Polynucleotide capture materials and methods of use thereof

Abstract

To provide methods for processing polynucleotide-containing biological samples, and materials for capturing polynucleotide molecules such as RNA and / or DNA from such samples.SOLUTION: The RNA and / or DNA is captured by polyamideamine (PAMAM (Generation 0)) bound to a surface, such as the surface of magnetic particles. The methods and materials have high binding ability and high releasing efficiency of RNA and DNA, and thereby permit quantitative determinations.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claim This application claims the benefit under 35 USC 119(e) of U.S. Provisional Application No. 60 / 959,437, filed July 13, 2007, which is incorporated by reference in its entirety. The technology described herein relates generally to methods for processing biological samples, and more particularly to materials that capture polynucleotide molecules, such as RNA and DNA, from said samples and enable their quantitative measurement. [Background technology]

[0002] The analysis of biological samples, such as clinical samples or food test samples, to determine the presence of pathogens such as viruses or the presence of specific genes generally involves the detection of one or more polynucleotides present in the sample.One type of detection is qualitative detection, which involves determining the presence or absence of target polynucleotides and / or determining information related to the type, size, presence or absence of mutations, and / or sequence of target polynucleotides.Another type of detection is quantitative detection, which involves determining the amount of specific polynucleotides present in a sample, for example, expressed as concentration or absolute amount by mass or volume.Detection can also include both qualitative and quantitative aspects.However, quantitative detection is generally more difficult than simply qualitatively determining the presence or absence of polynucleotides. Many detection methods involve the use of enzymes. For example, some detection methods involve amplifying the polynucleotide by polymerase chain reaction (PCR) or related amplification techniques. Other detection methods that do not amplify the polynucleotide being detected also use enzymes. However, the function of enzymes used in such techniques can be inhibited by the presence of substances (known as inhibitors) that accompany the polynucleotide in many biological samples, particularly clinical samples. Inhibitors can, for example, interfere with the efficiency and / or specificity of the enzyme.

[0003] Today, polynucleotide detection is moving towards ever faster and ever more sensitive techniques. For example, rapid and accurate diagnosis of viral infections is crucial for accurate patient management by directing the administration of appropriate antiviral therapy, eliminating unnecessary antibiotic use, and monitoring individualized versus prescribed regimens. Given the significant advantages of sensitivity, specificity, and time to results, polynucleotide detection (or nucleic acid testing) has become the presumptive international standard for viral diagnosis. However, the application of nucleic acid testing for routine diagnosis of viral targets has been limited to large clinical laboratories or large hospital laboratories due to the high cost, complexity, and level of competence required to perform such tests. Although significant improvements have been made in recent years, successful detection of RNA viruses in particular often requires laborious extraction methods that rely on the use of toxic chemicals. Furthermore, RNA molecules can be very unstable and therefore require delicate handling during their measurement. To date, these challenges have been overcome using large, expensive, and time-consuming robotic devices. With the ongoing demands on clinical medicine, laboratories performing diagnostic testing on patient samples see substantial benefits in being extremely high throughput; the shorter the time to reach a diagnostic result for a given sample, the better served the laboratory. In some cases, the smaller the actual sample being tested, the more quickly the test can be performed. More recently, there has been an increasing need for small, easy-to-use, low-cost automated platforms for the extraction of high-quality RNA from viral targets in clinical specimens.

[0004] Correspondingly, therefore, the need to be able to isolate minute quantities of polynucleotides from complex biological samples in a manner that effectively avoids the presence of inhibitors or reduces their deleterious effects is more important than ever. Furthermore, given the availability of a variety of stand-alone automated amplification devices, it is desirable to be able to routinely and reliably extract from unprocessed clinical samples quantities of polynucleotides that are amenable to amplification in terms of purity and quantity. The discussion of the background art herein is provided to explain the state of the art and should not be construed as an admission that anything mentioned herein was previously published, known, or part of the common general knowledge as of the priority date of any of the appended claims. Throughout the description and claims, the term "comprise" and its variants, such as "comprising" and "comprises," is not intended to exclude other additives, ingredients, integers or steps. Summary of the Invention [Problem to be solved by the invention]

[0005] The technology described herein generally provides methods for processing biological samples, and more specifically, materials that allow for the capture of polynucleotide molecules, such as RNA and DNA, from said samples and their quantitative measurement. [Means for solving the problem]

[0006] The methods and materials described herein are applicable to numerous test targets, and are particularly applicable to RNA-based test targets, such as influenza (A and B), RSV, HSV, CMV, adenovirus, and enterovirus. The technology described herein provides excellent capture and recovery of not only RNA but also DNA through the use of microparticles with high RNA and DNA binding capacities, e.g., 100 μg / mg beads and >90% release efficiency. In an exemplary embodiment, 8-10 μg of RNA can be extracted from overnight cultures. The methods described herein enable very rapid (15-20 minutes, including lysis) RNA extraction from cellular or viral material in a single-vessel process. The methods described herein involve a streamlined procedure with a small number of steps (e.g., 6) that progress from raw samples to purified RNA. Thus, the methods provide highly effective pretreatment of RNA from raw biological samples, thereby enabling PCR to be performed on them. The methods and processes are applicable across a variety of sample matrices used to collect raw samples, as well as clinical buffers, e.g., M4, UTM, and Todd Hewitt Broth. Suitable targets that have assays used in clinical trials and that can be the subject of the sample preparation methods described herein include, but are not limited to, Chlamydia trachomatis (CT); Neisseria gonorrhea (GC); Group B Streptococcus; HSV; HSV typing; CMV; Influenza A&B; MRSA; RSV; TB; Trichomonas; Adenovirus; Bordetella; BK; JC; HHV6; EBV; Enterovirus; and Mycoplasma pneumoniae (M. pneumoniae).

[0007] One aspect of the present invention relates to methods for processing one or more RNA and / or DNA compounds (e.g., concentrating and / or isolating RNA and / or DNA compound(s) from inhibitory compounds (e.g., hemoglobin, peptides, fecal matter, humic acid, mucus matter, DNA-binding proteins, or carbohydrates) that may inhibit the detection and / or amplification of the RNA and / or DNA compounds). In some embodiments, the method includes contacting a sample containing RNA and / or DNA compounds with a PAMAM (generation 0) that selectively binds (e.g., retains) the RNA and / or DNA compounds as opposed to inhibitors. The PAMAM (generation 0) is typically bound to a surface (e.g., the surface of one or more particles). The PAMAM (generation 0) retains the RNA and / or DNA compounds, and the RNA and / or DNA compounds can be separated from the inhibitor, for example, by washing the RNA and / or DNA compounds bound to the surface with the compounds. After separation, the association of the RNA and / or DNA compounds with the PAMAM (generation 0) can be disrupted, releasing (e.g., separating) the RNA and / or DNA compounds from the compounds and the surface.

[0008] The present disclosure provides a method for isolating polynucleotides from a cell-containing sample, the method comprising contacting the sample with a lysis solution and a plurality of binding particles coated with PAMAM (generation 0) so that the polynucleotides are released from the cells and bind to the PAMAM (generation 0), thereby creating a solution containing the polynucleotide-bound binding particles and remaining cellular material; clustering the polynucleotide-bound binding particles; removing the solution containing the remaining cellular material; washing the binding particles; and releasing the polynucleotides from the binding particles.

[0009] The disclosure further includes a method for concentrating RNA from a sample containing a polymerase chain reaction inhibitor, the method comprising contacting 500 μl to 1 ml of the sample with a plurality of RNA-binding particles, wherein the binding particles are configured to selectively retain RNA in the sample relative to the polymerase chain reaction inhibitor; concentrating the plurality of particles having one or more polynucleotides bound thereto to a working volume of 50 nanoliters to 5 microliters; and releasing the one or more polynucleotides into <30 μl of solution. The present disclosure further includes a composition comprising: a carboxyl-modified microparticle; and PAMAM (generation 0) bound to one or more of the carboxylic acid groups on the microparticle with one or more amine groups per molecule. The disclosure further includes a kit comprising a number of sealed tubes, each containing a lysis buffer; a tube containing lyophilized microparticles bound to PAMAM (Generation 0); a tube containing a liquid wash reagent sufficient to analyze a number of samples; and a tube containing a liquid release reagent sufficient to analyze a number of samples, wherein each component of the kit is stored in an airtight container. The present disclosure further includes a kit comprising: a first airtight container enclosing a number of tubes, each tube containing freeze-dried microparticles bound to PAMAM (Generation 0); a second airtight container enclosing a number of reagent holders, each holder including a tube containing a liquid dissolution agent; a tube containing a liquid washing reagent; and a tube containing a liquid release reagent. The disclosure further includes a method of making a polynucleotide retention member, the method comprising washing a number of microspheres with carbonate and MES buffers; preparing sulfo-NHS and EDAC; incubating the microspheres with sulfo-NHS and EDAC for 30 minutes; washing the microspheres with MES and borate buffers; contacting the microspheres with PAMAM(O) for 8 to 10 hours; and washing off unbound PAMAM(O) from the microspheres. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of an exemplary method described herein. [Figure 2] FIG. 1 is a schematic diagram of the processing of DNA affinity beads as described in further detail herein. [Figure 3] FIG. 1 shows the PCR curve of EV13 RNA extracted from nasal swabs. [Figure 4] FIG. 1 shows the PCR curve of EV13 RNA extraction in M4 medium. [Figure 5] FIG. 1 shows a comparison of PCR curves from RNA obtained from plasma and from RNA extracted from buffer. [Figure 6] FIG. 1 shows RNA extraction using beads. [Figure 7] FIG. 1 shows RNA extraction from plasma. [Figure 8] FIG. 10 is a diagram illustrating extraction sensitivity. [Figure 9] 1 is a flow chart of a method for producing PAMAM (generation 0) coated microparticles. Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description of the Invention Analysis of biological samples often involves determining whether one or more polynucleotides (e.g., DNA, RNA, mRNA, or rRNA) are present in the sample. The techniques described herein have the potential to measure both RNA and DNA present in a sample. For example, a sample can be analyzed to determine whether RNA of a particular pathogen is present, as well as to determine whether DNA of other pathogens or the same pathogen is present. If present, the RNA or DNA, together or separately, can indicate the corresponding disease or condition. Thus, the technology described herein relates to polynucleotide-binding materials and their use in isolating polynucleotides, such as DNA and RNA, from biological samples. The materials, along with methods for using the materials, provide for rapid and reliable extraction of RNA and DNA from many different biological samples, including quantitative measurement of both RNA and DNA. Such methods are commonly referred to as "sample preparation" methods. This term refers to the liberation, extraction, enrichment, and / or isolation of target organism RNA and / or DNA from an unprocessed sample containing bound target RNA and / or DNA in the form of cells, such as samples obtained directly from patients or from agricultural products or food. The liberated target RNA and / or DNA are then rendered suitable for amplification and / or detection in a central step of the method.

[0012] The terms DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), along with polynucleotides, as used herein can refer to individual molecules or populations of molecules that are distinguishable, for example, by all sharing a specific nucleotide sequence, or they can refer collectively to DNA or RNA molecules with distinct sequences. For example, a biological sample from a human patient can contain DNA from the patient's cells with one sequence and DNA or RNA from pathogen cells with a sequence different from that of the patient's DNA. Thus, the sample is said to contain DNA and RNA (or, collectively, polynucleotides) even if there are distinct (chemically distinct) DNA (or RNA) molecules in the sample. The methods described herein can be used to collectively isolate DNA and RNA molecules from both the patient's and pathogen's cells in such a sample. However, generally, in such instances, it is typically the pathogen's DNA or RNA of interest, selectively amplified from the total DNA and RNA ultimately isolated from the sample. DNA and RNA most suitable for extraction using the methods described herein are less than 7.5 Mbp in size. However, it will be apparent that larger DNA and RNA molecules are more susceptible to extraction and detection by the methods described herein.

[0013] Typically, biological samples are complex mixtures. For example, samples can be provided as blood samples, tissue samples (e.g., nasal, buccal, anal, or vaginal swabs), biopsy aspirates, lysates, fungi, or bacteria. The RNA and / or DNA to be measured is usually contained within particles (e.g., cells such as white blood cells or red blood cells), tissue fragments, bacteria (e.g., gram-positive or gram-negative bacteria), fungi, or spores. One or more liquids (e.g., water, buffer, blood, plasma, saliva, urine, cerebrospinal fluid (CSF), or organic solvents) are generally part of the sample and / or added to the sample during processing. The materials and methods described herein are compatible with a variety of clinical matrices, including at least blood, urine, CSF, swabs, and plasma. Methods for analyzing biological samples include releasing RNA and / or DNA from particles (e.g., bacteria) in the sample, amplifying one or more of the released RNA and / or DNA (e.g., by polymerase chain reaction (PCR)), and determining the presence (or absence) of the amplified polynucleotide(s) (e.g., by fluorescence detection). Clinical samples present various challenges, particularly for detecting target RNA and DNA using PCR or similar techniques. Target nucleic acids may be present at concentrations as low as 10 copies per milliliter against a background of millions or even billions of competing copies of nucleic acids (e.g., from a patient's normal cells). Furthermore, various other biochemical entities present in clinical samples inhibit PCR. For example, inhibitors may prevent isolation of RNA or DNA from the sample by being trapped by materials designed to retain RNA or DNA. If the inhibitor concentration is not reduced relative to the RNA or DNA being measured, the assay may produce false-negative results. Examples of these inhibitors, depending on the biological sample, include necrotic cell matter such as membrane fragments, humic acid, mucus, hemoglobin, other proteins such as DNA-binding proteins, salts, DNases, feces, meconium, urea, amniotic fluid, blood, lipids, carbohydrates, and polysaccharides. For example, such inhibitors may reduce the efficiency of DNA and RNA amplification by PCR and other enzymatic techniques for determining the presence of DNA and RNA.

[0014] Therefore, an effective sample preparation method should result in enrichment of target RNA or DNA and minimize the presence of inhibitors. The methods described herein can increase the concentration of DNA and / or RNA measured and / or decrease the concentration of inhibitors relative to the concentration of DNA and / or RNA measured. In addition, some target organisms, such as cells of Gram-positive bacteria (e.g., group B streptococci), are very difficult to lyse, meaning that lysis conditions must be very stringent. Such microorganisms may require chemicals such as mutanolysin for further lysis, as well as higher temperatures for optimal lysis. Such conditions can be adjusted using the materials and methods described herein.

[0015] Sample preparation method A typical sample preparation method can be performed in a processing chamber containing a multitude of particles (e.g., beads, microspheres) configured to retain the RNA and / or DNA of a sample under a first set of conditions (e.g., a first temperature and / or a first pH), release the RNA under a second set of conditions (e.g., a second, higher temperature and / or a second, more basic pH), and release the DNA under a third set of conditions (e.g., a third, different temperature and / or a third, more basic pH than those used in the first and second conditions). Generally, DNA and RNA are selectively retained relative to inhibitors that may be present in the sample. A typical sample preparation method is shown in FIG. 1. The various reagents mentioned with respect to FIG. 1 are described in further detail elsewhere herein. At 100, a processing tube 101, e.g., a standard laboratory 1.7 ml microcentrifuge tube, contains a biological sample including a liquid 109, e.g., an aqueous solution, and cellular material 111, where at least a portion of the cellular material can contain RNA and / or DNA of a target of interest. The biological sample can be any of the biological samples described elsewhere herein, and processing tube 101 can be any tube or suitable container described in further detail. While the method is illustrated with respect to FIG. 1, it should be understood that the method is not limited to being performed in a tube. The sample and various reagents can also be introduced into, mixed, and reacted within a chamber of a microfluidic device, such as a microfluidic cartridge, as described in further detail in U.S. Patent Application No. 11 / 281,247, filed November 16, 2005 (incorporated herein by reference).

[0016] A solution 107 of microparticles 105 is contained in a first pipette tip 103, which is introduced into a process tube and contacted with the biological sample contained therein. As described in further detail herein, the surface of the particles 105 is modified to have PAMAM(O) attached thereto to retain RNA and / or DNA in preference to inhibitors in the solution. As described in further detail herein, the solution 107 can be a lysis solution. As described elsewhere herein, in addition to various enzymes, the lysis solution can contain a surfactant. Thorough mixing of the microparticles, solution, and biological sample can be achieved simply by expelling the solution containing the microparticles from the pipette tip and roughly mixing the two solutions, or by mechanical or manual agitation of the process tube 101. A first pipette tip 103 is placed on top of the processing chamber 101, for example, manually by a user or by an automated pipetting head, as described in U.S. Provisional Patent Application No. 60 / 959,437, filed July 13, 2007, which is incorporated herein by reference. At 110, the same process tube 101 is used to incubate the microparticles, biological sample, and lysis agent, as shown, using heat, e.g., from an external heat source, to lyse the cells in the biological sample and liberate RNA and / or DNA. Under these conditions, DNA molecules bind to the appropriately configured surface of the microparticles, as described in further detail herein. Typically, the particles retain RNA and / or DNA from a liquid having a pH of about 9.5 or less (e.g., about 9.0 or less, about 8.75 or less, about 8.5 or less). It should also be noted that DNA binding to the affinity microparticles occurs simultaneously with the lysis process, and that binding is not adversely affected by the presence of detergents and, in some cases, lytic enzymes in the lysis solution. The temperature selection is determined by the temperature required to lyse the cells of interest; heating is not required to bind RNA or DNA to the particles. Generally, cells with stronger cell walls (e.g., Listeria or Bacillus anthracis) require higher temperatures. For example, for Chlamydia testing, a temperature of 37°C is used for 5-10 minutes for lysis and binding, and for Group B Streptococcus testing, a temperature of 60°C is used for 5-10 minutes. Generally, the liquid is heated to a temperature insufficient to boil the liquid in the presence of particles.

[0017] At 120, the microparticles are concentrated or clustered, and the remaining solution containing the remaining cellular material 125 is removed, for example, with a second pipette tip 123. Clustering means that the microparticles are concentrated in one location in the process tube, touching each other, instead of being uniformly distributed throughout the solution. If the microparticles are magnetic, clustering the microparticles can be accomplished, for example, by placing a magnet 121 near the outside of the process chamber 101 and moving the magnet up and down outside the chamber. The magnet attracts the magnetic particles, pulling them toward the inside of the wall of the process chamber adjacent to the magnet. As is typically done, the pipette tip 123 is used to remove as much of the remaining solution (sometimes referred to as the supernatant or the solution with remaining cellular material) as possible without picking up a significant amount of the microparticles. Typically, the pipette tip can be slid into the processing chamber 105 without contacting the microparticles. In this manner, the microparticles are concentrated by presenting a smaller amount of solution. The pipette tip 123 may be different from the pipette tip 103 or it may be the same tip. In some embodiments, less than 10 microliters of solution is left with the particles after removal of the solution containing the remaining cellular material. Typically, this is achieved by compacting the microparticles into a small pellet and positioning the pellet away from the location where the pipette is inserted to remove the supernatant. Positioning the pipette relative to the bottom of the tube is also important so that most of the supernatant is removed. The pipette tip should be fairly close to the bottom of the tube (within 1-2 mm), but the pipette tip should not completely seal against the bottom of the tube. A star-shaped design can also be used at the bottom of the lysis tube (as described in U.S. Provisional Patent Application No. 60 / 959,437, filed July 13, 2007, which is incorporated herein by reference), but the placement of this design relative to the location of the magnet is important to ensure that the sliding of densely packed particulates is not inhibited and that the crevices between the vertices of the star-shaped design do not trap particulates.

[0018] At 130, a wash solution 131 is introduced into the processing chamber 101 containing the densely packed microparticles using a third pipette tip 133. The wash solution can include, for example, a buffer such as Tris-EDTA containing a surfactant such as 1% Triton X100 and a final pH of 8.0. Typically, for a sample volume of 2 ml or less, the volume of the wash buffer is 100 microliters or less. The wash solution is used to wash away any non-DNA and non-RNA molecules, such as inhibitors, that may have bound to the microparticles. The wash solution is selected to selectively wash away non-RNA and non-DNA molecules while leaving the RNA and / or DNA molecules bound to the microparticles in place. The pipette tip 133 can be a different tip from one or both of the pipette tips 103 and 123, or either tip can be reused. To separately release the RNA and DNA from the particles, the wash solution 131 is replaced with an alkaline (pH ∼9.0) release solution, e.g., a buffer with a different pH than the wash solution. This can be done by pipetting as much of the wash solution as possible (e.g., leaving a residual volume of <5 microliters) and then dispensing the release buffer using a new pipette tip. If the same tip is used, it is necessary to expel the liquid sufficiently to avoid dilution of the release solution. For example, at 140, release solution 141 is added to the processing chamber 101 so that the RNA bound to the microparticles can be released from these microparticles. Generally, PAMAM (generation 0) on the particles (as described in further detail herein) most efficiently releases RNA at a pH of approximately 9. As a result, the RNA can be released from the particles into the surrounding liquid. Optionally, heat can be applied to the processing tube to facilitate the release of RNA, such as by heating the solution to 85°C. Generally, the liquid is heated to a temperature insufficient to boil in the presence of the particles. In some embodiments, the temperature is 100°C or less (e.g., less than 100°C, about 97°C or less). In some embodiments, the temperature is about 65°C or more (e.g., about 75°C or more, about 80°C or more, about 90°C or more). In some embodiments, the temperature is maintained for about 1 minute or more (e.g., about 2 minutes or more, about 5 minutes or more, about 10 minutes or more). In some embodiments, the temperature is maintained for about 30 minutes (e.g., about 15 minutes or less, about 10 minutes or less, about 5 minutes or less). In some embodiments, the process tube is heated to about 65-90°C (e.g., to about 70°C) for about 1-7 minutes (e.g., about 2 minutes). In other embodiments, heating is at 85°C for 3 minutes. In yet other embodiments, heating is at 65°C for 6 minutes. Generally, lower temperatures require longer heating times. Alternatively, or in combination, the particles retaining the RNA are heated to release the RNA without the aid of a release solution. If only heating is used to release the RNA, the release solution can be the same as the wash solution.

[0019] Typically, RNA from a 2 ml sample will be released into about 20 microliters or less (e.g., about 10 microliters or less, about 5 microliters or less, or about 2.5 microliters or less) of liquid following the lysis, binding, and washing procedures described elsewhere above. Although release of RNA is described with heating, RNA can be released without heating. For example, in some embodiments, the release solution has an ionic strength, pH, surfactant concentration, composition, or a combination thereof that causes release of RNA from the retention member without the need for heating. For example, during sample preparation wash-and-release (typically DNA release) procedures, excessive shear forces caused by rapid fluid movement during aspirate-and-dispense mixing can dislodge PAMAM (generation 0) from the particle surface, which can then inhibit downstream PCR. During the mixing process, the volume of back-and-forth movements should be limited to 1-20 microliters with a pipette, with each aspirate-and-dispense operation taking 1-10 seconds, and fewer than 10 aspirate-and-dispense operations. At 150, the microparticles, which now have essentially no bound RNA, can be concentrated or enriched in a manner similar to that described for 120, in this case to facilitate removal of the release fluid containing dissolved RNA. For example, the magnetic beads can be collected inside the processing chamber wall by bringing a magnet 121 into close proximity to the outside of the processing chamber. In Figure 1, magnet 121 is used to concentrate the microparticles in both steps 120 and 150, but it will be apparent that different magnets can be used in the two examples.

[0020] If the sample contains both RNA and DNA and it is desired to measure a specific RNA and a specific DNA, the procedures at 140 and 150 described herein can be repeated using a second release solution designed to release DNA. As explained in more detail in U.S. patent application Ser. No. 12 / 2006, filed on even date herewith, entitled "POLYNUCLEOTIDE CAPTURE MATERIALS, AND METHODS OF USING SAME," solutions designed to release DNA generally have a pH of about 12 or higher. This procedure is advantageous because RNA and DNA have different pK a and therefore are eluted from the surface of non-covalently bound particles at different pHs. Similar considerations for release conditions (temperature, reagent concentration, etc.) as for RNA apply to release of DNA. It should also be noted that up to this point, all of the processing steps have been performed in one vessel. This is advantageous for a number of reasons. First, unnecessary liquid transfer steps inevitably result in the loss of some of the target material. Additional liquid transfer steps add to the total protocol time. It should be noted that performing all liquid processing in one vessel is not an easy task, primarily due to the residual volumes left behind between successive liquid transfers. This becomes even more difficult when the final elution volume is very low, e.g., less than 30 microliters, or less than 20 microliters, or less than 10 microliters, or less than 5 microliters. Nevertheless, very good yields can be obtained using the protocol described here.

[0021] The RNA and / or subsequently the DNA liberated from the microparticles can be drawn up into the fourth pipette tip 153, respectively, in a solution of release liquid. Pipette tip 153 need not be different from all of pipette tips 103, 123 and 133, thus allowing for the reuse of these tips. Although it is preferred to use magnetic beads, non-magnetic beads can also be used herein and separated, for example, by centrifugation, instead of using a magnet. In certain embodiments, the ratio of the volume of the original sample introduced into the processing tube to the volume of liquid into which the RNA or DNA was released is at least about 10 (e.g., at least about 50, at least about 100, at least about 250, at least about 500, at least about 1,000). In some embodiments, RNA or DNA from a sample having a volume of about 2 ml can be retained in the processing tube and, after binding and washing, released into about 4 microliters or less (e.g., about 3 microliters or less, about 2 microliters or less, about 1 microliter or less) of liquid. In some embodiments, the sample has a volume that is at least about 10 times greater than the concentrated volume of the binding particles to which the RNA or DNA is bound. In other embodiments, the sample has a volume between 100 μl and 1 ml, and the packed particles occupy an effective volume of less than 2 microliters. The liquid from which the RNA or DNA is released typically contains at least about 50% (e.g., at least about 75%, at least about 85%, at least about 90%, or at least about 95%) of the RNA or DNA, respectively, present in the sample 109. Thus, for example, about 8-10 μg of DNA can be liberated from 1 ml of overnight culture, and about 2-4 μg of DNA can be extracted from one buccal swab. The concentration of RNA or DNA present in the release liquid can be higher than the corresponding concentration in the original sample because the volume of the release liquid is typically less than the volume of the original liquid sample. For example, the DNA concentration in the release liquid can be at least about 10-fold higher (e.g., at least about 25-fold higher, at least about 100-fold higher) than the DNA concentration in the sample 109. The inhibitor concentration present in the liquid from which the RNA or DNA is released is generally sufficiently lower than that obtained from the crude sample to increase the efficiency of RNA or DNA amplification relative to the inhibitor concentration in the original fluid sample.

[0022] In general, while the methods and materials described herein work well (usually for routine applications only) for a wide range of sample sizes and reagent volumes in most practical applications (considering the size of most biological samples for diagnostic analysis), the resulting volume of RNA- and / or DNA-bound packed particles ranges from 2 to 3 μl (before release) and is independent of sample volume, up to approximately 2 ml. Generally, the amount of microparticles required depends on the amount of RNA and / or DNA in the sample. Given the particle binding efficiency, it has been found that 0.5 mg of particles is sufficient for most manual applications and most associated automated pipetting, regardless of sample size. Thus, for example, for samples with volumes between 0.5 microliters and 3 milliliters, the packed particle volume is 2 to 3 μl. For example, for Chlamydia, sample sizes are typically 1 ml, and 0.5 mg of particles is sufficient. For other applications, DNA from a 2 ml sample can be extracted with 0.5 mg of particles, or in some cases, 1 mg of beads can be used. For smaller samples, eg, samples having a volume of 50 μl, it is still typical to use only 0.5 mg of particles. During manual operation, solutions can be pipetted up and down multiple times, e.g., 10, 15, or 20 times, to mix them during various steps. Such procedures are acceptable for the release step as well as the washing step. Vortexing is also useful for these steps. However, for automated processing, no mixing steps are tolerated, and the number of mixing operations is minimized because this can cause PAMAM(O) to dislodge and inhibit downstream PCR. The methods described herein offer highly effective sample preparation for PCR, providing the ability to detect as few as 25 copies of RNA or DNA from one milliliter of clinical sample. Because elution volumes are as low as three microliters, RNA or DNA is present at high concentrations. Residual sample liquid and / or wash volumes in the concentrated microspheres are also small, thereby minimizing dilution by sample or wash buffer and minimizing inhibition from the remaining sample.

[0023] The time interval between introduction of the polynucleotide-containing sample into the processing tube 101 and release of the RNA or DNA into the release solution is typically 10-30 minutes, typically about 15-20 minutes, and can be 15 minutes or less (e.g., about 10 minutes or less, about 5 minutes or less). These times include the lysis time (together with the sample binding time) and are extremely rapid. Separate release of RNA and DNA from a single sample simply requires the addition of an additional release step, shown at 140 in FIG. 1. Optionally, at 160 in FIG. 1, the released RNA or DNA in solution can be neutralized by contacting it with a neutralizing solution 165 (e.g., an equal volume of 25-50 mM Tris-HCl buffer (pH 8.0)). For example, the RNA or DNA in solution in pipette tip 153 can be released into a second processing chamber or container 161, such as a standard laboratory PCR tube, in which the neutralizing solution is present. The PCR tube can be removed and introduced into a PCR machine for further analysis. Typically, solutions for extracting RNA are nearly neutral, so a separate neutralization step is not necessary. The RNA or DNA in solution in vessel 161 is ready for amplification and detection by PCR. Furthermore, the process described above is extremely reliable and robust, achieving a 7 log dilution (10 to 10 of target RNA or DNA). 7 Allows for quantitative assay of extracted RNA or DNA across a range of copies / ml of sample.

[0024] The method of Figure 1 has been shown to be effective in both manual and automated formatting. The method shown in Figure 1 can be practiced with a reagent holder in which the processing chambers can be positioned and in which appropriate amounts of microparticles, lysis solution, washing solution, release solution, and neutralization solution can be provided, each of which can be accessed by one or more pipette tips for use as shown in Figure 1. An exemplary reagent holder is described in U.S. Provisional Patent Application No. 60 / 959,437, filed July 13, 2007, which is incorporated herein by reference. The magnet used to concentrate the magnetic microparticles shown in Figure 1 can be the magnetic separation device described in U.S. Provisional Patent Application No. 60 / 959,437 (filed July 13, 2007), which is incorporated herein by reference. In regard to heating the processing chamber 101 shown in FIG. 1, the heater assembly described in U.S. Provisional Patent Application No. 60 / 959,437 (filed July 13, 2007), which is incorporated herein by reference, can be used. The method illustrated in FIG. 1 is optimally used to prepare highly pure and concentrated RNA or DNA for use in low volume (e.g., 4 μl) PCR reactions that can be performed, for example, in a microfluidic cartridge, such as the microfluidic cartridge described in U.S. Provisional Patent Application No. 60 / 959,437, filed July 13, 2007, which is incorporated herein by reference.

[0025] Figure 2 shows a schematic of the sample preparation method at the molecular level. A typical magnetic particle 201 with a diameter of 1 μm is shown at 210. Attached to the surface of particle 201 are molecules 205 that have binding affinity for polynucleotides in the solution surrounding the particle. The molecules 205 are typically attached by covalent bonds. Such molecules are described in further detail herein and, in some embodiments, are PAMAM molecules (generation 0). Moving from 210 to 220, the magnetic particles are incubated in a solution containing RNA and / or DNA at a pH of 4-8, below the pKa of the molecules 205. At 220, particle 201 is shown bearing polynucleotides (i.e., DNA and / or RNA) that are molecules 211 bound to affinity molecules 205. Various other non-specifically bound substrates 213, represented by small ovals, cigars, and curves, are also shown. 2, particles 201 with both bound RNA and / or DNA molecules 211 and non-specifically bound molecules 213 are washed to remove non-specifically bound substrate, leaving particles coated with affinity molecules 205 and bound RNA and / or DNA molecules 211. Moving from 230 to 240, the RNA and / or DNA molecules 211 are released from the surface of the particles by increasing the pH of the solution surrounding the particles to pH 9 (RNA) and subsequently to pH 12-13 (releasing the DNA). The released RNA and / or DNA molecules can be collected separately in a ready-to-use format for PCR. Although the samples and various solutions described herein are described as having microliter-scale volumes, other volumes can be used. For example, processing tubes having surfaces (e.g., particles) configured to selectively retain RNA and / or DNA as opposed to inhibitors can have large volumes (e.g., tens of microliters or more, at least about 1 milliliter or more). In some embodiments, processing tubes are benchtop-scale, and other solutions are correspondingly scaled up.

[0026] Polynucleotide Capture Materials Suitable polynucleotide affinity molecules are those that provide a very high density of positive charges at low pH, allowing them to strongly attract and bind polynucleotides, including RNA and DNA, from clinical lysates within minutes.

[0027] An exemplary embodiment of the materials herein uses polyamidoamine (PAMAM) generation 0, product number 412368, available from Sigma-Aldrich Chemical Company ("Sigma-Aldrich"). This material, referred to herein as "PAMAM(generation 0)" or "PAMAM(O)" of "PAMAM(GO)," is a dendrimer whose molecules have the following structure: [ka] PAMAM 0th generation

[0028] The core of the molecule is ethylenediamine doubly substituted on both nitrogen atoms with acetyl groups, each of which is itself reacted with an ethylenediamine monomer to yield an amino-substituted amide group. However, PAMAM(O) forms suitable for use herein are not limited to products available from Sigma-Aldrich. The dendrimeric nature of PAMAM(O) is at least partially controlled by the degree of dendrimerization possible during its synthesis, allowing for a wide range of possible morphologies. Thus, many variations of PAMAM(O) with different numbers of substitution units are suitable for use herein. Generally, there is a range of sizes of dendrimer molecules (or PAMAM(O) derivatives) suitable for polynucleotide capture. Smaller sizes do not adequately capture RNA or DNA, while larger sizes hold RNA or DNA too tightly, preventing easy release. Furthermore, different monomers from ethylenediamine can be used to prepare PAMAM variations suitable for use herein. Such monomers include, but are not limited to, 1,2-propylenediamine, 1,3-propylenediamine, 1,2-butylenediamine, 1,3-butylenediamine, and 1,4-butylenediamine.

[0029] PAMAM molecules suitable for use herein may also be characterized by molecular weight. In particular, PAMAM(O) has a molecular weight of 516. Other suitable PAMAM molecules have molecular weights in the range of 500-600 Da. Since PAMAM(O) can itself act as an inhibitor of enzymatic processes such as DNA and RNA amplification, it is important that it be used in such a way that it is not present in solution with the released RNA and / or DNA, an aspect that is explained in more detail in the Examples below.

[0030] supporting material When used, PAMAM(O) is typically immobilized (e.g., bound) to the surface of a solid support, such as carboxylated beads or magnetic or non-magnetic beads. In many embodiments, such solid supports include microparticles, such as beads and microspheres. The terms microparticle, bead, and microsphere can be used interchangeably herein. Particles are typically made of a material to which PAMAM(O) can readily bind. Exemplary materials from which such particles can be fabricated include polymeric materials that can be modified to allow ligands to be attached. Typically, such solid supports can themselves be derivatized to provide surface functional groups that readily bind PAMAM(O) molecules, forming chemical bonds between the surface and the PAMAM(O). A frequently used and desirable surface functional group is a carboxylic acid (—COOH) group. Exemplary polymeric materials that have, or can be modified to have, carboxyl and / or amino groups available for binding PAMAM(O) include, for example, polystyrene, latex polymers (e.g., polycarboxylate-coated latex), polyacrylamide, polyethylene oxide, and derivatives thereof. Polymeric materials that can be used to make suitable particles are described in U.S. Patent No. 6,235,313 to Mathiowitz et al., which is incorporated herein by reference. Other materials include glass, silica, agarose, and aminopropyltriethoxysilane (APES) modified materials.

[0031] During the reaction process between carboxylated particles, such as magnetic particles, and PAMAM(O) molecules, all available amine groups on the PAMAM(O) molecule, e.g., one of the six available amine groups in the aforementioned Sigma-Aldrich product, are consumed in reaction with COOH groups on the particle surface to form carbodiimide bonds (e.g., U.S. Patent Application No. 11 / 281,247, p. 40). The remaining total number of amine groups, e.g., five groups in the aforementioned Sigma-Aldrich product, are available for protonation. In some embodiments, the synthesis protocol involves washing a number of microspheres with carbonate and MES buffers; preparing sulfo-NHS and EDAC; incubating the microspheres with sulfo-NHS and EDAC for 30 minutes; washing the microspheres with MES and borate buffers; contacting the microspheres with PAMAM(0) for 8-10 hours; and washing off unbound PAMAM(0) from the microspheres. An example of a synthesis protocol for producing microparticles conjugated with PAMAM(0) is provided in the Examples below. There are various sources of beads or particles that can be coupled to PAMAM(O) and used in the methods described herein, such as Seradyn Magnetic carboxyl-modified magnetic beads (Part #3008050250, Seradyn), Polysciences BioMag carboxyl beads, Dynal polymer-encapsulated carboxyl-coated magnetic beads, and Polybead carboxylate-modified microspheres (available from Polyscience (Cat. No. 09850)). The high density of PAMAM(O) molecules on the bead surface allows for the use of small amounts of beads (0.5 mg) for a 1 milliliter clinical sample, enabling binding to low concentrations of target RNA or DNA (<100 copies) in a background of billions of copies of other polynucleotides.

[0032] In some embodiments, at least some (e.g., all) of the particles are magnetic. In other embodiments, most (e.g., all) of the particles are non-magnetic. Magnetic particles are advantageous because centrifugation is generally not required to separate the magnetic particles from the solution in which they are suspended. Typically, the particles have an average diameter of about 20 microns or less (e.g., about 15 microns or less, about 10 microns or less). In some embodiments, the particles have an average diameter of at least about 4 microns (e.g., at least about 6 microns, at least about 8 microns). Magnetic particles herein typically have an average diameter of about 0.5 microns to about 3 microns. Non-magnetic particles herein typically have an average diameter of about 0.5 microns to about 10 microns. The particle density is typically at least about 10 7 particles / milliliter (e.g., about 10 8 or about 10 9 For example, a processing area of ​​about 1 microliter total volume present in a microfluidic device configured for use in sample preparation would be about 10 3 It may contain beads. In some embodiments, at least some (e.g., all) of the particles are solid. In some embodiments, at least some (e.g., all) of the particles are porous (e.g., the particles can have channels formed at least partially therein).

[0033] The microparticles described herein are not only suitable for use in processing tubes that are handled by manual pipetting operations, but can also be used in microfluidic devices such as sample concentrators, thereby allowing for the processing of even sub-microliter elution volumes when required. PAMAM(O)-bound microparticles are particularly effective at capturing and releasing RNA and, in some embodiments, the mass ratio of RNA captured by bound particles to the mass of bound particles prior to contact with RNA is 5-20%. In other embodiments, this ratio is 7-12%. In yet other embodiments, this ratio is about 10%, which corresponds to, for example, 100 μg of RNA per 1 mg of particles. PAMAM(O)-conjugated microparticles are particularly effective at capturing RNA and / or DNA consistently over a wide range of concentrations, thereby enabling quantitative analysis of RNA and / or DNA. In some embodiments, the conjugated particles convert 90% or more of the RNA or DNA released from cells into solution and exposed to the conjugated particles into target RNA or DNA. 7 Capture over 1 ml of copies / sample. In some embodiments, the binding particles release greater than 90% of the bound DNA when particular release conditions are used.

[0034] Sample Preparation Kit The PAMAM(O)-coated microparticles can be provided to the user in solid form, such as lyophilized form, or in solution. However, whatever the intended use, it is desirable to provide the reagents in a form that is ready for use by the user without any preparatory steps. The microparticles produced by the methods described herein can be lyophilized by methods known in the art to produce microparticles having the sizes and characteristics described herein. In each of the kits described herein, if the kit is used only to measure RNA compounds, a neutralizing reagent is not required. Therefore, a neutralizing reagent may be provided, but is provided as an option. A neutralizing reagent is typically used when the kit is used to measure DNA or when the kit is used to measure both RNA and DNA. The microparticles can also be provided in kit form, for example, with other reagents used in sample preparation. One embodiment of the kit includes a number of sealed tubes, for example, 24, each containing a lysis buffer; a tube containing lyophilized microparticles bound to PAMAM(0); a tube containing a liquid washing reagent sufficient to analyze a number of samples; a tube containing a liquid neutralization reagent sufficient to analyze a number of samples; and a tube containing a liquid release reagent sufficient to analyze a number of samples, with the kit components stored in an airtight container. Other numbers of tubes available in kit form include 12, 25, 30, 36, 48, 50, 60, and 100. Still other numbers are also permissible and are not inconsistent with the description herein.

[0035] Furthermore, in other embodiments of such kits, the container containing the lyophilized microparticles may further contain particles of a reagent selected from the group consisting of proteinase K; proteinase K and mutanolysin; and proteinase K, mutanolysin and an internal control DNA. In cell-specific lysis applications, additional enzymes are often used. In another embodiment, the kit includes a first airtight container enclosing a number of tubes, e.g., 24, each containing lyophilized microparticles having PAMAM(0) bound thereto; a second airtight container enclosing a number of reagent holders, each holder including a tube containing a liquid lysis agent, a tube containing a liquid washing reagent, a tube containing a liquid neutralization reagent, and a tube containing a liquid releasing reagent. Other numbers of tubes available in kit form include 12, 25, 30, 36, 48, 50, 60, and 100. Still other numbers are permissible and consistent with the teachings herein. Furthermore, in other embodiments of such kits, the tube containing the lyophilized microparticles may further contain particles of a reagent selected from the group consisting of: proteinase K; proteinase K and mutanolysin; and proteinase K, mutanolysin and an internal control DNA. In cell-specific lysis applications, additional enzymes are often used.

[0036] DNA binding and elution conditions When assessing the effectiveness of a DNA capture material, one factor to consider is the material's pK a The pK of HA, an acid, is a is pK a =-log 10 K a (In the formula, K a =[H + ][A - ] / [HA]) HA⇔H + +A - The equilibrium constant of the solution is pH (=-log 10 [H + ]) is the pK of the acid a At equilibrium, the acid is 50% dissociated when the pKa of the material is equal to the pH of the solution. Therefore, knowing the pKa of a material provides an indication of the pH below which the material is primarily dissociated (anionic form), and above which the material is primarily ionized. The pKa of an amino group is defined in terms of its conjugate base as follows: R-NH3, the protonated amine + is in dissociation equilibrium: R-NH3 + ⇔H + +R-NH2 The pK a is log 10 K a (In the formula, K a =[H + ][R-NH2] / [R-NH3 + ] is given by

[0037] Due to the trivalent nature of the nitrogen atom and the conditions of dendrimerization, each PAMAM(O) molecule contains a mixture of primary and tertiary amine groups. Therefore, the PAMAM(O) molecule has the pK a Multiple pKs spanning a range of values ​​that roughly match the range of values avalues, and as is evident from, for example, Table 12.2 of Organic Chemistry, 2nd Ed., Allinger, et al., Eds., Worth Publishers, Inc. (1976), generally their pK a values ​​range from 10 to 11. However, according to information provided by Dendritech of Midland, Michigan, the manufacturer of PAMAM(O), PAMAM actually has a pK ranging from 5.5 (for tertiary amines in the interior of the molecule) to 9.5 (for primary amines on the surface of the PAMAM molecule). a The academic paper citing this data is Tomalia, et al., Angew. Chem. Int. Ed. Engl, 29, 138-175 (1990), page 160, right column. The amine group of PAMAM(O) has a pK a PAMAM(O) is effective as a DNA binder in at least some of the methods described herein because it has a pK of 5-9. Thus, at low pH, it is typically positively charged and its pK a At lower pH values, they can even carry multiple positive charges per molecule, resulting from protonation of the amine groups, and therefore can bind strongly to polynucleotides such as DNA and RNA, which typically contain polyanions (which have a predominantly negative charge) in solution.

[0038] During the use of PAMAM(O) molecules in the methods described herein, the pH of the binding buffer (typically Tris) used to bind free DNA to the particles simultaneously with cell lysis is approximately 7 to 8. At this pH, all amines (six possible groups per PEI molecule available from Sigma) remain protonated (positively charged), strongly attracting negatively charged DNA molecules and binding them to the beads. The PAMAM(O) molecule is resistant to harsh chemicals such as lysozyme degradation, protease enzymes (a mixture of endo- and exoproteases such as proteases that cleave peptide bonds), surfactants, heating to 95 °C, and substances that can bind to RNA and DNA during lysis, for example, it is advantageous because it does not react with them. Thus, cell lysis and RNA and / or DNA binding can be combined into one (simultaneous) step, thereby saving time and at least one processing step. The strong binding of RNA and / or DNA molecules to PAMAM(O) enables rapid washing of affinity beads coated with PAMAM(O) using a washing solution to remove PCR inhibitors. Release of RNA and / or DNA from the affinity beads is effected by increasing the temperature in the presence of a release reagent. Since the amount of beads used is very small (<1 μl), RNA and / or DNA can be released to a final volume of about 3 microliters. The released RNA and / or DNA is neutralized using a neutralizing reagent to a final volume of 5 - 50 microliters, and thus preparation for downstream PCR is complete.

[0039] Typically, the amount of sample introduced is about 500 microliters or less (for example, about 250 microliters or less, about 100 microliters or less, about 50 microliters or less, about 25 microliters or less, about 10 microliters or less). In some embodiments, the sample amount is about 2 microliters or less (for example, about 0.5 microliters or less). PAMAM(O) exhibits an excellent recovery rate of RNA and DNA, based in part on its high binding capacity and high release efficiency. Generally, the mass of RNA or DNA retained on the particles relative to the particle mass is about 25 or less (for example, about 20 or less, about 10 or less). For example, in some embodiments, about 1 gram of particles retains about 100 milligrams of RNA or DNA, and similar ratios can be obtained when used in smaller amounts (for example, binding capacity of RNA or DNA ~100 μg / mg beads).

[0040] Other devices for DNA capture In other embodiments, the solid support can be configured as a retention member (e.g., a porous member such as a column, filter, porous membrane, microporous filter, or gel matrix having a number of openings, such as pores and / or channels, through which the RNA and / or DNA must pass) through which the sample material (containing RNA and / or DNA) must pass. Such a retention member can be formed from a number of appropriately shaped surface-modified particles. In some embodiments, the retention member comprises one or more filter membranes formed of polymers that have also been surface-modified and can be used to retain RNA and / or DNA, such as those available from Osmonics, Inc. In some embodiments, the retention member is configured as multiple surfaces (e.g., partitions or baffles) across which the sample passes. The partitions or baffles are modified to retain RNA and / or DNA in preference to, for example, PCR inhibitors. Such retention members are typically used when the microparticles are non-magnetic.

[0041] When a sample solution passes through a processing region containing such a retention member (suitably modified to selectively retain RNA and / or DNA), the RNA and / or DNA are retained, while the liquid and other solution components (e.g., inhibitors) exit the processing region less retained (e.g., unretained). Typically, such a retention member retains at least about 50% of the RNA and / or DNA molecules (e.g., at least about 75%, at least about 85%, at least about 90%) present in the sample entering the processing region. The processing region is typically maintained at a temperature of about 50°C or below (e.g., 30°C or below) during sample introduction. Processing can continue by washing the retention member with a wash solution to separate remaining inhibitors from the RNA and / or DNA retained by the retention member.

[0042] In some embodiments, the sample preparation methods described herein are performed in a microfluidic device, such as a microfluidic cartridge configured to introduce a sample and capture RNA and / or DNA molecules from the sample onto a solid support contained therein. Exemplary microfluidic cartridges are described in U.S. Patent Application Publication Nos. 2006 / 0166233 and WO2008 / 061165, both of which are incorporated herein by reference. Such cartridges may include one or more actuators configured to move microdroplets of various liquid solutions within the cartridge, a chamber configured to lyse cells in the sample, and one or more channels and associated valves configured to direct, block, or divert liquid flow within the cartridge. Although sample preparation has been described as a series of operations performed in one location, such as a processing tube or microfluidic cartridge, other configurations can be used. For example, in some embodiments, the retention member having the polynucleotide affinity material can be removed from the region where DNA and / or RNA capture occurs, and subsequent processing can occur elsewhere. For example, the retention member can be contacted with a mixture containing DNA and / or RNA and an inhibitor at one location and then moved to another location where the RNA and / or DNA is removed from the retention member.

[0043] Other advantages of the DNA capture materials described herein The extraction reagents and sample preparation methods described herein offer superior performance compared to currently available off-the-shelf kits for sample preparation. Advantages of the materials and methods herein include: A streamlined sample preparation procedure that has fewer steps (only 6 steps from processing sample to purified RNA and / or DNA) and uses fewer vessels than other procedures. In addition to affinity beads, extraction control (cell, plasmid, or naked) DNA can also be included. The internal control DNA can be included in the lysis reagent and eluted into the final released DNA so that it is co-purified along with other DNA (e.g., target DNA) present in the clinical sample. During amplification of the eluted DNA, the internal control DNA can also be amplified and subsequently detected using a different fluorophore than the target DNA. This provides further confirmation that the sample preparation process worked as required. The description herein has included an analysis of the properties and uses of PAMAM (Generation 0) coated microparticles. It will be apparent to those skilled in the art that other affinity molecules may be suitable for use in the methods described herein, as described elsewhere (e.g., U.S. Patent Application Publication No. 2006-0166233, incorporated herein by reference).

[0044] Examples of specific embodiments of the present invention are listed below. (1) A method for isolating polynucleotides from a cell-containing sample, comprising: contacting the sample with a lysis solution and a plurality of binding particles coated with PAMAM (Generation 0), causing polynucleotides to be released from the cells and bound to the PAMAM (Generation 0), thereby creating a solution containing the binding particles with bound polynucleotides and remaining cellular material; clustering the polynucleotide-bound binding particles; Removing the solution containing the remaining cellular material; Washing the bound particles; Releasing the polynucleotides from the bound particles; The method comprising: (2) The method described in (1), wherein the polynucleotide is DNA. (3) The method described in (1), wherein the polynucleotide is RNA. (4) The method of (1), wherein the polynucleotide has a size of less than 7.5 Mbp. (5) The method described in (1), wherein PAMAM (generation 0) is covalently bound to the surface of the binding particle. (6) The method according to (1), wherein the particles are made of a polymer material selected from the group consisting of polystyrene, latex polymer, polyacrylamide, and polyethylene oxide. (7) The method described in (6), wherein the polymer material is modified to provide one or more carboxyl groups, which provide binding sites for PAMAM (generation 0). (8) The method described in (1), wherein the particles have an average diameter of about 0.5 microns to about 10 microns. (9) Particles are approximately 10 7 ~10 9 The method of (1), wherein the particles are present at a density of 10 ... (10) The method of (1), wherein at least some of the particles are magnetic. (11) The method according to (1), wherein PAMAM (generation 0) is configured to bind to the polynucleotide preferentially over a polymerase chain reaction inhibitor. (12) The method according to (11), wherein the polymerase chain reaction inhibitor comprises at least one of hemoglobin, peptides, fecal matter, humic acid, mucus, DNA-binding proteins, or carbohydrates. (13) The method according to (1), wherein contacting the sample with a dissolution solution and a plurality of binding particles comprises incubating the sample with the solution and the particles at 60°C for 5 to 10 minutes. (14) The method of (13), wherein the contacting comprises heating the sample to a temperature, the temperature being insufficient to boil the liquid in the presence of the plurality of particles during heating. (15) The method described in (1), wherein the washing solution contains Tris-EDTA and 1% Triton X100 (pH 8.0). (16) The method according to (15), wherein the cleaning solution contains a surfactant. (17) The method of (1), wherein releasing the polynucleotide comprises heating the particles at 85°C for 3 minutes in the presence of a release solution. (18) The method according to (15), wherein the polynucleotide comprises an RNA molecule and the release solution has a pH of >9. (19) The method according to (15), wherein the polynucleotide comprises a DNA molecule and the release solution has a pH of >12. (20) The method according to (15), wherein the polynucleotide comprises a mixture of DNA and RNA molecules and two successive release solutions are used, the first release solution having a pH in the range of 9 to 12 and the second release solution having a pH in the range of 12 to 14. (21) The method of (1), wherein the method does not involve centrifugation of the particles. (22) The method according to (1), wherein the time required to complete the contacting, concentrating, removing, washing, and releasing is 10 to 30 minutes. (23) The method of (1), wherein the sample has a volume at least about 10 times greater than the concentration volume of the binding particles having polynucleotides bound thereto. (24) The method described in (1), wherein the sample has a volume of 0.5 microliters to 3 milliliters. (25) The method of (1), wherein the contacting, concentrating, removing, washing, and discharging are all performed in one container. (26) The method of (1), further comprising neutralizing the released polynucleotides, thereby preparing polynucleotides ready for PCR. (27) A method for concentrating RNA from a sample containing a polymerase chain reaction inhibitor, comprising contacting 500 μl to 1 ml of the sample with a plurality of RNA-binding particles, the binding particles configured to selectively retain RNA in the sample relative to the polymerase chain reaction inhibitor; concentrating the plurality of particles having one or more polynucleotides bound thereto to a working volume of 50 nanoliters to 5 microliters; Releasing one or more polynucleotides into <30 μl of solution; The method comprising: (28) A composition comprising: a carboxyl-modified microparticle; and PAMAM (generation 0) bound to one or more of the carboxylic acid groups on the microparticle with one or more amine groups per molecule. (29) multiple closed tubes, each containing lysis buffer; a tube containing freeze-dried microparticles conjugated with PAMAM (generation 0); a tube containing sufficient liquid wash reagent to analyze multiple samples; and A kit comprising tubes containing sufficient liquid releasing reagent to analyze multiple samples, wherein the components of the kit are stored in an airtight container. (30) The kit according to (29), wherein the number is 24. (31) The kit according to (30), wherein the tube containing the freeze-dried microparticles further contains particles of a reagent selected from the group consisting of proteinase K; proteinase K and mutanolysin; and proteinase K, mutanolysin and internal control DNA. (32) A first airtight container enclosing a plurality of tubes, each tube containing freeze-dried microparticles having PAMAM (Generation 0) bound thereto; a second airtight container enclosing a plurality of reagent holders, each holder containing a tube containing a liquid dissolving agent; a tube containing a liquid cleaning reagent; and the second airtight container comprising a tube containing a liquid releasing reagent; Kit including: (33) The kit according to (32), wherein the number of tubes is 24. (34) The kit of (33), wherein each tube containing freeze-dried microparticles further contains particles of a reagent selected from the group consisting of proteinase K; proteinase K and mutanolysin; and proteinase K, mutanolysin, and internal control DNA. (35) A method for producing a polynucleotide holding member, comprising: Washing the microspheres with carbonate and MES buffer; Prepare sulfo-NHS and EDAC; Incubate the microspheres with sulfo-NHS and EDAC for 30 min; Wash the microspheres with MES and borate buffer; The microspheres were contacted with PAMAM(O) for 8–10 h; washing unbound PAMAM(O) from the microspheres. [Example]

[0045] Sample preparation method The following six steps can be performed in approximately 20 minutes for a single sample and approximately 30 minutes for a batch of 12 samples using the reagent kit described in further detail herein. These steps can also be easily automated in the system described in U.S. Provisional Patent Application No. 60 / 959,437, filed July 13, 2007, which is incorporated herein by reference. These steps are also outlined in Figure 1 and described elsewhere herein.

[0046] One exemplary method is as follows: 1. Mix ~500 μl of clinical sample with lysis buffer and 500 μl of affinity magnetic beads with PAMAM(O) bound to their surface. Kits for detecting viruses such as EV13 contain a small amount of well-dissolved lytic enzyme in lysis buffer. 2. Incubate the mixture of sample, lysis buffer, and beads at room temperature to 60°C for 5 to 10 minutes to lyse the cells and allow the RNA and / or DNA to bind to the affinity beads. 3. Separate the magnetic beads and remove as much of the supernatant as possible. 4. Wash the beads with washing reagent. The RNA and / or DNA is released from the beads by heating the beads to 85° C. for 3 minutes in the presence of approximately 5.3 microliters of release solution. 6. Remove the released RNA and / or DNA and neutralize the solution with a neutralizing reagent, such as Tris buffer, to prepare ready-to-use RNA and / or DNA for PCR.

[0047] Another exemplary method is as follows. Sample: Mix 500 μl of plasma with 500 μl of Prep Buffer or mix a dip swab in 1 mL of RNA Prep Buffer. Optionally, the mixture can be pre-filtered. Incubate at 60°C for 10 min; optionally (swabs only) purify with proteolytic enzymes and perform RNA and / or DNA capture with PAMAM (GO)-coated affinity beads (which are magnetic) as described in further detail herein. Optionally, if it is desired to measure RNA, treat the mixture with DNAse (e.g., 7 U DNase for 10 minutes at 37°C). Wash the RNA-bound beads with 100 μl (2X) of wash reagent as described in further detail herein. The RNA is released from the beads by heating (85°C; 3 min) in the presence of a releasing reagent, as described in further detail herein, thereby freeing RNA ready for RT-PCR. [Example]

[0048] Application to various matrices The procedures described herein are useful for a variety of sample matrices, including both clinical and non-clinical samples, as illustrated by the following non-exhaustive list: Nasal swab CSF Nasal swab in M4 Nasal swab placed in UTM ·plasma [Example]

[0049] Representative results Figure 3 shows the use of the RNA extraction reagent, PAMAM(O), and the method described in further detail herein to isolate and purify Enterovirus 13 (EV13) RNA from nasal swabs using the lysis buffer described elsewhere herein. The graph shows the 2X10 4PCR curves are shown for various samples spiked with 1 mL of RNA Prep buffer, 2000, 200, 50, and 20 copies per mL. RNA was released to 10 μl, but only 2 μl of the released RNA was used for RT-PCR. RT-PCR was performed using a Qiagen RT-PCR kit. The PCR curves are plotted from left to right in decreasing order of concentration, ascending from the axis. Figure 4 illustrates the use of the RNA extraction reagent PAMAM(O) and the method described in further detail herein to isolate and purify enterovirus 13 (EV13) RNA from nasal swabs collected in M4 medium. The graph shows PCR curves for various samples spiked at 1000, 100, and 50 copies / mL of sample, respectively. The PCR curves ascend from the axis in decreasing concentration order from left to right.

[0050] Figure 5 shows a comparison of RNA extraction using PAMAM(O) beads for buffer samples and plasma. EV13 RNA 500 copies / 1 mL was used for both buffer and plasma samples, according to the methods described in more detail herein. Figure 6 shows DNA extraction using PAMAM(O) beads. 2.5 pg of DNA spiked into M4 buffer or the lysis buffer described herein was extracted using the method for extracting RNA from M4 recovery buffer, as described in further detail herein. Figure 7 shows the PCR curve of RNA extraction from a 500 μl plasma sample containing 200 copies of EV13 RNA using 7U DNAse treatment. Figure 8 shows the sensitivity of this method. Probit analysis indicates that the LoD is 50 copies / 200 μl CSF. [Example]

[0051] Typical Protocol for RNA Extraction from Dried Swab and THB Samples in M4, IX TCEP Buffer

[0052] Sample preparation pretreatment (only swab samples require filtration) JPEG0007789048000002.jpg43170

[0053] RNA extraction and PCR preparation JPEG0007789048000003.jpg223170 [Example]

[0054] Typical Protocol for Extraction of RNA from Plasma Samples RNA extraction and PCR preparation JPEG0007789048000004.jpg244169 [Example]

[0055] How to prepare 2X TCEP buffer for RNA extraction As described in further detail herein, the procedure of this example provides a method suitable for preparing up to 50 mL of 2X TCEP buffer (20 mM Tris-HCl (pH 7.0), 2% Tx-100, 10 mM TCEP) for use in RNA extraction. Below is a list of the reagents used in this method. 1M Tris-HCl (pH 7.0) 100% Triton X-100 (Tx-100) TCEP (tris(2-carboxyethyl)phosphine hydrochloride) ·Ultra pure water

[0056] Below is a list of the equipment used in this method. Laminar flow hood Serological pipette holder Serological pipettes Vortexer ·Appropriately sized container A new sterile graduated cylinder Appropriate personal protective equipment (PPE) Product Label

[0057] Personnel performing this procedure should be knowledgeable in buffer preparation methods, have good pipetting skills, prepare solutions in a laminar flow hood for sterility using standard laboratory sterile techniques, and be careful not to contaminate the stock solutions. Personnel should wear gloves and lab coats at all times.

[0058] Preparation of 50 mL of 2X TCEP buffer (20 mM Tris-HCl pH 7.0, 2% Tx-IOO, 10 mM TCEP) JPEG0007789048000005.jpg86169 [Example]

[0059] Typical manufacturing method of RNA-affinity magnetic microspheres This procedure provides a suitable method for preparing one batch of PAMAM(GO)-coated magnetic microspheres, commonly referred to as RNA-affinity magnetic microspheres. One batch consists of a 1-10 ml synthesis, yielding 6 mL of RNA-affinity magnetic microspheres. A flowchart of this method is shown in Figure 9. Below is a list of the equipment used in this method. Vortexer ·Microcentrifuge Magnetic rack 1.7mL microcentrifuge tube 4-ounce sample container 50mL conical tube 15mL conical tube ·Centrifuge pH meter ·pipette Pipette tips Ultrasonic dismembrator dH20 cleaning bottle Task Wiper ·Balances Inspection markers Gloves and lab coat Orbital shaker Label tape Pipette holder Serological pipettes

[0060] Personnel performing this procedure must be able to use a microbalance, pipettes, pH meter, ultrasonic dismembrator, and microcentrifuge, have knowledge of buffer preparation methods, and have good pipetting techniques. Personnel must always wear gloves, lab coats, and eye protection. Hearing protection must be worn during the sonication process. All solutions are prepared in a laminar flow hood.

[0061] Procedure - How to prepare buffer solutions JPEG0007789048000006.jpg43170

[0062] How to prepare 70mL of buffer SN-B (50mM MES buffer (pH6.1), 0.15% Triton X-100) JPEG0007789048000007.jpg53170

[0063] Preparation of 50 mL of SN-G buffer solution (50 mM Tris (pH 7.5), 0.1% Triton X-100) JPEG0007789048000008.jpg53170

[0064] Preparation of 10 mL of SN-H buffer solution (50 mM MES (pH 6.1), without Tx) JPEG0007789048000009.jpg48170

[0065] Steps taken on Day 1 include: JPEG0007789048000010.jpg60170

[0066] JPEG0007789048000011.jpg76170

[0067] JPEG0007789048000012.jpg151170

[0068] JPEG0007789048000013.jpg32168

[0069] JPEG0007789048000014.jpg76170

[0070] Steps taken on Day 2 include: JPEG0007789048000015.jpg32170

[0071] JPEG0007789048000016.jpg38170

[0072] JPEG0007789048000017.jpg48170

[0073] The foregoing description is intended to illustrate various aspects of the present technology. The examples set forth herein are not intended to limit the scope of the appended claims. While the invention has now been fully described, it will be apparent to those skilled in the art that many changes and modifications can be made thereto without departing from the spirit and scope of the appended claims.

Claims

1. a plurality of magnetic beads coated with PAMAM (generation 0) dendrimers covalently bound to carboxyl groups on the plurality of magnetic beads via one or more carbodiimide bonds; and lytic enzymes, A container comprising: the container is configured to receive a biological sample containing polynucleotides and to allow the biological sample to contact the plurality of beads; the plurality of magnetic beads in the container are configured to reversibly bind to polynucleotides of the biological sample, thereby generating a set of polynucleotide-retaining beads; The container, and, discharge liquid Including, the set of polynucleotide-retaining beads is configured to release the polynucleotides when contacted with the release solution and heated; system.

2. 10. The system of claim 1, wherein the plurality of magnetic beads and the lytic enzyme are in dry form in the container prior to receiving the biological sample in the container.

3. The system of claim 1, wherein the PAMAM (generation 0) dendrimer comprises six amine groups prior to covalent attachment to the plurality of magnetic beads.

4. The system of claim 1, wherein the PAMAM (generation 0) dendrimer has a molecular weight of less than 600 Da before being covalently attached to the plurality of magnetic beads.

5. The system of claim 1, wherein the PAMAM (generation 0) dendrimer comprises at least four dendrimer branches terminated in primary amines prior to covalent attachment to the plurality of magnetic beads.

6. The system of claim 1, wherein the PAMAM (generation 0) dendrimer comprises at least five amine groups when covalently attached to a plurality of magnetic beads.

7. 10. The system of claim 1, wherein the container further comprises a sample treatment control polynucleotide in dry form prior to receiving the biological sample in the container.

8. The system of claim 1 , wherein the lytic enzyme comprises proteinase K.

9. A kit comprising a plurality of containers, at least one of said containers comprising: a plurality of magnetic beads coated with PAMAM (generation 0) dendrimers covalently bound to carboxyl groups on the plurality of magnetic beads; and, lytic enzymes, Including, the at least one container is configured to receive a biological sample containing polynucleotides and to allow the biological sample to contact the plurality of magnetic beads; and the plurality of magnetic beads in the at least one container are configured to reversibly bind to polynucleotides of the biological sample through ionic bonds with the PAMAM (generation 0) dendrimer, thereby generating a set of polynucleotide-retaining beads; the set of polynucleotide-retaining beads is configured to release the polynucleotides when contacted with a release solution and heated; The kit.

10. 10. The kit of claim 9, wherein the PAMAM (generation 0) dendrimer contains six amine groups prior to covalent attachment to carboxyl groups on the magnetic beads.

11. 10. The kit of claim 9, wherein the PAMAM (generation 0) dendrimer comprises at least four dendrimer branches terminated in primary amines prior to covalent attachment to carboxyl groups on the magnetic beads.

12. 10. The kit of claim 9, wherein the PAMAM (generation 0) dendrimer comprises at least five amine groups when covalently attached to carboxyl groups on the magnetic beads.

13. 10. The kit of claim 9, wherein the PAMAM (generation 0) dendrimer comprises at least three dendrimer branches terminated in primary amines when covalently attached to carboxyl groups on the magnetic beads.

14. 10. The kit of claim 9, wherein at least one container further comprises a sample treatment control polynucleotide prior to receiving the biological sample in said container.

15. 10. The kit of claim 9, wherein the lytic enzyme comprises proteinase K.

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