Using a porous capillary membrane to quantify the products of the rotating circular amplification technique

Abstract

Using a porous capillary membrane to quantify the products of the rotating circular amplification technique

Description

#x200f- #x200f#x200fUsing a #x200f#x200fmembrane#x200f#x200f#x200f#x200fhair#x200f#x200f#x200fpermeable#x200f Rotating#x200f Related references This application claims the benefit of UK Patent Application No. 1507376.0 filed on 30 April 2015, the entire application of which is incorporated herein for all relevant purposes. Background Non-invasive prenatal testing (NIPT) uses a maternal blood sample or other sample taken non-invasively from a pregnant woman to provide information about the condition or health of the fetus. Early detection of the condition or health of the fetus allows for medical intervention and treatment. The rotating circular amplification (RCA) technique is useful for analyzing cell-free DNA in maternal blood. However, quantifying RCA products with statistical conditions can be challenging. On a practical level, although the total number of products in an RCA reaction may be considerably high to provide adequate statistical conditions, the molar concentration of specific RCA amplicons in the reaction may be quite low, limiting the variety of quantification methods that can be used. For example, RCA products in a sample could, in theory, be detected by labeling the RCA products, placing the sample on the surface of a glass slide, and counting the number of labeled products on the slide. However, simply placing the product containing the RCA-labeled products on a glass slide, allowing the RCA-labeled products to spread over the surface, and then counting those RCA-labeled products that adhere to the slide can take several hours, and not all of the RCA-labeled products reach the slide and are counted. The use of filtration on a solid surface for the quantification of labeled RCA products is described here. As will be described in detail below, these methods can facilitate the analysis of samples containing RCA products. Summary A method of analyzing a sample is provided. In certain embodiments, the method may include: (a) filtering a liquid sample containing products of a rotating circular amplification (RCA) technique using a porous capillary membrane whereby a range of RCA products is produced on the membrane; wherein the sample contains at least a first pool of RCA products and a second pool of RCA products, wherein the first and second pools of labeled RCA products are identifiably labeled; and (b) determining the amount of the first labeled pool of RCA products and the amount of the second labeled pool of RCA products in a region of the membrane. In some embodiments, the step of determining the amount can include counting the number of first and second labeled RCA products in that region. In other embodiments, the step of determining the amount can include detecting an expression signal from that region. In either case, the method can provide an estimate of the number of first and second pools of RCA products in the sample. Depending on how the method is implemented, the use of a porous capillary membrane can enable the quantification of essentially all RCA products in a sample (and not just those that are spread on the slide surface), which in turn increases the accuracy, sensitivity, and reproducibility of the assay. In addition, the use of a porous capillary membrane allows the assay to be performed in minutes rather than hours compared to some alternative methods, which, as mentioned above, can be performed by pipetting the sample onto a glass slide and then incubating the slide for a long period of time in the hope that the RCA products will spread and adhere to the slide surface. The laboratory section of the present application reports in agreement with this issue that performing the present method can result in counting 2.5 times the RCA products after 90 seconds (which is equal to the time it takes for the sample to be placed on the membrane and then for the sample to pass through the membrane). This time can be compared to 16 hours of incubation on a glass slide.Additionally, the use of membranes can help reduce background by allowing potential sources of background (e.g., fluorescent oligonucleotide molecules that are not coupled to an RCA product or the like) to pass through the membrane (especially if the membrane is washed after using RCA products). This method has found particular use in the analysis of samples that have a relatively wide range of RCA product concentrations (e.g., 10 to 10M RCA product in a volume of 50 to 200 μl or more) which in many cases can be relatively low concentrations, as mentioned above, the membrane helps to concentrate the RCA products on the membrane surface. To put it another way, without the use of a membrane and instead of using an alternative method, a 50 μl sample is spread on the surface of a glass microscope slide and even if all the RCA products can be made to adhere to the slide, the RCA products are so spatially separated on the slide that a single field of view can only show an inadequate number of RCA products, making the process time-consuming and inefficient to count by microscopy or other means without any reasonable accuracy. Depending on how the method is implemented, the membrane may aid in concentration and support the RCA products while, at the same time, providing a means by which the RCA products can be washed to reduce background.Additionally, the membrane can serve as a microscopy slide, especially if it is transparent or can be made transparent by adding a wetting agent. In summary, it is believed that the present method provides a rapid, highly accurate, sensitive, and reproducible method for quantifying RCA products - a product that is itself already produced without bias in that each target molecule is seen with only one RCA product in the sample (unlike PCR where each target molecule is amplified in an unknown number of copies and different target molecules are amplified in different amounts). Thus, the present method should prove to be invaluable for applications where rapid, accurate, and precise measurements of the mass of DNA molecules in a sample are essential. In particular, the present method should prove to be valuable for noninvasive prenatal diagnostic testing, where rapid, accurate, and precise measurements of the mass of DNA molecules present at relatively low concentrations in the cell-free fraction of the maternal bloodstream are critical. These and other potential features and benefits are outlined in the descriptions below. Brief explanation of the images Those skilled in the art will understand that the drawings described below are for illustrative purposes only. These drawings and drawings are not intended to limit the scope of the present teachings in any way. Figure 1 schematically shows some of the steps of the present method. Figure 2 schematically illustrates some of the principles of the present method. Figure 3 is a graph showing the number of counts per image on the y-axis and samples, incubation time, type of glass plate or aluminum oxide on the x-axis. Figure 4 is a histogram showing some of the results of the experiments described in Example 2. Figure 5 is a plot showing the ratio between counts for the two channels in relation to cell line composition. Full description Before various embodiments are described, it is to be understood that the subject matter of this disclosure is not limited to the specific embodiments described and may vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present disclosure is limited only by the claims set forth herein. The section headings used herein are for purposes of categorization only and should not be construed as limiting the subject matter described. While the present disclosure has been described in connection with various embodiments, it is not intended that the present disclosure be limited to such embodiments. On the contrary, the present disclosure encompasses various alternatives, modifications, and similar matters that will be apparent to those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by one skilled in the art to which this disclosure relates. Although any methods and materials similar or equivalent to those described herein may be used to practice or test the present subject matter, certain exemplary methods and materials are described herein. The citation of any publication is for the purpose of disclosure prior to the filing date of the application and should not be construed as implying that the present claims cannot take precedence over such publications. Furthermore, the dates of publications provided may differ from the actual publication dates and may need to be independently verified. As will be apparent to those skilled in the art reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that can be readily separated or combined with features of several other embodiments without departing from the scope or spirit of the present disclosure. Each method recited may be performed in the order of events recited or in any other order that is reasonably possible. All patents and publications, including all disciplines disclosed within such patents and publications, are expressly incorporated herein by reference. Before describing the sample expressions in detail, the following definitions are intended to illustrate the meaning and scope of the terms used in the text description. It should be noted that the singular forms of words used herein and in the claims include plural references unless the context clearly dictates otherwise. For example, the term "a primer" refers to one or more primers, such as a single primer and multiple primers. It should be noted again that the claims may be written in such a way that they do not list any selective agents. In such cases, this statement is intended to be used as a basis for reference for the use of such exclusive words as "exclusively," "only," and the like in connection with the listing of the claimed agents or the use of a "negative" limitation. As described herein, the term "filtration" includes the act of passing a liquid containing an analyte (e.g., the products of a rotating circular amplification technique) through a filter in such a way that some of the analyte is retained by the filter. In filtration, at least some of the liquid moves from one side of the filter to the other. The term "rotating circular amplification technique" or "RCA" as used herein refers to thermometric amplification that produces linear copies of a circular nucleic acid template using a separate isolated polymerase. RCA is well known in the field of molecular biology and has been described in various publications including, but not limited to, Lizardi et al (Nat. Genet. 1998 19:225-232), Schweitzer et al (Proc. Natl. Acad. Sci. 2000 97:10113-10119), Wiltshire et al (Clin. Chem. 2000 46:1990-1993) and Schweitzer et al (Curr. Opin. Biotech 2001 12:21-27), which are incorporated herein by reference. As used herein, the term "products of the rotating circular amplification technique" includes the replicated products of a rotating circular amplification reaction. The term "fluorescently labeled rotating circular amplification products" as used herein refers to rotating circular amplification products that have been fluorescently labeled by, for example, coupling a fluorescently labeled oligonucleotide to the rotating circular amplification products or by other means (e.g., by incorporating a nucleotide into the product during amplification). The term "porous capillary membrane" as used herein includes membranes that are relatively compact in individual capillaries that form the thickness of the membrane, i.e., that extend from one side of the membrane to the other, thereby allowing the passage of liquid except for particles from one side of the membrane to the other. Examples of porous capillary membranes include, but are not limited to, anodic aluminum oxide membranes (see below), nanochannel glass membranes, semipermeable membranes, and polytetrafluoroethylene. Nanochannel glass membranes are made of glass and have a large number of uniformly shaped channels with diameters ranging from 15 microns to 15 nanometers (see, for example, Tonucci et al., Advances in Nanophotonics II, AIP Conference Proceedings, 2007 959: 59-71; Pearson et al, Science 1995 270: 68-70 and Tonucci et al., Science 1992 258: 783-785, also U.S. Patents 5,306,661; 5,332,681; 5,976,444; 6,087,274; 6,376,096; 6,483,640; and 6,599,616, which are incorporated by reference).Semipermeable membranes are made of a transparent polymer (e.g., polycarbonate, polyethylene terephthalate, or polyimide, or the like) containing pores having a diameter in the range of 0.01 um to 30 um that are formed by a combination of charged particle bombardment (irradiation) and chemical etching. Other desirable porous membranes include, but are not limited to, amorphous fluoropolymers such as NAFION™, TEFLON AF™, FEFLON FEIP™, and CYTOP™ (DuPont Fluoroproducts, Fayetteville, NC). As will be appreciated, a porous capillary membrane can have a surface (e.g., a coating (glaze) or a chemically modified surface) that is different from the material from which the membrane is made. For example, the surface of the porous capillary membrane may have altered charge characteristics or hydrophobic or hydrophilic characteristics. In some embodiments, the surface may be coated with aminosilane, polylysine, or another compound to provide a positive charge that helps retain RCA products on the surface.Alternatively or additionally, the surface may have thin layers of metal (e.g., titanium, gold) deposited thereon, which can be bonded to other elements that alter the characteristics of the filter surface. The term "anodic aluminum oxide membrane" as used herein refers to a non-porous membrane structure with a discrete structure that is produced when Al is anodized in a specific acidic medium. The inner diameter of the pores in the membrane, the distance between the centers of adjacent pores in the membrane, and the distance between the edges of adjacent pores in the membrane can be controlled by the deposition voltage, the type of acid, and other parameters. An anodic aluminum oxide membrane is nearly transparent when wet. Anodic aluminum oxide membranes, their characteristics, and detailed studies of such membranes are described in various publications, including but not limited to: Li et al (Chem. Mater 1998 10: 2470-2480), Santos et al (Trends on Analytical Chemistry 2013 44: 25-38), Ingham et al (Biotechnology Advances 30 2012 1089-1099), and Poinern et al. (Materials 2011 4: 487-526), ​​whose materials are incorporated herein by reference.Andovik aluminum oxide membranes are commercially available under the trade name ANOPORE™ from, for example, SPI Supplies (West Chester, PA) and other vendors such as Sykera Technologies Inc (Longmont, CO) and Signma-Aldrich (St. Louis, MO) and can be purchased with a retaining ring. The term "region" as used herein refers to a region of a membrane or a region of an image that includes a continuous or discontinuous area. For example, if a method involves quantifying RCA-labeled products in a region, e.g., counting the number of RCA-labeled products in a region, the region in which RCA products are quantified can be a single, continuous space or multiple discontinuous spaces. The term "imaging" as used herein includes the process by which optical signals from the surface of an object are detected and stored as data associated with a location (e.g., "pixel"). A digital image of an object can be reconstructed from this data. A membrane region can be imaged using a single image or one or more images. The term "single labeled RCA products" includes single RCA molecules that are labeled. The term "quantification" as used herein includes methods in which individually recovered RCA products are counted as well as methods that involve measuring an aggregate signal from multiple RCA products. In methods that involve measuring the concentration of an aggregate signal, individual RCA products need not be recovered. The amount of RCA products that can be expressed using any suitable unit. In some cases, the amount of RCA products can be expressed as the number of individually recovered RCA products that have been counted. The term "counting" as used herein includes determining the number of individual objects in a larger set. In embodiments, "counting" requires tracking individual signals from individual objects in a polarity (other than the collective signal from the polarity of the objects) and then determining how many objects are present in the polarity by counting the individual signals. In the context of the present methods, "counting" can be accomplished by determining the number of individual signals in a signal array. The term "transparent" as used herein refers to the state in which an object is optically transparent at the wavelength used. For fluorescence microscopy, "transparent" means that the object is transparent to one or both of the emission and emission spectra of a fluorophore. As discussed in more detail below, certain membranes are transparent only when wetted. Such membranes are considered transparent even though the dry form of such membranes may not be transparent. The term "array" as used herein refers to an array of RCA products consisting of a collection of individual RCA products on a planar surface, where the RCA products are spatially separated from each other on the surface (to the extent that the Poisson distribution of the array is truly random). A "random" array includes an array in which the elements, e.g., RCA products, are distributed on the surface of a subsurface at positions that are not previously determined. In some cases, the distribution of RCA products on a random array can be described by Poisson statistics, such that, for example, the distribution of distances between RCA products of a random array can be approximated using the Poisson distribution. In other words, the RCA products can be distributed randomly, e.g., at locations that are not predetermined on a membrane. Other meanings of these and other words may be mentioned throughout the text of the specification. Before describing the present method in detail, it is recognized that the present method can be performed using any support that acts as a filter for RCA products. Such supports should have a low background signal at the wavelengths used in the analysis and a suitable pore size to allow rapid passage of the liquid stream and capture the RCA products. Suitable supports can be made of organic or inorganic porous materials, including materials such as porous metals, ceramics, homogeneous films (e.g., polymers) and heterogeneous solids (polymer blends, mixed glasses). Porous ceramic membranes can be made of inorganic materials (e.g., alumina, titania, zirconia oxides, doubly crystallized silicon carbide). See, for example, the PamChip sold by Pamgene (The Netherlands), Wu et al, Nucleic Acids Res. 2004 32: e123 and Anthony et al Biotechniques. (2003) 34:1082-6, 1088-9.The porous polymer membranes of the sample can be made of cellulose acetate, nitrocellulose, cellulose esters (CA, CN and CE), polysulfone (PS), polyether sulfone (PES), polyacrylonitrile (PAN), polyamide, polyimide, polyethylene and polypropylene (PE and PP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF) and polyvinyl chloride (PVC). Thus, in particular embodiments of the method, the method may include: (a) filtering a liquid sample containing products of the rotating circular amplification (RCA) technique using a capture support that acts as a filter for the RCA products, thereby producing an array of RCA products on the support; wherein the sample contains at least a first stack of RCA products and a second stack of RCA products, wherein the first and second stacks of labeled RCA products are identifiably labeled; and (b) determining the accumulated amount of the first labeled RCA products and the accumulated amount of the second labeled RCA products in a region of the membrane. The following description shows a model implementation in which a porous capillary membrane is used. Porous capillary membranes are an example of a support for capture that can be used. The following description illustrates the present method by example. As mentioned above, a method of analyzing a sample is provided. In particular embodiments, the method may include: (a) filtering a liquid sample containing products of the rotating circular amplification (RCA) technique through a porous capillary membrane resulting in the production of an array of RCA products on the membrane; (b) fluorescently labeling the RCA products before or after step (a); and (c) determining the amount of individual labeled RCA products in a region of the membrane, resulting in an estimate of the number of labeled RCA products in the sample. The method can be performed in various ways, an example of which is schematically shown in FIG. 1 . Referring to FIG. 1 , particular embodiments of the method include: filtering a liquid sample 2 containing fluorescently labeled products of the rotating circular amplification (RCA) technique 4 through a porous capillary membrane 6 (e.g., an anodic aluminum oxide membrane). The filtration step concentrates the particles and results in an array 8 of RCA products on the membrane 6.In the embodiment, the next step is shown to involve tracking the particles as they are on the membrane. In some embodiments, this step can generate an image 10 of the array 8. As is evident, tracking can be performed using any suitable fluorescent detector, e.g., a fluorescent microscope, scanner, using a high-magnification CMOS or CCD detector, or using a PMT, and the like. Finally, the amount of labeled RCA products in the membrane area is determined, e.g., by counting individual RCA products recovered, or by measuring the accumulated signal, etc. This determination provides an estimate of the number of labeled RCA products 12 in the sample 2. In certain embodiments, and depending on how the method is performed, the filter can be wetted, e.g., with a wetting agent (e.g., immersion in oil or glycerol to wet the membrane and make it transparent). In these embodiments, the porous capillary membrane can be clarified by using a wetting agent after the RCA products are filtered and before the number of RCA products is determined.In some embodiments, the RCA products can be labeled after the filtration step. Additionally, in some cases, an image of the array may not need to be generated and stored. In these embodiments, analysis of the array can be performed during imaging or immediately thereafter. . In some embodiments where the analysis involves counting, the fluorescent tracer used should be suitable for recovering the various RCA products on the membrane. In some embodiments, the fluorescent tracer can have a magnification of less than 10 um, such as less than 5 um or less than 1 um. In embodiments involving measurement of an accumulated signal, the fluorescent tracer need not have this magnification. In each embodiment, the pores of the capillary membrane should be of sufficient size to prevent RCA products from passing through the pores. For example, in embodiments, the pore diameter of the capillary membrane can be no more than 50% of the average diameter of the RCA products, while in some embodiments it can be no more than 20% of the average diameter of the RCA products, and in some embodiments it can be no more than 10% of the average diameter of the RCA products. Thus, in the filtration of a sample using a porous capillary membrane, the RCA products should remain on top of the membrane and should not completely enter or pass through the pores. In certain embodiments, the porous capillary membrane does not comprise a tethering agent (e.g., a tethering oligonucleotide, an antibody or streptavidin or the like) that specifically binds to or binds to RCA products. In such embodiments, the RCA products do not bind to the porous capillary membrane via a specific (e.g., strand-specific) exchange. In such embodiments, the porous capillary membrane does not comprise a polarity of different strand-specific tethering agents (e.g., oligonucleotides) that are tethered to the membrane at pre-identifiable locations in the form of a microarray as described in US2006022813. In some embodiments, instead of generating an optical signal, no chemical reaction (e.g., a reaction that results in the breaking or formation of a covalent bond) is required on the membrane after the RCA products are filtered through the membrane. As is known, the products of rotating circular amplification (RCA) were not altered (e.g., not exposed to a temperature of at least 90oC for 2 minutes) before filtration through the membrane. Figure 2 shows some principles of an exemplary embodiment of this method. In a first step, a sample containing fluorescently labeled RCA products is placed in a container, e.g. a canister containing a membrane, e.g. at the bottom of the container. The sample is concentrated by applying pressure which causes the liquid phase of the sample to be drawn through the membrane. The RCA products are retained in the membrane bottom in an array at a concentration of e.g. at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000 or at least 10,000 / mm2. If the membrane is not transparent, then after the addition of a wetting agent the membrane becomes transparent and the RCA products can be detected, e.g. by imaging. The RCA products can also be held in place using a holder, such that the membrane can be transported and / or stored in a location where it can be re-read in the event of a positive result. In some embodiments, the wetting agent, in addition to making the membrane transparent, also acts as a preservative. The array can be analyzed from either side of the membrane, for example, through the membrane (as shown in Figure 2).As is apparent, if the membrane is read from "above", e.g., from the same side as the RCA products, the membrane need not be transparent. In some embodiments, the membrane can be dried prior to being transparent and analyzed. The analysis zone can contain at least 10, e.g., at least 100, at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, or at least 200,000 or more RCA products. If desired, the RCA products can be labeled while they are bound to the membrane, and in some embodiments, the membrane can be washed after the array of labeled RCA products is generated and before analysis, e.g., with water or an aqueous buffer containing salt. This washing step can reduce background because potential sources of background (e.g., labeled nucleotides or labeled oligonucleotides that are not bound to an RCA product) can be washed through the filter so that they are not with the filter when the filter is analyzed. If necessary, other agents, e.g., anti-fading agents that enhance fluorescence or the like, can be added to the membrane before analysis to reduce background and increase signal or the like. Similarly, if necessary, the labeled RCA products can be bound (covalently or non-covalently) to the membrane before analysis, if desired.The chemical principles for attaching biomolecules to a surface are well known, and in certain cases, RCA products can be made using a modified nucleotide or a primer that has a group that is specifically reactive with the membrane surface, thus ensuring that only RCA products stick to the surface. In certain embodiments, the filtration step is performed by (1) placing the sample in a porous membrane; and (2) applying a force that causes the sample to move through the membrane. The force applied to the sample may be an active force (e.g., a centrifugal force, a negative pressure, or a positive pressure) or a passive force (e.g., through capillary action (e.g., using blotting paper) or evaporation). As noted above, the inner diameter of the pores in the membrane, the distance between the centers of adjacent membranes in the membrane, and the distance between the edges of adjacent pores in the membrane can be controlled by the deposition voltage, the type of acid, and other parameters (see, for example, Poinern, supra). In some embodiments, the inner diameter of the pores in the membrane may be in the range of 5 nm to 500 nm, e.g., 4 nm to 250 nm, 4 nm to 50 nm, 50 nm to 100 nm, 100 nm to 200 nm, or 200 nm to 500 nm. Independently, the average distance between the centers of adjacent pores in the membrane should be in the range of 50 nm to 1000 nm, e.g., 50 nm to 420 nm, 50 nm to 100 nm, 100 nm to 250 nm, 250 nm to 500 nm, or 500 nm to 1000 nm. The average distance between the edges of adjacent pores in the membrane can range from 10 nm to 500 nm, 10 nm to 50 nm, 50 nm to 200 nm, or 200 nm to 500 nm. It can be understood that the values ​​of diameter and average distance between pores presented here are typical and such values ​​can vary depending on the application. In some embodiments, the pore diameter and the distance between adjacent pore centers can be optimized to obtain labeled RCA products that are individually retrievable by the detection system used. In particular, in certain cases, the pores in the membrane can have a relatively large diameter (e.g., a diameter in the range of 100 nm to 500 nm) and the average distance between adjacent pore centers in the membrane can be relatively large (e.g., in the range of 200 nm to 1000 nm, e.g., 200 nm to 420 nm), such that each labeled RCA product is drawn to the entrance of a pore (an effective "blocking" pore) and the distance between adjacent labeled RCA products is retrievable by fluorescence microscopy. These optimal distances can be reduced using a very high-magnification fluorescence microscope (Huang et al, Annu Rev. Biochem. 2009 78: 993–1016). The membrane used can have a suitable thickness, for example in the range of 20 μm to 500 μm or 50 μm to 200 μm, and if desired, as mentioned above, it can contain one or more support structures (e.g. a support ring) to maintain the integrity of the membrane during use. As noted above, the present method can be used in protocols that require accurate quantitative determination of the number of RCA products in a sample, particularly a sample that has a variable concentration of RCA products (e.g., from 10 to 10M which can be a relatively low concentration, e.g., 5,000 to 1M RCA products in a volume of 50 to 200 μl or more) and the statistical magnification required to detect a difference can be achieved by counting only at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, or at least 200,000 or more RCA products. As will be described in more detail below, this method has particular utility in copy number analysis and in non-invasive prenatal diagnostic testing devices. In some embodiments, the sample may have multiple stacks of RCA products (e.g., two, three, or four or more stacks of RCA products, such as a first stack of RCA products and a second stack of RCA products) that are identifiably labeled in different stacks of RCA products, meaning that each member of each stack of RCA products can be individually tracked and counted even when the stacks are mixed. Fluorescent labeled pairs that are suitable for detection in the subject method include Cy-3 and Cy-5 (Amersham Inc., Piscataway, NJ), Quasar 570 and Quasar 670 (Biosearch Technology, Novato CA), Alexafluor555 and Alexafluor647 (Molecular Probes, Eugene, OR), BODIPY V-1002 and BODIPY V1005 (Molecular Probes, Eugene, OR), POPO-3 and TOTO-3 (Molecular Probes, Eugene, OR), and POPRO3 TOPRO3 (Molecular Probes, Eugene, OR). Other suitable detectable and detectable labels can be found in Kricka et al. (Ann Clin Biochem. 39:114-29, 2002).For example, RCA products can be labeled with any combination of ATTO, ALEXA, CY or dimeric cyanine dyes such as YOYO, TOTO, etc. Additional labels may be provided. Exemplary methods for making identifiable stacks of labeled RCA products are described, for example, in Patent Nos. PCT / US2014 / 06771 filed November 26, 2014 and PCT / US2014 / 067719 filed November 26, 2014, the contents of which are incorporated herein by reference. In some cases, a stack of RCA products can be identifiable by applying multiple labels, which increases the likelihood of partitioning. For example, in some cases, the RCA products are from stacks that may each be labeled with two identifiable dyes (e.g., both Cy3 and Cy5). When read, such RCA products will have two labels that are distinguishable from those RCA products labeled with a single dye (e.g., Cy3 or Cy5). In some embodiments, the first stack of RCA products may represent a "test" stack of labeled RCA products and the second stack of RCA products may represent a "reference" stack of RCA products such that the number of first RCA products can be compared. For example, in some embodiments, the first stack of RCA products may correspond to a first chromosomal region (e.g., a first chromosome such as chromosome 21) and the second stack of RCA products may correspond to a second chromosomal region (e.g., a second chromosome such as chromosome 13 or 18 or a region different from the first chromosome) and the number of first stacked RCA products and second stacked RCA products may be counted and compared to determine whether there is a difference in the copy number of the regions (indicating that the test region has a duplication or deletion). In some embodiments, the sample includes at least a first stack of RCA products and a second stack of RCA products, wherein the first and second stacks of labeled RCA products are identifiable during the labeling step.In these embodiments, the methods comprise quantifying (e.g., counting) the first labeled RCA products in a region of the membrane and quantifying (counting) the second labeled RCA products in a region (the same region or a different region) of the membrane, thereby providing an estimate of the amount of accumulation of the first and second RCA products in the sample. This embodiment can further comprise comparing the number of the first RCA products in the sample with the number of the second RCA products in the sample. In some embodiments of the methods, the methods can comprise imaging the first and second stacks of labeled RCA products to produce one or more images (e.g., one image or a first image and a second image, respectively) and optionally (1) determining the amount of labeled RCA products in the one or more images, thereby providing an estimate of the amount of the first and second stacks of labeled RCA products in the sample. The first and second stacks of labeled RCA products can be separately detected using known methods (e.g., using appropriate filters, etc.). Detection of the RCA products can be performed serially, in which case, e.g., separate images can be obtained for each label and the RCA products from each stack can be analyzed serially. Detection of the RCA products can also be performed substantially simultaneously (e.g., in which case, a single image representing both stacks of RCA products can be obtained and the RCA products from each stack can be analyzed substantially simultaneously).These embodiments of the methods can further include comparing the number of first RCA products in the sample with the number of second RCA products in the sample. This step of the methods can include counting at least 1,000 (e.g., at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 500,000 to 1M or more) labeled RCA products in the first stack and counting at least 1,000 (e.g., at least 5,000, at least 10,000, at least 20,000 or at least 50,000, at least 100,000, at least 500,000 to a maximum of 1M or more) labeled RCA products in a region of the membrane, thereby ensuring that the difference in copy number can be measured with statistical accuracy. In embodiments, the methods can further comprise detecting a difference between the amount of the first RCA products in the sample and the amount of RCA products in the sample, where the difference has a P-value of at least 0.95, at least 0.98, or at least 0.99. In some embodiments, the method can be used to detect at least a 1% difference, at least a 2% difference, at least a 3% difference, or at least a 5% difference, with a P-value greater than 0.95, 0.98, or 0.99. For example, in embodiments involving a maternal sample from a pregnant woman, the fetal fragility of the sample can be included in this calculation. As noted above, in some cases the sample analyzed using this method can be a sample of cell-free DNA obtained from blood, for example from the blood of a pregnant woman. In these embodiments, this method can be used, for example, to detect abnormal chromosomes in a developing fetus (as described above) or to calculate the fragility of fetal DNA in the sample. Copy number abnormalities that can be detected using this method include, but are not limited to, trisomy 21, trisomy 13, trisomy 18, trisomy 16, XXY, XYY, XXX, monosomy X, monosomy 21, monosomy 22, monosomy 16, and monosomy 15. Other copy number abnormalities that can be detected using the present method are listed in the table below. Abnormal chromosome X-linked disease XO Turner syndrome Y XXY Klinefelter syndrome Y XYY Two-syndrome YY XXX Trisomy syndrome XY XXXX Four-syndrome XY Deletion Xp21 Duchene's / Becker syndrome, congenital adrenal hyperplasia, Cui syndrome Y Deletion Xp22 Steroid sulfatase disorder Y Deletion Xp26 X-linked lympho-olfactory disease 1 1p (Somatic) Monosomy Trisomy Neuroblastoma 2 Monosomy Trisomy 2q Growth retardation, developmental and cerebral delay, and dwarfism 3 Monosomy Trisomy (Somatic) Non-Hodgkin lymphoma 4 Monosomy Trisomy (Somatic) Acute lymphoblastic leukemia (ANLL) 5 5p Cri du chat; Lejeune syndrome 5 5p (Somatic) Monosomy Trisomy Myelodysplastic syndrome 6 Monosomy Trisomy (Somatic) Malignant connective tissue tumor 7 7q11.23 deletion William syndrome 7 Monosomy Trisomy Monosomy 7 syndrome Childhood; Somatic: Adrenal adenoma; Myelodysplastic syndrome 8 8q24 deletion.1 Langer-Giedon syndrome 8 monosomy trisomy myelodysplastic syndrome; Warkany syndrome, somatic: Kiewit syndrome 9 monosomy 9p Alfi syndrome 9 monosomy 9p partial trisomy Rethore syndrome 9 trisomy complete trisomy 9 syndrome; mosaic trisomy 9 syndrome 10 monosomy trisomy (somatic) ALL or ANLL 11 11p- Aniridia; Wilms tumor 11 11q- Jacobson syndrome 11 monosomy (somatic) Hereditary mylenoid trisomy (ANLI,MDS) 12 monosomy trisomy (somatic) CLL, JUvenile granulosa cell tumor (JGCT) 13 13q- 13q syndrome; Orbeli syndrome 13 13q14 deletion Retinoblastoma 13 Monosomy Trisomy Patau syndrome 14 Monosomy Trisomy (Somatic) Myeloid disorder (MDS, ANLL, atypical CML) 15 15q11-q13 deletion Prader-Willi, Angelman syndrome 15 Trisomy (Somatic) Myeloid and lymphoid hereditary e.g. MDS, ANLL, ALL, CLL) 16 16q13.3 deletion Rubenstein-Taybi 3 Monosomy Trisomy (Somatic) Papillary intestinal cell carcinoma (malignant) 17 17p-(Somatic) 17p syndrome in malignant myeloid 17 17q11.2 deletion Smith-Magenis 17 17q13.3 Miller-Dieker 17 Monosomy Trisomy (Somatic) Renal cortical adenoma 17 17p11.Trisomy 2-12 Charcot-Marie Tooth syndrome type 1; HNPP 18 18p- Partial monosomy 18p syndrome or Grouchy Lamu Thieffry syndrome 18 18q- Grouchy Lamy Salmon Landry syndrome 18 Monosomy trisomy Edwards syndrome 19 Monosomy trisomy 20 20p- Trisomy 20p syndrome 20 deletion 20p11.2-12 Alagille 20 20q- Somatic: MDS, ANLL, polycythemia vera, chronic neutophilic leukemia 20 Monosomy trisomy (somatic) Papillary intestinal cell carcinoma (malignant) 21 Monosomy trisomy Down syndrome 22 deletion 22q11.2 DiGeorge syndrome, velocardiofacial syndrome, conotruncal anomaly face syndrome, autosomal dominant Opitz G / BBB syndrome, Caylor cardiofacial syndrome 22 Monosomy trisomy Trisomy 22 complete. Exemplary methods for manufacturing RCA products and labeling them are described, for example, in patent applications PCT / US2014 / 06771 filed November 26, 2014 and PCT / US2014 / 067719 filed November 26, 2014, the contents of which are incorporated herein by reference. Composition A composition is also provided. In some embodiments, the composition can comprise: a) a porous capillary membrane, e.g., an anodic aluminum oxide membrane, and b) an array of fluorescently labeled RCA products on a membrane surface. The array can comprise at least 1,000 labeled RCA products (e.g., at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 500,000, or at least 1M labeled RCA products), and the labeled RCA products can be distributed across the membrane surface in a random manner at a concentration of, e.g., at least 10, at least 500, at least 1,000, at least 5,000, or at least 10,000 / mm2. As described above, the composition can comprise a porous capillary membrane; and at least two stacks of fluorescently labeled RCA products on a membrane surface wherein the different stacks of labeled RCA products are identifiably labeled. Further details and embodiments of this composition can be found in the Methods section of this disclosure. Kits Kits are also provided for practicing the methods of the subject disclosure as described above. In these embodiments, a kit can contain at least: reagents for circularizing the selected segment in a strand-specific manner and then performing a rotating circular amplification technique on the circularized products. For example, one or more restriction enzymes, a ligase, and one or more oligonucleotides that can act as a spacer for circularizing the products, a polymer from the isolated spacer for amplifying the circularized products by RCA, one or more labeled oligonucleotides for labeling the RCA products, and a porous capillary membrane, e.g., an anodic aluminum oxide membrane as described above. The various components of the kit can be in different compartments or specific compatible components can be pre-packaged in a single compartment if desired. Further details and components of this kit can be found in the Methods section of this disclosure. In addition to the components mentioned above, the kits of this subject matter can further include instructions for using the kit components to perform the methods of the subject matter, e.g., sample analysis instructions. Instructions for performing the methods of this subject matter are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, e.g., paper or plastic, etc. Similarly, the instructions can be provided in the kits as a packaging label, on a label of the kit container or components thereof (e.g., with the package or within the package), etc. In other embodiments, the instructions are in the form of an electronic data file on a suitable computer-readable medium, e.g., CD-ROM, diskette, etc. Again, in some embodiments, the original instructions are not in the kit but the instructions are accessible from a remote source, e.g., via the Internet. An example of this embodiment is a kit that includes a website address where the instructions can be viewed and / or the instructions can be downloaded.In the case of instructions, the method of accessing the instructions is included on an appropriate layer. Appearances What follows is an example of this method. In some embodiments, the method may include: (a) filtering a liquid sample containing fluorescently labeled rotating circular amplification (RCA) products using an anodic aluminum oxide membrane, whereby an array of RCA products is formed on the membrane; and (b) quantifying the amount of labeled RCA products in the region of the membrane, thereby providing an estimate of the amount of labeled RCA products in a sample. In some embodiments, the quantifying step may include counting the number of first and second labeled RCA products in the region. In other embodiments, the quantifying step may include detecting an expression signal from the region. In embodiments, step (a) of the method can be performed by: (1) placing the sample on an anodic aluminum oxide membrane and (2) applying a force that forces the sample through the membrane. In such embodiments, the force can be an active force (a centrifugal force, negative pressure, or positive pressure) or a passive force (which may be provided by capillary action or evaporation). In some embodiments, the method can include washing the porous anodic aluminum oxide membrane between steps (a) and (b). In some embodiments, the step of determining the amount can include counting at least 1,000 labeled RCA products in the image area. In embodiments, the inner diameter of the pores in the membrane can be in the range of 5 nm to 500 nm and independently, the average distance between the centers of adjacent pores in the membrane can be in the range of 50 nm to 1000 nm. In embodiments, the average distance between the edges of adjacent pores in the membrane can be in the range of 10 nm to 500 nm. In some embodiments, the sample can include at least a first stack and a second stack of labeled RCA products, wherein the first and second stacks of labeled RCA products are identifiable. In such embodiments, the method can further comprise imaging the first and second stacks of labeled RCA products to produce one or more images (e.g., a first image and a second image, respectively). These methods can further consist of (1) counting the number of labeled RCA products in the first image area and (2) separately counting the number of labeled RCA products in a region, for example, the same region as the second image, thereby providing an estimate of the number of first and second clusters of RCA products in the sample.This further embodiment may include comparing the number of first labeled RCA products in the sample with the number of second labeled RCA products in the sample. As mentioned, a composition can comprise: a) a porous capillary membrane; and b) an array of fluorescently labeled rotating circular amplification (RCA) products on a membrane surface. In some of these embodiments, the porous capillary membrane can be a porous anodic aluminum oxide membrane. In some embodiments, the array can comprise at least 1,000 RCA products. In some embodiments, the composition can comprise multiple arrays of fluorescently labeled RCA products, where different arrays are located on the membrane surface and are identifiably labeled. Descriptions of each of these agents, as well as selection agents that may be present in the composition, can be found in the previous description. Also provided is a kit comprising: a) a porous capillary membrane; and b) reagents for circularizing DNA and performing the rotating circular amplification (RCA) technique; and c) reagents for labeling RCA products. Descriptions of each of these components, as well as selection factors that may be present in the kit, can be found in the previous description. Embodiment 1 is a method of analyzing a sample comprising: (a) filtering a liquid sample containing products of the rotating circular amplification (RCA) technique through a transparent porous capillary membrane, resulting in the concentration of the RCA products and the production of an array of RCA products on the membrane; (b) fluorescently labeling the RCA products before or after step (a); and (c) counting the number of individual labeled RCA products in an area of ​​the membrane, resulting in an estimate of the number of labeled RCA products in the sample. Embodiment 2. The method of Embodiment 1, wherein the transparent porous capillary membrane is a porous anodic aluminum oxide membrane. Embodiment 3. The method of embodiment 1 or 2, wherein step (b) is performed by coupling fluorescently labeled oligonucleotides to the RCA products, before or after step (a). Embodiment 4. The method of any preceding embodiment, wherein the method comprises imaging a region of the membrane to produce one or more images and counting the number of individual labeled RCA products in the one or more images. Embodiment 5. The method of any preceding embodiment, wherein step (a) is performed by: (1) placing the sample on a transparent porous capillary membrane and (2) applying a force that forces the sample through the membrane. Embodiment 6. The method of Embodiment 5, wherein the force is an active force or a passive force. Embodiment 7. The method of embodiment 6, wherein the active force is a centrifugal force, negative pressure, or positive pressure. Embodiment 8. The method of embodiment 6, wherein the passive force is applied by capillary action or evaporation. Embodiment 9. The method of any of the preceding embodiments, further comprising washing the transparent porous capillary membrane between steps (a) and (b). Embodiment 10. The method of any preceding embodiment, wherein the counting step comprises counting at least 1,000 labeled RCA products in the membrane region. Embodiment 11. The method of any preceding embodiment, wherein the inner diameter of the pores in the membrane is in the range of 2 nm to 500 nm. Embodiment 12. The method of any preceding embodiment, wherein the average distance between adjacent pore centers in the membrane is in the range of 50 nm to 1000 nm. Embodiment 13. The method of any preceding embodiment, wherein the average distance between the edges of adjacent pores in the membrane is in the range of 10 nm to 500 nm. Embodiment 14. The method of any preceding embodiment, wherein the sample comprises at least a first stack of RCA products and a second stack of RCA products, wherein the first and second stacks of labeled RCA products are detectably labeled in step (b). Embodiment 15. The method of embodiment 11, wherein the method comprises counting the number of first labeled RCA products in a region of the membrane and counting the number of second labeled RCA products in a region of the membrane, thereby providing an estimate of the number of first and second aggregated RCA products in the sample. Embodiment 16. The method of Embodiment 15, further comprising comparing the number of first RCA products in the sample with the number of second RCA products in the sample. Examples The following examples are provided to illustrate how the present invention is made and used to provide those skilled in the art with an illustration of how the invention is made and used, and are not intended to limit the scope of what the inventors of the invention intended, nor are they intended to be all or only experiments that have been performed. Example 1 Using an AAO filter Introduction The first step in many molecular diagnostic applications is the conversion of a patient's biological material, such as DNA, into an active biomarker. The DNA is extracted, often purified, and further converted into surrogate molecules, such as rotating circular products (RCPs), which can be measured or quantified. The detection of these surrogate molecules is usually accomplished by specific binding of fluorescent dyes. Different dyes can be attached to different surrogate markers, allowing for the detection of distributions even in complex mixtures. Spreading onto flat surfaces, such as glass plates or slides, allows the counting of labeled molecules by microscopy or scanning. A particular challenge arises when the sample concentration in a solution is low and it is difficult to disperse a sufficient number of molecules for accurate counting. Coating glass slides with agents such as poly l-lysine (PLL), aminosilanes, or their equivalents has been shown to be an effective method for improving the dispersion of surrogate markers that are mostly DNA-containing (see, for example, PCT / US2014 / 06771 filed November 26, 2014 and PCT / US2014 / 067719 filed November 26, 2014). This improves the dispersion efficiency somewhat, however, many molecules remain in solution and are not included in subsequent analysis. In an attempt to increase the dispersion of fluorescently labeled biomarkers on a smooth surface, the following filtration method has been developed. Materials and methods Devices: Aluminum oxide membranes are bonded to suitable plate structures that are either fabricated in-house or purchased from Ibidi (part # 81201). Aminosilane-coated plates (96-Well Glass Bottom Plates SuperAmine, part# M96M) Purchased from ArrayIt Corporation. Two separate rotating circular amplification products were generated using methods previously described (PCT / US2014 / 06771). One RCA reaction was labeled by coupling an additional tracer oligonucleotide containing a 5' Atto 550 tag, the other RCA reaction was labeled in a similar manner with 5' Atto 647N. The RCA reactions were mixed in a 50:50 mixture at a concentration of 50 fM. For glass plates, 100 ul of the reaction was sampled into 3 wells of the plate and incubated at room temperature for 16 hours. Another set of 3 wells was incubated for 90 seconds. After incubation, the wells were rinsed twice with 2x SSC and allowed to dry. For membranes, 100 ul of samples were added to the membrane end plates and the plates were then placed on blotting paper (VWR part# 28298-022). Samples were passed through the aluminum oxide membrane in approximately 90 seconds. The membrane end plate was removed from the blotting paper and 2 drops of anti-fade reagent (Life Technologies) were added to each well to make it clear. Imaging for both formats was performed with an Olympus X81 microscope with a 20X objective and a Hamamatsu Orca 4.0lt camera. Imaging was performed with a 9×9 image tiling arrangement to ensure good coverage of the entire end of each well. Images were analyzed and RCA products were counted using software developed for this purpose. Results The results are summarized in Figure 3. With incubation for 16 hours, spreading on glass plates resulted in average counts of 2,000 to 4,000 per image for Atto 550-labeled RCA products. For Atto 647N, the average counts per image were between 2,000 and 3,000. Incubation for 90 seconds on glass plates resulted in average counts of between 500 and 2,500 per image for each of the two channels. In contrast, incubation / spreading on aluminum oxide plates resulted in average counts of 6,000 to 10,000 per image for Atto 500-labeled RCPs and between 6,000 and 9,000 for 674N-labeled RCA products. The data show that spreading on an aluminum oxide membrane results in approximately 4 times more counts while spreading on a glass plate has a 90 second time interval. If the incubation time for the glass plate is increased to 16 hours, the result is an increase in RCA products detected on the glass plate, although still 2.5 times less than when placed on aluminum oxide for 90 seconds. Example 2 Multiple tracking methods Materials and methods Devices: Aluminum oxide membranes with 20nm pores were bonded to suitable 96 well plate structures custom-made by 4titude Ltd, UK. In this multiplex experiment, DNA mixtures from 2 cell lines were used as genetic starting materials. The cell line DNA contained either 2 copies of chromosome 21 (normal genetic production) or the cell lines contained 3 copies of chromosome 21 (trisomy 21). DNA extracted from the cell lines was mixed in ratios of 100:0, 95:5, 90:10, 0:100. Each DNA mixture was first digested using restriction enzymes, ligated and transferred to a test set and subsequently enzymatically amplified by RCA as previously described (see WO2015083001 and WO2015083002). Two chromosome-specific tracer oligonucleotides (Atto 550 for chromosome 18, Atto 647 for chromosome 21) were added to each sample and allowed to anneal. The chromosome-specific RCA products in each sample were then fluorescently labeled. After labeling, 100 μl of sample was added to the membrane end plates and the plates were then placed on a vacuum manifold (Supleco part #66879-U). Samples were passed through the aluminum oxide membrane in approximately 90 seconds. The membrane end plate was washed twice with 400 μl of 0.5X SSC and then allowed to dry. Three hundred μl of wetting agent / preservative was then applied to each well to make them transparent and to attach the RCA products to the membrane. Imaging was performed with an Olympus X81 microscope with a 20X objective and a Hamamatsu Orca 4.0lt camera. Imaging was performed with a 10×10 image tile array to cover the entire end of each well. Images were analyzed and RCA products were counted using software developed for this purpose. Results The results are summarized in Figure 4. The average count per image for all 91 samples in the experiment ranged from 3000 to just over 5000 per image. The histograms clearly show the difference in the ratio of counts between channels 550 and 647 at the 0:100 mix ratio (last 22 replicates), however it is very difficult to distinguish the difference in the ratio between the 0, 5 and 10% samples by numbers alone. Figure 5 is a plot showing the ratio of counts for the two channels in relation to the cell line mix composition. The trend is clearly visible in this graph, showing the change in the relative ratio of counts following the ratio of input cell line DNA samples. The data show that spreading on the aluminum oxide membrane results in approximately 4 times more counts while spreading on the glass plate has a 90 second time interval. If we increase the incubation time for the glass plate to 16 hours, the result is an increase in RCA products detected on the glass plate, although they are still 2.5 times less than when placed on aluminum oxide for 90 seconds. Claims 1. A sample analysis method consisting of: (a) filtering a liquid sample containing products of the rotating circular amplification (RCA) technique using a porous capillary membrane, whereby a range of RCA products is produced on the membrane; wherein the sample contains at least a first pool of RCA products and a second pool of RCA products, wherein the first and second pools of labeled RCA products are identifiably labeled; and (b) determining the accumulated amount of the first labeled RCA products and the accumulated amount of the second labeled RCA products in a region of the membrane. #x200f2.#x200f#x200f #x200fThe method of claim 1, wherein the step of determining the value of (b) comprises counting the number of first and second labeled RCA products in the area. #x200f3.#x200f#x200f #x200fThe method of claim 1, wherein the step of determining the value (b) comprises tracking the accumulated signal from the region. 4. The method of any preceding claim, wherein the porous capillary membrane is a porous anodic aluminum oxide membrane. 5. The method of any preceding claim, wherein the RCA products are detectably labeled by coupling fluorescently labeled oligonucleotides to the RCA products prior to step (a). 6. The method of any preceding claim, wherein the method comprises imaging a region of the membrane to produce one or more images and determining the number of individual labeled RCA products in the one or more images. 7. The method of any of the preceding claims, wherein step (a) is performed by: (1) placing the sample on a porous capillary membrane; and (2) Applying force to force the sample through the membrane. 8. The method of claim 7, wherein the force is an active force or a passive force. 9. The method of claim 8, wherein the active force is a centrifugal force, negative pressure, or positive pressure. 10. The method of claim 8, wherein the passive force is applied by capillary action or evaporation. 11. The method of any of the preceding claims, further comprising washing Capillary membrane between steps (a) and (b). 12. The method of any preceding claim, wherein the counting step comprises counting at least 1,000 labeled RCA products in the membrane region. 13. The method of any preceding claim, wherein the inner diameter of the pores in the membrane is in the range of 2 nm to 500 nm. 14. The method of any preceding claim, wherein the average distance between adjacent pore centers in the membrane is in the range of 50 nm to 1000 nm. 15. The method of any preceding claim, wherein the average distance between the edges of adjacent pores in the membrane is in the range of 10 nm to 500 nm. 16. The method of any preceding claim further comprising comparing the amount of first RCA products in the sample with the number of second RCA products in the sample. 17. The method of any preceding claim, wherein the porous capillary membrane is clarified using a wetting agent between steps (a) and (b). #x200f18.#x200f#x200f #x200fA sample analysis method consists of the following: (a) filtering a liquid sample containing products of the rotating circular amplification (RCA) technique using a support that acts as a filter for the RCA products, whereby a range of RCA products is produced on the membrane; wherein the sample contains at least a first pool of RCA products and a second pool of RCA products, wherein the first and second pools of labeled RCA products are identifiably labeled; and (b) determining the accumulated amount of the first labeled RCA products and the accumulated amount of the second labeled RCA products in a region of the membrane. #x200f19.#x200f#x200f #x200fThe method of claim 18, wherein the step of determining the value of (b) comprises counting the number of first and second labeled RCA products in the area. #x200f20.#x200f#x200f #x200fThe method of claim 18, wherein the determining step (b) comprises tracking the accumulated signal from the region. 21. The method of any of claims 18-20, wherein the porous capillary membrane is a porous anodic aluminum oxide membrane. 22. The method of any of claims 18-21, wherein the RCA products are detectably labeled by coupling fluorescently labeled oligonucleotides to the RCA products prior to step (a). 23. The method of any of claims 18-22, wherein the method comprises imaging a region of the membrane to produce one or more images and determining the number of individual labeled RCA products in the one or more images. 24. The method of any of claims 18-23, wherein step (a) is performed by: (1) placing the sample on a porous capillary membrane; and (2) Applying force to force the sample through the membrane. 25. The method of claim 24, wherein the force is an active force or a passive force. 26. The method of claim 25, wherein the active force is a centrifugal force, negative pressure, or positive pressure. 27. The method of claim 25, wherein the passive force is applied by capillary action or evaporation. 28. The method of any of claims 18-27, further comprising washing Capillary membrane between steps (a) and (b). 29. The method of any of claims 18-28, wherein the counting step comprises counting at least 1,000 labeled RCA products in the membrane region. 30. The method of any of claims 18-29, wherein the inner diameter of the pores in the membrane is in the range of 2 nm to 500 nm. 31. The method of any of claims 18-30, wherein the average distance between adjacent pore centers in the membrane is in the range of 50 nm to 1000 nm. 32. The method of any of claims 18-31, wherein the average distance between the edges of adjacent pores in the membrane is in the range of 10 nm to 500 nm. 33. The method of any of claims 18-32, further comprising comparing the amount of first RCA products in the sample with the number of second RCA products in the sample. 34. The method of any of claims 18-33, wherein the porous capillary membrane is clarified using a wetting agent between steps (a) and (b). Abstract A method of analyzing a sample is provided. In certain embodiments, the method may include: (a) filtering a liquid sample containing products of the rotating circular amplification (RCA) technique using a porous capillary membrane, whereby a range of RCA products is produced on the membrane; wherein the sample contains at least a first pool of RCA products and a second pool of RCA products, wherein the first and second pools of labeled RCA products are identifiably labeled; and (b) determining the amount of the first labeled pool of RCA products and the amount of the second labeled pool of RCA products in a region of the membrane.

Claims

CLAIMS 1. A method of sample analysis, comprising: (a) filtering a liquid sample containing rolling circle amplification (RCA) products using a porous capillary membrane, thereby producing an array of the RCA products on the membrane; wherein the sample contains at least a first population of RCA products and a second population of RCA products, wherein the first and second populations of labeled RCA products are distinguishably labeled; and (b) determining the amount of the first labeled population of RCA products and the amount of the second labeled population of RCA products in an area of the membrane.

2. The method of claim 1, where determining step (b) includes counting the numbers of first and second labeled RCA products in the area.

3. The method of claim 1, where determining step (b) includes detecting an aggregate signal from the area.

4. The method of any prior claim, wherein the porous capillary membrane is a porous anodic aluminum oxide membrane.

5. The method of any prior claim, wherein the RCA products are distinguishably labeled by hybridizing fluorescently labeled oligonucleotides to the RCA products, prior to step (a).

6. The method of any prior claim, wherein the method comprises imaging an area of the membrane to produce one or more images and determining the number of the individual labeled RCA products in the one or more images.

7. The method of any prior claim, wherein step (a) is done by: (i) placing the sample on the porous capillary membrane; and (ii) applying a force that moves the sample through the membrane.

8. The method of claim 7, wherein the force is an active force or a passive force.

9. The method of claim 8, wherein the active force is a centrifugal force, negative pressure or positive pressure.

10. The method of claim 8, wherein the passive force is applied by capillary action or evaporation.

11. The method of any prior claim, further comprising washing the porous capillary membrane between steps (a) and (b).

12. The method of any prior claim, wherein the counting step comprises counting at least 1,000 labeled RCA products in the area of the membrane.

13. The method of any prior claim, wherein the interior diameter of the pores in the membrane is in the range of 2 nm to 500nm.

14. The method of any prior claim, wherein the average distance between the centers of adjacent pores in the membrane is in the range of 50 nm to 1000 nm.

15. The method of any prior claim, wherein the average distance between the edges of adjacent pores in the membrane is in the range of 10 nm to 500 nm16. The method of any prior claim, further comprising comparing the amount of first RCA products in the sample to the number of second RCA products in the sample.

17. The method of any prior claim, wherein the porous capillary membrane is made transparent using a wetting agent between steps (a) and (b).

18. A method of sample analysis, comprising: (a) filtering a liquid sample containing rolling circle amplification (RCA) products using a capture support that acts as a filter for the RCA products, thereby producing an array of the RCA products on the support; wherein the sample contains at least a first population of RCA products and a second population of RCA products, wherein the first and second populations of labeled RCA products are distinguishably labeled; and (b) determining the amount of the first labeled population of RCA products and the amount of the second labeled population of RCA products in an area of the membrane.

19. The method of claim 18, where determining step (b) includes counting the numbers of first and second labeled RCA products in the area.

20. The method of claim 18, where determining step (b) includes detecting an aggregate signal from the area.

21. The method of any of claims 18-20, wherein the porous capillary membrane is a porous anodic aluminum oxide membrane.

22. The method of any of claims 18-21, wherein the RCA products are distinguishably labeled by hybridizing fluorescently labeled oligonucleotides to the RCA products, prior to step (a).

23. The method of any of claims 18-22, wherein the method comprises imaging an area of the membrane to produce one or more images and determining the number of the individual labeled RCA products in the one or more images.

24. The method of any of claims 18-23, wherein step (a) is done by: (i) placing the sample on the porous capillary membrane; and (ii) applying a force that moves the sample through the membrane.

25. The method of claim 24, wherein the force is an active force or a passive force.

26. The method of claim 25, wherein the active force is a centrifugal force, negative pressure or positive pressure.

27. The method of claim 25, wherein the passive force is applied by capillary action or evaporation.

28. The method of any of claims 18-27, further comprising washing the porous capillary membrane between steps (a) and (b).

29. The method of any of claims 18-28, wherein the counting step comprises counting at least 1,000 labeled RCA products in the area of the membrane.

30. The method of any of claims 18-29, wherein the interior diameter of the pores in the membrane is in the range of 2 nm to 500nm.

31. The method of any of claims 18-30, wherein the average distance between the centers of adjacent pores in the membrane is in the range of 50 nm to 1000 nm.

32. The method of any of claims 18-31, wherein the average distance between the edges of adjacent pores in the membrane is in the range of 10 nm to 500 nm33. The method of any of claims 18-32, further comprising comparing the amount of first RCA products in the sample to the number of second RCA products in the sample.

34. The method of any of claims 18-33, wherein the porous capillary membrane is made transparent using a wetting agent between steps (a) and (b). ABSTRACT A method of sample analysis is provided. In certain embodiments, the method may comprise: (a) filtering a liquid sample containing rolling circle amplification (RCA) products using a porous capillary membrane, thereby producing an array of the RCA products on the membrane; wherein the sample contains at least a first population of RCA products and a second population of RCA products, wherein the first and second populations of labeled RCA products are distinguishably labeled; and (b) determining the amount of the first labeled population of RCA products and the amount of the second labeled population of RCA products in an area of the membrane.

Images