HK1150166B - Apparatus, system, and method for purifying nucleic acids - Google Patents

Description

Devices, systems, and methods for purifying nucleic acids

Technical Field

The present invention relates to the purification of chemical substances, and more particularly, to devices, methods and systems for performing chemical purification and analysis. More specifically, the devices, methods and systems provided by the present invention have particularly useful applications in the purification and analysis of nucleic acids, particularly microfluidic devices for performing such purification and analysis. The invention can be used in the fields of analytical chemistry, forensic chemistry, microfluidics and micro-devices, medicine and public health.

Background

The development of semiconductor manufacturing technology to create highly miniaturized chemical devices (Beach, strittatter et al 2007) has revolutionized analytical chemistry, particularly by providing tools to identify chemical species present at low concentrations in complex mixtures with extremely high precision and accuracy. This revolution has had a significant impact on the fields of chemical processing, medicine, forensic science, and defense, where the device can provide a fast, portable, and economical biological detector. Examples of such devices include devices for the collection and identification of microparticles (Wick 2007), systems for the detection of molecular contaminants (Knollenberg, Rodier et al 2007) and devices for the detection of proteins (Terry, Scudder et al 2004; Deshmukh 2006). Other devices utilize Polymerase Chain Reaction (PCR) in automated systems using fluidic techniques to isolate and/or amplify nucleic acids. Examples of such devices are those marketed by Qiagen (Hilden, Germany), Roche (Basel, Switzerland), Applied Biosystems (Foster City, Calif.), Idaho Technologies (salt lake City, Utah) and Cepheid (Sanvell, Calif.).

However, as with any analytical method, the preparation of the sample prior to processing is critical to achieving good results. The presence of too many complex factors and the concentration of substances that can mask the analyte of interest makes efficient detection almost impossible. This problem is of particular concern when attempting to analyze the nucleic acid content of cell lysates, which are extremely complex heterogeneous mixtures (Colpan 2001). The task of preparation becomes more difficult when dealing with portable analytical devices, as these devices are intended for applications where common laboratory support equipment such as centrifuges and separation columns are not available. In these cases, some means for filtering the original sample, such as a blood or urine sample, is critical to provide meaningful results. Current devices based on fluidic technology (particularly the Qiagen devices described above) use soft, compliant glass filters that require a scaffold. The filter has a small pore size, typically about 1-3 microns, to enable efficient capture of nucleic acids from the sample. Due to this small aperture, the filter is therefore also thin, typically less than 2 mm thick, thereby reducing the resistance to fluid flow as the sample is forced through the small aperture. In the Qiagen procedure, the sample is typically mixed with a chaotropic agent, such as guanidine, and the mixture is passed through a glass filter using centrifugal force, wherein the fluid flows in only one direction. Nucleic acid was attached to a glass filter; it was washed off with ethanol or isopropanol and then released using 10 millimolar (10mM) Tris buffer at pH about 8(pH 8.0) or water. However, small pore sizes limit the amount of sample that can be processed due to the resistance created by fluid flow and the possibility of clogging by larger flow rates. Thus, devices such as Qiagen devices may be easily damaged or easily rendered ineffective. In addition, these characteristics limit sample input, sample type that can be tested, large volume samples, concentration factors, and simple fluid integration.

Larger glass filters have been used to provide pre-treatment filtration of samples. For example, U.S. Pat. No. 4,912,034 (Kalra, Pawlak et al 1990) describes an immunoassay for detecting a target analyte in a liquid sample, which includes an optional prefilter assembly made of glass fibers. However, the device is not a microfluidic device and does not explicitly or implicitly indicate that a glass frit plate (glass frit) can be used as a filter prior to a micro-scale PCR reaction. U.S. patent No. 4,923,978 (McCormack 1990) describes the prior use of glass fiber filters to remove undesirable proteins and protein-DNA complexes from aqueous DNA samples, but mentions in a devastating manner that such filters have lower binding capacity (see column 2). Indeed, U.S. patent No. 4,923,978 claims a distinct material for performing such filtration. U.S. patent No. 6,274,371 (Colpan2001) describes silica gel, alumina and diatomaceous earth as preferred filters for removing undesirable contaminants from cell lysates prior to nucleic acid analysis. Us patent No. 6,800,752 (Tittgen 2004) describes the use of a chromatographic material for separating a mixture comprising nucleic acids, wherein the material comprises a support and an ion exchanger function, wherein the support comprises a fibrous material, such as a plastic filter plate (plastic frit), on a support.

However, in view of the above, there remains a need to provide fluidic devices that can effectively isolate and identify nucleic acids that overcome the deficiencies of current generation devices. The present invention meets these needs and others.

Disclosure of Invention

In a first aspect of the invention, there is provided a method of isolating nucleic acids from a mixture containing the nucleic acids and extraneous material. Suitable nucleic acids for use in the present invention include microbial DNA and human genomic DNA. In one embodiment, the method of the invention comprises passing the mixture through a frit filter plate under conditions effective to separate the nucleic acids from the extraneous material. In a more particular embodiment, the frit filter plate is a sintered frit filter plate. In some embodiments, the glass frit has a pore size of about 2 microns to about 220 microns; in some more particular embodiments, the glass frit filter plate has a pore size of about 150 microns to about 200 microns; in yet other more particular embodiments, the glass frit filter plates have a pore size of about 2 microns to about 100 microns; more particularly, the glass frit has a pore size of about 40 microns to about 75 microns; yet other more particular embodiments include those where the glass frit filter plate has a pore size of from about 2 microns to about 20 microns. In another embodiment, the method of the invention comprises passing the mixture through a frit filter plate, thereby producing a first filtered mixture, and then passing the first filtered mixture through a second frit filter plate under conditions effective to separate nucleic acids from the first filtered mixture.

In a second aspect of the invention, there is provided a device for the filtration separation of nucleic acids from a mixture containing said nucleic acids and extraneous matter. In some embodiments, the device comprises a cavity having an inlet and an outlet; and at least one frit filter plate having a pore size of about 2 microns to about 220 microns disposed in the cavity, the frit filter plate being disposed at a location between the inlet and the outlet. In some more particular embodiments, the frit filter plate is a sintered frit filter plate. In other embodiments, the frit filter plate is a sintered frit filter plate. In some embodiments, the glass frit has a pore size of about 2 microns to about 220 microns; in some more particular embodiments, the glass frit filter plate has a pore size of about 150 microns to about 200 microns; in yet other more particular embodiments, the glass frit filter plates have a pore size of about 2 microns to about 100 microns; more particularly, the glass frit has a pore size of about 40 microns to about 75 microns; yet other more particular embodiments include those where the glass frit filter plate has a pore size of from about 2 microns to about 20 microns.

A third aspect of the invention provides a fluidic device for identifying one or more nucleic acids from a mixture containing the nucleic acids and extraneous material. In some embodiments, such fluidic devices of the present invention comprise: an inlet, an outlet, and at least one fluid reaction chamber in communication with both the inlet and the outlet and located between the inlet and the outlet. The apparatus also includes at least one frit filter plate disposed at a location(s) adjacent to and in fluid communication with the inlet and the reaction chamber(s). The frit filter plate has a pore size of about 2 microns to about 220 microns. The mixture entered the device through the inlet and passed through a frit filter plate to exit there as filtered product and then into the fluid reaction chamber. At least one fluid reagent distributor is disposed between the frit filter plate and the reaction chamber(s) in fluid communication therewith. In a more particular embodiment, the frit filter plate is a sintered frit filter plate. In some embodiments, the glass frit has a pore size of about 2 microns to about 220 microns; in some more particular embodiments, the glass frit filter plate has a pore size of about 150 microns to about 200 microns; in yet other more particular embodiments, the glass frit filter plates have a pore size of about 2 microns to about 100 microns; more particularly, the glass frit has a pore size of about 40 microns to about 75 microns; yet other more particular embodiments include those where the glass frit filter plate has a pore size of from about 2 microns to about 20 microns. In another embodiment, the fluidic device comprises a heater adjacent to the frit filter plate(s).

These and other aspects and advantages will become apparent upon reading the following description with reference to the accompanying drawings.

Drawings

FIG. 1 is a schematic diagram of a glass frit filter plate device ("filter module") for purifying nucleic acids according to the present invention.

Fig. 2A and 2B are diagrams of the filter of the present invention. FIG. 2A is an exploded view of a glass frit filter plate device ("filter module") for purifying nucleic acids of the present invention. Fig. 2B is a cross-sectional view of the same device.

FIG. 3 is a schematic of a glass frit filter plate device ("cavity") for purifying nucleic acids of the present invention.

FIG. 4 is a schematic view of a microarray device of the present invention.

FIG. 5 is a flow chart of a method for purifying and identifying nucleic acids of the present invention.

FIG. 6 is a graph illustrating the improved performance characteristics of the device of the present invention as shown in FIGS. 2A and 2B by measuring sample material (100 μ L at 1X 10) as a function of PCR cycle⁵The concentration of individual cells/mL bacillus anthracis cells present in whole blood). Solid line (a) shows the results for samples treated according to the present invention; dashed line (B) shows a sample processed using a commercially available device from Qiagen; dashed lines (C) and (D) are untreated sample and negative control.

FIG. 7 is a graph illustrating the improved performance characteristics of the device of the present invention as shown in FIG. 3 by measuring sample material (500 μ L at 1X 10) as a function of PCR cycle⁴Cells of bacillus anthracis present in sputum at a concentration of one cell/mL) was demonstrated. Solid line (a) shows the results for samples treated according to the present invention; dashed line (B) shows the untreated sample; the dotted line (C) is a negative control.

Detailed Description

The present invention provides methods and devices for at least partially purifying nucleic acids from mixtures of nucleic acids in combination with other substances such as proteins, small molecules, cell membrane fragments, and the like.

As used herein, "nucleic acid" refers to naturally occurring or synthetic individual nucleic acids and polymeric strands of nucleic acids, including DNA and RNA (including analogs thereof), or variants thereof, particularly those of any length known to occur naturally. Examples of nucleic acid lengths of the invention include, but are not limited to, lengths suitable for PCR products (e.g., about 50 base pairs (bp)) and human genomic DNA (e.g., on the order of from about thousands of base pairs (Kb) to billions of base pairs (Gb)). Thus, it is understood that the term "nucleic acid" encompasses both natural or artificial individual nucleic acids and combinations of nucleotides, stretches of nucleotides (stretch) and small fragments thereof (e.g., expressed sequence tags or gene fragments), as well as longer strands, as exemplified by genomic material, including individual genes, and even whole chromosomes. In a more specific embodiment, the nucleic acid is from a pathogen such as a bacterium or virus. The pathogens include pathogens harmful to humans and animals. In the case of pathogens that are harmful to humans, in some embodiments, the pathogen is a pathogen that is used as a biological weapon, including naturally occurring pathogens that have been weaponized. In some embodiments, the nucleic acid comprises microbial DNA. In one embodiment of the invention, the microbial DNA is from Bacillus anthracis. In other embodiments, the nucleic acid is from a human or animal. In some embodiments, the nucleic acid comprises human genomic DNA.

In a first aspect of the invention, there is provided a method for separating nucleic acids from a mixture containing said nucleic acids and extraneous material. In some embodiments, the methods of the present invention comprise passing the mixture through a cavity having an inlet and an outlet; wherein the inlet and outlet are the same and the cavity is provided with at least one porous filter under conditions effective to substantially separate the nucleic acids from extraneous material. As used herein, "extraneous material" refers to all substances in a sample other than nucleic acids. Examples of such extraneous materials include, but are not limited to, proteins, starches, lipids, metal ions, and larger cellular structures such as membrane fragments. As used herein, the phrase "substantially isolated" refers to an isolation that provides, in some embodiments, a nucleic acid that is at least 30% pure relative to the extraneous material, in more particular embodiments at least 50% pure relative to the extraneous material, in more particular embodiments at least 70% pure relative to the extraneous material, in more particular embodiments at least 95% pure relative to the extraneous material, and in more particular embodiments at least 99% pure relative to the extraneous material.

In the various embodiments of the invention described herein, the frit filter plates are made from standard materials using standard methods known to those of ordinary skill in the art, or are commercially available as described below. In some embodiments, the glass frit has a thickness that is substantially in the range of about 1 mm to about 20mm, more specifically in the range of about 2 mm to about 5 mm, and even more specifically in the range of about 2 mm to about 3 mm. Exemplary frit filter plate pore sizes suitable for use in the present invention, including the various embodiments described herein, range from about 2 microns to about 200 microns. In a more particular embodiment, the pore size is from about 150 microns to about 200 microns. In yet other more particular embodiments, the pore size is from about 2 microns to about 100 microns, more particularly from about 40 microns to about 75 microns. Other embodiments include those having a pore size of about 2 microns to about 20 microns. For applications involving microbial DNA, the glass frit filter plate pore size is preferably from about 10 microns to about 15 microns. Larger frit filter plate pore sizes are useful for human genome applications. Suitable frit filter plates are composed of sintered glass, are commonly used in chemical glassware, and are commercially available from Robu (Germany). The selection and manufacture of such frit filter plates is known to those of ordinary skill in the art.

In other embodiments, the frit filter plate is replaced by, or used in conjunction with, a porous filter. As used herein, "porous filter" refers to any material that allows the selective passage of substances contained in at least one liquid. More specifically, "porous filters" refer to those materials that are capable of substantially removing nucleic acids from a liquid containing the nucleic acids. Examples of suitable porous filters include, but are not limited to, filter paper configured to capture nucleic acids (e.g., FTA paper available from Whatman), glass fibers, glass beads, beads with a Charge switch technology coating available from Invitrogen, alumina filters, and porous monolithic polymers. Such materials and products are familiar to those of ordinary skill in the art. In some embodiments, the porous filter is a frit filter plate. In other embodiments, the frit filter plate is a sintered frit filter plate. Other materials suitable for providing the filtering function of the frit or sintered frit filter plates are polysiloxane and XTRABIND (Xtrana, inc., brookfield, colorado). The construction of the material to perform the filtering function of the present invention will be apparent to those of ordinary skill in the art.

In one embodiment, the above-described frit filter plate is packaged in a frit filter plate support or fluidic module. A cross-sectional view of one exemplary embodiment of the support or module is shown at 1000 in fig. 1. Wherein a frit filter plate (1004) as described above is placed in the housing (1008). The housing comprises an inlet (1012), through which inlet (1012) a fluid mixture comprising a nucleic acid of interest enters the housing and interacts with a frit filter plate as described herein, producing a first filtered mixture that passes through the housing outlet (1016). After exiting the filter holder or module, the first filtered mixture may enter other chambers in fluid contact with the outlet as described below or into a collector. Some embodiments include an optional heater as shown at 1020. The design and manufacture of such devices is known to those of ordinary skill in the art.

A second embodiment of this aspect of the invention is shown in figures 2A and 2B. The design and manufacture of such devices is known to those of ordinary skill in the art. Fig. 2A shows an exploded view of one embodiment of a frit filter plate holder (2000) comprising an upper shell (2002) and a lower shell (2004). The lower housing contains a groove (2006), described in more detail in fig. 2B, in which groove (2006) one or more of the above-described frit filter plates (2008) are disposed. A frit filter plate is loaded into both housings using gaskets (2010, 2012). The lower housing (2004) also includes an inlet (not shown) through which material containing nucleic acids to be separated is introduced to the frit filter plate and an outlet (2014) from which waste material and purified nucleic acids exit the filter module. Fig. 2B shows a cross-sectional view of a frit filter plate housing (2000). In this figure, in addition to the elements just described, an inlet (2018) and channels that direct the flow of matter through the frit filter plate and outlet are shown.

Another aspect of the present invention provides an apparatus for separating nucleic acids from a mixture containing the nucleic acids and extraneous materials. One embodiment of such a filter of the present invention is shown at 3000 in fig. 3. In this figure, the cavity (3004) has a first opening (3008) and a second opening (3012) through which the mixture containing nucleic acids passes and an outlet to discharge the at least partially dissolved mixture. Between the inlet and outlet there is a frit filter plate (3018) as described above that extends axially through at least a portion of the interior volume of the cavity to the extent shown at 3016. In some embodiments, more than one such frit filter plate is used. In other embodiments, at least one of the frit filter plates is made of sintered glass. The design and manufacture of such devices is known to those of ordinary skill in the art.

In some more specific embodiments, one end of the cavity has a frustoconical shape, and the chamber is sized to be mountable at a tip of a pipetting instrument, such as a pipette tip, such that the substance is first absorbed through the second opening, passes through the frit filter plate, is filtered, and then remains in the chamber portion above the frit filter plate. In some embodiments, a sample remaining in a portion of the chamber above the frit filter plate returns through the frit filter plate and through the second opening (3012).

In another embodiment, the pipette tip described above is used in conjunction with a heating device configured to heat the frit filter plate to facilitate separation of nucleic acids from the mixture. In a more specific embodiment, the heater is sized to fit within the pipette tip. The design and manufacture of such devices is known to those of ordinary skill in the art.

In other embodiments, the pipette tip is coupled to an electronic pipettor or automated pipetting station to control the flow rate through the frit filter plate. In some embodiments, the electronic pipettor is a handheld device. The design, manufacture, and operation of such devices are known to those of ordinary skill in the art.

In some embodiments of the invention, more than two porous filters are used in combination. In a more specific embodiment, the layers have different pore sizes. Without wishing to be bound by any particular theory of action, the larger porous filter pore size traps larger particles and thus may function as a prefilter. For example, a 40 micron to 60 micron porous filter can be used in series with a 10 micron to 15 micron porous filter to filter out human genomic DNA from a sample (e.g., blood) and thereby isolate microbial DNA. The use of a 40-60 micron porous filter to remove large amounts of human DNA allows low copy microbial DNA to better bind to a 10-15 micron porous filter and perform more stable analyses because human genomic DNA is no longer present in concentrations large enough to cause significant interference (e.g., by whole genome amplification). The porous filter may be located at the pipette tip as described above, with a thickness and diameter of about 5 millimeters (mm) each. In some embodiments, two or more porous filters are bonded together to form a substantially monolithic structure. The design, manufacture, and operation of such devices are known to those of ordinary skill in the art.

In a more specific embodiment using a pipette tip to provide the filter, frit filter plate(s) with larger pore sizes are provided closer to the pipette tip inlet. Again, without wishing to be bound by any particular theory of action, it will be appreciated by those of ordinary skill in the art that placing a larger pore size filter closer to the pipette tip inlet may provide a more uniform distribution of nucleic acids bound within the frit filter plate. By comparison, one of ordinary skill in the art would expect that if not, the nucleic acid would tend to adhere to the frit in the area closest to the pipette tip opening, since initial contact of the nucleic acid is more likely to occur as soon as the nucleic acid enters the frit.

Yet another aspect of the invention provides a microfluidic device for analyzing nucleic acids. One embodiment of the microfluidic device is shown at 4000 in fig. 4. A frit filter plate holder (4002) as described herein is provided. Upstream there is a frit filter plate holder in fluid communication with an elution buffer source (4004), a guanidine hydrochloride (Gu) reservoir (4006), and a Gu mixing column (4008), the flow of liquid from the Gu reservoir and Gu mixing column being controlled by a valve (4010). The Gu mixing tower is also in fluid communication with an ethanol-air source (4012). One of ordinary skill in the art will recognize that chaotropic agents other than Gu may also be used in the present invention. There is additionally a bead tissue grinder (4014) and electrical contacts (4020) in fluid communication with a sample collection column (4016), which sample collection column (4016) is in turn in fluid communication with an inlet check valve (4018). The output of these elements is controlled by valve 4022.

With continued reference to fig. 4, downstream of the frit filter plate holder (4002) is a waste tank (4024) to which flow is controlled by a valve (4026). Downstream flow from the frit filter plate holder is also controlled by a second valve (4028), said second valve (4028) controlling flow to the elution column (4030) and the check valve (4032) along the first branch; and a flow along a second branch of the flow path to another valve (4034). Further downstream of valve 4034 are one or more PCR reagent reservoirs 4036 and a valve 4038 leading to a PCR chamber 4040. Downstream of the PCR chamber is a valve (4042), which valve (4042) together with valve 4046 controls the flow of liquid from the PCR chamber to the microarray chamber (4050), which microarray chamber (4050) is also in fluid communication with a mix and flush buffer reservoir (4048) and a waste container (4052). The design and manufacture of such devices is known to those of ordinary skill in the art.

The operation of the apparatus described with reference to figure 4 is illustrated in the flow chart of figure 5. After obtaining an original sample (5002), such as a sputum sample containing cells of interest, the cells are lysed (5004) using bead-milled tissue mill 4014 and the mixture is passed through frit filter plate 4002 to purify the nucleic acid (5006) for amplification (5008) by PCR chamber 4040 and detection (5010) by microarray chamber 4050.

Without being bound by any particular theory or role, the present invention meets the above needs by providing a rigid, self-supporting frit filter plate structure that is thicker and thus provides high bonding capability, contains larger pores and thus provides low fluid resistance, higher flow rates and high resistance to particles in clinical and environmental samples, and is neither comprised of loose materials (e.g., silica gel, diatomaceous earth, glass spheres) nor comprised of brittle, fragile materials (e.g., fiber filters, membrane filters, silicon microstructures) and thus can provide handling and packaging under harsh conditions and simplified manufacturing.

Examples

The following examples are provided to illustrate certain aspects of the present invention and to assist those skilled in the art in practicing the invention. These examples are in no way to be construed as limiting the scope of the invention in any way.

I. Arrangements for using the apparatus of the invention

Referring to fig. 2A and 2B, a protocol for performing the purification and detection of the present invention is provided below.

1. A frit filter plate was inserted into the holder body (one of four different pores: fine, medium, coarse and extra coarse). The housing is fixed.

2. 500 μ L of the sample (10)⁴copies/mL) was mixed with 500. mu.L of 6M guanidine (pH 6.5).

3. The mixture (1mL) was passed through a frit filter plate using a 1mL syringe at a flow rate of 100. mu.L/min. The samples were washed by manually passing air through a frit filter plate using a 5mL syringe.

4. Using a 1mL syringe, 1mL of 70% ethanol (EtOH) was injected at a rate of 1 mL/min to wash the bound nucleic acids. Air was manually passed through the frit filter plate using a 5mL syringe to clean the EtOH.

5. Elution buffer (10mM Tris, pH 8.0) was carefully injected into the frit filter plate holder using a 1mL syringe at a rate of 100. mu.L/min until the buffer was first seen in the outlet tube.

6. A heater (heat block) was placed under the frit filter plate holder and heated at 70 ℃ for 3 minutes.

After 7.3 minutes, the elution buffer was continued to pass through the frit filter plate holder. Fractions (50. mu.L to 100. mu.L) were collected for PCR analysis.

8. The frit filter plate holder was rinsed with 1mL of 10% bleach (bleach dilution time not exceeding 1 week), 5mL of 10% Tris-HCl (pH 8.0), and 5mL of water. The frit was replaced.

Second embodiment of the invention

Referring to fig. 3, a protocol for performing the purification and detection of the present invention is provided below.

1. To 500. mu.L of 6M guanidine in A vial was added 500. mu.L of the sample. And vortex mixed.

2. A1.2 mL pipette tip with a frit filter plate built in was attached to an electronic pipettor (Gilson Concept).

3. The electronic pipettor is set to speed 1 (lowest speed). 1mL of the sample mixture in the A vial was aspirated. The sample mixture was pushed completely through the frit filter plate. The bolus injection of the sample mixture allowed it to sit immediately above the frit filter plate.

4. The sample was placed back in vial a. The bolus of sample mixture caused it to be expelled completely back into the bottle.

5. Steps 3 and 4 were repeated four times.

6. The electronic pipettor is set to speed 5 (highest speed). 1mL of 70% ethanol in the B vial was aspirated to wash the bound nucleic acids on the frit filter plate. This was repeated four times.

7. Traces of ethanol were removed by placing the tip above the ethanol solution. Air was drawn off five times to dry the frit filter plates.

8. A C vial containing 100. mu.L of 10mM Tris-HCl (pH 8.0) was placed in a heater set at 70 ℃. Heat for 5 minutes.

9. The electronic pipettor is set to speed 1. The elution buffer was aspirated and placed back into the C vial and repeated five times to remove the nucleic acids from the glass frit filter plate.

Demonstration of the Excellent results of the invention

The results of two experiments using the protocol are shown in fig. 6 and 7, where samples of bacillus anthracis (Ba) in whole blood and sputum were treated using the materials and methods of the present invention.

In fig. 6, the samples processed using the methods and devices of the present invention (fig. 2A and 2B) (curve a) outperformed the samples processed using the devices commercially available from Qiagen (curve B). Curves C and D are untreated input sample and negative control, respectively. In the experiment, the input of 100. mu.L in whole blood was 10⁵Perml of Bacillus anthracis cells, passed through a medium (pore size 10 μm to 15 μm) frit filter plate as described herein or into a Qiagen apparatus, approximately 50 μ L of the eluate was collected and analyzed using PCR. As shown, after approximately 25 cycles of PCR amplification, the samples treated using the materials and methods of the present invention clearly and significantly outperformed the results obtained with samples treated using prior art devices.

FIG. 7 illustrates the results of another experiment using the method and apparatus (FIG. 3) in which a sample of Bacillus anthracis in sputum was processed using the materials and methods of the present invention. The samples treated using the method and apparatus of the present invention (curve a) outperformed the untreated samples (curve B). Curve C is a negative control. In this experiment, the input of 500. mu.L in sputum was 10⁴Perml of Bacillus anthracis cells, passed through a medium glass frit filter plate as described herein, and about 100. mu.L of the eluate was collected and analyzed using quantitative, real-time PCR.As shown, after approximately 27 cycles of PCR amplification, the samples treated using the materials and methods of the present invention clearly and significantly outperformed the results obtained for the untreated samples.

The above-described methods and apparatus can be successfully used to identify DNA and/or RNA from the various organisms listed in Table 1, which are present in the various matrices (i.e., sample types) listed in Table 2.

TABLE 1

Viral Equine Encephalitis (VEE)

Vaccinia virus

Plague bacillus

Bacillus anthracis

Adenoviral vectors

Streptococcus pyogenes

Chlamydia pneumoniae

Influenza A

Influenza B

Mixture of adenovirus and streptococcus pyogenes

Mixture of influenza A and adenovirus

The following were effectively analyzed using the method and apparatus of the present invention:

TABLE 2

water/TE (10mM Tris-HCl,

1.0mM EDTA buffer)

Cotton swab extract (swab extracts)

Phlegm (phlegm)

Nose washing water

Whole blood

Conclusion

Compared to the prior art, the present invention provides several advantages: 1) simplified methods and devices that do not require centrifugation or high pressure to move the fluid; 2) a modular device for fluid integration into a complete analytical system; 3) large pore sizes that can handle complex samples while maintaining low fluid resistance; 4) high extraction and elution efficiency due to the fact that the fluid can move from two directions; 5) the rigidity of an additional supporting structure is not needed; and 6) the device can be reused for continuous sampling durability. Although specific embodiments and examples are described herein, it will be appreciated by those of ordinary skill in the art that many different embodiments of the invention can be made without departing from the spirit or scope of the disclosure. For example, other materials with affinity for nucleic acids may be used for the frit, or the frit may be modified to better attract nucleic acids and better attract nucleic acids without the use of chaotropic salts such as guanidine. The frit filter plate may contain immobilized antibodies to extract microorganisms and toxins. Other variations will be apparent to persons of ordinary skill in the art.

Bibliography

The following references are incorporated by reference in their entirety into this specification, which are suitable for various purposes.

Beach, R.A., Strittmatter, R.P. et al (2007) Integrated micropump analysis Chip and Method of Making the Same US7,189,358.

Colpan, M. (2001) Process and device for the Isolation of cell components, Such as Nucleic Acids, from 20 Natural Sources (method and apparatus for isolating cellular components Such as Nucleic Acids from 20 Natural Sources) US 6,274,371.

Deshmukh, A.J, (2006). Method and Automated Fluidic System for detecting proteins in Biological samples US 6,989,130.

Kalra, k.l., Pawlak, k.et al (1990). Immunoassay Test Device and method US 4,912,034.

Knollenberg, B.A., Rodier, D.et al (2007) Molecular contamination monitoring System and Method (Molecular contamination monitoring System and Method) US7,208,123.

McCormack，R.M.(1990).Process for Purling Nucleic Acids US4,923,978.

Terry, B.R., Scrudder, K.M. et al (2004) Method and Apparatus for high density Screening of Bioactive Molecules US 6,790,652.

Tittgen, 1(2004), Chromatography Material and a Method Using the same (Chromatography Material and methods of Using the same) US 6,800,752.

Wick, C.H, (2007). Method and System for Detecting and recording submicron Sized Particles (Method and System for Detecting and recording submicron Particles). US7,250,138.

Claims (8)

1. A method of separating a nucleic acid from a mixture comprising the nucleic acid and extraneous material, the method comprising:

flowing the mixture through the interior volume of the cavity; the cavity having disposed therein at least one frit filter plate to which the nucleic acid is bound, the frit filter plate being disposed in the cavity such that the mixture flows through the frit filter plate under conditions effective to sufficiently separate the nucleic acid from the extraneous matter, thereby retaining the nucleic acid on the frit filter plate, and

eluting the nucleic acid bound to the frit filter plate,

the glass material filter plate is a sintered glass material filter plate, has a pore size of 2-220 micrometers, and has a thickness of 1-20 mm.

2. The method of claim 1, wherein the nucleic acid comprises microbial DNA and RNA.

3. The method of claim 1, wherein the nucleic acid comprises human genomic DNA and RNA.

4. The method of claim 1, wherein the mixture produced after flowing the mixture through the frit filter plate is a first filtered mixture, and the method further comprises flowing the first filtered mixture through a second frit filter plate under conditions effective to separate the nucleic acid from the first filtered mixture.

5. The method of claim 1, wherein the glass frit filter plate has a pore size of 150-200 microns.

6. The method of claim 1, wherein the glass frit filter plate has a pore size of 2 microns to 100 microns.

7. The method of claim 6, wherein the glass frit filter plate has a pore size of 40-75 microns.

8. The method of claim 6, wherein the frit filter plate has a pore size of 2-20 microns.