allow the agents to be

concentrated into relatively small portions of the fluid. This may provide a higher concentration of the agent within a portion of the fluid, or decrease losses as the agent undergoes processing due to a decrease of contact area between the agent and the membrane.

Certain aspects of the invention relate to using a chamber for positioning or manipulating an agent, such as genomic DNA. In some aspects, the chamber is minimally comprised of an inlet port, a porous membrane that allows fluid but not the agent of interest to pass through, and a plurality of channels positioned on a side of the porous membrane opposite the inlet port. The chamber may be operated in a first mode where a fluid containing agents is introduced into the chamber through the inlet port and flowed through the porous membrane in the chamber. Fluid may be introduced through one or more of the channels to move a portion of the fluid towards a peripheral portion of the membrane. The desired agents may then be positioned on the central portion of the membrane. Flow may be reversed through the inlet port to move any agents positioned on the membrane out of the chamber in central streamlines that exit the chamber through the first fluid port.

The invention is based in part on devices with chambers (referred to herein

interchangeably as a "reaction chamber" or a "fluidic chamber") that may be used to concentrate a fluid sample, which may contain various agents, to a smaller volume of fluid. Concentrating samples may prove useful when relatively small volumes are available for analysis.

Additionally or alternatively, concentrating a sample may prove useful in introducing a sample from a macro-scale environment, such as from where a sample may have been collected, to a micro-scale or nano-scale environment, such as where analysis may be performed on the sample. In one embodiment, the device is configured to isolate, purify, and then process various types of samples, including, but not limited to DNA from microorganisms.

FIG. 1 illustrates one embodiment of a device 100 for preparing a fluid sample. The device includes a body 30 with a chamber 10 and an inlet 12. As shown, the inlet 12 may be centrally positioned at the top of the chamber 10 and is configured to move fluids into and/or out of the chamber 10. The device 100 also includes a porous membrane 14 that allows fluid but not the agents of interest to pass therethrough. A plurality of channels 16, 18, 20 are coupled to the bottom of the chamber. As illustrated, the inlet 12 is positioned on a first side of the membrane 14 and the plurality of channels 16, 18, 20 are positioned on a second side of the membrane, where the first side is opposite the second side. As set forth below, the channels 16 may be used to direct flow, typically introduced through the inlet 12, in different directions.

Embodiments of the chamber may be constructed with different configurations and dimensions, some examples of which are discussed herein. By way of example, the chamber 10 may provide a diffusive flow pathway between the inlet port 12 and the flow region, which, in many embodiments, may laterally spread the flow of fluid introduced through the inlet port to promote even distribution of agents about the porous membrane.

The chamber 10 may be shaped differently according to various embodiments. In one illustrative embodiment, the chamber 10 includes a diffuser portion 8 which is typically designed to smoothly widen or diffuse flow that enters the flow region from the inlet port without subjecting agents to excessive shear forces. As shown in FIG. 1, in one embodiment, the diffuser portion 8 of the chamber 10 is substantially frustoconical in shape. In one embodiment, the chamber 10 has a symmetric, truncated cone shape with substantially linear sides that form an angle of about 60 degrees with a line that extends along a central axis of the inlet port 12. It is to be appreciated, however, that the chamber may include walls that are angled differently, or that are gently curved instead of being linear, as aspects of the invention are not limited in this regard. In one embodiment, the chamber 10 has a symmetric, truncated cone shape with substantially linear sides that form an angle of about 45 degrees with a line that extends along a central axis of the inlet port 12. According to some embodiments, the chamber may include flat sides, appearing more like a truncated pyramid. Other embodiments may also include asymmetric chambers.

The inlet port 12 is typically positioned in the central portion of the chamber and is configured to direct a flow of fluid orthogonally toward the porous membrane 14 of the chamber 10. According to other embodiments, however, the inlet port 12 may be offset to one side of the chamber. Additionally or alternatively, the inlet port may direct fluid flow toward the membrane at an angle, instead of orthogonally. It is also to be appreciated that embodiments of the chamber may include a plurality of inlet ports positioned about the diffuser portion 8.

The chamber and/or inlet port, when described as being substantially opposed to the membrane 14, are understood to be positioned to direct fluid to impinge on a surface of the membrane. That is, at least a portion of the fluid flow is directed to intersect with the membrane 14.

The porous membrane 14 (also referred to herein as a substrate or a filter) is typically positioned to receive fluid flow that is introduced to the chamber from the inlet port 12, as shown in FIG. 1, such that a fluid sample passing therethrough may be received on the membrane 14. The membrane typically has a threshold size that relates to the porosity of the membrane and that describes the size or molecular weight of agents or other constituents that are prevented from passing therethrough. According to some embodiments, the membrane 14 has a threshold size that prevents the passage of cells, of genomic DNA, of proteins, and the like, although other threshold sizes are possible, as aspects of the invention are not limited in this respect. Some examples of membranes include ultrafiltration membranes. According to many embodiments, the membrane may be chosen such that it does not have an affinity for agents that may be processed in the chamber and thus does not prevent the agent from being removed from the chamber.

As set forth in more detail below, the membrane 14 may comprise a removable filter material that is held by a frit or support body 28, as shown in FIG. 1. Some operating protocols may utilize a membrane 14 with different threshold sizes, or that are constructed differently, and may benefit from being removable from the chamber. According to some embodiments, the membrane itself is relatively stiff, such that a support body may not be required.

In one embodiment, the chamber 10 may include a body section 6 that defines a wall of the chamber 10 that lies between the membrane 14 and the diffuser portion 8. As shown in FIG. 1, the body section 6 of the chamber 10 is substantially cylindrical in shape and extends for a relatively short distance between the diffuser portion 8 of the chamber 10 and the membrane 14. In other embodiments, the chamber 10 may be shaped differently, or the diffuser portion 8 of the chamber 10 may extend directly to the membrane 14, such that there is no body section 6 at all in the chamber 10. A plurality of channels 16, 18, 20 are positioned adjacent the membrane 14, and as shown in FIG. 1, the channels 16, 18, 20 are positioned on a second side of the membrane 14 (i.e. on a side of the membrane opposite the inlet 12) and they each extend outwardly from the membrane 14. The particular embodiment illustrated in FIG. 1 includes a first channel 16 which extends outwardly from a central portion of the membrane 14, a second channel 18 which extends outwardly from a peripheral portion of the membrane 14, and a third channel which also extends outwardly from a peripheral portion of the membrane 14. In one illustrative

embodiment, the first channel 16 extends outwardly from a central portion of the membrane, and the second and third channels 18, 20 extend outwardly from peripheral portions. As illustrated, the third channel 20 may be positioned on a peripheral portion of the membrane opposite the second channel 18.

The plurality of channels 16, 18, 20 are configured to be connected to an external pump or valve that controls the proportion of flow that passes though the channels. Any vacuum (or positive pressure) produced by the external pump, in turn, causes a vacuum (or pressure) in one or more selected channels 16, 18, 20 to move the fluid sample in the chamber. For example, if a vacuum is applied within the first channel 16, fluid within the chamber 10 will move into the first channel 16 and agents will collect along the central portion of the porous membrane 14. If a vacuum is applied within the second channel 18, fluid within the chamber 10 will move into the second channel 18 and agents may collect along the peripheral portion of the porous membrane 14, and similarly, if a vacuum is applied within the third channel 20, fluid within the chamber 10 will move into the third channel 20 and agents may collect along the peripheral portion of the porous membrane 14. As set forth in more detail below, in one embodiment, a vacuum may be applied within the first channel 16 to initially move the fluid sample and its agents toward the central portion of the membrane 14 (i.e. toward the first zone 40 of the membrane). Thereafter, a vacuum may be applied within the second channel 18 and/or the third channel 20 to move undesired agents and/or debris towards the peripheral portion of the membrane 14 (i.e. toward the second zone 50 of the membrane 14), thus isolating the desired agents on the central portion of the membrane 14. It is contemplated that a vacuum may also be applied within the first channel 16 at the same time that a vacuum is being applied within the second and third channels 18, 20. Flow may be reversed through the first channel 16 to move the desired agents on the central portion of the membrane out of the chamber 10. In one embodiment, when a vacuum is applied within the first channel 16, the fluid flows substantially normal or perpendicular to the membrane 14 such that the desired agents in the fluid sample press against the central zone of the membrane. When a vacuum is applied within the second and/or third channels 18, 20, the fluid may flow with a tangential component toward the peripheral portion of the membrane 14.

In one embodiment, the membrane 14 includes at least a first zone 40 and a second zone 50, where the first zone 40 is the central portion of the membrane 14 and the second zone is the peripheral portion of the membrane 14. In one embodiment, the second zone 50 substantially surrounds the first zone 40, and the second zone 50 may be substantially annular shaped. Other shapes are also contemplated, and in one embodiment, there may be a plurality of second zones 50 as the invention is not necessarily so limited. In one embodiment, the first zone is substantially circular shaped, although other shapes are also contemplated.

As shown in FIG. 1, in one embodiment, there is a barrier 60 which separates the first zone 40 of the membrane 14 from the second zone 50 of the membrane 14. The barrier 60 is configured to prevent movement of the sample through or within the membrane, to help to fluidly isolate the first zone 40 from the second zone 50. Thus, when a vacuum is drawn in the second zone 50 (i.e. with second and/or third channel 18, 20), the barrier 60 may be configured to prevent movement of the sample already positioned on the first zone 40 of the membrane 14.

It is recognized that the barrier 60 could be formed in a variety of different ways. For example, in one embodiment, the barrier 60 may be formed by a weld on the membrane material. The first and second zones 40, 50 of the membrane 14 may be made of one continuous membrane material with a weld formed therein to isolate the first zone 40 from the second zone 50. In another embodiment, the first and second zones 40, 50 may be formed of at least two membrane materials and another type of barrier 60, such as, but not limited to added layers of the membrane material, or other types of objects which physically separate the two zones 40, 50 may be employed.

The size and shape of the membrane 14 and the barrier 60 may vary, but as shown in FIG. 6, in one embodiment, the membrane 14 includes a substantially annular shaped portion and the barrier is substantially annular in shape. It is also contemplated that the barrier 60 may be formed from an annular washer-like component. It is further contemplated that the barrier may be formed with a sealant.

As shown in FIG. 1, in one illustrative embodiment, the body 30 further includes a collar 70 extending upwardly from the chamber inlet 12. The collar 70 has a passageway 72 extending therethrough, and the passageway 72 has an inlet 74 configured to receive the fluid sample. In one illustrative embodiment, the collar inlet 74 is larger than the chamber inlet 12. Such a configuration enables the fluid sample to be more easily dispensed into the device 100, while also preventing disturbance to the reaction chamber 10. For example, the collar inlet 74 may be configured to receive a robotic probe which is configured to dispense a fluid sample into the device 100. In one illustrative embodiment, at least a portion of the collar passageway is frustoconical in shape as it narrows from the collar inlet 74 to the chamber inlet 12. However, other shapes are also contemplated as the present invention is not necessarily limited in this respect. In one embodiment, the collar passageway 72 is configured to act as a reservoir to hold a fluid, such as a buffer. As set forth below, the passageway 72 may be sized and shaped to hold a volume of the fluid sufficient to perform a particular function. For example, in one embodiment, the reservoir passageway 72 is configured to hold a volume that is at least approximately five times larger than the volume of the chamber 10. In one embodiment, the collar 70 includes a restriction 76 which separates the larger reservoir portion of the passageway 72 from the chamber inlet 12. This restriction 76 isolates the chamber 10 such that the reservoir fluid can be replaced without disturbing the chamber 10.

In one embodiment, the device 100 may also be equipped with features to regulate temperature in the chamber 10. According to one embodiment, a frit 28 that lies below and supports the membrane 14 is made of a thermally conductive material, like stainless steel, and may be heated or cooled by an external source, like a thermoelectric module, to regulate temperature. Additionally or alternately, fluid may pass through the chamber 10 to cool or heat the chamber. The chamber may also be equipped with other devices, like a radiant heater that heats fluid in the chamber through non-contact methods, or like an inline heater that heats fluids entering the chamber which, in turn, may help maintain uniform temperature conditions throughout the chamber volume.

Broadly speaking, the plurality of channels 16, 18, 20 are configured to receive fluid that has passed through the membrane from the flow region. As set forth below, the flow through the various channels 16, 18, 20 can be varied to control the movement of the fluid sample and the agents contained within the fluid sample. It is however to be appreciated that the channels 16, 18, 20 may be used to accomplish other effects, such as heating and/or cooling of the flow region, as discussed herein. FIGS. 2A-2D illustrate a plurality of sample preparation steps that may be performed with the device 100 illustrated in FIG. 1. FIG. 2A illustrates a washing step in which a buffer is passed through the chamber 10 and that buffer passes through both the first and second zones 40, 50 of the membrane 14. In particular, as illustrated, a vacuum may be applied within the first channel 16, the second channel 18, and the third channel 20. The fluid buffer may already be positioned within the reservoir portion of the passageway 72.

FIG. 2B illustrates an injecting step in which a fluid sample is injected into the device 100. In one illustrative embodiment, a robotic probe 80 is used to inject or dispense the fluid sample into the device 100. As illustrated, the device 100 may be filled with the buffer fluid when the fluid sample is being injected into the device 100. The type of fluid sample may vary, as the invention is not necessarily limited in this respect, but in one embodiment, the fluid sample contains DNA. In one embodiment, the sample may include a suspension of cells (such as but not limited to bacteria, yeast, molds, and/or mycoplasma). In one embodiment, the sample includes isolated nucleic acids varying in length from about 0.01 megabases to about 1 megabase in a fluid having a volume between about 10 μΐ _^ to about 100 μΐ _^ . In another embodiment, the sample includes isolated nucleic acids varying in length from about 0.01 megabases to about 0.1 megabases, and in another embodiment, the sample includes isolated nucleic acids varying in length from about 0.1 megabases to about 1 megabase.

In one embodiment, focused flow techniques may be employed during the injection step. In particular, the buffer fluid surrounding the probe 80 is utilized to focus the flow of the sample in the chamber 10. For example, a vacuum may be applied within the first channel 16 at a first flow rate. As mentioned above, this will cause the fluid in the chamber to move toward the central portion of the membrane 14. The fluid sample is injected into the device at a second flow rate. In one embodiment, the first flow rate is greater than the second flow rate, such that the buffer surrounding the probe 80 also moves toward the membrane. The flow rate of the buffer toward the membrane is approximately equal to the difference between the first flow rate and the second flow rate. This surrounding sheathed buffer flow may act to focus the flow of the sample toward the membrane 14 by constraining the sample towards the central portion of the membrane. In one particular embodiment, the first flow rate is approximately 200 microliters/minute, and the second flow rate is approximately 100 microliters/min, thus the resulting flow rate of the surrounding buffer is approximately 100 microliters/min. In another embodiment, the first flow rate is approximately 100 microliters/minute, and the second flow rate is approximately 50 microliters/min, thus the resulting flow rate of the surrounding buffer is approximately 50 microliters/min.

FIG. 2C illustrates an incubation step where there may be no fluid flow either into or out of the device 100. In one embodiment, the temperature of the chamber is increased using one of the above-described techniques. For example, the temperature of the chamber 10 may be increased to approximately 37° C.

Thereafter, another washing step may be performed as shown in FIG. 2A. In this washing step, the undesired cellular debris may be moved into the second peripheral zone of the membrane and the desired agents in the fluid sample, such as for example, the DNA sample, may be retained on the central portion of the membrane. As shown in FIG. 2A, a vacuum may be applied in the first central zone 40 of the membrane 14 to keep the bigger desired agents in the fluid sample on the first central zone 40 of the membrane 14. As also shown in FIG. 2A, a vacuum may also be applied in the peripheral second zone 50 of the membrane 14 to move smaller undesired components in the fluid sample away from the first central zone 40. As mentioned above, the barrier 60 may be configured to prevent the desired agents in the fluid sample from migrating from the first zone into the second zone. The device may be configured such that the larger particles/agents remain on the central portion of the membrane, whereas the smaller particles/agents move into the peripheral portion of the membrane.

The steps shown in FIGS. 2A-2C may be repeated one or more times. For example, a restriction enzyme may thereafter be injected into the chamber, as shown in FIG. 2B, and then the incubation step shown in FIG. 2C may be repeated. In one embodiment, a bacteria sample may be introduced into the chamber and the bacteria sample may remain on the membrane 14 while undergoing cell lysis, DNA extraction, DNA digestion, and/or DNA labeling.

FIG. 2D illustrates the additional step of ejecting the desired sample from the device. As shown, this may be done by reversing the flow of fluid and applying a positive pressure through the first channel 16 such that the sample that has collected on the central portion of the membrane moves up through the chamber and out of the device. The barrier 60 may be configured to prevent the undesired debris, etc. in the second zone of the membrane from migrating over into the first central zone, thus the undesired debris may remain within the device 100. Once the desired sample has been removed from the device, the undesired debris, etc. that may have accumulated along the peripheral portion of the membrane may be removed from the device. FIGS. 3-5 illustrate one embodiment of a first portion 200 of a device for preparing a sample which is made from a solid starting material, and FIGS. 6 and 7 illustrates another embodiment of a first portion 300 of the device for preparing a sample which is injection molded. FIG. 6 illustrates a second portion 310 of the device for preparing a sample. The first portion 200, 300 and the second portions 310, when coupled together, may form a device which is substantially equivalent to the device 100 described above and shown in FIGS. 1-2.

Accordingly, like components have been given identical reference numbers with a prime (') added.

As shown in FIGS. 3-5, the first portion 200 may include a body 30' with both the chamber 10' and the collar 70' formed within the body 30' . In this embodiment, there is a narrow restriction 76' that separates the chamber 10' from a reservoir portion of the collar passageway 72'. In one embodiment, the restriction 76' is at least three times the height of the chamber 10' . In another embodiment, the restriction 76' is at least five times the height of the chamber 10' .

As shown in FIGS. 6 and 7, the portion 300 may include a body 310 with the plurality of channels 16', 18', 20' formed within the body 310, and the membrane 14' may be configured to be coupled to the body 310 of the portion 300. This body 310 (which includes the channels 16', 18', 20') may be formed separately from the chamber body. One portion may be configured to be disposable and another portion may be configured to be reusable. In this particular illustrative embodiment, the first and second portions 300, 310 are configured to form a plurality of separate devices with chambers 10' for preparing a sample. In one embodiment, there are four isolated chambers 10' which are separated with an O-ring seal 202. Other configurations and numbers of chambers 10' are also contemplated, as the present invention is not so limited. For example, it is contemplated that a device with such a configuration may be used with a fluid dispensing device that includes a plurality of robotic probes 80.

As shown best in FIG. 5, the body 30' may include a plurality of downwardly extending feet 210 spaced apart around the perimeter of the chamber 10' . A plurality of passages 220 may be formed between the feet 210, and the passages 220 may be configured such that fluid can pass from the chamber 10' into the second zone 50 of the membrane 14' .

As shown in FIG. 6, in one illustrative embodiment, one sheet of membrane material may form a plurality of membranes 14' in adjacent chambers 10'. It is also contemplated that multiple membrane sheets may form the plurality of membranes. As mentioned above, these first and second portions 300, 310 illustrated in FIGS. 3-7, when coupled together, may form a device which is substantially equivalent to the device 100 described above and shown in FIGS. 1-2. In one embodiment, the first portion 300 is configured as a disposable component. The first portion 300 may be a one-piece component in which the reservoir 70, restriction 76, chamber 10 and feet 210 are integrally formed, and for example, the first portion 200 may be molded. In another embodiment, the second portion 310 is configured as a disposable component.

Fluid flow may be controlled through the chamber during the various steps with different configurations of pumps and valves. According to some embodiments, flow is controlled by a first variable flow rate pump in fluid communication with the first channel 16 and by a second variable flow rate pump that is in fluid communication with the second and third channels 18, 20. It is to be appreciated, however, that other arrangements of pumps (either pressure or vacuum) and valves may be used to control flow through the chamber in various modes of operation, as aspects of the invention are not limited in this respect. Additionally, aspects of the invention are not limited to any one type of pump or valve.

Embodiments of the chamber may be operated by a controller that receives information for a particular operating protocol and, in turn, controls pumps and/or valves to run the system automatically to complete the protocol. The term 'automatically', as used herein, refers to a system that is capable of switching between modes of operation without the intervention of an operator, or to a system that is otherwise capable of altering operating conditions, such as flow rates or temperatures without manual operator intervention, such as by following a predefined operating protocol or by controlling the system to predetermined set points. The controller and operating protocol combination may be implemented in any of numerous ways. For example, in one embodiment, the controller and operating protocol combination may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. It should be appreciated that any component or collection of components that perform the functions described herein can be generically considered as one or more controllers that control the functions discussed herein. The one or more controllers can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above. The one or more controllers may be included in one or more host computers, one or more storage systems, or any other type of computer that may include one or more storage devices coupled to the one or more controllers.

In this respect, it should be appreciated that one implementation of the embodiments of the present invention comprises at least one computer-readable medium (e.g., a computer memory, a floppy disk, a compact disk, a tape, etc.) encoded with an operating protocol in the form of a computer program (i.e., a plurality of instructions), which, when executed by the controller, performs the herein-discussed functions of the embodiments of the present invention. The computer-readable medium can be transportable such that the treatment protocol stored thereon can be loaded onto any computer system resource to implement the aspects of the present invention discussed herein. In addition, it should be appreciated that the reference to an operating protocol or controller which, when executed, performs the herein-discussed functions, is not limited to an application program running on a host computer. Rather, the term operating protocol is used herein in a generic sense to reference any type of computer code (e.g., software or microcode) that can be employed to program a processor to implement the herein-discussed aspects of the present invention.

The device may also comprise one or more sensors that receive information from the chamber or channels used to connect the chamber to other portions of the device. Such sensors may receive information regarding pressure, temperature, flow rates, and the like, in any portion of the chamber or device. The device may also receive information for detectors that are used to analyze or detect the presence of an agent in a portion of the device.

Analysis of a Nucleic Acid Sample

According to some aspects, the present invention provides devices that allow polymers of any length, including nucleic acids containing entire genomes, to be stretched into a long, linear conformation for further analysis. Polymers are loaded into a device and run through the structures, propelled by, inter alia, physical, electrical or chemical forces. Stretching is achieved by, e.g., applying shear forces as the polymer passes through the device. Because the forces are applied continuously, it is possible to stretch out polymers to a length that is equal to or greater than the active area of the apparatus, i.e., where information about the polymer is collected as the polymer is analyzed. For example, if a video camera or laser illuminated volume is focused on the region of the chip where spreading occurs, unlimited lengths of DNA molecules can be monitored, i.e., much larger than the video image or the laser illumination volume. Since multiple molecules may be stretched in succession, extremely high throughput screening, e.g., screening of more than one molecule per second, may be achieved.

An extended labeled polymer may be moved past at least one detection station, at which labeled units of the polymers interact with the station to produce an object-dependent impulse. As used in this application, "moves past" refers to embodiments in which the station is stationary and the extended polymer is in motion, the station is in motion and the extended polymer is stationary, and the station and extended polymer are both in motion. As used herein, a

"detection station" is a detection arrangement that detects physical quantities and/or properties of a polymer. Such properties include emission of energy or light of one or more wavelengths, wherein such emission is the result of laser interaction with the polymer or a sequence- specific probe hybridized to the polymer, including one or more fluorophores attached thereto. A detection station includes detection instruments, such as a camera, CCD camera or a silicon- intensified camera, a laser and optical detector combination, a light and optical detector combination, confocal fluorescence illumination and detection, or any other suitable detection instrument. This process is discussed in greater detail in U.S. Patent No. 6,696,022 which is herein incorporated by reference in its entirety.

Certain devices of the invention are used in conjunction with methods for analyzing the extended polymers by detecting signals referred to as object-dependent impulses. An "object- dependent impulse," as used herein, is a detectable physical quantity which transmits or conveys information about the structural characteristics of at least one unit-specific marker of an extended polymer. A unit- specific marker, as used herein, can either be a measurable intrinsic property of a particular type of individual unit of the extended polymer, e.g., the distinct absorption maxima of the naturally occurring nucleobases of DNA (the polymer is intrinsically labeled), or a compound having a measurable property that is specifically associated with one or more individual units of a polymer (the polymer is extrinsically labeled). A unit-specific marker of an extrinsically labeled polymer may be a particular fluorescent dye with which all nucleobases of a particular type, e.g., all thymine nucleobases, in a DNA strand are labeled. Alternatively, a unit-specific marker of an extrinsically labeled polymer may be a fluorescently labeled oligonucleotide of defined length and sequence that hybridizes to and therefore "marks" the complementary sequence present in a target DNA. Unit-specific markers may further include, but are not limited to, sequence specific major or minor groove binders and intercalators, sequence- specific DNA or peptide binding proteins, sequence specific PNAs, etc. The detectable physical quantity may be in any form that is capable of being measured. For instance, the detectable physical quantity may be electromagnetic radiation, chemical conductance, radioactivity, etc. The object-dependent impulse may arise from energy transfer, directed excitation, quenching, changes in conductance (resistance), or any other physical changes. In one embodiment, the object-dependent impulse arises from fluorescence resonance energy transfer ("FRET") between the unit-specific marker and the station, or the environment surrounding the station. In preferred embodiments, the object-dependent impulse results from direct excitation in a confined or localized region, or epiillumination of a confocal volume or slit-based excitation is used. Possible analyses of polymers include, but are not limited to: determination of polymer length, determination of polymer sequence, determination of polymer velocity, determination of the degree of identity of two polymers, determination of characteristic patterns of unit-specific markers of a polymer to produce a "fingerprint", and characterization of a heterogeneous population of polymers using a statistical distribution of unit-specific markers within a sample population.

There are numerous methods and products available for analyzing polymers as described in PCT Publication No. WO 98/35012, which is incorporated herein by reference in its entirety.

The genetic information of all cellular and some viral organisms is encoded in long polymeric chains of DNA. The ultimate resolution of base-by-base DNA sequence is unique for each organism. The identity and interrelatedness of organisms however can be determined by lower resolution detection of repeated elements in their genetic code. Genomic technology is capable of optically characterizing the length of individual restriction endonuclease digested molecules, and identifying the spatial location of fluorescent labels tagged to repeated sequence motifs on each molecule of DNA. Genomic technology has wide ranging applicability in identification of bacterial genomic DNA.

The ability to optically resolve discrete sites of fluorescent labeling requires physical manipulation of each molecule such that it is stretched into a fully elongated, linearized conformation. Previous studies have demonstrated the efficacy of stretching long fragments of bacterial genomic DNA in microfluidic devices with combined shear and elongational flows. In two-dimensional tapered funnels etched to 1 μιη depth, DNA stretching is strongly affected by the funnel taper, and confinement of the DNA to the 1 μιη deep channel serves to pre-stretch the DNA, resulting in more uniform stretching. This ability to elongate single fragments of DNA in continuous flow conditions is highly advantageous over alternative DNA elongation methods because of the ease of sample manipulation, high sample throughput, and simplicity of the microfluidic device.

The sensitivity of Genomic Sequence Scanning (GSS™) technology as a detection and identification method is limited by DNA detection throughput, resolution of length information, and the range of fragment lengths that can be fully extended. By careful consideration of the stretching funnel geometry, the inventors have significantly improved these factors. First, as set forth below, the relationship between the effects of fluid velocity, funnel taper, and fragment length range have been correlated to allow for design of funnel geometries that stretch a desired range of fragment lengths at any desired fluid velocity. Second, as set forth below, novel funnel geometries have been designed that maintain the tension of stretched DNA molecules during detection. This increases analyzable molecule throughput by stretching a higher percentage of molecules to the fully extended state and eliminating relaxation and shear-induced molecular tumbling during detection. Finally, funnel geometries that maintain tension in the DNA detection channel dictate continuous acceleration of the molecule. Correct molecule analysis requires corrections of this acceleration. Because the acceleration profile is dictated by the funnel geometry, the acceleration correction can be determined from the funnel taper.

As set forth in greater detail in U.S. Patent No. 6,696,022, it is generally desirable for the polymer sample to be in a stretched elongated state. However, a polymer sample is typically in a lower-energy, more coiled conformation. Therefore, aspects of the present invention are directed to devices with elongation structures that include tapered channels that are designed to stretch the polymer sample and then cause the polymer sample to remain in a stretched conformation.

As set forth in greater detail below, aspects of the present invention are directed to devices with tapered channels with at least a first zone having a first tapered shape and a second zone having a second tapered shape, where the second tapered shape is different than the first tapered shape. As discussed below, in one illustrative embodiment, the first tapered shape includes an increasing strain rate taper, and the second tapered shape includes a constant strain rate taper. The increasing strain rate taper may be configured to stretch, straighten out and/or elongate the polymer sample. The constant strain rate taper may be configured to substantially maintain the elongated shape of the polymer sample to prevent the polymer from returning to a more coiled and/or hairpin shape. By way of background, as discussed in U.S. Patent No. 6,696,022, a constant shear rate, or change in average velocity with distance in the channel, is defined as S: du/dx= S where x is the distance down a substantially rectangular charnel, and u is the average fluid velocity in the x direction, which is computed from the overall fluid flow (Q) and the cross sectional area, A, of the channel as follows: u=Q/A

In one embodiment where the channel cross-section is rectangular, the channel may be defined by a constant height, H and width, W such that the cross-sectional area A=HW, and the average fluid velocity is given by: u=Q/HW

Applying the boundary condition that the fluid flow must be continuous (i.e., incompressible), Q is constant. Hence, u is inversely proportional to W. This relationship can be substituted into the original expression for S to determine a relationship between the shear rate and the width:

S = du/dx =Q/H d/dx (1/W) = (-Q/HW ² ) (dW/dx) dW/dx = (-SH/Q)(W ² ) Integrating this expression, it is found that:

W=(SHx/Q + C) ^{" 1} where C is a constant of integration determined by the original width of the channel

(boundary condition). Similar calculations may readily be completed by those of skill in the art for non-rectangular channel shapes. When no net momentum transfer occurs in the height axis, i.e., when the velocity profile in the z-axis has been established, the shear rate from the width profile results in a stretching force. Illustrating in the case of a Newtonian fluid, the stress tensor, t^, required to compute the force is easily expressed in terms of the shear rate:

F= / / - tyz,dzdx = / / ^(du/dx)dzdx = / / -μ Sdzdx where μ is the solution viscosity. In these equations, x is the direction of motion, y is the width, and z is the height. The surface over which the shear rate needs to be integrated is that of the channel wall, which results in:

F = μ HLS where L is the length of the channel wall, approximately the length of the channel in which 15 the constant shear is maintained.

The structures for stretching DNA of the present invention ("elongation structures") comprise two components: a delivery region and a region of polymer elongation. The delivery region is a wider channel that leads into and out of the region of polymer elongation. The region of elongation comprises a tapered channel (i.e. a funnel).

Funnel structures are tapered channels that apply elongational forces in a

regular and continuous manner as the polymer flows down the channel. The particular elongational forces are defined by the type of channel structure and shape. The channel may include a tapered channel that begins at a given width and continuously decreases to a second width, creating an increasing elongational force in the funnel portion of the channel defined by: du/dx = (-Q/H)(dW/dx)(l/W ² )

In one embodiment of the invention, for at least a portion of the channel, the width of the channel decreases linearly so that dW/dx is constant; in this embodiment, the shear, du/dx, thus increases as W decreases. In this embodiment, the angle of the funnel as measured from the continuation of a straight wall is preferably between 1° and 75°, with a most preferred value of 26.6° for DNA in a low viscosity solution such as TE (10 mM TRIS, 1 mM EDTA) buffer, pH 8.0. Starting widths for the linear funnel embodiment preferably range from 1 micron to 1 cm, with ending widths preferably in the range of 1 nm to 1 mm depending on the polymer in question, and in one embodiment, values of 50 microns and 5 microns, respectively, for DNA.

In one embodiment, at least a portion of the channel may also be configured such that the width decreases at an increasing rate as fluid passes down the channel, resulting in an increase in strain rate as the channel is traversed. Such tapers may offer especially good protection against natural relaxation of the polymer, since as time passes and the molecules move down the channel, they face increasing counter-forces to their tendency to recoil. Furthermore, the increasing force taper allows some design flexibility; any polymer that will encounter elongational forces large enough to cause the polymer to stretch in the taper and will not encounter elongational forces large enough to cause the polymer to break in the taper can be successfully run through the taper and stretched. There is no need to find the ideal or threshold force for the polymer, only an effective range. The inventors have appreciated that, in situations involving pressure driven fluid flow, increasing strain rate tapers may yield uniform and reproducible elongation compared to sudden-onset or continuous strain-rate tapers. The gradual onset of polymer deformation achieved in increasing strain rate tapers allows for more thorough sampling of conformational space for each polymer, thus avoiding trapping of molecules in partially extended states.

In one embodiment, at least a portion of the tapered channel is designed such that the strain rate is constant. The value of the constant strain rate required to achieve an adequate force to completely stretch the polymer over the course of the channel will vary based on the length of that channel.

The strain rate of the funnel can be determined by measuring the distance between two known points on a strand of DNA. For example, concatamers of λ DNA are used as standards for elongational force measurements. A unique sequence on each concatamer is fluorescently tagged with a hybridization probe. The interprobe distance on the concatamer is thus the length of a single λ DNA molecule (48 kilobases). The physical distance between the probes is determined using video microscopy or time-of-flight measurements. The physical distance for λ DNA in native solution is 14.1 μιη. This value is compared with the actual measured physical distance. For instance, if the measured distance is 15.0 μιη, then the strain rate can be calculated from the amount of stretching that the DNA has experienced in the stretching structures. The predicted elongational force on the DNA, as measured by the velocity of the DNA and the dimensions of the channel, is matched with the elongation of the DNA and its intrinsic nonlinear stiffness.

Now turning to the figures, as shown in FIG. 8, in one illustrative embodiment, the device 410 includes an elongation structure that is formed into a chip. As shown, the device 410 may include four ports and may include a sample loading port 430, two sheath buffer channels 440, 450, the elongation structure 460 (may also be called the DNA stretching funnel), and a waste port 470. As illustrated, in one embodiment, there is a delivery channel 432 between the sample loading port 430 and the elongation structure, and there are two opposing buffer channels 440, 450 that also lead into the elongation structure.

The DNA stretching funnel 460 geometry was optimized based upon experimental results. In one illustrative embodiment, the structure of the funnel 460 is divided into two distinct regions or zones. The first zone 462 may be defined as the stretching portion of the funnel, and the second zone 464 may be defined as the detection region. The first zone 462 may have a first tapered shape, and the second zone 464 may have a second tapered shape, different from the first zone. Distinct taper definitions may be required for each region. The overall funnel geometry may therefore be fully described by three characteristic widths (wi = width of first end of tapered channel, = width of tapered channel at transition between first zone 462 and second zone 464, and W ₃ = width of tapered channel at the second end of the tapered channel), two characteristic lengths (/ ,= length of first zone, and h = length of second zone), and two taper definition equations. These geometries are detailed in FIG. 8B.

Previous studies have demonstrated that DNA stretching is most uniform when the initial DNA extension occurs gradually in an increasing- strain rate funnel. In some embodiments, the geometry of the first zone 462 (the stretching portion) of the funnel may be described by the following equations:

I

w(x) 2

Equation 1

Where the width of the channel (w(x)) is a function of the distance along the funnel (//J, the initial funnel width (w _/ ) and the width at the transition between the stretching and detection portions of the funnel (¼¾).

In some embodiments, the geometry of the first zone 462 of the funnel may be described by the following equations: w(x) = Equation 2

2v..w..x

Where the width of the channel w(x) is a function of the distance along the detection channel (x), the width at arbitrary position i (wi), and the fluid velocity at arbitrary position i (v . Fj, which describes the geometrical taper coefficient for an increasing strain rate funnel, is a function of the distance along the detection channel (x) the fluid velocity at distance x (v _x ), the funnel width at distance x (w _x ), and the strain rate at distance x (έ _χ ). The funnel taper geometry can be solved to provide a desired strain rate at any given fluid velocity.

Previously described DNA stretching funnels utilized a parallel walled detection channel, which imposed a constant-velocity fluid profile in this region. For such funnels, W3 = W2- This type of second zone 464 configuration is illustrated in FIG. 8B. In one experimental study, a novel detection channel geometry was also investigated, in which the increasing strain rate funnel transitions smoothly into a tapered detection channel with a constant-strain rate taper. This unique second zone 464 tapered configuration is also illustrated in FIG. 8B. In some embodiments, the geometry of the second zone 464 (the detection region) of the funnel may be described by the following equations: w(x)

I ■ V il

^ Equation 3 w w ₃ - 1

Where the width of the channel w(x) is a function of the distance along the detection channel (x), the width at the interface with the stretching portion of the funnel (¼¾), and the final funnel width (w ₃ ).

In some embodiments, the geometry of the second zone 464 of the funnel may be described by the following equations:

F

w(x) =— Equation 4 Where the width of the channel w(x) is a function of the distance along the detection channel (x) and the constant strain rate taper coefficient E ₂ . F ₂ is a function of the fluid velocity at distance x (v _x ), the funnel width at distance x (w _x ), and the strain rate (ε _χ ).

FIG. 8C illustrates a DNA sample passing through the elongation structure 460 and being stretched in the tapered channel. Several funnel profiles were investigated to determine the effects of geometry on DNA extension under multiple operating conditions. The tested funnel parameters are summarized in FIG. 9.

In one embodiment, sheathing buffer flows were used to constrain the DNA stream to the center of the stretching funnel. A fluidic circuit model was used to design the dimensions of resistive elements in the microfluidic structure. The driving constraint was to normalize the width of the DNA stream at the nominal point of detection to 0.25 μιη. This served multiple purposes; the DNA stream could precisely be targeted to the projected laser points in the detection channel, and all DNA molecules were subjected to a uniform flow stream in the center of the funnel, thus avoiding variations in velocity and fluidic path length near the outer walls of the funnel. Operational parameters for tested devices, including predicted driving pressures and bulk flow through the DNA injector path were derived from the fluidic models.

The microfluidic chips were fabricated by Micralyne Inc. (Edmonton, Alberta, Canada). All channels were etched in 500 μιη thick fused silica to a depth of 1 or 2 μιη by reactive ion etching. Through holes for fluidic access were machined by ultrasonic drilling and channels were sealed by fusion bonding to a 170 μιη thick cover wafer. The finished wafers were then diced to form individual devices.

Each chip to be tested was bonded to a custom acrylic manifold (Connecticut Plastics, Wallingford, CT) using UV-curable adhesive (Dymax 140M, Dymax Corporation, Torrington, CT). To prepare chips for use, the devices were first wetted with 100 mM NaOH to remove any residual debris or surface contamination from the fluidic channels. The chips were subsequently flushed with water and TE buffers prior to use. All solutions presented to the microfluidic devices were passed through a 0.2 μιη syringe filters (Millipore, Billerica, MA) prior to use.

A DNA sample, Escherichia coli K12 (MG1655) was purchased from ATCC (American Type Culture Collection, Manassas, VA). Bacterial genomic DNA was prepared in a mini- reactor as described previously. The purified genomic DNA was restriction digested using Notl enzyme, and tagged with fluorescent labeled bis-PNA tags. The prepared DNA sample was eluted at -0.5 - 1.5 ng / μΐ. Immediately prior to stretching on the microfhiidic device 410, a 10 μΐ aliquot of DNA was gently mixed with POPO- 1 intercalator (Life Technologies, Grand Island, NY) to uniformly stain the DNA backbone. Sample concentration was quantified by ethidium bromide- stained gel prior to intercalation in order to standardize a ratio of three nucleic acid base pairs per dye. The extent to which a given fragment of DNA is stretched in the microfhiidic funnel may depend on the ratio of dye to DNA, therefore for all comparisons presented in one experiment were performed such that a common stock of intercalated DNA was used for each experiment set.

Single-molecule data acquisition was performed as previously described. A 5 μΐ sample of prepared, intercalated DNA was pipetted into the sample port 430 on a device 410 to be tested (see FIG. 8A). The loaded sample concentration typically varied from 0.5 - 1.5 ng/μΐ DNA. The chip was then mounted to a custom confocal fluorescence microscope. Three laser detection points were projected into the detection channel of the device. Two 455 nm spots elicited fluorescence from the POPO-1 stained DNA backbone. These spots were separated by 20 μιη along the detection channel. A third detection point at 532 nm excited ATTO dyes attached to sequence specific probes. This detection point was fixed at 5 μιη before the first of the 455 nm points. Fluorescence emission was collected in three discrete channels using a lOOx oil-immersion microscope objective. Fluorescence signals were spectrally separated using a multi-pass dichroic element, and the individual fluorescence signals were collected through fiber coupled avalanche photodiodes (APDs, Perkin Elmer). For all experiments, data acquisition was normalized to 2 bins per micron. Spatial resolution between neighboring fluorescent events was therefore maintained regardless of the fluid velocity within the detection channel (see FIG. 9).

All data analysis was performed using custom software developed at PathoGenetix. Collation of DNA backbone and tag fluorescence signals was performed using a first software application. Clustering of molecules based on the sequence- specific tags was performed using a second software application. An example of a "tag" is a sequence- specific nucleic acid probe. Single molecule backbone fluorescence morphology was analyzed using a scripted program. This program was used to detect the incidence of elevated fluorescence along the POPO- intercalated DNA backbone. An event was defined by 3 or more consecutive bins with a fluorescence intensity greater than 1.5 times the average backbone fluorescence. Molecules with detected elevated events were characterized by the location of the event along the trace. Events could occur at the leading edge of the molecule, at the trailing edge of the molecule, or located within the length of the molecule. These events were categorized as hairpin, relaxation, or overlap events, respectively.

Detection sensitivity in genomic technology devices can be directly correlated to the detection throughput of well stretched, analyzable molecules. The first variable addressed to improve detection throughput was to simply increase the driving fluid velocity of the detection funnel. The influence of funnel geometry must be considered when changing the driving velocity in genomic technology devices.

The primary attribute affecting the extension of DNA in genomic technology funnels is the strain rate of the accelerating fluid that surrounds the DNA. The strain rate (ε) is defined by the change in fluid velocity over a given distance along the axis of the funnel:

e = dV/dx Equation 5

Where the velocity scales inversely with the width of the funnel

V _X = V ₀ ^- Equation 6

For a given funnel geometry, as the final velocity at the exit of the funnel is increased, the peak strain rate also increases proportionally (FIG. 11A). As shown in FIG. 9, a CV 7.5 funnel (constant velocity funnel) driven at 7.5 μιη/ms achieves a peak strain rate of 5 This value increases to 10 and 20 μ8 ^_1 at 15 and 30 μιη/ms respectively. This directly impacts the peak tension experienced by a given DNA fragment, and therefore limits the range of well stretched molecules that can be achieved.

The peak tension on a single elongated molecule of DNA (T^) can be predicted by:

Τ _παχ = Χ _o i ² ^ Equation 7

Where ζ _ι is the parallel drag coefficient (also known as the molecular drag coefficient), L _mo i is the extended length of the molecule in microns, and [ε _ι ^ is the average strain rate of the funnel device, defined by:

/ ^• \ Δν v _f - v _;

25 { £ ) =— =— Equation 8

\ / Ax L _f where v is the fluid velocity at the entrance (i) or exit (f) of a funnel of length (L). ζ _Ά has been previously estimated in computational models to be 0.61 centiPoise (cP). Using this value, the peak tension was calculated for all molecule lengths up to 200 um in constant velocity detection funnels (CV7.5). As the fluid velocity increased from 7.5 um/ms to 15 and 30 um/ms, the peak tension on a molecule of a given length increased dramatically. The inventors predict that an 80 μιη fragment would be well stretched at 7.5 μιη/ms (35 pN tension), but would overstretch dramatically at 30 μητ/ms (141 pN).

The relationships between fluid velocity, strain rate, tension, and molecule extension in the CV7.5 funnel are demonstrated in FIG. 11. A sample of E. coli genomic DNA digested with Notl was prepared as described above and was loaded to the CV7.5 device. Molecule stretching data was acquired at fluid velocities of 7.5, 15, and 30 μιη/ms. At 7.5 μητ/ms, DNA fragments ranging from 40 -100 μιη in length appear to be well stretched - fragments appear as discrete bursts with uniform average backbone fluorescence intensity. Molecules shorter than 40 μιη appear under stretched, with a distribution of higher average fluorescence intensities. Molecules longer than 100 μιη (the 358 and 361 kb fragments predicted in this digest) appear to have somewhat lower average backbone fluorescence, indicating the onset of overstretching.

At 15 and 30 μιη/ms fluid velocities, the fragment distribution appears to shift. Short fragments appear to stretch to the same observed length, but longer fragments are significantly overstretched as peak tension overcomes the 60 pN threshold. The range of well- stretched fragment lengths is obviously compromised by simply increasing the driving velocity in a fixed funnel geometry.

The inventors predicted from equations 7 and 8 that the average strain rate, and therefore tension on a molecule would scale inversely with the length of the funnel - At a fixed fluid velocity, a longer funnel would produce less tension on a molecule. This effect is demonstrated in computational models in FIG. 11. The same E. coli digest was run on the CV 30 device (constant velocity device) at 30 μιη/ms, demonstrating a similar DNA stretching pattern as observed at 7.5 μιη/ms in the CV7.5 device. DNA throughput in stretching funnels can therefore be increased without loss of long, analyzable fragments if the length of the funnel is adjusted to achieve a consistent strain rate.

As demonstrated above, longer stretching funnels may be required to accommodate high fluid velocities in genomic technology devices. This poses a technical difficulty in that the fluidic resistance (R _¾ ) of the microchannel scales with the length of the funnel. Reducing the geometry to the simple case of a rectangular straight channel, YluL

R _h = Equation 9

Where μ is fluid viscosity, and L, w, and h are the length, width, and height of the channel respectively. Because the microfluidic device 410 may be driven by applying vacuum at the DNA waste port 470, the device 410 may be limited to a 1 atmosphere pressure drop along the channel. In a 1 μιη deep device, this may imposes a constraint on the maximum channel length, fluid strain, and fluid velocity. To achieve higher fluid velocities in longer stretching funnels, the inventors explored devices with differing etch depths.

The interaction of the channel floor and ceiling may have complex effects on the stretching of DNA in fluidic devices. Constraining the coiled, unstretched DNA into the narrow confines of a 1 μιη channel serves to pre-extend the DNA, due to shear interactions caused by poiseuille flow in the channel. Once extended, the rate of DNA relaxation back to the condensed state is also slowed in shallow channels. Conversely, flow in high-shear structures can lead to decreased stretching efficiency due to imposed molecular tumbling. Channel etch depth may therefore have beneficial and adverse effects that must be balanced in the design of a DNA stretching device.

To determine the effects of channel etch depth on high fluid velocity DNA stretching, the inventors compared two constant-velocity funnels designed for optimal stretching at 30 μιη/ms (CV30a, CV30b, FIG. 9). The optimal fluid velocity was achieved at 25 psi in the 1 μιη funnel, but at 7 psi in the 2 μιη funnel. Again, the E. coli Notl digest was processed on both devices. The confocal laser spots were positioned 50 μιη from the onset of the constant velocity zone. Roughly equivalent DNA stretching was observed on both devices - well resolved comet plots were achieved without evidence of DNA overstretching (FIG. 11). To determine subtle effects on a molecule-by-molecule basis, the inventors utilized the software algorithm described above to determine if each molecule in a given selection was well stretched, or if there was evidence of additional fluorescent signal at the leading edge, trailing edge, or middle of the molecule, thus indicating hairpinning, relaxation, or molecule overlap respectively. The inventors individually isolated clusters of molecules at -40, 60, 80, and 100 μιη in three discreet paired experiments for both the 1 μιη and 2 μιη deep devices.

In both devices, the efficiency of observing well- stretched molecules increased with the length of the molecule. The 2 μιη deep device may appear to be more efficient in delivering well stretched DNA, across all lengths. The incidence of overlapping molecules was essentially equivalent in both devices, indicating that the molecular occupancy in the detector was uniformly low in all experiments. The 1 μπι deep device caused significantly more molecules to display elevated fluorescence in the leading edge of the molecule, indicating that molecular tumbling was enhanced in the higher- shear 1 μπι channel. The 2 μπι channel however showed uniformly higher (although not statistically significant) incidence of elevated fluorescence in the trailing molecule edge, suggesting more rapid relaxation of molecules in the deeper channels.

In summary, retention of well stretched molecules in the 2 μπι channels was no worse than observed in the 1 μπι channels. Because of the benefit of being able to operate devices at lower vacuum pressures, further experiments with high velocity funnels were performed in 2 μπι deep devices. These observations helped explain the complex effect of shear flow on the observation of DNA stretching. The inventors observed that stretching of DNA is substantially uniform across a long range of molecule lengths as controlled by the elongational flow established by the stretching funnel geometry. Many potentially analyzable molecules must be rejected from further analysis, however due to hairpinned or relaxed conformational states, both of which are influenced by shear flow.

While modeling strain rate as a function of the distance along the stretching funnel, the inventors note that once a molecule passes from the increasing strain region (i.e. the first zone 462) of the funnel into the constant velocity detection zone (i.e. the second zone 464, or the constant strain rate region) the fluid strain rate drops to zero, which suggests that tension along the molecule also decreases. In prior publications, the inventors have explored different stretching funnel geometries, and determined that an increasing strain rate profile yielded more uniform DNA stretching than did a funnel with constant strain rate. The inventors decided to test whether the combination of an increasing strain rate stretching funnel with a constant strain rate detection zone could improve stretching efficiencies by maintaining tension on each molecule during detection. This may be known as a compound constant strain rate funnel and data for such a device is shown in the last two columns in FIG. 9. The funnel taper profile for this detection region is described above, and the resulting strain rate profiles for 30 μιη/ms constant velocity and constant strain rate funnels are shown in FIG. 11.

The relative tension profile along a molecule experiencing extensional flow may be modeled with a known funnel taper, assuming the DNA molecule is a rigid rod with a known length. The velocity of the molecule at any given position within the funnel is the average of the fluid velocity at every point along the extended molecule. In accelerating flow, the head of the molecule is therefore moving more slowly than the surrounding fluid, while the tail of the molecule moves more quickly than the surrounding fluid. The drag of the surrounding fluid elements therefore exerts tension on the molecule. The magnitude of that tension at any point along the molecule is proportional to the integral of the difference between the molecule velocity and the fluid velocity along the length of the molecule.

The inventors computed the relative tension profile on a 100 μπι long molecule positioned with the head of the molecule at the origin of the constant velocity region, as well as 50 and 100 microns within the channel. At the 0 μπι position, the molecule is fully constrained by the increasing strain rate region of the funnel, and displays a nearly parabolic tension profile. As the molecule precedes 50 μπι along the detection zone, the inventors appreciate that the tension profile has shifted towards the tail of the molecule and the overall tension has dropped significantly. With the head of the molecule at 100 μπι, it is fully within the constant velocity zone. At this point, the molecule as a whole travels at the surrounding fluid velocity and tension along the molecule has reduced to zero. From these observations, the inventors note that every molecule analyzed in a constant velocity detection zone is subjected to a complex, dynamic cycle of stretching and relaxation. The state of stretching and relaxation is also highly sensitive to the positioning of the detection point within the constant velocity zone.

By comparison, a similar model was built for a funnel comprising the increasing strain rate stretching region (i.e. the first zone 462) with the constant strain rate detection zone (i.e. the second zone 64). For this design, the inventors defined the desired point of detection to be 150 μπι from the transition between the two taper profiles. The overall length of the detection zone (i.e. the second zone 464) was defined to be approximately 350 μπι. Molecules up to 150 μπι in length would therefore be fully constrained within the constant strain rate region (i.e. the second zone 464) of the funnel during the entire detection process. The computational model of this funnel design yields a truly parabolic tension distribution that is identical regardless of position varying 50 μπι in either direction from the nominal detection point. This suggests that a DNA molecule will experience constant tension during the entire detection process.

As an initial observation, the E. coli Notl digest was run on the CV30 and CS30 devices (FIG. 13). The detection point was set at the 50 μπι position on the CV30 device and the 150 μπι position on CS30. Again, at first inspection of the comet plots, both devices provide excellent stretching of DNA, and all predicted fragment lengths were detected without obvious overstretching. Both data sets were processed through backbone filters, and the inventors recognize that the percentage of well stretched fragments was greater through all observed molecule clusters in the constant strain rate funnel as compared to the constant velocity funnel.

To further characterize the improvement in DNA stretching in constant strain rate detection channels, the inventors repeated the stretching comparisons in CV30 and CS30 devices while varying the detector spot position up to 100 μπι in each device. The CV30 funnel was run at 0, 50, and 100 μπι from the onset of the constant velocity region. The CS30 funnel was run with the detection point at 100, 150, and 200 μπι from the transition from increasing strain rate to constant strain rate tapers. Both devices were run at fixed vacuum to achieve 30 μιη/ms at 0 μπι and 150 μπι spot positions respectively. All data sets were again process through the software to tabulate the different mechanisms by which individual molecules would fail backbone fluorescence intensity filtration.

A significant improvement in the percentage of well- stretched molecules was detected in CS30 (constant strain funnel) compared to CV30 (constant velocity funnel). In both devices, the percentage of good molecules increased with molecule length, due to increased peak tension on longer molecules. The percentage of good molecules was uniform regardless of spot position for each isolated collection of molecule lengths. This is in stark contrast to what is observed in the CV funnel. There, we observed that more molecules pass backbone intensity filters when the spot position is at 0 μπι, and decreases with the distance from the origin. The inventors also observed that the intra-experimental reproducibility in the percentage of well stretched molecules was substantially improved in the CS funnel compared to the CV funnel. The frequency of overlapped molecules again was uniformly low in all samples, indicating that the sample concentration was well-controlled for all experiments.

The percentage of molecules exhibiting hair pinned or relaxed morphologies

dramatically demonstrated the difference in stretching in CS (constant strain) and CV (constant velocity) funnels. In the CS30 funnel, molecules tended to have fewer observed hairpins with increasing molecule length. The percentage of observed hair pins was independent of spot position. In the CV funnel, however, there was a significant dependence of the number of observed hair pins on the detection point in the funnel. For example, for an 80 μπι fragment, only -30 % had hairpinned conformations when the detection point was at the origin of the constant velocity channel, but this increased to nearly 60% when the detection point was shifted 100 μπι down the channel. This indicates that as molecules are allowed longer time in the constant velocity, no-tension portion of the channel, there is increased opportunity for shear- induced tumbling of the leading end of the molecule. This effect was eliminated in the constant strain rate funnel shown in FIG. 8B. The remaining hairpinned molecules likely represent the percentage of molecules that are not provided sufficient cumulative strain to resolve complex initial conformations - these molecules are likely never fully extended in the first place.

The CV30 funnel (constant strain funnel) also produced a uniformly high incidence of molecules exhibiting high fluorescence on the trailing edge. The number of observed relaxed molecules was highly variable form run to run, but appeared more pronounced with the spot position 100 μπι from the onset of the constant velocity zone. Relaxed molecules was nearly eliminated in the CS funnel, as would be expected if the molecules were held under constant tension.

In total, use of the CS funnel (shown in FIG. 8B) as opposed to the prior art CV funnel at a given fluid velocity results in nearly two-fold improvement in the recovery of well- stretched, analyzable molecules by eliminating shear-induced molecular tumbling and relaxation.

Acceleration Correction in Constant Strain Rate Detection Channels. One ramification of detecting stretched DNA molecules in constant-tension conditions is that the molecules are continuously accelerating during the time of observation. In genomic analysis, DNA molecules are stretched to reveal the locations of sequence- specific fluorescent probes. For accelerating molecules, the spatial resolution between closely located probes will be greater at the leading end than the trailing end, due to the decreasing residence time for each segment of DNA at the detection spot. This imposes an acceleration- dependent bias on the optical signal generated from each molecule.

The detection channel may include multiple detection points. In some embodiments, the detection channel includes two backbone spots that are spaced from one another along the detection channel. The backbone spots detect fluorescence from the intercalator fluorophore. The detection channel also includes two tag spots that are spaced from one another along the detection channel. The tag spots detect fluorescence from the fluorescently labeled tags. The spatial distance between the two backbone spots is fixed and known, as is the spatial distance between the two tag spots. The position of the first backbone spot determines the position of the remaining spots within the detection channel. Each spot could be a laser spot or any other suitable detection arrangement. It should be appreciated that any number of backbone spots and/or tag spots may be used, as this aspect is not so limited. The constant- strain detection channel geometry retains molecules under constant tension with a well-defined acceleration. The form of this acceleration can be derived analytically from the funnel geometry. Obtaining this acceleration may be critical to two related stages of data processing. In the first stage (position acceleration correction), the time and position

dependence of the acceleration can be used to determine the entrance and exit time of the molecule in the tag spot, based on the entrance and exit time of the molecule in the backbone spots. In the subsequent stage (trace acceleration correction), the time dependence of the acceleration can also be used to convert the fluorescence pattern from the time domain in which it is acquired to the positional domain along the molecule. The "time domain" is also referred to herein as the intensity versus time trace or the observed trace. The "positional domain along the molecule" is also referred to herein as the distance domain or the intensity versus distance trace. The acceleration correction is used to properly convert the intensity versus time trace that was obtained during detection into an intensity versus distance trace by shifting each data point in the observed trace by an acceleration correction.

An approximation of the exact acceleration correction Ax _c is given by:

I ²

Ax _c ≡ τ{\ - τ) Equation 10

2x,

Where Ax _c is the difference in the distance a molecule would travel assuming a constant velocity compared to the distance traveled in the accelerating flow in the tapered channel. L is the length of the molecule. x _tag is a funnel-geometry derived parameter, and is the distance of the point of detection from the theoretical asymptotic origin of the constant strain portion of the channel. The length of the molecule L is measured from observations of the intercalator backbone signal. The velocity of the molecule is estimated from the time-of-flight of the intercalator signal from the first backbone spot to the second backbone spot. This estimated velocity of the molecule, along with a measured dwell time in the first backbone spot, is then used to calculate the length of the molecule L. This calculated length L approximates the actual length of the molecule, as it corrects any acceleration-dependent bias. x _tag values for the CS30 and CS50 devices are presented in FIG. 9.

τ is the time for the passage of the molecule through a detection spot. In some embodiments, the detection spot may be one of the tag spots, τ = 0 corresponds to the time at which the leading end of the molecule enters the detection spot, τ = 1 corresponds to the time at which the trailing end of the molecule leaves the detection spot. Values of τ between 0 and 1 correspond to the times at which the remaining portions of the molecule between the leading end and the trailing end enter the detection spot. For example, a value of τ = 0.1 may correspond to the time at which a portion of the molecule that is located behind the leading end of the molecule enters the detection spot, and a value of τ = 0.2 corresponds to the time at which another portion of the molecule that is located even further behind the leading end of the molecule enters the detection spot.

As such, each intensity data point plotted in the observed intensity versus time trace receives a unique τ value ranging from 0 to 1, inclusive. Because the acceleration correction is a function of τ, the value of the acceleration correction will differ among the data points in the observed trace. Thus, when the acceleration correction is applied to the observed trace, each data point in the observed trace will shift by an amount that depends on the acceleration correction value that is associated with that specific data point. For example, the intensity point associated with τ = 0 will not shift at all, since the value of the acceleration correction at τ = 0 is 0. Similarly, the intensity point associated with τ = 1 also will not shift, since the value of the acceleration correction at τ = 1 is also 0. Finally, the maximum value (amplitude) of the acceleration correction Ax _cmax occurs at τ =1/2, as shown in Equation 11 below.

Ar ~ J ² Equation 11

Thus, the intensity point associated with τ = 1/2 will shift by the greatest amount during the application of the acceleration correction.

As an example of the application of the acceleration correction to experimental data, a sample of E. coli Notl digest was prepared and run on the CS 30 device. The average tag fluorescence traces were plotted for groups of molecules with similar observed length (FIG. 14A, top traces). As a DNA molecule has equal probability of entering the stretching funnel in either orientation, the observed average tag fluorescence traces are expected to appear symmetrical about the center of the average molecule. To exemplify the acceleration induced asymmetry of the raw traces, the fluorescence trace was also inverted and superimposed on the original. This highlights significant asymmetry in each of the selected examples. The amplitude of that asymmetry increases with the length of the selected fragment. For each average trace, acceleration correction was applied using Equation 10. The resulting corrected forward and reverse oriented traces are plotted in FIG. 14A (bottom traces). This demonstrates that the matched traces are superimposable. FIG. 14B shows the acceleration correction amplitude along the length of each molecule. Acceleration correction depends only on the tag spot position x _tag , and the length of the molecule, L. The amplitude of the acceleration correction is, to a good approximation, proportional to the squared fragment length. This dependency is shown in FIG. 14C. The maximum error to this approximation in the tag spot positions with 120 μιη molecules is less than 0.05 μιη.

The effect of acceleration dependent signal bias can be readily observed in the average fluorescent signal generated from a cluster of similarly sized molecules. FIG. 15A shows the average tag signal observed in an 85 μιη cluster of molecules. Individual molecules generated from a single restriction digest fragment can enter the detection funnel in either a "head first" or "tail first" orientation, with equal probability. Any average trace signal is therefore expected to be symmetrical assuming sufficient detected molecules. When the average signal trace, however, is superimposed on its reverse, the inventors observe that discrete peaks are not directly matched, but appear to be somewhat phase shifted. The amount of this phase shift correction can be computed directly from observed data on a fragment by fragment basis in automated software. Once molecules are corrected for acceleration, the average traces become directly superimposable with its reverse (FIG. 15B).

The acceleration correction can be calculated for all molecules within the working fragment range for samples processed on either the CS30 (closed circles) or CV30 devices (open circles, FIG. 15C). The detection spot position was set at the 150 μιη position in the CS chip and 50 μιη in the CV chip, as described previously. All molecules in the CS device required correction for acceleration, but that correction scaled predictably with fragment length. More strikingly, all analyzed molecules also required acceleration correction in the CV30 device. The slope of the required acceleration correction was comparable to what was required in the CS funnel, but the onset of acceleration correction was shifted towards longer molecules. In the CV funnels, molecules longer than the distance from the onset of the parallel walled channel to the detection point will project into the accelerating strain portion of the funnel. Therefore shorter molecules would be expected to travel at constant velocity but longer molecules would still experience acceleration, thus requiring correction. These observations were repeated using three paired comparisons of CS and CV funnels, and yielded reproducible fits for the acceleration correction in the slope (FIG. 15D) and intercept (FIG. 15E). Assuming that acceleration correction is required regardless of funnel type, there are significant advantages to applying such corrections in the constant strain rate geometry over the constant velocity geometry. First, the requirement of acceleration correction is uniform for all analyzable molecule lengths. In the constant velocity funnel, the need for acceleration correction is dependent on the precise location of the detection spot in the detection channel. This causes different acceleration correction regimes depending on individual molecule length. Second, because molecules in constant velocity channels experience relaxation of the trailing end, the acceleration correction must accommodate for this distortion as the tail accelerates towards the head of the molecule. Finally, because molecules are observed under constant tension conditions and competing effects of shear-induced tumbling and relaxation are minimized in the CS funnels, the required acceleration correction can be predicted directly for all lengths of DNA from the funnel taper profile.

The inventors have uncovered several developments in the understanding of the behavior of DNA extension in mixed flow microfluidic funnels. There are several key observations. First, DNA extension is dictated by the tension applied to each molecule. This allows for the taper of a stretching funnel to be tailored to any desired fluid velocity and range of molecule lengths. Second, the etched depth of the funnel has competing effects on the efficiency of DNA stretching. The benefits of pre-extension of DNA and reduced rate of molecular relaxation in shallow channels, however may be outweighed by the benefits of reduced fluidic resistance and shear-induced molecular tumbling experienced in deeper channels. Furthermore, application of constant tension detection channels, as the third significant improvement, eliminates any disadvantage incurred by complex shear flow and permits observation of single molecules under constant tension conditions.

Detection under constant tension conditions clearly provides a better physical basis for understanding the mechanics of DNA stretching in extensional flow. Similarly, stretching DNA in constant tension provides a uniform, geometry based framework for the prediction of molecular acceleration. Acceleration correction in constant velocity funnels is much more complicated, as it depends on the length of a DNA molecule, the distance of the detection point from the onset of the constant velocity channel, and also must accommodate any acceleration of the trailing end of the molecule towards the leading end and the molecule begins to relax.

All of the modifications to funnel design serve to significantly improve the throughput of analyzable, well- stretched molecules. According to one embodiment, the ultimate evolution of the funnel design in 2 μηι etch depth is the CS50 device. This permits uniform DNA stretching at 50 μιη/ms at -10 psi vacuum. The length range of this device at 50 μιη/ms is comparable to that of the CV7.5 at 7.5 μΓη/ms, representing a 6.66 fold improvement in throughput due to velocity. Changing from a 1 μιη to 2 μιη etch depth served to double the bulk flow through the DNA injector onto the device, contributing an additional 2 fold improvement. Transitioning from the constant velocity detection channel to the constant strain rate channel also contributes about 2 fold improvement in retention of stretched molecules due to reduced relaxation and hairpinning. In all, through better understanding of DNA stretching in fluidic two-dimensional funnels, the work presented here demonstrates nearly 25 fold improvement in molecule throughput in a simple fluidic device. The throughput improvements demonstrated here are also compatible with microfluidic devices designed to improve molecule throughput by

electrokinetic stacking of DNA onto semi-permeable polymer gels and elimination of molecule overlap by fractionation of short, information poor molecules. Improvements in throughput in genomic technology enhances its applicability of detection of rare pathogens in complex bacterial mixtures and in detection from low starting masses of bacterial isolates.

On-Chip Intercalation of Nucleic Acids

According to yet some other aspects, the invention provides methods and systems, including microfluidic devices, useful for uniformly labeling nucleic acids with an agent such as but not limited to a backbone stain even if the amount and concentration of nucleic acid being stained is unknown and/or is varied from sample to sample. As a result, the invention facilitates the labeling of nucleic acids since it avoids the need to quantitate nucleic acids prior to labeling.

These and other aspects of the invention are described herein in the context of Direct Linear Analysis (DLA) or Genome Sequence Scanning (GSS™), for exemplary purposes. However it is to be understood that the methods and systems of the invention are not so limited and may be used in a variety of nucleic acid applications.

DLA or GSS™ involves analyzing a polymer such as a nucleic acid in a linear manner (i.e., starting from one end and moving along the length of the nucleic acid). The analysis detects signals along the length of the nucleic acid and determines their position along the length of the nucleic acid and their intensity. The signals are typically those from sequence nonspecific backbone stains and sequence- specific agents such as oligonucleotides. Signals from sequence non-specific backbone stains are used to detect a nucleic acid and to visualize its length. Signals from sequence- specific agents are used to derive sequence information about the nucleic acid which in turn can be used to determine the identity of the nucleic acid and/or its relatedness to other nucleic acids and/or its source including its microbial source.

Importantly, DLA or GSS™ analyzes individual nucleic acids, yielding a profile for each analyzed nucleic acid. This is in contrast to methods that analyze a plurality of identical (or near-identical) nucleic acids. Accordingly, DLA or GSS™ can be performed on directly harvested nucleic acid samples without an intervening in vitro amplification (e.g., PCR step).

Uniform labeling of a nucleic acid across its backbone via a backbone specific (rather than a sequence specific) stain is important for DLA or GSS™ due to its use of the backbone staining to visualize the nucleic acid and to approximate its location (including the location of its "head" and "tail" ends) and its length. Thus, the invention, in certain aspects, provides methods and systems for uniform labeling of nucleic acids by sequence non-specific stains.

The invention therefore, in part, contemplates the labeling of individual nucleic acids as they move through a region populated by an intercalator. The nucleic acids are therefore labeled in this manner while they are in flow. This is in contrast to methods in which relatively immobile nucleic acids are labeled with an intercalator in a reaction vessel (such as a test tube or an Eppendorf tube) and then, after labeling, are placed in flow for analysis. The nucleic acids may or may not be pre-labeled with a sequence-specific agent (such as oligonucleotides). In some important embodiments, the nucleic acids are labeled with a sequence- specific agent while relatively immobile in a reaction vessel, and then are placed to flow through a solution that comprises an intercalator. The intercalator is provided in solution, such as a buffered solution. The solution, as its name implies, is a liquid rather than a solid or a semi-solid (such as a gel).

In some important instances, DLA or GSS™ is performed using mono-cyanine dyes as intercalator s. Mono-cyanine dyes are characterized by lower affinity to DNA than their bis counterparts, discussed below, but otherwise share similar chemistries and spectral properties. Use of mono-cyanines, in some important instances, reduces the likelihood of intercalator- induced condensation, precipitation and aggregation, thereby eliminating or reducing the possibility of a single dye binding two molecules of DNA, and thus crosslinking them.

Examples of mono-cyanines include but are not limited to the PO-PRO intercalators such as but not limited to PO-PRO- 1 and PO-PRO-3. In some instances, the intercalator is positively charged at the pH at which the nucleic acid is analyzed. This pH is typically in the range of about 6-8. In some embodiments, the buffer is TE (tris-EDTA) buffer pH 8. The region through which the nucleic acids flow may be in a microfluidic device or in a capillary coupled to a microfluidic device. The region is one which the nucleic acid flows through as it progresses towards an elongation region (where the nucleic acid is stretched) and ultimately an interrogation region (where the nucleic acid is exposed to a laser and where signals from the nucleic acid are detected), both of which are typically integral to the microfluidic device. Thus in some instances the region may be located partially or wholly within the elongation region. The fluid in the region moves through the region once a force such as a hydrodynamic or electrokinetic force is applied.

In some aspects, the nucleic acid and intercalator travel in essentially the same direction but in separate streams (or flows). The streams are arranged so that the nucleic acid stream is sheathed or surrounded by the intercalator stream, in some aspects. Labeling of the nucleic acid is thought to occur through the diffusion of the intercalator from its stream(s) to that of nucleic acid. Importantly, the streams are laminar in nature. Labeling in this manner has been found, in accordance with the invention, to ensure that the nucleic acids are uniformly and adequately labeled (and not overlabeled) regardless of the amount of nucleic acids in the sample. This is partly because the nucleic acids travel through regions comprising the intercalator individually. As a result it is possible to control the amount of nucleic acid that flows through the intercalator regions and ultimately to control the ratio of nucleic acid to intercalator without the need to determine the amount or concentration of nucleic acid in the sample. This means that the intercalator can be used at a concentration that is sufficient for adequate labeling of the nucleic acid but which is not introducing increased background signal or otherwise impeding the analysis.

Accordingly, the invention addresses issues that can arise when too little or too much intercalator is used in the labeling of a nucleic acid. When too little intercalator is used, the nucleic acid being analyzed may be under-labeled, potentially resulting in the nucleic acid not being detected in whole or in part. When too much intercalator is used, it can attach to the interior surfaces of the microfluidic device, thereby causing significant background signal. In addition, fused silica of which the microfluidic device may be comprised is also negatively charged, and positively charged intercalator at high concentrations can bind to it, causing enhanced fluorescence in the fluidics, as well as sticking of the nucleic acid to the intercalator- coated fused silica surface. DNA condensation can be reduced through the use of mono-cyanine dyes such as PO-PRO. Thus, it was found in accordance with the invention that, in some instances, that cross- linking of DNA to other moieties such as other DNA or to surfaces such as glass surfaces within a microfluidic chip can be eliminated or reduced if the intercalation reaction was performed with a "mono-intercalator" such as but not limited to PO-PRO intercalators (e.g., PO-PRO-1 or PO- PRO-3). As used herein, a mono-intercalator intends a moiety that binds (typically non- covalently) with a DNA (or other nucleic acid) at a single site rather than at two sites, such as two adjacent sites. This is to be contrasted with a bi-intercalator (or bis-intercalator), such as a bis-cyanine intercalator, which binds to two separate but adjacent sites on a DNA. FIG. 16 illustrates such binding by a mono-intercalator and a bis-intercalator. FIG. 17 illustrates that on- chip intercalation using PO-PRO-1 eliminates DNA aggregation and surface sticking.

Mono-cyanine intercalators have reportedly lower affinity for DNA compared to their bis-counterparts. However, this reduced affinity did not impact the use of such mono-cyanine intercalators in the methods of the invention which involve sheathing of DNA with flow streams comprising excess intercalator. Thus, some embodiments of the invention utilize mono-cyanine intercalators in view of (a) their lower propensity to aggregate DNA and (b) the sheathing buffer scheme of the microfluidic devices contemplated by the invention.

Certain methods and devices of the invention are premised in part on the ability to perform on-chip intercalation without knowledge of and thus without dependence on the concentration of the intercalator used and the DNA being labeled. This is a significant advancement over the prior art methods that typically required that intercalator and DNA concentrations be known. In contrast, the methods of the invention can achieve intercalation even in the absence of such information. This means that samples may be processed without first measuring DNA content. This may be particularly useful for rare samples or samples with limited amount of DNA.

The ability to intercalate DNA from solutions of ranging DNA concentration is shown in the Examples. Briefly, solutions of varying DNA concentration were introduced into a microfluidic device having an elongation funnel, as described herein, and exposed to a fixed concentration of a mono intercalator in the sheathing buffer streams. In the Examples, 250 μΜ PO-PRO-1 was used. A uniform stretching coefficient for the 250.5 kB fragment was observed through a 32-fold dilution of stock DNA solution. This evidences that on-chip intercalation can be performed using a fixed intercalator concentration and a varying concentration of DNA, the latter of which may be known or unknown, without any appreciable effect on stretching. Accordingly, the data so generated can be used in linear DNA analysis methods, such as those described in published U.S. patent application US-2012-0283955-A1.

The Examples further show experiments performed to determine optimal conditions for intercalation by varying the concentration of intercalator in the sheathing buffer. Briefly, DNA was run through a microfluidic device described herein with intercalator at concentrations ranging from 50 - 500 nM in the sheathing buffer streams. For each condition, fragment lengths (in μιη) were determined for all restriction fragments by identifying each clustered fragment by its signature trace of site-specific probes. (Protozanova et al., Analytical Biochemistry, 2010. 402: p. 83-90.) When the measured fragment length (in μιη) was plotted against the known fragment length in kb, characteristic quadratic stretching curves were observed (FIG. 19A). The extensibility of DNA increased with increasing PO-PRO- 1 concentration.

The quadratic relationship between DNA extension and polymer length suggested that DNA extension was coupled to the tension on each molecule. The mechanism of DNA extension in GSS™ stretching funnels has been well characterized (Griffis et al., Lab Chip, 2013. 13(2): p. 240-51 ; Larson et al., Lab Chip, 2006. 6(9): p. 1187-99). The molecule velocity within the stretching funnel is the average of the fluid velocity along the length of the molecule. In the constant strain-rate funnel, the average fluid velocity occurs at the center of mass of the extended polymer, and fluid velocity increases linearly along the length of the molecule. The difference in velocity between the molecule and the fluid surrounding it at each segment along the length of the molecule results in a drag profile where the difference in velocities is greatest at the ends of the molecule and is zero at the center of the molecule. This results in a parabolic tension profile along the molecule, where the cumulative strain is maximal at the center of the molecule, and approaches zero at the ends. This is in contrast to the uniform tension profile generated in traditional strain loading techniques such as optical tweezers or AFM.

As discussed above, the peak tension Τ^ _χ along a molecule of length L ^ is

approximately:

1 ₂ .

^max ^{= ~} ^ L _mol £(x) where ^ is the strain rate in the stretching funnel, and ^ is the molecular drag coefficient (0.61 cP). The average tension is approximately 2/3 that value. The stretching coefficient for each fragment cluster, defined as the ratio of the measured length in μπι to the known polymer length in kb, was plotted for experiments spanning a range of intercalator concentrations from 50 nM to 500 nM (FIG. 19B). These plots indicate that the DNA stretching coefficient is linearly dependent on the tension applied to each molecule. The slope of these plots does not vary with intercalator concentration, but the y axis intercept does increase with increasing intercalator concentration. These findings were based on stretching experiments performed with a mono intercalator (PO-PRO-1). DNA intercalated with the bis intercalator POPO-1, in contrast, had a significantly lower slope and that slope varied with intercalator concentration (FIG. 19C).

Accordingly, uniform DNA stretching can be achieved between experiments using on- chip intercalation, by fixing the concentration of intercalator added to the sheathing ports of the microfluidic device. From the data presented here, intercalation and DNA extension are determined by the intercalator concentration, but are unaffected by the concentration of DNA in a given sample. This differs from the DNA concentration dependency on stretching behavior observed using standard in-tube intercalation with POPO-1.

The Examples provide experiments that validate on-chip intercalation, particularly where a mono intercalator (e.g., PO-PRO-1) is introduced to a microfluidic chip sheathing buffer.

In the methods provided herein, individual DNAs are intercalated (one at a time) as each enters the DNA stretching funnel. These methods are preferably carried out, in some instances, using a mono intercalator such as PO-PRO-1, in order to eliminates or reduce DNA sticking to the glass chip. The methods therefore provide that DNA extension can occur independently of the DNA concentration using on-chip intercalation. The methods further provide that DNA extension is dependent on intercalator concentration, which can be fixed independently of knowing the DNA concentration.

Certain aspects and embodiments of the invention are exemplified in the context of intercalation of double stranded nucleic acids such as double stranded DNA. However, it is to be understood that the methods and devices described herein may be used for rapid,

concentration-independent reaction of polymers with other ligands.

Various of the methods and systems of the invention will now be described in greater detail. These methods and systems allow for sufficient concentrations of labeling agents such as intercalators to be used in order to adequately label large masses of nucleic acids, thereby limiting or avoiding adverse effects that can occur when higher concentrations of labels are used (including for example over-intercalation) in the presence of lower masses of DNA. These methods and systems therefore facilitate analysis on a broad range of sufficiently intercalated DNA masses, among other things. Several implementations of microfluidic devices and their methods of use, in accordance with the invention, are described below.

In one aspect, the invention provides a capillary coupled to a microfluidic device that supports hydrodynamic or electrophoretic loading of nucleic acids in the presence of intercalator. In this aspect, the nucleic acid is exposed to the intercalator during the loading process (e.g., through the capillary that is used to load the nucleic acid onto the microfluidic device.

This aspect provides a system comprising a microfluidic device coupled to one end of a capillary comprising an intercalator. An example of such a system is shown in FIG. 20. As shown, one end of the capillary is coupled to the microfluidic device and the other end can be placed in a vessel that comprises the sample to be analyzed. The coupling of the capillary to the microfluidic device may be permanent or temporary. The capillary may be made of plastic (including PEEK, Ultem, or cyclic oleofins) or glass, although it is not so limited. The capillary length is not limiting. Capillary limiting diameter should be small to minimize diffusion distance. In some embodiments, the capillary may be about 15 cm long and have an internal diameter of about 150-500 microns.

"A capillary comprising an intercalator" means the capillary houses a fluid that comprises an intercalator. The capillary comprises the intercalator prior to entry of the nucleic acid into capillary (i.e., the intercalator is present in the fluid held by the capillary). The capillary may be loaded with the fluid comprising the intercalator using hydrodynamic force (or pressure), for example by applying a vacuum downstream in the fluid path connected to the capillary. Thus, in some embodiments, the system comprises a vacuum downstream of the capillary. The vacuum may be coupled to a waste reservoir or to another inlet or outlet in the system including the microfluidic device that comprises a microfluidic chip. As used herein, the terms inlet and inlet port are used interchangeably, and the terms outlet and outlet port are used interchangeably. The capillary may be loaded with the fluid comprising the intercalator using electrokinetic force, for example by placing a cathode downstream of the capillary and placing an anode upstream of the capillary as shown in FIG. 21. The cathode may be in a waste reservoir and the anode may be in the vessel housing the sample prior to analysis also as shown in FIG. 21.

In various of the systems provided herein that incorporate electrokinetic force, the capillary and/or the microfluidic device comprises an electroosmotic flow (EOF) suppressor. EOF is the motion of liquid induced by an applied potential across a capillary tube, a microchannel, or any other fluid conduit. EOF is most significant in small channels, and can occur in buffered as well unbuffered (e.g., water) solutions. It is preferable to reduce or eliminate EOF during DLA and GSS™ and other microfluidic applications. An EOF suppressor is an agent that suppresses (partially or, more preferably, completely) EOF.

Examples include but are not limited to water soluble methylhydroxyethyl derivative of cellulose, polyvinylalcohol, polyvinylpyrrolidone, polyethyleneglycol or Triton X-100. Other EOF suppressors are known in the art and the invention intends to embrace their use as well.

In some embodiments, the capillary and/or the microfluidic device is coated with an EOF suppressor on one or more of its interior surfaces (i.e., those surfaces that contact the solutions comprising the intercalator and/or nucleic acid. In some embodiments, the EOF suppressor is simply present in the solution comprising the intercalator and/or the nucleic acid. Thus, the solution housed by the capillary and/or the microfluidic device may comprise intercalator and buffer, and optionally also an EOF suppressor.

The intercalator is used at a concentration suitable for labeling the nucleic acid at a frequency of about one intercalator (molecule) per about three base pairs. In the electrokinetic loading scheme, the concentration of intercalator used could be in excess of the local concentration of DNA base pairs in order to saturate their appropriate binding sites. The opposite motilities of free intercalator and DNA in the presence of an electric field would serve to continuously separate the DNA from free intercalator.

The buffer may be any buffer having buffering capacity in the neutral pH range. An non-limiting example is tris buffer. Typically, the solution will also include divalent ion chelators such as EDTA in order to reduce the activity of nucleases such as DNase. Buffered solutions may include TE or Tris-Borate-EDTA (TBE). One of ordinary skill will be able to select an appropriate buffered solution for the particular application.

The invention therefore also provides, in another aspect, a method for uniformly labeling a nucleic acid with an intercalator comprising (1) providing a capillary coupled to a microfluidic device, wherein the capillary comprises an intercalator, (2) placing a first end of the capillary in a vessel comprising a nucleic acid, and (3) applying hydrodynamic or electrokinetic force sufficient to move the nucleic acid from the vessel through the capillary and into the

microfluidic device. The flow of the nucleic acid through the capillary is laminar as is the flow of the intercalator. In this as well as other aspects of the invention, the nucleic acid may be a double stranded nucleic acid such as but not limited to double stranded DNA. It will be understood that, when the methods and systems of the invention are used to label a nucleic acid with for example a sequence specific agent such as an oligonucleotide, the nucleic acid may be a single stranded nucleic acid such as single stranded DNA or RNA.

Depending on the embodiment, the capillary and chip are typically pre-filled with intercalator using hydrodynamic force. For example, a vacuum could be applied to ports 53 and 61 for capillary or chip loading respectively. The hydrodynamic or electrokinetic forces are also used to move the nucleic acid.

In the case of the hydrodynamic force, a vacuum (or negative pressure) may be applied downstream of the capillary (i.e., in a direction away from the sample vessel). In this embodiment, the nucleic acid is drawn through the intercalator, taking advantage of the parabolic flow profile in the narrow channel to sheath the nucleic acid with a thin layer of intercalator near the walls of the capillary, permitting rapid diffusion and staining of the nucleic acid.

In the case of the electrokinetic force, a cathode is present in a waste reservoir and an electrode is present in the vessel. In this embodiment, the nucleic acid and the intercalator will have opposite motilities in the presence of an electric field and therefore intercalator will be driven through the approaching front of the nucleic acid, staining the nucleic acid as it loads onto the chip.

Typically, the nucleic acid and intercalator move through the capillary and/or the microfluidic device in separate laminar streams, with the intercalator comprising solution sheathing the nucleic acid solution.

Two embodiments are illustrated in FIGs. 20 and 21. FIG. 20 illustrates a hydrodynamic loading approach while FIG. 21 illustrates an electrokinetic loading approach. In FIG. 20, the sample loop and the capillary are prefilled with intercalator in buffer. The sample loop is a relatively deep channel on the microfluidic chip (e.g., it may have a depth of about 20 microns). The available end of the capillary is then placed into the DNA sample of interest for

hydrodynamic loading. The parabolic Poiseuille flow profile draws DNA through the intercalator present in the sample loop (including the capillary), sheathing it with a layer of fluorophore, and establishing conditions for transverse lateral diffusion. (Krylova et al., 2009) This approach has several aspects of interest. First, replenishment of intercalator between runs is simplified, as the intercalator is loaded through the same path as the sample (i.e., intercalator loads through the same end as does the nucleic acid, thereby preventing

contamination of the next sample by flushing the preceding sample back onto the chip with the next bolus of intercalator). It is also expected that the short time between intercalation and interrogation/detection limits the amount of nucleic acid condensation that can occur, in contrast for example to intercalation of nucleic acids in a reaction vessel prior to loading into the device itself. In view of this distinction, the intercalation of the invention may be thought of as being "real-time" intercalation since intercalation occurs during the process of introducing the nucleic acid into the microfluidic device, into the elongation region of the device and/or through this region, as will be described in more detail below. In addition, the small cross- sectional volumes of the capillary (e.g., about 150 micron internal diameter) and the 20 μπι cross channel sample loop may locally isolate individual nucleic acids, constraining them and thereby limiting condensation. In addition, low level adhesion of intercalator at the interior surfaces of the capillary or microfluidic chip surface may serve as a reservoir for intercalator replenishment with each loading cycle.

FIG. 21 illustrates the same microfluidic chip and capillary setup as in FIG. 20 except that the hydrodynamic loading is replaced with electrokinetic loading and intercalation.

(Krylovaet al. 2009) In the approach illustrated in FIG. 21, TE buffer containing intercalator is loaded hydrodynamically into the sample loop. The nucleic acid sample is then introduced to the capillary, with an anode in the sample vessel and a cathode at the cross flush waste reservoir. When current is applied, electrophoresis occurs. Negatively charged nucleic acid travels onto the chip, toward the waste, while positively charged intercalator migrates toward the sample vessel. The resulting cross flow assures a continuous stream of intercalator over the nucleic acid, as well as potential displacement of poorly bound intercalator. Because of the intrinsically charged surfaces in the microfluidic devices (e.g., those made from fused silica, among others), electroosmotic flow (EOF) may compromise electrokinetic nucleic acid loading. To address this possibility, the device may be pre-coated with an EOF suppressor or an EOF suppressor may be included in the intercalator containing solution.

In another implementation of in-capillary intercalation, intercalator is included in a leading buffer, and the sample in a sample buffer is loaded onto the chip by isotachophoresis (ITP). (Jung, Bharadwaj et al. 2006; Shackman and Ross 2007) The loading condition for this approach is established similarly to the electrokinetic scheme described above. Differential anionic mobilities between the leading and sample buffers focus nucleic acids at the moving interface between the buffer types, resulting in the concentration of nucleic acids at the interface between the buffers. Additionally, the inclusion of intercalator as a cationic component of the leading buffer allows for simultaneous intercalation. It has been found that if the intercalator is positively charged then although the nucleic acids concentrate at the interface between the buffers, the intercalator travels across the region of concentrated nucleic acids. In these embodiments, nucleic acid intercalation can be combined with concentration of the nucleic acids.

The invention also contemplates performing the intercalation reaction within the confines of shallow-etched microfluidic channels. This is referred to herein as "on-chip intercalation". In some embodiments, the intercalation reaction may be confined to a 2 micron deep channel. It could however be performed in deeper channels (e.g., on the order of 100 micron microchannels). This approach can provide, in some instances, more precise control over kinetics of the intercalation reaction. Due to laminar flow, short diffusion distances, and small mass diffusion coefficients, the extent of completion of the intercalation reaction can be closely monitored and adjusted. Embodiments of this approach are illustrated in FIGs. 22, 24 and 26. These approaches have been shown to provide rapid, uniform intercalation on a microfluidic device.

One embodiment provides a microfluidic device comprising a microfluidic chip that itself comprises a sample inlet port, a sheath inlet port, an elongation region, and a waste reservoir downstream of the elongation region, wherein the sheath inlet port and the elongation region comprise an intercalator prior to introduction of a nucleic acid sample. As illustrated in FIG. 22, the chip may comprise two sheath inlet ports and preferably they are positioned on opposite sides of the sample inlet port. As used herein, when two sheath inlet ports are positioned on opposite sides of a sample inlet port, this means that one of the two sheath inlet ports is on one side of the sample inlet port and the other sheath inlet port is on the other side of the sample inlet port. This is illustrated in FIG. 22. The positioning of the sheath inlet ports preferably is equidistant from the sample inlet port, as the sheath fluid from such ports is used to position the nucleic acids centrally within the elongation and interrogation and detection regions of the chip. The sample inlet port, as its name implies, is the port through which the sample, including the nucleic acid, enters the chip. Similarly, the sheath inlet port is the port through which the sheath fluid enters the chip. The elongation region is the region on the chip through which the nucleic acid travels and in the process becomes elongated (or stretched). Elongation regions are known in the art and reference may be made to published application US-2004- 0166025-A1 and Griffis et al., Lab Chip, 2013. 13(2): p. 240-51 for examples of elongation regions. In some embodiments, the elongation region is a funnel that narrows in the direction of the interrogation/detection region.

In the interrogation/detection region, the nucleic acid is exposed to light of one or more wavelengths, typically as provided by a laser. In some embodiments, the interrogation region comprises three lasers, two of which are tuned to excite two different fluorophores on different sequence-specific agents bound to the nucleic acid, and one of which is tuned to excite the intercalator. As an example, the PO-PRO-1 intercalator is excited using a 488 nm argon laser, and the remaining two lasers excite fluorophores in the green and red spectrums. Signals from the nucleic acid are detected by one or more detectors in the interrogation/detection region. The detectors include a detector for the intercalator signals and optionally a detector for signals from the sequence- specific agents bound to the nucleic acid (e.g., fluorescently labeled

oligonucleotides). In an important embodiment, the intercalator is a mono-cyanine dye such as PO-PRO-1.

These embodiments envision the placement and presence of intercalator containing solution in the microfluidic chip prior to the introduction of the nucleic acids. The intercalator solution may be continually applied to the microfluidic chip through the sheath inlet port(s). As noted in FIG. 22, a vacuum (or negative pressure) may be applied downstream of the elongation and interrogation/detection regions. The hydrodynamic force moves the nucleic acid, intercalator and solution through the chip towards a waste reservoir downstream of the detection region. In some instances, the nucleic acid contacts the intercalator at the elongation region. In some instances, the nucleic acid contacts the intercalator as it enters the interrogation region, after it has been elongated. Depending on the nature of the elongation region and the particular chip, it is also possible that the elongation region is integral to the interrogation region.

This chip configuration may be used in a method for uniformly labeling a nucleic acid with an intercalator that comprises (1) providing a microfluidic device comprising a microfluidic chip comprising a sample inlet, a sheath fluid inlet, an elongation region, and a waste reservoir downstream of the elongation region, (2) introducing intercalator into the microfluidic device through the sheath fluid inlet, (3) introducing a nucleic acid into the microfluidic chip through the sample inlet, and (4) applying hydrodynamic force sufficient to move the nucleic acid from the sample inlet and the intercalator from the sheath fluid inlet through the elongation region to the waste reservoir, wherein flow of the nucleic acid and intercalator is laminar. It will be understood that in some embodiments, the intercalator is flowed through and is present in the microfluidic device prior to introduction of the nucleic acid. As discussed herein, the microfluidic chip may comprise two sheath fluid inlets positioned on opposite sides of the sample inlet, and intercalator may be introduced into the microfluidic chip through both sheath fluid inlets.

This approach allows nucleic acids to be exposed to intercalator individually, meaning that the nucleic acids are not in physical contact with each other and even more preferably are sufficiently separated from each other so that each nucleic acid has relatively equivalent exposure to intercalator. The nucleic acids may be exposed to the intercalator while under tension (i.e., while they are entering and/or within the elongation region. The method may also comprise modulating the time the nucleic acid is exposed to the intercalator by modulating fluid velocity through the chip and/or the geometry of the elongation region. In some instances, the intercalator is present at a concentration ranging from about 50 nM to about 500 nM, about 100 nM to less than about 10 μΜ, or from about 50 nM to about 10 μΜ. The nucleic acid and intercalator may move through the elongation region in separate streams.

FIG. 22 illustrates an embodiment of sheath flow intercalation. In this embodiment, a high concentration of intercalator (relative to the concentration of DNA base pairs present in the given fluid volume) in tris-EDTA (TE) is introduced onto the chip through the sheath inlet ports. Intercalation at concentration ranging from 100 nM to less than 10 microns are suitable. In one embodiment, the intercalator PO-PRO-1 is used at a concentration near 300 nM. As nucleic acid is injected into the elongation region, it is sheathed with intercalator-containing buffered solution. Diffusion of the intercalator into the central stream (which comprises the sample and thus the nucleic acid) then initiates staining of the nucleic acid.

There are several advantages to this mode of intercalation. First, nucleic acids are exposed to the intercalator individually (i.e., one at a time), minimizing intermolecular aggregation. Second, the nucleic acid is maintained under tension during the reaction process, minimizing intramolecular aggregation via charge-negation. Third, the reaction time between nucleic acid and intercalator is a function of the fluid velocity and elongation region (e.g., funnel) geometry, and thus it can be tightly controlled. This mode of intercalation has been tested and its feasibility has been demonstrated as illustrated in FIG. 23. While some degree of incomplete staining of individual nucleic acids was observed, this was thought due to the short reaction times. This can be addressed and intercalation could be enhanced by decreasing the velocity of the sheath and sample moving through the chip and/or by increasing intercalator concentration with the proviso that at intercalator concentrations greater than 10 μΜ (e.g., for PO-PRO-1) rapid coating of the microfluidic device can occur, leading to increased background fluorescence. As discussed herein, an EOF suppressor may be used to reduce non-specific binding of the intercalator and the consequent background fluorescence.

Alternatively, another chip geometry may be used to facilitate intercalation. An example of one such geometry is illustrated in FIG. 24. That geometry is generally described as a microfluidic device comprising a microfluidic chip comprising a sample inlet, an intercalator inlet, two sheath fluid inlets an elongation region, and a waste reservoir downstream of the elongation region, wherein the intercalator inlet comprises two channels ("intercalator channels") extending therefrom and merging with, and positioned on opposite sides of, a channel extending from the sample inlet ("sample channel") and the first and second sheath fluid inlets are positioned on opposite sides of, and feed into, the elongation region.

Using this geometry, a method may be used for uniformly labeling a nucleic acid with an intercalator that comprises (1) providing a microfluidic device comprising a sample inlet, an intercalator inlet, two sheath fluid inlets, an elongation region, and a waste reservoir downstream of the elongation region, wherein the intercalator inlet comprises two intercalator channels extending therefrom and merging with the sample channel wherein the intercalator channels are positioned on opposite sides of the sample channel; and the sheath fluid inlets are positioned on opposite sides of, and feed into, the elongation region, (2) introducing a nucleic acid into the microfluidic device through the sample inlet, (3) introducing intercalator into the microfluidic device through the intercalator inlet, and (4) applying hydrodynamic force sufficient to move the nucleic acid from the sample inlet and the intercalator from the intercalator inlet through the elongation region to the waste reservoir, wherein flow of the nucleic acid and intercalator is laminar.

FIG. 24 provides an example of this on-chip intercalation approach. In this example, separate ports are loaded with nucleic acid, intercalator and sheath fluid (or running buffer, as the terms are used interchangeably). The nucleic acid (or sample) stream is nested between two adjacent intercalator streams within the microfluidics as shown in FIG. 25. Subsequently, the three flows pass through the elongation region (e.g., a stretching funnel). This scheme takes advantage of the fluidic extension of nucleic acid in the presence of intercalator (as does the approach of FIG. 22), it uses the 2 μπι etch depth in the chip channel to limit condensation, and it also incorporates an additional incubation channel to increase the time in which the nucleic acid is exposed to the intercalator. Additionally, downstream flow focusing can also be used to prolong the intercalation reaction until completion, in order to reduce intercalator concentrations to suitable levels.

In yet another embodiment, separate ports are again loaded with nucleic acid, intercalator and sheath fluid. This embodiment is illustrated in FIG. 26. The fluidics of this embodiment route the intercalator and nucleic acid streams into two separate perpendicular channels, vertically offset from each other. At their intersection, laminar flow preserves the parallel planar relationship between the two, and the total diffusion distance between the intercalator stream and the nucleic acid stream is reduced to half the channel height (i.e., the geometry consists of two crossing channels of height H and at the intersection of the two channels the total height is 2H). Due to small mass diffusion coefficients, this allows for near instantaneous onset of the intercalation reaction, and provides uniform access between the nucleic acid and the intercalator, regardless of the position of any given nucleic acid in the sample fluidic layer.

The microfluidic devices and configurations provided by the invention are intended to promote in-capillary or on-chip intercalation in order to enhance non-specific labeling of nucleic acids, regardless of the concentration of nucleic acid present in a given sample. All of these devices operate under laminar flow conditions. Device geometries have been optimized to maximize diffusion rates by performing the intercalation reactions in small volume microfluidic channels. As discussed herein, these devices and methods may also be used to promote and/or enhance binding between nucleic acids and other binding partners such as oligonucleotides, ligands, enzymes, and the like.

The invention may be practiced using a variety of intercalators.

Examples of mono intercalators include PO-PRO-1, BO-PRO- 1, YO-PRO-1, TO-PRO- 1, JO-PRO-1, PO-PRO-3, BO-PRO-3, YO-PRO-3, TO-PRO-3, TO-PRO-5.

The invention further contemplates use of other DNA-ligand interactions as well.

Examples of other intercalators that may be used in some instances include cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red).

Examples of other intercalators that may be used in the methods and systems described herein include phenanthri dines and acridines (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer- 1 and -2, ethidium monoazide, and ACMA); minor grove binders such as indoles and imidazoles (e.g., Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneous nucleic acid stains such as acridine orange (also capable of intercalating), 7-AAD, actinomycin D, LDS751, and hydroxystilbamidine.

In various embodiments of the invention, it may be preferable to use intercalators and/or other backbone stains that are not capable of crosslinking DNA and other nucleic acids, either covalently or non-covalently. The use of such intercalators or backbone stains reduces or eliminates DNA non-specific adhesion to glass surfaces such as those in a microfluidic chip or device.

The methods provided herein may involve the use of agents that bind to a nucleic acid in a sequence- specific manner. "Sequence- specific" when used in the context of a nucleic acid means that the agent recognizes a particular linear (or in some instances quasi-linear) arrangement of nucleotides. "Specific binding" means the agent binds with greater affinity to a particular nucleic acid or a region within a particular nucleic acid than it does to other nucleic acid or other nucleic acid regions. It should be possible to achieve conditions under which the agent binds predominantly (or only) to its cognate nucleic acid sequence. Such "stringent hybridization conditions" are known in the art. (See for example Maniatis' Handbook of Molecular Biology.)

Any agent that is capable of recognizing a nucleic acid with sequence specificity (whether through complementary nucleic acid hybridization or through structural recognition based on particular nucleotide sequence can be used as a sequence- specific agent. In some instances, the agent is nucleic acid in nature and binds to a target nucleic acid via Watson-Crick binding. In other instances, the nucleic acid agent can bind to a target nucleic acid via

Hoogsteen binding, thereby typically forming a triplex. In some instances, the nucleic acid probes can form both Watson-Crick and Hoogsteen bonds with the nucleic acid target. BisPNA probes, for instance, are capable of both Watson-Crick and Hoogsteen binding to a nucleic acid. The sequence specific agents may be nucleic acids, including oligonucleotides, nucleic acid aptamers, PNAs such as bisPNAs, pcPNAs, ssPNAs, LNAs, DNAs, RNAs, co-nucleic acids such as DNA-LNA co-nucleic acids, siRNA, shRNA, proteins or peptides including antibodies and antigen-binding antibody fragments, etc.

The sequence- specific agents may be inherently or intrinsically labeled with a detectable label. Typically, for the sake of convenience, the detectable label is selected so that it can be detected using the same detector type used to detect the intercalator signals. Thus, typically, the detectable labels are fluorophores. More specifically, the detectable label may be selected from the group consisting of directly detectable labels such as a fluorescent molecule (e.g., fluorescein, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, allophycocyanin (APC), fluorescein amine, eosin, dansyl, umbelliferone, 5- carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), 6 carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy- X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL), 5-(2'- aminoethyl) aminonaphthalene-1- sulfonic acid (EDANS), 4-acetamido-4'- isothiocyanatostilbene-2, 2'disulfonic acid, acridine, acridine isothiocyanate, r-amino-N-(3- vinylsulfonyl)phenylnaphthalimide-3,5, disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin, 7-amino-4-methylcoumarin, 7- amino-4-trifluoromethylcouluarin (Coumarin 151), cyanosine, 4', 6-diaminidino-2-phenylindole (DAPI), 5', 5"-diaminidino-2-phenylindole (DAPI), 5', 5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red), 7-diethylamino-3- (4'-isothiocyanatophenyl) -4-methylcoumarin diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydro-stilbene-2, 2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, 4-dimethylaminophenylazophenyl-4'- isothiocyanate (DABITC), eosin isothiocyanate, erythrosin B, erythrosin isothiocyanate, ethidium, 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), QFITC (XRITC),

fluorescamine, IR144, IR1446, Malachite Green isothiocyanate, 4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine, pararosaniline, Phenol Red, B-phycoerythrin, o-phthaldialdehyde, pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron . RTM. Brilliant Red 3B-A), lissamine rhodamine B sulfonyl chloride, rhodamine B, rhodamine 123, rhodamine X, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101, tetramethyl rhodamine, riboflavin, rosolic acid, and terbium chelate derivatives). Other detectable labels include an electron spin resonance molecule (such as for example nitroxyl radicals), a chemiluminescent molecule (e.g., chemiluminescent substrates), a radioisotope (e.g., P32 or H3, 14C, 1251 and 1311), an optical or electron density marker, an enzyme, an enzyme substrate, a biotin molecule, a streptavidin molecule, an electrical charge transferring molecule (i.e., an electrical charge transducing molecule), a chromogenic substrate, a semiconductor nanocrystal, a semiconductor nanoparticle (such as quantum dots described for example in U.S. Patent No. 6,207,392 and commercially available from Quantum Dot

Corporation and Evident Technologies), a colloid gold nanocrystal, a ligand, a microbead, a magnetic bead, a paramagnetic particle, a quantum dot, an affinity molecule, a protein, a peptide, nucleic acid, a carbohydrate, an antigen, a hapten, an antibody, an antibody fragment, and a lipid. They are not so limited however.

Sorting of Nucleic Acids

According to yet some other aspects, the invention provides, inter alia, methods and devices for rapid analysis of nucleic acid samples, including but not limited to genomic nucleic acid samples regardless of source. The methods and devices provided herein facilitate analysis, including sequencing of nucleic acid samples by directing analysis and sequencing resources towards nucleic acids and nucleic acid fragments of interest, rather than analyzing and sequencing nucleic acids and fragments that are not of interest. In some instances, those of interest may be in the minority.

With the advent of new generation sequencing (NGS) technologies, the spectrum of applications based on sequencing has progressively widened in view of the increased throughput and decreased cost. However, even with such high throughput (measured currently in tens of gigabases per run), NGS-based applications can still benefit from limiting analysis to only relevant DNA fragments. This approach is referred to as targeted resequencing. Applications that utilize or would benefit from targeted resequencing are described below.

Targeted resequencing simplifies functional analysis of genomes. Sequencing de novo of a complete genome is still far from routine, especially for the NGS technologies, most of which have short read lengths. Short reads are difficult to combine into a contiguous genome because of common or nearly common elements that are longer than a single read length (e.g., repeats or rrn operons). Analyzing genomic regions that are 5' and 3' of these common or near common elements requires reads that are longer than these elements or other techniques that may be slower, manual and expensive.

For the vast majority of applications, especially in human diagnostics,

pharmacogenomics, etc., de novo sequencing is not necessary. Instead, sequencing is typically used to identify genetic differences between individuals, using a process called targeted resequencing. In this approach, only portions of a genome that include the genes of interest are sequenced (Ng et al., 2009; Shendure & Aiden, 2012; Glaser 2013). Some techniques exist to identify and isolate the genomic fragments of interest. An example of such a technique is exome sequencing. This approach focuses on analyzing coding sequences or exon regions.

Targeted resequencing simplifies analysis of microbiomes. This application is explained in greater detail below.

Microbiome Analysis. Most of living mass in this world consists of bacteria. Bacterial populations including dozens to millions of species inhabit soil and air, food and water, surface of tables in restaurants and surface of walls in hospital wards, skins and intestines of animals, humans included. Every population is a dynamic system that responds with changes of its composition to every change of conditions. The bacteria constituting the populations may compete with each other or be symbiotically connected. These bacteria can be beneficial or pathogenic for the hosts they inhabit (Sachs et al. 2011).

Bacterial populations are fundamental to the existence of biomes that include every form of life. However, most of the studies so far have been centered on the human- oriented aspects. For example, among the properties of soil-dwelling bacteria, the ones attracting most interest are the ability of some to generate antibiotics and the mechanisms of the antibiotic resistance of the others (D'Costa et al. 2006). Another type of study is search for new functions, such as enzymes that efficiently deconstruct plant cellulosic biomass to employ them in the biofuel production (Hess et al. 2011).

Human-inhabiting microbiomes attract ever growing attention as they are directly relevant to human health and well-being. Bacterial populations inhabiting skin, nose, lungs, and guts of humans possibly influence various body processes such as formation of atherosclerotic plaque (Koren et al. 2011). Another example is formation of a complex microbiome in the airways of cystic fibrosis patients that influences the disease progress (Cox et al. 2010;

Zemanick et al. 2010). Intestinal microbial community is rich and plastic (Arumugam et al. 2011; Koren et al. 2011; Walter et al. 2011). It is easily modified by external influences such as diet or antibiotic treatment (Dethlefsen et al. 2008; Goodman et al. 2011). These modifications may have immediate and dangerous effects on human health, for example, by promoting growth of pathogens such as Clostridium difficile (Bartlett 2002; Cohen et al. 2010).

Studies of microbial populations were gaining steam in the last decade, facilitated by the development of the NGS technologies and related methods. The two main methodologies are based on sequencing housekeeping genes and on shotgun sequencing. The other co-existing techniques are more laborious, or more expensive, or less accurate. For example, the most direct approach based on culturing microbiome components includes analysis of tens of thousands of colonies that must be grown both under aerobic and anaerobic conditions and still does not reproduce exactly the composition of the gut microbiota (Goodman et al. 2011). Isolated bacteria grow differently in cultures, and many cannot be grown at all. Even using gnotobiotic mice to achieve the most adequate conditions cannot achieve accurate quantitative

representation (Goodman et al. 2011).

Microbiome analysis using housekeeping genes. This approach is based on genes that code the most vital functions of bacteria cells and therefore evolve at a much slower pace than less important elements. These genes include conservative sequences that may be used to design primers for DNA synthesis capable of hybridizing to the genes of many different bacteria.

Therefore, a limited set of primers can be used with a bacterial mixture. Once the sections of the housekeeping genes defined by primers are sequenced, the sequences are used to identify the bacteria species and their copy numbers to quantify the proportions of the species. The most popular housekeeping genes used in this analysis are the genes coding RNA of ribosomes (rRNA).

The housekeeping genes, coding the rRNA for the 5S, 16S, and 23S ribosome particles, are combined in rrn operon as well as the tRNA molecules required for protein synthesis (Klappenbach et al. 2001). Of these genes, the one coding 16S rRNA is used most often for the sequencing-based applications. This gene has most sequences determined for different bacteria; as of 4/10/11, the 16S rRNA database included 1,962,952 sequences.

The whole 16S rRNA sequence is 1.4 kb long. It includes 7 conservative sites that are used for DNA priming (Liu et al. 2007). These sites are used to generate amplicons of various lengths (FIG. 28). The regions V3 and V6 are most often used in microbiome studies. For example, for the bacteria inhabiting human intestine the average amplicon lengths of V3 and V6 regions are 145 and 59 bp, respectively (Dethlefsen et al. 2008). The longer the length of the region used for analysis, the higher the specificity of bacteria identification that can be achieved (Liu et al. 2007; Turnbaugh et al. 2009). The whole 16S rRNA gene can be sequenced in a single run only by a classic Sanger sequencer, such as ABI 3730x1 system. However, this method has the lowest throughput (192 full length 16S rRNA sequences per run) and, therefore, the highest cost of analysis. The highest throughput can be achieved with Illumina sequencing system (1.8xl0 ⁸ reads of 100 bp length per run for GAIIx system). However, because of short read length, this system can be used for only V6 regions. Only with the recent 150PE flow cell does the Illumina system become applicable for V3 region analysis also. 454 pyrosequencing system has lower throughput than Illumina system, but higher throughput than ABI sequencer (1.4xl0 ⁶ reads of 300-500 bp length for Genome Sequencer FLX system). To decrease costs, multiple samples can be pooled for analysis in a single run. Up to 286 samples can be pooled for a 454 sequencing system (Hamady et al. 2008). Even more samples can be combined for an Illumina system analysis.

Selection of the system for microbiome analysis is a compromise between specificity and sensitivity of detection of bacteria, because the sequencing systems with higher throughput have shorter read lengths. The longer the length of the genomic sequence used for analysis, the higher the specificity in bacteria identification that can be achieved (Turnbaugh et al. 2009). The larger the number of reads per sample, the lower the proportion of bacteria that can be detected.

A bacterium identified by a sequence-based method is referred to as an operational taxonomic unit (OTU), because the gene sequence-based recognition of uncultivated microbial populations is not equivalent to traditional taxonomic classification (Dethlefsen et al. 2008). Therefore, strictly speaking terms such as "species" or "strain" are not appropriate. OTUs can be defined in various ways and at different levels of resolution.

Full-length 16S rRNA sequences offer the highest possible degree of taxonomic resolution using this gene, but the cost of dideoxy Sanger sequencing is high (Dethlefsen et al. 2008). Less phylogenetic information is available and therefore less specific identification of bacteria is possible from a single pyrosequencing read typically covering 150-230 bp

(Dethlefsen et al. 2008). In a human gut microbiome sample, for example, on average 53% of the reads could be assigned to a genus and 80% to a phylum (Arumugam et al. 2011). This relatively low specificity is only partially due to the short reads. Another problem is insufficient information content of the sequence database because abundant, uncharacterized bacterial taxa, novel species even at the family level, are still found in the human gut (Dethlefsen et al. 2008). Environmental microbiomes are even more diverse and less studied (Walter et al. 2011). In this case, the advantage of highly conservative genes is a higher chance to exhibit similarities to sequences available in a database. If higher specificity in bacterial identification is required, other markers, such as cpn60, can be used for studies (Hill et al. 2010).

Both the clone library and current NGS technologies include PCR amplification as a standard step of the sample preparation process (Metzker 2010); therefore, every NGS technology may be prone to the PCR-related artifacts, such as biased amplification. This problem is reduced for the shorter sequences required for the NGS technologies (Dethlefsen et al. 2008). With careful selection of primers no erroneous data has been generated either for the V6 or V3 variable regions or even for full-length 16S rRNA sequences (Dethlefsen et al. 2008).

Microbiome analysis using shotgun sequencing. In this approach, total genomic DNA of the sample is isolated and fragmented, every short fragment is sequenced, and then the short reads are connected using overlaps (Hess et al. 2011; Qin et al. 2010). The assembled contigs can be used, for example, for analysis of a bacterial population by comparison with available databases of genes (Qin et al. 2010) or for mining the microbiomes for the genes coding enzymes with new functions (Hess et al. 2011). Typically, hundreds of Gigabases of sequences are obtained in these studies, several percents of which are possible to connect into contigs with a median length of 0.9-1.6 kb.

The power of this approach is that it does not require preliminary assumptions and generates information about the gene pool and enzymatic functions of the studied bacterial populations. The weakness of this approach is that it provides very limited information about the bacteria composing the microbiome. However, a paradigm presumes that the collection of the enzymes is more important for a microbiome than the particular composition of bacteria carrying these enzymes (Qin et al. 2010). Analysis of microbiomes using shotgun sequencing requires generating massive amount of data. Although the Illumina GAIIx system generates 18 Gb data per run, which is on par with all the known bacterial genome sequences combined, dozens of its runs are required to generate data for a single shotgun genomic study. And these data, once generated, require extensive computer power to generate contigs. The invention provides in part a novel approach for carrying out targeted resequencing including analysis of microbiomes or other bulk nucleic acid populations. The approach of various aspects and embodiments of the invention utilizes Genome Sequence Scanning™ (or GSS™) which is described in greater detail herein. This approach is more general than existing approaches in the sense that one can select the subset of nucleic acid fragments to analyze further based on virtually any genetic marker or element. In addition, this approach is also automated, making for less operator error.

More specifically, in various methods of the invention, nucleic acids such as but not limited to genomic DNA may be prepared by standard methods and then may be analyzed using GSS™. Tagged DNA fragments (e.g., DNA fragments having one or more sequence- specific probes hybridized thereto) are analyzed by algorithms such as those described in published US patent application US 20120283955, the entire contents of which are incorporated by reference herein.

An example of such an algorithm is the "molecular classifier" algorithm (Meltzer et al., 2011). This algorithm compares profiles of single nucleic acids, such as DNA fragments, to a database of profiles. In some instances, the algorithm compares fluorescence intensity profiles generated by non-specific probes . If the desired signature is detected, the DNA fragment corresponding to that signature is re-directed (also referred to herein as "sorted") using a selector chip or a selector module. FIG. 31A provides a schematic of such operation performed by a chip or module. It will be clear that the acquisition of the profile and its analysis must be performed while the DNA fragment is still in transit in the microfluidic chip. If the analysis is too slow, the DNA fragment will exit the chip without being redirected (sorted). Redirected DNA fragments are thus captured and available for further on-chip or off-chip analysis, including targeted resequencing. If need be, the length of the chip may be extended or velocity of the DNA can be decreased before sorting.

It is to be understood that the signatures can be used to identify DNA fragments of interest. Additionally or alternatively, they can be used for genome assembly, if desired.

Alternatively, certain short sequences may be specifically targeted (FIG. 3 IB). These shorter sequences may belong to conservative regions of the genome such as but not limited to housekeeping genes. An example of such a sequence and its use to select fragments is described in greater detail herein. It is to be understood that various aspects of the invention contemplate nucleic acid fragment selection based on virtually any detectable sequence, region or element, and that the examples provided herein are intended for illustration of the invention.

Various aspects of the invention further contemplate that sorting of nucleic acids can be performed using any number of detectable sequences, regions or elements provided that they are distinguishable from each other.

Genome Sequence Scanning™. Genome Sequence Scanning™ (GSS™) technology, a.k.a. Direct Linear Analysis (DLA) technology, is a proprietary technology developed by Pathogenetix (PGX, formerly U.S. Genomics) for large-scale analysis of long DNA molecules and ideally suits analysis of bacterial genomes (Chan et al. 2004; Protozanova et al. 2010). The technology has been applied to detection of biopathogens in aerosols, analysis of clinical isolates of S. aureus, and speciation and typing of multiple strains of various pathogens using a single reagent set.

GSS™ includes isolation of genomic DNA from a bacterial sample, specific digestion with a restriction endonuclease (RE), tagging the DNA with a fluorescent sequence- specific probe, measuring the distribution pattern of probes along the DNA fragments (a signature, essentially a map of probes), and then comparing the signatures against a database for identification (FIG. 29) (Protozanova et al. 2010). GSS™ analyzes long DNA fragments (100- 300 kb) and the probes hybridize to short (6-8 bases) sequences; therefore, every DNA fragment carries dozens of the probes, the positions of which depend on unique underlying genomic sequence. Typically, RE digestions generate several long DNA fragments per every genome. The signatures are information-rich and highly specific. The information content is further increased by the simultaneous use of two different-colored probes recognizing different motifs, therefore generating two independent barcodes for every fragment (FIG. 30).

Because the probes hybridize to short sequences, they bind with high probability to any genomic DNA. Therefore, unlike approaches that require specific reagents for detection of each pathogen, GSS™ employs a single reagent set to create the barcodes which can then be used to detect and identify thousands of strains from hundreds of species. The fragments of genomic DNA and the signatures generated depend on the selection of the probes and of the restriction enzyme; we call the combination of the probes and of the enzyme the signal-generating (SG) set. The RE is selected to produce the maximal number of fragments of genomic DNA in the length range between 80 and 350 kb. The probes are selected to maximize specificity of the barcodes, which typically requires high density of cognate sites. The same GSS™ instrument can be used with different SG sets, which can be optimized for different applications. SG sets for typing are finely tuned to maximize sensitivity to small genomic differences within a group of similar genomes. SG sets for speciation cover wide range of different genomes. And SG sets for detection of targets in presence of biobackground are biased to produce longer fragments of genomic DNA. However, although optimization improves GSS™ performance, even not optimized SG sets perform well and combine the ability to identify many species with high typing discrimination.

The databases used for identification can be generated either in silico from sequenced genomes or measured using cultured isolates of any bacterium. Only organisms with templates in the database can be identified. However, signatures of all microorganisms in a sample can be measured; therefore, detection of the presence of unknown microbes is possible. Also different strains of a species in the database generally carry sufficient information overlap with a newly detected strain and it can therefore its species origin or source can be determined even if the particular profile is not specifically included in the database.

GSS™ technology has potential for microbiome analysis. Because it employs the same reagent set for multiple bacteria and detects any single DNA fragment from the sample, any bacterial mixture can be processed and measured using GSS™. GSS™ can be used to detect certain target microorganisms in the bacterial mixture. Only closely related microorganisms, such as Escherichia coli and certain Shigella species, demonstrate detectable relatedness because of their genomic similarity. Otherwise, only strains of the same species exhibit sufficient resemblance for identification in the absence of exact matches in the database.

Because of the lack of signature similarity between different species, applicability of GSS™ to microbiome analysis requires presence in the database of at least some strain of the species detected in a sample. However, microbiomes in general include poorly studied bacteria, which usually have no sequenced relatives at strain level (Dethlefsen et al. 2008; Walter et al. 2011). Populating a database by direct measurements of reference samples is also impractical, because even similar microbiomes, such as guts of different humans, include very different bacterial populations (Arumugam et al. 2011; Dethlefsen et al. 2008). Some aspects of the invention permit combining the generality of analysis based on sequencing of either complete housekeeping genes or their portions with the specificity of GSS™ that can identify bacteria down to species or even strain level.

In one aspect, the invention provides a method that comprises

(1) providing a population of nucleic acid fragments,

(2) labeling the fragments with a nucleic acid probe that is specific for a housekeeping gene or other genetically conserved sequence (i.e., the specific probe),

(3) labeling the fragments with a nucleic acid probe that is a non-specific probe (i.e., the non-specific probe), wherein the specific probe and the non-specific probe are labeled with different detectable labels,

(4) analyzing individual nucleic acid fragments for the presence of signal from the

specific and non-specific probes,

(5) obtaining, for fragments having signal from the specific probes, a profile of signals from the specific and non-specific probes along the length of the fragment,

(6) separating fragments having signal from the specific probes from fragments lacking signal from specific probes, and optionally

(7) sequencing fragments having signal from non-specific probes.

The profile obtained in step (5) can be used to identify the source of the fragment (e.g., the species and/or strain of bacteria). In some embodiments, the source of the fragment is identified by sequencing nucleic acid regions adjacent to the binding site of the specific probe.

In some embodiments, the specific and non-specific probes are labeled with the same detectable label and are the signals from these are distinguished from each other based on intensity.

In some embodiments, the method comprises steps (1) through (4), or steps (1) through (5), or steps (1) through (6), or steps (1) through (7). In some embodiments, the method comprises steps (1) through (4) and (6) and optionally (7) also.

In some embodiments, the method further comprises fragmenting a population of nucleic acids in order to form the population of nucleic acid fragments. The population of nucleic acids will typically be obtained from a cell population such as a cell population from a biological sample (e.g., a gut sample, a lung sample, a stool sample, and the like). As used herein, labeling fragments with a probe means that the fragments are exposed to the probe and, if the fragment comprises the probe's binding site, then the probe binds to the fragment. The probe's binding site may be a sequence complementary to the probe's sequence. Although shown above as two separate steps, labeling of the fragments with the specific and non-specific probes may occur simultaneously.

As used herein, fragments having signal from a probe means fragments having probes bound to them.

As used herein, a profile of signals from the probes along the length of the fragment means an intensity versus length (or distance) plot for an individual nucleic acid fragment. Examples of such a profile are provided in FIG. 31.

As used herein, a probe that is labeled with a detectable label is a probe that is conjugated to a detectable label. The detectable label may be a fluorophore but it is not so limited.

Detectable labels include fluorophores such as organic fluorophores (e.g., TMR or fluorescein), polystyrene beads impregnated with fluorophores such as organic fluorophores, dendrimers carrying conjugated fluorophores, and the like. Detectable labels also include inorganic fluorescent moieties such as quantum dots.

Various embodiments are described in greater detail below.

Specific Probes: Specific probes are typically specific for conserved gene or nucleic acid regions. An example of a conserved gene is a housekeeping gene. In some embodiments, the housekeeping genes belong to the rrn operon. In some embodiments, the housekeeping gene encodes 16S rRNA.

The specific probe should be designed such that it binds only to its target. Typically, this means that it will be sufficiently long and therefore will only find complementarity to its target and not to other nucleic acid regions that by chance have the same sequence. As will be discussed below, this characteristic is in contrast to the non-specific probe used in certain methods described herein. There may be multiple copies of the housekeeping gene of interest. As a result, fragments may contain one or more binding sites for the specific probe and/or a genome may contribute one or more fragments having a specific probe binding site to the population of fragments. Finally, housekeeping genes tend to be conserved across species and strains of species and therefore it is possible to analyze a number of species and strains using the same probes. This facilitates the analysis.

The specific probes are labeled with a detectable label. The detectable label may be a fluorophore. The detectable label on the specific probe typically is different from the detectable label on the non-specific probe. For example, the specific probe may be labeled with a fluorophore emitting in the red wavelength range and the non-specific probe may be labeled with a fluorophore emitting in the green wavelength range.

In some embodiments, the specific probes are labeled with multiple detectable labels. This results in the intensity of signal from such probes to be greater than the intensity of signal from the non-specific probes. Intensity alone may therefore be used to distinguish between a specific probe signal and a non-specific probe signal.

FIG. 31 shows profiles having signals from specific and non-specific probes. As will be apparent, the peaks corresponding to the specific probes can be distinguished from the peaks corresponding to the non-specific probes based on color, number of peaks (e.g., usually only one specific peak and multiple non-specific peaks from a single fragment), and intensity of peak (e.g., a higher intensity peak from a specific probe compared to a non-specific probe).

At least two schemes exist that achieve the goals of specific binding and high intensity signals. These are PD-loops (Demidov et al. 2001) and single flap hybridization (Das et al. 2010). In both schemes, exceptional specificity is achieved by the combination of several independent events, such as hybridization of probes or single strand nicking, within a narrow region around the tagging site. Both schemes permit introduction of at least 2 fluorophores per site.

PD-loops: PD-loops are formed by hybridization of two bisPNA probes in close proximity to each other on a target DNA (FIG. 32). If the binding sites of the two bis PNA probes are within 12-14 bases, they displace a DNA strand that cannot re-hybridize with its complementary DNA sequence between the probes. Therefore, two short bisPNAs displace a long DNA strand and maintain its single stranded conformation. If a non-bisPNA

oligonucleotide complementary to the displaced DNA strand is added, it can hybridize to the displaced DNA strand. Although the bisPNAs are short and may have other cognate sites on a long DNA fragment, the formation of the complete PD-loop structure which requires both bisPNAs hybridized in close proximity is a very rare event and can ensure unique tagging even in very large genomes (Demidov et al. 2001). Because the whole process is performed under non-denaturing conditions, the non-bisPNA oligonucleotides can hybridize only if both bisPNAs are bound to the DNA fragment. If only the non-bisPNA oligonucleotide carries fluorophores, the fluorescent signal can be generated only if all three components are in place. Therefore, PD- loop tagging ensures extremely high specificity, far surpassing the specificity that can be achieved with any single probe.

Specificity of this method can be improved even further if the bisPNA probes have short cognate sequences (6-7 bases) and cannot form stable complexes except if both are present and in close proximity to each other (Phillips et al. 2005).

BisPNA requires a polypurine target site to hybridize. The presence of two polypurine runs of sufficient length in close proximity is relatively rare. To assess if this approach is possible, for example, with the 16S rRNA gene, we studied sequences of 10 genes in 9 bacteria: Acidaminacoccus fermentas VR4, Bacteroides fragilis YCH46, Escherichia coli K12 MG1655, Escherichia coli 0157:H7 TW14359, Prevotella ruminicola 23, Ruminococcus albus 7, S.

enterica Typhimurium LT2 (2 different genes), Shigella flexneri 2a str. 2457T, Staphylococcus aureus Mu3. This set includes representatives of commensal and pathogenic intestinal microflora, gram-positive and gram-negative bacteria, different and similar (E. coli and S.

flexneri) species, and two strains of the same species (E. coli). Therefore this set is

representative of the bacteria that can be encountered in a gut microbiome. We have identified sufficiently conservative regions that are the same in every bacterium, and searched for the sites suitable for formation of PD-loops. We found two suitable regions, starting at 1215 bp and 1531 bp (FIGS. 33 and 34, respectively) that could be used for PD-loop formation.

Single strand flap hybridization: In this method illustrated in FIG. 35, one of the DNA strands is specifically cut by a nicking enzyme (Das et al. 2010). This may be carried out using an artificial Type II restriction endonuclease, which retains the ability to bind specifically to its cognate DNA sequence while its ability to cut DNA backbone is inactivated in one of its domains (thereby resulting in nicks in the DNA rather than double- stranded cuts). After nicking, the 3 '-terminus at the DNA cut is extended using a polymerase that retains displacement activity, but lacks 5' - 3' exonuclease activity (such as Vent (exo) polymerase). As a result, the DNA strand with a 5 '-terminus at the DNA cut is displaced by the extended DNA chain (FIG. 35B). If an oligonucleotide complementary to the displaced strand is added, it can hybridize with the displaced DNA strand. Even though the nicking enzyme may generate nicks at some other sites, the oligonucleotide probe is not designed to be complementary to the DNA in their vicinity. Because the whole process is performed under non-denaturing conditions, the oligonucleotide can hybridize only with the single stranded flaps. The specificity of this technique is based on the combination of two requirements - the cognate site for the nicking enzyme must be in close proximity to the DNA sequence that is complementary to the oligonucleotide probe. Only the oligonucleotide probe carries detectable labels such as fluorophores; hence, only the specific complexes with all three components are detected.

To assess if this approach is possible, for example, with the 16S rRNA gene, we studied the sequences of the same set of 10 genes in 9 bacteria, described in the previous section, which is representative of the bacteria that can be encountered in gut microbiome. We have identified sufficiently conservative regions that are the same in every bacterium, and searched for the sites suitable for formation of single strand flaps. We were able to find a suitable region, starting at 351 bp that can be used with Nt.BsmAI nicking enzyme.

Nicking enzymes that can be used in various aspects of the invention include but are not limited to those commercially available and those engineered from Type II restriction enzymes.

Non-specific probe: The non-specific probes are probes that bind specifically to more common sequences in the genome. Accordingly, the binding sites for these probes are not specific for any particular gene or region within the genome. The binding sites for these probes are about 8 bases in length. The probe themselves may be longer particularly if they are for example bis-PNA probes. In some instances, the binding site of the non-specific probes is chosen so that it occurs multiple times per 100 kb of genomic DNA, including for example 10- 15 times per 100 kb of genomic DNA.

Nucleic acid fragmentation: Genomic DNA is fragmented, typically before the labeling process. Typically, the fragmentation generates at least some fragments that are at least 50 kb or at least 100 kb in length. Preferably, some fragments range in length from about 50 kb to about 350 kb. Fragmentation may be performed in any number of ways. One example is restriction enzyme digestion in which one or more restriction enzymes may be used and they may be used simultaneously or consecutively. Other methods for DNA fragmentation, such as controlled shearing in narrow channels, can be used.

The fragmented genomic DNA is then labeled with the two types of probes described above and is subjected to standard GSS™ measurement. As a result of the measurement, the presence of every DNA fragment is detected together with the presence and positions of the bound probes of both types. Detection of the specific (housekeeping gene) probe signals the presence of the housekeeping gene and determines its position on the DNA fragment. For every DNA fragment, the positions of the multiple non-specific (short binding site) probes are determined, thereby generating the map of the cognate sites.

The detection of the specific (housekeeping gene) probe initiates a sorting impact (deflection) that redirects the DNA fragment bound to the specific probe into an alternative exit channel (FIG. 31). This way, the DNA fragments containing the housekeeping genes with the surrounding genomic DNA are separated from the bulk DNA, accumulated, and optionally used for further analysis including sequencing.

An exemplary use of the technology is the analysis of microbiomes. The selected DNA fragments include housekeeping genes (typical size of the operons <10 kb) with long lengths of adjacent genomic DNA (50-350 kb). Moreover, every DNA fragment is accompanied by the map of the second probe (i.e., the short non-specific sites). This arrangement permits further analysis such as sequencing of sections of the 16S rRNA genes using one of the standard techniques, and therefore exploits the power of detectable relatedness of the conservative sequences even between distant relatives. This analysis however typically permits identification no better than down to family or genus level. However, the genomic sequences adjacent to the conserved regions carry more detailed information that may permit identification and discrimination between bacteria according to species and strain. Importantly, by using GSS™ analysis before-hand, most of the bulk DNA is removed, which facilitates application of shotgun sequencing. The availability of bisPNA maps accompanying every DNA fragment helps the assembly of contigs, further helping shotgun sequencing. Finally, the bisPNA maps alone in combination with the housekeeping genes may be sufficient for better specificity of the analysis of bacterial populations, even without shotgun sequencing.

The technique may be used for the analysis of bacterial mixtures. It may also be used to analyze mammalian cell populations.

In a mammalian cell context, one or more housekeeping genes may be targeted in a single experiment (i.e., more than one specific probe type may be used, each specific for a particular housekeeping gene). For example, multiple housekeeping genes of a mammalian genome can be selectively tagged and separated through the proposed process, and then optionally subjected to more detailed analysis (such as sequencing) of the entire genes and their adjacent regions.

Another possible application of the technology is in cancer diagnostics. In this embodiment, the specific probes can be specific for known oncogenes or other genomic cancer markers rather than housekeeping genes. The GSS™ -based sorting selects only the DNA fragments including these specific genes, eliminating the bulk of the DNA and simplifying the analysis. Simplification of the DNA material is especially important in the detection of malignancies, typically accompanied by multiple genomic distortions involving shuffling of large DNA segments, which are different for different cells even within the same tumor (Chaffer and Weinberg 2011; Hanahan and Weinberg 2011; Stratton 2011).

For analysis of the populations of mammalians with known genome sequence, the technique can be used in its simplest form. In this case, the size and probe signature of restriction fragments containing genes of interest (or target genes) can be determined in silico using the genomic sequence. No specific probe tagging is required and DNA selection can be performed solely on the basis of the signature generated by the non-specific probes.

On-fly sorting: The prototype for on-chip sorting of DNA, including the chip, electronics, and software, have been developed and successfully tested. It successfully separated BAC 12M9 (185 kb) from Lambda phage DNA (48 kb) or from a Digital DNA™ octamer (80 kb) in real time.

In the prototype chip, a mixture of DNA molecules of different lengths was injected into the entrance (inlet) port, marked "DNA mix" in FIG. 36A. The DNA molecules were carried hydrodynamically through the microfluidic taper labeled "I" (see also FIG. 36B), where they were stretched and their optical signals were detected. These signals were analyzed in real time. In this simple prototype, only the DNA lengths were measured; however, complete GSS™ information can be used in a similar way to distinguish between DNA fragments of the same length, but with different underlying genomic sequences. Accordingly, DNA fragments can be analyzed on the basis of binding of one or more non-specific probes, wherein the pattern of binding of the non-specific probes provides the identity of the DNA fragment.

The following is a description of a use of the chip. If a shorter DNA fragment is detected, no action is performed and the fragment proceeds further towards the port labeled "Waste" in the scheme. However, if a longer DNA molecule is detected, an electric pulse is applied between the positive and negative electrodes inserted into the ports labeled "Anode" and "Cathode," respectively. As a result, the longer fragment is directed into the sorting zone labeled "Π" (see also FIG. 36C) towards the port labeled "BAC."

This chip was designed for GSS™ measurements with enhanced accuracy. The first microfluidic taper, preceding the sorting, was designed to stretch a wide range of molecule lengths. Its performance can be enhanced by introduction of the sheath flow delivered through the ports labeled "Buffer" (see also the microfluidic configuration in FIG. 36B). After the DNA fragments are sorted and accumulated in the different zones, vacuum driving the flow is switched from the port "Waste" to the port "λ DNA". As a result, shorter DNA molecules are directed to the λ stretching funnel, optimized for short DNA molecules, and measured with high resolution. Then vacuum is switched from the port "λ DNA" to the port "BAC". As a result, longer DNA molecules are directed to the BAC stretching funnel, optimized for long DNA molecules, and measured with high resolution.

For the techniques described herein, simpler chips may be used that do not require secondary stretching and rather just comprise reservoirs to accumulate the sorted fragments. Such chips have been developed and tested.

The sorting system, including the chip, flow controls, electronics, and software, can be added to the existing prototypes and future devices as an option, which does not require other modifications of the system.

It is to be understood that the sorting applications of the invention do not require the use of droplets or immiscible fluid types. Thus, the sorting applications occur without

compartmentalizing DNA fragments of interest in a droplet such as an oil in water droplet or the like.

The invention contemplates in various aspects and embodiments that DNA fragments of interest may be sorted using electric or hydrodynamic forces. In electrodynamic applications, charges are applied to the stream in which the DNA fragments exist in order to direct the fragments into waste or collection reservoirs. In hydrodynamic applications, the balance of flows is distorted in order to direct the fragments into waste or collection reservoirs. This latter approach is shown in FIGS. 37 A and 37B. FIGS. 37 A and 37B illustrate a microfluidic chip (labeled I) that stretches probe bound DNA fragments and performs GSS™ measurements. A fluid comprising DNA is injected into the device (or system) and is immediately sheathed with two symmetric flows of a carrying buffer (labeled F _IA and F _ro ). These focusing flows center the DNA in the middle of the channel and carry it through the taper, through which the flow accelerates and thereby unwinds the DNA. In their stretched conformation, DNA molecules are conveyed across the spots of laser light (labeled L), which excites fluorescence of the DNA- bound non-specific and (if any) specific probes. This fluorescence is detected in the detection module (labeled D) while the DNA passes through. As a result, the fluorescence intensity profile is detected that is characteristic of the distribution of the probes along the DNA which in turn is defined by the underlying genomic sequence of the DNA. The fluorescence intensity pattern detected for the DNA molecule is called a trace (or a profile).

While the DNA molecule continues to travel, including into and through the microfluidic subsystem II, its trace may be compared with a database that contains various templates of interest to identify the fragment of interest. The templates may be generated experimentally (and thus may be single traces or an average of multiple traces), or they may be generated non- experimentally (e.g., through prediction based on known sequence of the DNA fragment and the probes to be used). In another embodiment, the presence of specific probe is detected that identifies the fragments of interest.

In the normal state, the focusing flows of the subsystem II are also symmetric (F _HA =F _HB ) and the path of the DNA is directed to the waste port (labeled W). This state is maintained until the system detects a DNA molecule of interest. Once a it is detected, the balance of the sheathing flows changes (F _HA >F _IIB ), and the desired DNA molecule is directed to the collection port C.

It should be appreciated that various embodiments of the present invention may be formed with one or more of the above-described features. The above aspects and features of the invention may be employed in any suitable combination as the present invention is not limited in this respect. It should also be appreciated that the drawings illustrate various components and features which may be incorporated into various embodiments of the present invention. For simplification, some of the drawings may illustrate more than one optional feature or component. However, the present invention is not limited to the specific embodiments disclosed in the drawings. It should be recognized that the present invention encompasses embodiments which may include only a portion of the components illustrated in any one drawing figure, and/or may also encompass embodiments combining components illustrated in multiple different drawing figures.

It should be understood that the foregoing description of various embodiments of the invention are intended merely to be illustrative thereof and that other embodiments,

modifications, and equivalents of the invention are within the scope of the invention recited in the claims appended hereto.

EXAMPLES Example 1. On-chip intercalation with mono-cyanine intercalator.

This Examples demonstrates labeling of DNA using the mono-cyanine intercalator PO-

PRO-1.

Off-chip ("bulk") fluorescence binding assays were performed in a reaction vessel. These assays suggested that the intercalation reaction approached saturation near 250 nM Po- PPvO-1 in the presence of 50 nM DNA base pairs. At concentrations above 1 uM, the observed fluorescence intensity appeared to decrease, either due to aggregation of intercalator that in turn induced autoquenching of the fluorescent signal or due to aggregation and precipitation of the intercalated DNA.

Preliminary on-chip intercalation was then performed using 250 nM PO-PRO-1 in low ionic strength Tris-EDTA buffer. Initial microscopic observation indicated the presence of a single stream of intercalated DNA emerging from the DNA injection channel. No aggregation or surface adhesion was observed (FIG. 17). Use of the mono-intercalating dye promoted successful on-chip intercalation by eliminating DNA inter-molecular cross-linking and DNA adhesion to the chip surface(s). The off rate of PO-PRO is so high that if a DNA sample was intercalated in tube at 1: 1 dye-intercalator ratio, no observable fluorescence is observable in the detection channel due to diffusion of the intercalator away from the DNA (data not shown).

Example 2. On-chip intercalation is independent of DNA concentration.

For in-tube intercalation, solutions of varying DNA concentrations were incubated in a tube with a fixed concentration of the bis intercalator POPO-1 (1 μΜ POPO-1). The DNA was then introduced into a microfluidic device described herein. Each solution was introduced into the reaction chamber from which the DNA entered the elongation region (or elongation funnel), and 30 minute data sets were acquired. The stretching coefficient for the 250.5 kb fragment was determined as a representative value for each data set. We observed that the stretching coefficient decreased as the ratio of DNA to intercalator increased (FIG. 18, open triangles). Only a limited range of DNA to intercalator ratios were achieved, since at low dilutions of DNA, the frequency of molecules reaching the detector dropped precipitously (corresponding to the observed onset of fluorescence quenching and precipitation).

For on-chip intercalation, similar DNA solutions (of differing concentrations) were loaded to the type of microfluidic chip operating with 250 nM PO-PRO-1 in the sheathing buffer streams. In contrast to the in-tube intercalation, a uniform stretching coefficient was observed across a 32 fold dilution of the original stock DNA, when on-chip intercalation was used. (FIG. 18, closed circles)

Example 3. On-chip intercalation is independent of intercalator concentration.

This Example describes experiments performed to determine optimal conditions for intercalation by varying the concentration of intercalator in the sheathing buffer. A sample of E. coli K12 DNA digested with the Notl restriction enzyme was loaded to the injection port, and intercalator ranging from 50 - 500 nM was loaded with the sheathing buffer streams. Samples with no intercalator have been run on the instrument, but it is impossible to determine observed molecule lengths in the absence of intercalating dye. For each condition, fragment lengths (in μπι) were determined for all restriction fragments by identifying each clustered fragment by its signature trace of site-specific probes. (Protozanova et al., Analytical Biochemistry, 2010. 402: p. 83-90.) When the measured fragment length (in μπι) was plotted against the known fragment length in kb, characteristic quadratic stretching curves were observed (FIG. 19A). The extensibility of DNA increased with increasing PO-PRO-1 concentration.

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OTHER EMBODIMENTS

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or

configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding,"

"composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.