US20120283423A1 - Apparatus, system and method for purifying nucleic acids - Google Patents
Apparatus, system and method for purifying nucleic acids Download PDFInfo
- Publication number
- US20120283423A1 US20120283423A1 US13/541,353 US201213541353A US2012283423A1 US 20120283423 A1 US20120283423 A1 US 20120283423A1 US 201213541353 A US201213541353 A US 201213541353A US 2012283423 A1 US2012283423 A1 US 2012283423A1
- Authority
- US
- United States
- Prior art keywords
- glass frit
- micron
- glass
- nucleic acids
- pore size
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D39/00—Filtering material for liquid or gaseous fluids
- B01D39/14—Other self-supporting filtering material ; Other filtering material
- B01D39/20—Other self-supporting filtering material ; Other filtering material of inorganic material, e.g. asbestos paper, metallic filtering material of non-woven wires
- B01D39/2003—Glass or glassy material
- B01D39/2006—Glass or glassy material the material being particulate
- B01D39/201—Glass or glassy material the material being particulate sintered or bonded by inorganic agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/02—Burettes; Pipettes
- B01L3/0275—Interchangeable or disposable dispensing tips
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0681—Filter
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
Definitions
- This invention relates to the purification of chemical substances, and, more particularly, to devices, methods, and systems for performing chemical purification and analysis. More particularly, the devices, methods, and systems provided by the invention have particularly useful application in the purification and analysis of nucleic acids, and, more particularly to microfluidic devices for performing such purification and analysis.
- the invention has applications in the areas of analytical chemistry, forensic chemistry, microfluidics and microscale devices, medicine, and public health.
- PCR Polymerase Chain Reaction
- the filters have small pore sires, typically between about one- and three microns, to get efficient capture of the nucleic acids from the sample. Because of the small pores sires, the filters are also relatively thin, typically less than two millimeters thick to reduce fluid flow resistance when sample is forced though the small pore sires.
- a sample is mixed with a chaotropic agent, such as guanidine, and the mixture is passed through the glass filter using centrifugal force, in which fluid flows in only one direction.
- Nucleic acids bind to the glass filter; they are washed with ethanol or isopropanol, and subsequently released using a ten millimolar (10 mM) Tris buffer at a pH of about eight (pH 8.0) or water.
- a ten millimolar (10 mM) Tris buffer at a pH of about eight (pH 8.0) or water.
- the small pore sizes limit the amount of sample that can he processed, due to resistance created by fluid flow and potential for clogging created by greater flow rates.
- devices such as the Qiagen devices can be easily damaged or otherwise rendered ineffective easily.
- these characteristics limit sample input volume, the types of samples that can be examined, large-volume samples, concentration factors, and simple fluidic integration.
- U.S. Pat. No. 6,274,371 (Colpan 2001) describes silica gel, aluminum oxide, and diatomaceous earth as a preferred filtering agent for removing unwanted contaminants from cellular lysates prior to nucleic acid analysis.
- U.S. Pat. No. 6,800,752 (Tittgen 2004) describes using a chromatography material to separate mixtures comprising nucleic acids, in which the material includes carrier and ion exchanger functions wherein the carrier comprises a fibrous material on a support, such as a plastic frit.
- the present invention provides a method for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter.
- Suitable nucleic acids for use in the present invention include microbial DNA and human genomic DNA.
- the method of the invention comprises passing the mixture through a glass fit under conditions effective to separate the nucleic acids from the extraneous matter.
- the glass frit is a sintered glass frit.
- the glass frit has a pore size between about 2 microns and about 220 microns; in more specific embodiments, the glass fit has a pore size between about 150 microns and about 200 microns; in other more specific embodiments, the glass fit has a pore size between about 2 microns and about 100 microns; and still more specifically, the glass frit has a pore size between about 40 microns and about 75 microns; yet other more specific embodiments includes those in which the glass frit has a pore size between about 2 microns and about 20 microns.
- the method of the invention includes passing the mixture through a glass fit to produce thereby a first-filtered mixture and then passing the first-filtered mixture through a second glass fit under conditions effective to separate the nucleic acids from the first-filtered mixture.
- the present invention provides a device for filtering isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter.
- the device comprises a hollow chamber having an inlet and an outlet; and disposed therein at least one glass frit having a pore size of between about 2 microns and about 220 microns and arranged at a location intermediate the inlet and the outlet.
- the glass frit is a sintered glass frit.
- the glass fit is a sintered glass fit.
- the glass fit has a pore size between about 2 microns and about 220 microns; in more specific embodiments, the glass frit has a pore size between about 150 microns and about 200 microns; in other more specific embodiments, the glass fit has a pore size between about 2 microns and about 100 microns; and still more specifically, the glass frit has a pore size between about 40 microns and about 75 microns; yet other more specific embodiments includes those in which the glass frit has a pore size between about 2 microns and about 20 microns.
- the present invention provides a fluidic device for identifying one or more nucleic acids from a mixture of such nucleic acids and extraneous matter.
- the fluid device of the invention comprises: an inlet, an outlet, and at least one fluidic reaction chamber intermediate the inlet and the outlet and in communication with each of the inlet and the outlet.
- the device further comprise at least one glass fit arranged at a location (or locations) proximal to the inlet and the reaction chamber(s) and in fluidic communication with each of the inlet and reaction chamber(s).
- the glass frit(s) have a pore size of between about 2 microns and about 220 microns.
- the mixture enters the device through the inlet and passes through the glass fit to exit therefrom as a filtered product before entering the fluidic reaction chamber(s).
- At least one fluidic reagent dispenser is arranged intermediate the glass frit and the reaction chamber(s)and in fluidic communication therewith.
- the glass frit is a sintered glass fit.
- the glass frit has a pore size between about 2 microns and about 220 microns; in more specific embodiments, the glass frit has a pore size between about 150 microns and about 200 microns; in other more specific embodiments, the glass frit has a pore size between about 2 microns and about 100 microns; and still more specifically, the glass frit has a pore size between about 40 microns and about 75 microns; yet other more specific embodiments includes those in which the glass frit has a pore size between about 2 microns and about 20 microns.
- the fluidic device includes a heater proximal to the glass frit(s).
- FIG. 1 is a schematic illustration of a glass frit device (“a filter module”) for purifying nucleic acids in accordance with the present invention.
- FIGS. 2A and 2B are illustrations of a filter in accordance with the present invention.
- FIG. 2A is an illustration an exploded view of a glass frit device (“a filter module”) for purifying nucleic acids in accordance with the present invention.
- FIG. 2B is a cut-away illustration of the same device.
- FIG. 3 is a schematic illustration of a glass frit device (“a hollow chamber”) for purifying nucleic acids in accordance with the present invention.
- FIG. 4 is a schematic illustration of a microarray device in, accordance with the present invention.
- FIG. 5 is a flowchart of a process for purifying and identifying nucleic acids in accordance with the present invention.
- FIG. 6 is graph illustrating the improved performance characteristics of devices in accordance with the present invention as shown in FIGS. 2A and 2B as demonstrated by measuring fluorescence of a sample material (100 ⁇ L of Bacillus anthracis cells in whole blood at a concentration of 1 ⁇ 105 cells/mL) as a function of PCR cycles.
- the solid trace (A) shows the result for samples treated in accordance with the present invention; the dashed trace (B) shows sample treated using a device available commercially from Qiagen; dashed traces (C) and (D) are unprocessed sample and negative control.
- FIG. 7 is graph illustrating the improved performance characteristics of devices in accordance with the present invention as shown in FIG. 3 as demonstrated by measuring fluorescence of a sample material (500 ⁇ L of Bacillus anthracis cells in sputum at a concentration of 1 ⁇ 10 4 cells/mL) as a function of PCR cycles.
- the solid trace (A) shows the result for samples treated in accordance with the present invention; the dashed trace (B) shows unprocessed sample; dashed trace (C) is negative control.
- the present invention provides methods and devices for at least partially purifying nucleic acids from mixtures of such in combination with other substances, such as proteins, small molecules, cell membrane fragments, and the like.
- nucleic acids refers to individual nucleic acids and polymeric chains of nucleic acids, including DNA and RNA, whether naturally occurring or artificially synthesized (including analogs thereof), or modifications thereof, especially those modifications know to occur in nature, having any length.
- nucleic acid lengths that are in accord with the present invention include, without limitation, lengths suitable for PCR products (e.g., about 50 base pairs (bp)) and human genomic DNA (e.g., on an order from about kilobase pairs (Kb) to gigabase pairs (Gb)).
- nucleic acid encompasses single nucleic acids as well as stretches of nucleotides, nucleosides, natural or artificial, and combinations thereof, in small fragments, e.g., expressed sequence tags or genetic fragments, as well as larger chains as exemplified by genomic material including individual genes and even whole chromosomes.
- the nucleic acids are from a pathogen, such as bacteria or a virus. Such pathogens include those harmful to humans and animals. Of the former, in some embodiments, the pathogen is one that used as a biological weapon, including naturally occurring pathogens that have been weaponized.
- the nucleic acids comprise microbial DNA.
- the microbial DNA is from Bacillus anthracis.
- the nucleic acids come from humans or animals.
- nucleic acids comprise human genomic DNA.
- the present invention provides methods for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter.
- the methods of the invention comprise passing the mixture through a hollow chamber having an inlet and an outlet; wherein said inlet and outlet are the same and disposed therein at least one porous filter, under conditions effective to separate substantially the nucleic acids from the extraneous matter.
- extraneous matter refers to all materials that are distinct from the nucleic acids in the sample. Examples of such extraneous materials include, but are not limited to, proteins, starches, lipids, metal ions, and larger cellular structures such as membrane fragments.
- the phrase “separate substantially” as used herein refers to separations that, in some embodiments, provide the nucleic acids in at least 30% purity with respect to the extraneous materials, in more specific embodiments provide the nucleic acids in at least 50% purity with respect to the extraneous materials, in still more specific embodiments provide the nucleic acids in at least 70% purity with respect to the extraneous materials, in yet more specific embodiments provide the nucleic acids in at least 95% purity with respect to the extraneous materials, and in still yet more specific embodiments, provide the nucleic acids in at least 99% purity with respect to the extraneous materials.
- the glass frit is made from standard materials using standard methods as known to persons having ordinary skill in the art or available commercially as described below.
- the glass frit has a thickness substantially between about one millimeter and about 20 millimeters, more specifically between about two millimeters and about five millimeters, and still more specifically between about two millimeters and about three millimeters.
- Exemplary glass frit pore sizes suitable for use with the present invention, including the various embodiments described herein, are between about 2 microns and about 200 microns. In more specific embodiments, the pore size is between about 150 microns and about 200 microns.
- the pore size is between about 2 microns and about 100 microns, and still more specifically between about 40 microns and about 75 microns. Other embodiments include those for which the pore size is between about 2 microns and about 20 microns.
- a glass frit size of between about 10 microns and about 15 microns is suitable. Larger frit pore sizes can be used for human genomic applications. Suitable glass frits are composed of sintered glass and are typically used in chemistry glassware and are available commercially from Robu (Germany). The choice and manufacture of such glass frits will be understood by persons having ordinary skill in the art.
- the glass frit is replaced or used in conjunction with a porous filter.
- porous filter refers to any material that allows selective passage of at least one substance contained in a liquid. More specifically, “porous filter” refers to those materials capable of substantially removing nucleic acids from a liquid containing such nucleic acids. Examples of suitable porous filters include, but are not limited to, filter papers configured to trap nucleic acids (e.g., FTA paper, available from Whatman), glass fibers, glass beads, beads with the Charge Switch Technology coating, available from Invitrogen, aluminum oxide filters and porous monolithic polymers. Such materials and products are familiar to those having ordinary skill in the art.
- the porous filter is a glass frit.
- the glass frit is a sintered glass frit.
- suitable materials for providing the filtering function of the glass frit or sintered glass fit are silicone and XTRABIND (Xtrana, Inc., Broomfield, Colo.). The configuration of such materials to perform the filtering functions of the present invention will be apparent to persons having ordinary skill in the art.
- the above-described glass frit is packaged into a frit holder or fluidic module.
- a frit holder or fluidic module is shown in cut-away view at 1000 in FIG. 1 .
- a glass frit as just described above 1004
- the housing includes an inlet ( 1012 ) into which fluidic mixture including nucleic acids of interest enter the housing and interact with the glass frit as described herein to produce a first-filtered mixture which passes through an outlet of the housing ( 1016 ).
- the first-filtered mixture can proceed to other chambers in fluidic contact with the outlet, such as described below, or to a collector.
- An optional heater, shown at 1020 is included in some embodiments. The design and manufacture of such devices will be understood by those having ordinary skill in the art.
- FIGS. 2A and 2B A second embodiment of this aspect of the invention is shown in FIGS. 2A and 2B .
- the design and fabrication of such devices are known to those having ordinary skill in the art.
- FIG. 2A shows an exploded view of one embodiment of a frit holder ( 2000 ), which includes an upper housing body ( 2002 ) and a lower housing body ( 2004 ).
- the lower housing body includes a recess ( 2006 ), described in greater detail in FIG. 2B , into which is disposed one or more glass frits ( 2008 ) which are described above.
- the glass frit is seated in the housing bodies using gaskets ( 2010 , 2012 ).
- the lower housing body ( 2004 ) also includes an inlet (not shown) through which materials containing nucleic acids to be separated are introduced to glass frit, and outlet ( 2014 ) from which waste material and the purified nucleic acids exit the filter module.
- FIG. 2B shows a cut-away view of the frit housing ( 2000 ). There, in addition to the elements just described, the inlet ( 2018 ) is shown, along with channels for directing the flow of material through the glass frit and outlet.
- the present invention provides a device for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter.
- a hollow chamber ( 3004 ) having a first opening ( 3008 ) and a second opening ( 3012 ) through which a mixture comprising nucleic acids is passed and an outlet from which an at least partially resolved mixture exits.
- a glass frit ( 3018 ) as described above that extends axially through at least a portion of the interior volume of the hollow chamber, the extent to which is shown at 3016 .
- more than one such frit is used.
- at least one of the glass frits is made of sintered glass. The design and fabrication of such devices are known to those having ordinary skill in the art.
- one end of the hollow chamber has a fiustaconical shape the chamber is dimensioned to fit on the end of a pipetting instrument, e.g., as a pipet tip, so that materials are first taken up though the second opening, pass through the glass frit, are filtered and then retained in the portion of the chamber above the frit.
- a pipetting instrument e.g., as a pipet tip
- the sample retained in the portion of the chamber above the frit is passed back through the frit through the second opening ( 3012 ).
- the above-described pipet tip is combined with a heating device that is configured to heat the frit to facilitate separation of the nucleic acids from the mixture.
- the heater is dimensioned to fit within the pipet tip. The design and fabrication of such devices are known to those having ordinary skill in the art.
- the pipet tip is coupled with an electronic pipettor or robotic pipetting workstation to control the flow rate through the frit.
- the electronic pipettor is a hand-held device. The design, fabrication, and operation of such devices are known to those having ordinary skill in the art.
- each layer has a different pore size.
- larger porous filter pore sizes trap larger particles, and so can serve as a prefilter.
- a 40 micron-60 micron porous filter could be used in tandem with a 10 micron-15 micron porous filter to deplete human genomic DNA from a sample (e.g., blood) to isolate microbial DNA.
- the porous filter can be in a pipet tip as described above, having a thickness and diameter of about five millimeters (mm) each.
- two or more of the porous filters are fused together to form a substantially monolithic structure. The design, fabrication, and operation of such devices are known to those having ordinary skill in the art.
- the glass frit(s) having larger pore sizes are disposed closer to the pipet tip inlet.
- the larger pore sized filter nearer the pipet tip inlet can provide a more uniform distribution of nucleic acid binding within the frit.
- persons having ordinary skill in the art will expect that otherwise the nucleic acids will tend to bind to the frit at the area closest to the pipet tip opening, since the nucleic acids are more likely to make initial contact just as the nucleic acids enter the frit.
- the present invention provides a microfluidic device for analyzing nucleic acids in accordance with the present invention.
- a microfluidic device for analyzing nucleic acids in accordance with the present invention.
- FIG. 4 One embodiment of such a microfluidic device is show in FIG. 4 at 4000 .
- a fit holder ( 4002 ) as described herein is provided. Upstream, the frit holder in fluidic communication with a source of elution buffer ( 4004 ), a guanidine hydrochloride (Gu) reservoir ( 4006 ) and Gu mixing tower ( 4008 ), the flow from which Gu and Gu mixing tower are controlled by a valve ( 4010 ).
- the Gu mixing tower is further in fluidic communication with an ethanol-air source ( 4012 ).
- chaotropes other than Gu can be used with the present invention.
- a bead beater ( 4014 ) that is in fluidic communication with a sample collection tower ( 4016 ), which is in turn in fluidic communication with inlet check value ( 4018 ), and an electrical contact ( 4020 ). The output from these elements is controlled by valve 4022 .
- a waste tank ( 4024 ) downstream of frit holder ( 4002 ) is a waste tank ( 4024 ), the flow to which is controlled by a valve ( 4026 ). Downstream flow from the fit holder is also controlled by a second valve ( 4028 ), which controls flow to an elution tower ( 4030 ) and a check valve ( 4032 ) along a first branch; and to another valve ( 4034 ) along a second branch of the flow path.
- a second valve ( 4028 ) downstream from valve 4034 , are one or more reservoirs of PCR reagents ( 4036 ) and a valve ( 4038 ) which leads to a PCR chamber ( 4040 ).
- valve 4042 Downstream of the PCR chamber is a valve ( 4042 ), which, along with valve 4046 , controls flow from the PCR chamber to a microarray chamber ( 4048 ) that is also in fluidic communication with hybridization and wash buffer reservoir ( 4048 ) and a waste container ( 4052 ).
- a microarray chamber 4048
- hybridization and wash buffer reservoir 4048
- a waste container 4052
- the operation of the device described with respect to FIG. 4 is illustrated by the flowchart shown in FIG. 5 at 5000 .
- the cells are lysed ( 5004 ) using bead beater 4014 , and the mixture passed for purification of the nucleic acids ( 5006 ) through the fit 4002 for amplification ( 5008 ) by PCR chamber 4040 and detection ( 5010 ) by the microarray chamber 4050 .
- the present invention meets the needs described above by implementing a rigid, self-supporting fit structure that is relatively thick for high binding capacity, contains relatively large porosities for low fluid impedence, faster flow rates, and higher tolerance to particles in clinical and environmental samples, and consists of no loose material (e.g. silica gel, diatomaceous earth, glass beads) and no flimsy, delicate materials (e.g. fiber filters, membrane filters, silicon microstructures) for rugged operation and packaging and simplified manufacturing.
- no loose material e.g. silica gel, diatomaceous earth, glass beads
- delicate materials e.g. fiber filters, membrane filters, silicon microstructures
- FIGS. 6 and 7 The results of two experiment using such protocols are show in FIGS. 6 and 7 , where samples of Bacillus anthracis (Ba) in whole blood and sputum were treated using the materials and methods of the invention.
- the sample treated using a method and device ( FIGS. 2A and 2B ) of the invention outperformed a sample treated using a device available commercially from Qiagen (trace B).
- Traces C and D are the unprocessed input sample and negative control, respectively.
- 100 ⁇ L of Bacillus anthracis cells at 10 5 /mL in whole blood was input, either through a medium (10-15 ⁇ m pore size) frit as described herein or into a Qiagen device, and elution of about 50 ⁇ L were collected and analyzed using PCR.
- the sample treated using the materials and methods of the invention clearly and significantly outperform the results from the sample treated using the prior art device.
- FIG. 7 illustrates the result of another experiment using such a method and device ( FIG. 3 ), where a sample of Bacillus anthracis cells in sputum was treated using the materials and methods of the invention.
- the sample treated using the method and device of the invention (trace A) outperformed an unprocessed sample (trace B).
- 500 ⁇ L of Bacillus anthracis cells at 10 4 /mL in sputum was input through a medium flit as described herein, and an elution of about 100 ⁇ L was collected and analyzed using quantitative, real-time PCR.
- the sample treated using the material and method of the invention clearly and significantly outperform the results from unprocessed sample.
- the present invention offers several advantages over the prior art: 1) a simplified method and device requiring no centrifugation or high pressure to move fluids, 2) a modular device for fluidic integration into a complete analytical system, 3) large pore sizes to process complex samples while maintaining low fluid resistance; 4) high extraction and elution efficiencies since fluids can be moved from both directions; 5) rigidity that eliminates the need for additional support structures, and 6) durability that allows reusing the device for sequential samples.
- glass frit can be modified to better attract nucleic acids and nucleic acids without using a chaotropic salt such as guanidine.
- Glass frits can contain immobilized antibodies to extract microbes and toxins. Still other variations will be clear to those having ordinary skill in the art.
Abstract
Methods and devices for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter are provided. In one embodiment, the method of the invention comprises passing the mixture through a glass frit under conditions effective to separate the nucleic acids from the extraneous matter. In a more specific embodiment, the glass frit is a sintered glass frit.
Description
- This Application is a Continuation Application of U.S. patent application Ser. No. 12/793,253, filed on Jun. 3, 2010, which is a Continuation Application of U.S. patent application Ser. No. 11/933,113, filed on Oct. 31, 2007 now U.S. Pat. No. 7,759,112. The entirety of all of the aforementioned applications is incorporated herein by reference.
- This invention relates to the purification of chemical substances, and, more particularly, to devices, methods, and systems for performing chemical purification and analysis. More particularly, the devices, methods, and systems provided by the invention have particularly useful application in the purification and analysis of nucleic acids, and, more particularly to microfluidic devices for performing such purification and analysis. The invention has applications in the areas of analytical chemistry, forensic chemistry, microfluidics and microscale devices, medicine, and public health.
- The extension of semiconductor fabrication techniques to create highly miniaturized chemical devices (Beach, Strittmatter et al. 2007) has created a revolution in analytical chemistry, especially by providing a means for identifying chemical substances present in minute concentrations in complex mixtures with great precision and accuracy. This revolution has had noticeable impact in chemical processing, medicine, forensic science, and national defense, where such devices provide fast, portable, and economic biological detectors. Examples of such devices include devices for collecting and identifying particulates (Wick 2007), systems for detecting molecular contaminants (Knollenberg, Rodier et al. 2007), and devices for detecting proteins (Terry, Scudder et al. 2004; Deshmukh 2006). Other devices use fluidic technologies to isolate and/or amplify nucleic acids using Polymerase Chain Reaction (PCR) in an automated system. Examples of such devices are those sold commercially by Qiagen (Hilden, Germany), Roche (Basel, Switzerland), Applied Biosystems (Foster City, Calif.), Idaho Technologies (Salt Lake City, Utah), and Cepheid (Sunnyvale, Calif.).
- But as with any analytical process, preparing the sample prior to processing is critical to good performance. The presence of too many complicating factors and concentrations of substances that may mask analytes of interest can render robust detection all but impossible. This problem is of particular concern when attempting to analyze the nucleic acid content of cell lysates, which are extremely complex and heterogenous mixtures (Colpan 2001). The preparatory task is made still more difficult where portable analytical devices are concerned, since those devices are expected to be used in locations where common laboratory support equipment, such as centrifuges and separation columns, are not available. In those cases, some means for filtering a raw sample, such as a blood or urine sample, is critical to providing meaningful results. Current devices based on fluidic technologies, in particular the above-mentioned Qiagen devices, use glass filters that are soft and compliant, requiring a support matrix. The filters have small pore sires, typically between about one- and three microns, to get efficient capture of the nucleic acids from the sample. Because of the small pores sires, the filters are also relatively thin, typically less than two millimeters thick to reduce fluid flow resistance when sample is forced though the small pore sires. In the Qiagen procedure, typically a sample is mixed with a chaotropic agent, such as guanidine, and the mixture is passed through the glass filter using centrifugal force, in which fluid flows in only one direction. Nucleic acids bind to the glass filter; they are washed with ethanol or isopropanol, and subsequently released using a ten millimolar (10 mM) Tris buffer at a pH of about eight (pH 8.0) or water. But the small pore sizes limit the amount of sample that can he processed, due to resistance created by fluid flow and potential for clogging created by greater flow rates. Thus, devices such as the Qiagen devices can be easily damaged or otherwise rendered ineffective easily. Moreover, these characteristics limit sample input volume, the types of samples that can be examined, large-volume samples, concentration factors, and simple fluidic integration.
- Larger glass filters have been used to provide pre-processing 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 that includes an optional prefilter assembly made of glass fibers. However, this device is not amicrofluidic device and does not show or suggest the use of glass frits as a filter prior to microscale PCR reactions. U.S. Pat. No. 4,923,978 (McCormack 1990) describes prior uses of glass fiber filters to remove unwanted protein- and protein-DNA complexes from aqueous DNA samples, but in a disparaging manner noting that such filters have low binding capacities (see Column 2). Indeed, the '978 patent claims a very different material for performing such filtrations. U.S. Pat. No. 6,274,371 (Colpan 2001) describes silica gel, aluminum oxide, and diatomaceous earth as a preferred filtering agent for removing unwanted contaminants from cellular lysates prior to nucleic acid analysis. U.S. Pat. No. 6,800,752 (Tittgen 2004) describes using a chromatography material to separate mixtures comprising nucleic acids, in which the material includes carrier and ion exchanger functions wherein the carrier comprises a fibrous material on a support, such as a plastic frit.
- Nevertheless, there remains therefore a need to provide fluidic devices that are effective to isolate and identify nucleic acids that overcome the limitations of the current generation of such devices. The present invention meets these and other needs.
- In a first aspect, the present invention provides a method for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter. 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 glass fit under conditions effective to separate the nucleic acids from the extraneous matter. In a more specific embodiment, the glass frit is a sintered glass frit. In some embodiments, the glass frit has a pore size between about 2 microns and about 220 microns; in more specific embodiments, the glass fit has a pore size between about 150 microns and about 200 microns; in other more specific embodiments, the glass fit has a pore size between about 2 microns and about 100 microns; and still more specifically, the glass frit has a pore size between about 40 microns and about 75 microns; yet other more specific embodiments includes those in which the glass frit has a pore size between about 2 microns and about 20 microns. In another embodiment, the method of the invention includes passing the mixture through a glass fit to produce thereby a first-filtered mixture and then passing the first-filtered mixture through a second glass fit under conditions effective to separate the nucleic acids from the first-filtered mixture.
- In a second aspect, the present invention provides a device for filtering isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter. In some embodiments, the device comprises a hollow chamber having an inlet and an outlet; and disposed therein at least one glass frit having a pore size of between about 2 microns and about 220 microns and arranged at a location intermediate the inlet and the outlet. In more specific embodiments, the glass frit is a sintered glass frit. In other embodiments, the glass fit is a sintered glass fit. In some embodiments, the glass fit has a pore size between about 2 microns and about 220 microns; in more specific embodiments, the glass frit has a pore size between about 150 microns and about 200 microns; in other more specific embodiments, the glass fit has a pore size between about 2 microns and about 100 microns; and still more specifically, the glass frit has a pore size between about 40 microns and about 75 microns; yet other more specific embodiments includes those in which the glass frit has a pore size between about 2 microns and about 20 microns.
- In a third aspect, the present invention provides a fluidic device for identifying one or more nucleic acids from a mixture of such nucleic acids and extraneous matter. In some embodiments, the fluid device of the invention comprises: an inlet, an outlet, and at least one fluidic reaction chamber intermediate the inlet and the outlet and in communication with each of the inlet and the outlet. The device further comprise at least one glass fit arranged at a location (or locations) proximal to the inlet and the reaction chamber(s) and in fluidic communication with each of the inlet and reaction chamber(s). The glass frit(s) have a pore size of between about 2 microns and about 220 microns. The mixture enters the device through the inlet and passes through the glass fit to exit therefrom as a filtered product before entering the fluidic reaction chamber(s). At least one fluidic reagent dispenser is arranged intermediate the glass frit and the reaction chamber(s)and in fluidic communication therewith. In a more specific embodiment, the glass frit is a sintered glass fit. In some embodiments, the glass frit has a pore size between about 2 microns and about 220 microns; in more specific embodiments, the glass frit has a pore size between about 150 microns and about 200 microns; in other more specific embodiments, the glass frit has a pore size between about 2 microns and about 100 microns; and still more specifically, the glass frit has a pore size between about 40 microns and about 75 microns; yet other more specific embodiments includes those in which the glass frit has a pore size between about 2 microns and about 20 microns. In another embodiment, the fluidic device includes a heater proximal to the glass frit(s).
- These and other aspects and advantages will become apparent when the Description below is read in conjunction with the accompanying Drawings.
-
FIG. 1 is a schematic illustration of a glass frit device (“a filter module”) for purifying nucleic acids in accordance with the present invention. -
FIGS. 2A and 2B are illustrations of a filter in accordance with the present invention.FIG. 2A is an illustration an exploded view of a glass frit device (“a filter module”) for purifying nucleic acids in accordance with the present invention.FIG. 2B is a cut-away illustration of the same device. -
FIG. 3 is a schematic illustration of a glass frit device (“a hollow chamber”) for purifying nucleic acids in accordance with the present invention. -
FIG. 4 is a schematic illustration of a microarray device in, accordance with the present invention. -
FIG. 5 is a flowchart of a process for purifying and identifying nucleic acids in accordance with the present invention. -
FIG. 6 is graph illustrating the improved performance characteristics of devices in accordance with the present invention as shown inFIGS. 2A and 2B as demonstrated by measuring fluorescence of a sample material (100 μL of Bacillus anthracis cells in whole blood at a concentration of 1×105 cells/mL) as a function of PCR cycles. The solid trace (A) shows the result for samples treated in accordance with the present invention; the dashed trace (B) shows sample treated using a device available commercially from Qiagen; dashed traces (C) and (D) are unprocessed sample and negative control. -
FIG. 7 is graph illustrating the improved performance characteristics of devices in accordance with the present invention as shown inFIG. 3 as demonstrated by measuring fluorescence of a sample material (500 μL of Bacillus anthracis cells in sputum at a concentration of 1×104 cells/mL) as a function of PCR cycles. The solid trace (A) shows the result for samples treated in accordance with the present invention; the dashed trace (B) shows unprocessed sample; dashed trace (C) is negative control. - The present invention provides methods and devices for at least partially purifying nucleic acids from mixtures of such in combination with other substances, such as proteins, small molecules, cell membrane fragments, and the like.
- As used herein, “nucleic acids” refers to individual nucleic acids and polymeric chains of nucleic acids, including DNA and RNA, whether naturally occurring or artificially synthesized (including analogs thereof), or modifications thereof, especially those modifications know to occur in nature, having any length. Examples of nucleic acid lengths that are in accord with the present invention include, without limitation, lengths suitable for PCR products (e.g., about 50 base pairs (bp)) and human genomic DNA (e.g., on an order from about kilobase pairs (Kb) to gigabase pairs (Gb)). Thus, it will be appreciated that the term “nucleic acid” encompasses single nucleic acids as well as stretches of nucleotides, nucleosides, natural or artificial, and combinations thereof, in small fragments, e.g., expressed sequence tags or genetic fragments, as well as larger chains as exemplified by genomic material including individual genes and even whole chromosomes. In more specific embodiments, the nucleic acids are from a pathogen, such as bacteria or a virus. Such pathogens include those harmful to humans and animals. Of the former, in some embodiments, the pathogen is one that used as a biological weapon, including naturally occurring pathogens that have been weaponized. In some embodiments, the nucleic acids comprise microbial DNA. In one embodiment of the invention, the microbial DNA is from Bacillus anthracis. In other embodiments, the nucleic acids come from humans or animals. In some embodiments, the nucleic acids comprise human genomic DNA.
- In a first aspect, the present invention provides methods for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter. In some embodiments, the methods of the invention comprise passing the mixture through a hollow chamber having an inlet and an outlet; wherein said inlet and outlet are the same and disposed therein at least one porous filter, under conditions effective to separate substantially the nucleic acids from the extraneous matter. As used herein “extraneous matter” refers to all materials that are distinct from the nucleic acids in the sample. Examples of such extraneous materials include, but are not limited to, proteins, starches, lipids, metal ions, and larger cellular structures such as membrane fragments. The phrase “separate substantially” as used herein refers to separations that, in some embodiments, provide the nucleic acids in at least 30% purity with respect to the extraneous materials, in more specific embodiments provide the nucleic acids in at least 50% purity with respect to the extraneous materials, in still more specific embodiments provide the nucleic acids in at least 70% purity with respect to the extraneous materials, in yet more specific embodiments provide the nucleic acids in at least 95% purity with respect to the extraneous materials, and in still yet more specific embodiments, provide the nucleic acids in at least 99% purity with respect to the extraneous materials.
- In the various embodiments of the invention described herein, the glass frit is made from standard materials using standard methods as known to persons having ordinary skill in the art or available commercially as described below. In some embodiments, the glass frit has a thickness substantially between about one millimeter and about 20 millimeters, more specifically between about two millimeters and about five millimeters, and still more specifically between about two millimeters and about three millimeters. Exemplary glass frit pore sizes suitable for use with the present invention, including the various embodiments described herein, are between about 2 microns and about 200 microns. In more specific embodiments, the pore size is between about 150 microns and about 200 microns. In other more specific embodiments, the pore size is between about 2 microns and about 100 microns, and still more specifically between about 40 microns and about 75 microns. Other embodiments include those for which the pore size is between about 2 microns and about 20 microns. For applications involving microbial DNA, a glass frit size of between about 10 microns and about 15 microns is suitable. Larger frit pore sizes can be used for human genomic applications. Suitable glass frits are composed of sintered glass and are typically used in chemistry glassware and are available commercially from Robu (Germany). The choice and manufacture of such glass frits will be understood by persons having ordinary skill in the art.
- In other embodiments, the glass frit is replaced or used in conjunction with a porous filter. As used herein, “porous filter” refers to any material that allows selective passage of at least one substance contained in a liquid. More specifically, “porous filter” refers to those materials capable of substantially removing nucleic acids from a liquid containing such nucleic acids. Examples of suitable porous filters include, but are not limited to, filter papers configured to trap nucleic acids (e.g., FTA paper, available from Whatman), glass fibers, glass beads, beads with the Charge Switch Technology coating, available from Invitrogen, aluminum oxide filters and porous monolithic polymers. Such materials and products are familiar to those having ordinary skill in the art. In some embodiments, the porous filter is a glass frit. In further embodiment the glass frit is a sintered glass frit. Other suitable materials for providing the filtering function of the glass frit or sintered glass fit are silicone and XTRABIND (Xtrana, Inc., Broomfield, Colo.). The configuration of such materials to perform the filtering functions of the present invention will be apparent to persons having ordinary skill in the art.
- In one embodiment, the above-described glass frit is packaged into a frit holder or fluidic module. One exemplary embodiment of such a holder or module is shown in cut-away view at 1000 in
FIG. 1 . There, a glass frit as just described above (1004) in placed inside a housing (1008). The housing includes an inlet (1012) into which fluidic mixture including nucleic acids of interest enter the housing and interact with the glass frit as described herein to produce a first-filtered mixture which passes through an outlet of the housing (1016). After exiting the filter holder or module, the first-filtered mixture can proceed to other chambers in fluidic contact with the outlet, such as described below, or to a collector. An optional heater, shown at 1020, is included in some embodiments. The design and manufacture of such devices will be understood by those having ordinary skill in the art. - A second embodiment of this aspect of the invention is shown in
FIGS. 2A and 2B . The design and fabrication of such devices are known to those having ordinary skill in the art.FIG. 2A shows an exploded view of one embodiment of a frit holder (2000), which includes an upper housing body (2002) and a lower housing body (2004). The lower housing body includes a recess (2006), described in greater detail inFIG. 2B , into which is disposed one or more glass frits (2008) which are described above. The glass frit is seated in the housing bodies using gaskets (2010, 2012). The lower housing body (2004) also includes an inlet (not shown) through which materials containing nucleic acids to be separated are introduced to glass frit, and outlet (2014) from which waste material and the purified nucleic acids exit the filter module.FIG. 2B shows a cut-away view of the frit housing (2000). There, in addition to the elements just described, the inlet (2018) is shown, along with channels for directing the flow of material through the glass frit and outlet. - In another aspect, the present invention provides a device for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter. One embodiment of such a filter in accordance with the present invention is shown at 3000 in
FIG. 3 . There, a hollow chamber (3004) having a first opening (3008) and a second opening (3012) through which a mixture comprising nucleic acids is passed and an outlet from which an at least partially resolved mixture exits. Between the inlet and outlet is a glass frit (3018) as described above that extends axially through at least a portion of the interior volume of the hollow chamber, the extent to which is shown at 3016. In some embodiments, more than one such frit is used. In still other embodiments, at least one of the glass frits is made of sintered glass. The design and fabrication of such devices are known to those having ordinary skill in the art. - In some more specific embodiments, one end of the hollow chamber has a fiustaconical shape the chamber is dimensioned to fit on the end of a pipetting instrument, e.g., as a pipet tip, so that materials are first taken up though the second opening, pass through the glass frit, are filtered and then retained in the portion of the chamber above the frit. In some embodiments, the sample retained in the portion of the chamber above the frit is passed back through the frit through the second opening (3012).
- In another embodiment, the above-described pipet tip is combined with a heating device that is configured to heat the frit to facilitate separation of the nucleic acids from the mixture. In more specific embodiments, the heater is dimensioned to fit within the pipet tip. The design and fabrication of such devices are known to those having ordinary skill in the art.
- In still another embodiment, the pipet tip is coupled with an electronic pipettor or robotic pipetting workstation to control the flow rate through the frit. In some embodiments, the electronic pipettor is a hand-held device. The design, fabrication, and operation of such devices are known to those having ordinary skill in the art.
- In some embodiment's of the present invention, two or more porous filters are used in combination. In a more specific embodiment, each layer has a different pore size. Without wishing to be bound to any particular theory of action, larger porous filter pore sizes trap larger particles, and so can serve as a prefilter. For example, a 40 micron-60 micron porous filter could be used in tandem with a 10 micron-15 micron porous filter to deplete human genomic DNA from a sample (e.g., blood) to isolate microbial DNA. Removing the abundant human DNA with the 40 micron-60 micron porous filter allows better binding of the low copy microbial DNA to the 10 micron-15 micron porous filter and more robust analysis, since the human genomic DNA will not be present in concentrations large enough to interfere significantly (e.g., by whole genome amplification). The porous filter can be in a pipet tip as described above, having a thickness and diameter of about five millimeters (mm) each. In some embodiments, two or more of the porous filters are fused together to form a substantially monolithic structure. The design, fabrication, and operation of such devices are known to those having ordinary skill in the art.
- In more specific embodiments in which the filter is disposed with a pipet tip, the glass frit(s) having larger pore sizes are disposed closer to the pipet tip inlet. Again not wishing to be bound to any particular theory of action, but those persons having ordinary skill in the art will appreciate that arranging the larger pore sized filter nearer the pipet tip inlet can provide a more uniform distribution of nucleic acid binding within the frit. By way of comparison, persons having ordinary skill in the art will expect that otherwise the nucleic acids will tend to bind to the frit at the area closest to the pipet tip opening, since the nucleic acids are more likely to make initial contact just as the nucleic acids enter the frit.
- In yet another aspect, the present invention provides a microfluidic device for analyzing nucleic acids in accordance with the present invention. One embodiment of such a microfluidic device is show in
FIG. 4 at 4000. A fit holder (4002) as described herein is provided. Upstream, the frit holder in fluidic communication with a source of elution buffer (4004), a guanidine hydrochloride (Gu) reservoir (4006) and Gu mixing tower (4008), the flow from which Gu and Gu mixing tower are controlled by a valve (4010). The Gu mixing tower is further in fluidic communication with an ethanol-air source (4012). Those persons having ordinary skill in the art will realize that chaotropes other than Gu can be used with the present invention. In addition are a bead beater (4014) that is in fluidic communication with a sample collection tower (4016), which is in turn in fluidic communication with inlet check value (4018), and an electrical contact (4020). The output from these elements is controlled byvalve 4022. - With continuing reference to
FIG. 4 , downstream of frit holder (4002) is a waste tank (4024), the flow to which is controlled by a valve (4026). Downstream flow from the fit holder is also controlled by a second valve (4028), which controls flow to an elution tower (4030) and a check valve (4032) along a first branch; and to another valve (4034) along a second branch of the flow path. Continuing downstream fromvalve 4034, are one or more reservoirs of PCR reagents (4036) and a valve (4038) which leads to a PCR chamber (4040). Downstream of the PCR chamber is a valve (4042), which, along withvalve 4046, controls flow from the PCR chamber to a microarray chamber (4048) that is also in fluidic communication with hybridization and wash buffer reservoir (4048) and a waste container (4052). The design and fabrication of such devices are known to those having ordinary skill in the art. - The operation of the device described with respect to
FIG. 4 is illustrated by the flowchart shown inFIG. 5 at 5000. After obtaining the raw sample (5002), e.g., a sputum sample that contains cells of interest, the cells are lysed (5004) usingbead beater 4014, and the mixture passed for purification of the nucleic acids (5006) through the fit 4002 for amplification (5008) byPCR chamber 4040 and detection (5010) by themicroarray chamber 4050. - Without being bound to any particular theory or action, the present invention meets the needs described above by implementing a rigid, self-supporting fit structure that is relatively thick for high binding capacity, contains relatively large porosities for low fluid impedence, faster flow rates, and higher tolerance to particles in clinical and environmental samples, and consists of no loose material (e.g. silica gel, diatomaceous earth, glass beads) and no flimsy, delicate materials (e.g. fiber filters, membrane filters, silicon microstructures) for rugged operation and packaging and simplified manufacturing.
- The following Examples are provided to illustrate certain aspects of the present invention and to aid those of skill in the art in the art in practicing the invention. These Examples are in no way to be considered to limit the scope of the invention in any manner.
- Referring to
FIGS. 2A and 2B , a protocol for practicing purification and detection in accordance with the present invention is provided below. -
- 1. Insert a glass frit into holder (one of four different porosities: Fine, Medium. Coarse, and Extra Coarse). Tighten the housing.
- 2. Mix 500 μL of a sample (104 copies/mL) with 500 μL of 6M guanidine, pH 6.5.
- 3. Pass mixture (1 mL) through fit at a flow rate of 100 μL/min using a 1 mL syringe. Pass air manually through frit to purge sample using a 5 mL syringe.
- 4. Pass 1 mL of 70% ethanol (EtOH) to wash bound nucleic acid using a 1 ml, syringe at rate of 1 mL/min. Pass air through the fit manually to purge EtOH using a 5 mL syringe.
- 5. Carefully pass an elution buffer (10 mM Tris, pH 8.0) using 1 mL, syringe at 100 μl/min into the frit holder until buffer can be first seen in the outlet tubing.
- 6. Place heat block under the frit holder and heat at 70° C. for 3 min.
- 7. After 3 minutes continue to pass elution buffer through frit holder. Collect the fractions (50 μL-100 μL) for PCR analysis.
- 8. Flush the frit holder with 1 ml, of 10% bleach (bleach dilution no more than one week old), 5 mL of 10 mM Tris-HCl (pH 8.0), and 5 mL of water. Replace the
- Referring to
FIG. 3 , a protocol for practicing purification and detection in accordance with the present invention is provided below. -
- 1. Add 500 μL of sample to 500 μL of 6M guanidine in Vial A. Vortex to mix.
- 2. Attach a 1.2 mL pipet tip with an embedded frit to an electronic pipettor (Gilson Concept).
- 3. Set electronic pipettor to speed 1 (slowest speed). Aspirate the 1 mL of sample mixture in Vial A. Allow the sample mixture bolus to completely pass through the fit. The sample mixture bolus will establish itself immediately on top of the frit.
- 4. Dispense the sample back into Vial A. The sample mixture bolus will completely expel back into the vial.
- 5. Repeat steps 3 and 4 four times.
- 6. Set electronic pipettor to speed 5 (fastest speed). Aspirate and dispense 1 mL of 70% ethanol in Vial B to wash bound nucleic acids on frit. Repeat four times.
- 7. Remove traces of ethanol by positioning tip above the ethanol solution. Aspirate and dispense air five times to dry the frit.
- 8. Place Vial C containing 100 μL of 10 mM Tris-HCl (pH 8.0) into a heat block set at 70° C. Let heat for 5 minutes.
- 9. Set the electronic pipettor to speed 1. Aspirate the elution buffer and dispense back into Vial C five times to remove nucleic acids from the fit.
- The results of two experiment using such protocols are show in
FIGS. 6 and 7 , where samples of Bacillus anthracis (Ba) in whole blood and sputum were treated using the materials and methods of the invention. - In
FIG. 6 , the sample treated using a method and device (FIGS. 2A and 2B ) of the invention (trace A) outperformed a sample treated using a device available commercially from Qiagen (trace B). Traces C and D are the unprocessed input sample and negative control, respectively. In the experiment, 100 μL of Bacillus anthracis cells at 105/mL in whole blood was input, either through a medium (10-15 μm pore size) frit as described herein or into a Qiagen device, and elution of about 50 μL were collected and analyzed using PCR. As the Figure illustrates, after about 25 cycles of PCR amplification, the sample treated using the materials and methods of the invention clearly and significantly outperform the results from the sample treated using the prior art device. -
FIG. 7 illustrates the result of another experiment using such a method and device (FIG. 3 ), where a sample of Bacillus anthracis cells in sputum was treated using the materials and methods of the invention. The sample treated using the method and device of the invention (trace A) outperformed an unprocessed sample (trace B). Trace Cis a negative control. In the experiment, 500 μL of Bacillus anthracis cells at 104/mL in sputum was input through a medium flit as described herein, and an elution of about 100 μL was collected and analyzed using quantitative, real-time PCR. As the Figure illustrates, after about 27 cycles of PCR amplification, the sample treated using the material and method of the invention clearly and significantly outperform the results from unprocessed sample. - The above-described methods and devices were used successfully to identify DNA and/or RNA from a variety of organisms listed in Table 1 that were spiked into a variety of matrices (i.e., sample types) listed in Table 2.
-
-
- Viral Equine Encephalitis (VEE)
- Vaccinia virus
- Y. pestis
- B. anthracia
- Adenovirus
- S. pyogenes
- C. pneumoniae
- Influenza A
- Influenza B
- Mixture of Adenovirus+S. pyogenes
- Mixture of Flu A and Adenovirus
- The following materials were analyzed successfully using the methods and devices of the invention:
-
-
- Water/TE (10 mM Tris-HCl,
- 1.0 mM EDTA buffer)
- Swab extracts
- Sputum
- Nasal Wash
- Whole Blood
- The present invention offers several advantages over the prior art: 1) a simplified method and device requiring no centrifugation or high pressure to move fluids, 2) a modular device for fluidic integration into a complete analytical system, 3) large pore sizes to process complex samples while maintaining low fluid resistance; 4) high extraction and elution efficiencies since fluids can be moved from both directions; 5) rigidity that eliminates the need for additional support structures, and 6) durability that allows reusing the device for sequential samples. Although various specific embodiments and examples have been described herein, those having ordinary skill in the art will understand that many different implementations of the invention can be achieved without departing from the spirit or scope of this disclosure. For example, other materials with nucleic acid affinity could be used for the glass frit or the glass frit can be modified to better attract nucleic acids and nucleic acids without using a chaotropic salt such as guanidine. Glass frits can contain immobilized antibodies to extract microbes and toxins. Still other variations will be clear to those having ordinary skill in the art.
- The following references are incorporated herein by reference in their entirety and for all purposes.
- Beach, R. A., Strittmatter, R. P., et al. (2007). Integrated Micropump Analysis Chip and Method of Making the Same U.S. Pat. No. 7,189,358.
- Colpan, M. (2001). Process and device for the Isolation of Cell Components, Such as Nucleic Acids, from 20 Natural Sources U.S. Pat. No. 6,274,371.
- Deshmukh, A. J. (2006). Method and Automated Fluidic System for Detecting Protein in Biological Sample U.S. Pat. No. 6,989,130.
- Kalra, K. L., Pawlak, K., et al. (1990). Immunoassay Test Device and Method U.S. Pat. No. 4,912,034.
- Knollenberg, B. A., Rodier, D., et al. (2007). Molecular Contamination Monitoring System and Method U.S. Pat. No. 7,208,123.
- McCormack, R. M. (1990). Process for Purling Nucleic Acids U.S. Pat. No. 4,923,978.
- Terry, B. R., Scudder, K. M., et al. (2004). Method and Apparatus for High Density Format Screening for Bioactive Molecules. U.S. Pat. No. 6,790,652.
- Tittgen, I (2004). Chromatography Material and a Method Using the Same U.S. Pat. No. 6,800,752.
- Wick, C. H. (2007). Method and System for Detecting and Recording Submicron Sized Particles. U.S. Pat. No. 7,250,138.
Claims (21)
1-25. (canceled)
26. A device for isolating nucleic acids from a mixture containing such nucleic acids and extraneous matter, comprising:
a hollow chamber having disposed therein a first glass frit and a second glass frit, wherein said first glass frit and said second glass frit bind to nucleic acids and are fused together to form a substantially monolithic structure, wherein said first glass frit and second glass frit are rigid, self-supporting fits and are not modified with a material with nucleic acid affinity.
27. The device of claim 26 , wherein said first glass frit and said second glass frit have different porosity.
28. The device of claim 26 , wherein said first glass frit has a pore size of about 40 micron to about 60 micron and said second glass frit has a pore size of about 10 micron to about 15 micron.
29. The device of claim 26 , wherein each of said first glass frit and said second glass frit has a thickness of 1-20 mm.
30. The device of claim 29 , wherein each of said first glass frit and said second glass frit has a thickness of about 5 mm.
31. The device of claim 29 , wherein each of said first glass fit and said second glass frit has a diameter of about 5 mm.
32. The device of claim 26 , wherein said device is in the form of a pipet tip.
33. The device of claim 26 , wherein said device is in the form of a pipet tip having a pipet tip inlet for withdrawing a sample into said pipet tip.
34. The device of claim 33 , wherein said first glass frit has pore size that is larger than the pore size of said second frit and wherein said first glass frit is disposed closer to said pipet inlet than said second glass frit.
35. The device of claim 34 , wherein said first glass fit has a pore size of about 40 micron to about 60 micron and said second glass frit has a pore size of about 10 micron to about 15 micron.
36. The device of claim 26 , wherein said first glass frit and said second glass frit have a pore size between about 2 microns and about 220 microns.
37. A method for purifying nucleic acids from a sample, comprising:
passing a sample through the device according to claim 26 ;
washing said device with a washing buffer; and
eluting nucleic acids from said device with an elution buffer.
38. The method of claim 37 , wherein said first glass frit and said second glass frit have different porosity.
39. The method of claim 37 , wherein said first glass fit has a pore size of about 40 micron to about 60 micron and said second glass frit has a pore size of about 10 micron to about 15 micron.
40. The method of claim 37 , wherein each of said first glass fit and said second glass frit has a thickness of 1-20 mm.
41. The device of claim 37 , wherein said device is in the form of a pipet tip.
42. The method of claim 37 , wherein said device is in the form of a pipet tip having a pipet tip inlet for withdrawing a sample into said pipet tip.
43. The device of claim 42 , wherein said first glass frit has pore size that is larger than the pore size of said second frit and wherein said first glass frit is disposed closer to said pipet inlet than said second glass frit.
44. A method for purifying microbial DNA from a human sample, comprising:
(a) passing said sample through a first glass frit having a pore size of about 40 micron to about 60 micron to deplete human genomic DNA from said sample;
(b) passing a flow-through from step (a) through a second glass frit having a pore size of about 10 micron to about 15 micron;
(c) eluting microbial DNA from said second glass fit,
wherein said first glass frit and second glass fit are rigid, self-supporting frits and are not modified with a material with nucleic acid affinity.
45. The method of claim 44 , wherein said human sample is a blood sample.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/541,353 US20120283423A1 (en) | 2007-10-31 | 2012-07-03 | Apparatus, system and method for purifying nucleic acids |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/933,113 US7759112B2 (en) | 2007-10-31 | 2007-10-31 | Apparatus, system, and method for purifying nucleic acids |
US12/793,253 US8236553B2 (en) | 2007-10-31 | 2010-06-03 | Apparatus, system and method for purifying nucleic acids |
US13/541,353 US20120283423A1 (en) | 2007-10-31 | 2012-07-03 | Apparatus, system and method for purifying nucleic acids |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/793,253 Continuation US8236553B2 (en) | 2007-10-31 | 2010-06-03 | Apparatus, system and method for purifying nucleic acids |
Publications (1)
Publication Number | Publication Date |
---|---|
US20120283423A1 true US20120283423A1 (en) | 2012-11-08 |
Family
ID=40581474
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/933,113 Active US7759112B2 (en) | 2007-10-31 | 2007-10-31 | Apparatus, system, and method for purifying nucleic acids |
US12/793,218 Active 2028-05-30 US8236501B2 (en) | 2007-10-31 | 2010-06-03 | Apparatus, system and method for purifying nucleic acids |
US12/793,253 Active 2028-04-30 US8236553B2 (en) | 2007-10-31 | 2010-06-03 | Apparatus, system and method for purifying nucleic acids |
US13/541,353 Abandoned US20120283423A1 (en) | 2007-10-31 | 2012-07-03 | Apparatus, system and method for purifying nucleic acids |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/933,113 Active US7759112B2 (en) | 2007-10-31 | 2007-10-31 | Apparatus, system, and method for purifying nucleic acids |
US12/793,218 Active 2028-05-30 US8236501B2 (en) | 2007-10-31 | 2010-06-03 | Apparatus, system and method for purifying nucleic acids |
US12/793,253 Active 2028-04-30 US8236553B2 (en) | 2007-10-31 | 2010-06-03 | Apparatus, system and method for purifying nucleic acids |
Country Status (7)
Country | Link |
---|---|
US (4) | US7759112B2 (en) |
EP (2) | EP2215103B1 (en) |
JP (2) | JP5977921B2 (en) |
CN (2) | CN101883776B (en) |
CA (2) | CA2703950C (en) |
HK (1) | HK1150166A1 (en) |
WO (2) | WO2009058414A1 (en) |
Families Citing this family (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070017870A1 (en) | 2003-09-30 | 2007-01-25 | Belov Yuri P | Multicapillary device for sample preparation |
US7759112B2 (en) * | 2007-10-31 | 2010-07-20 | Akonni Biosystems, Inc. | Apparatus, system, and method for purifying nucleic acids |
US10125388B2 (en) | 2007-10-31 | 2018-11-13 | Akonni Biosystems, Inc. | Integrated sample processing system |
US20090111193A1 (en) | 2007-10-31 | 2009-04-30 | Cooney Christopher G | Sample preparation device |
US9428746B2 (en) | 2007-10-31 | 2016-08-30 | Akonni Biosystems, Inc. | Method and kit for purifying nucleic acids |
JP2011517773A (en) * | 2008-03-28 | 2011-06-16 | バイオティクス, インコーポレイテッド | Sample preparation device and analyte processing method |
WO2009121034A2 (en) * | 2008-03-28 | 2009-10-01 | Pelican Group Holdings, Inc. | Multicapillary sample preparation devices and methods for processing analytes |
US8846878B1 (en) | 2009-01-23 | 2014-09-30 | Cubrc Corporation | Method and device for isolating a protein sample |
CN105675496B (en) | 2009-09-21 | 2019-07-05 | 阿科尼生物系统公司 | Integrated barrel |
JP5773280B2 (en) * | 2010-06-18 | 2015-09-02 | 東洋紡株式会社 | Method for simultaneous lysis of mycobacteria and separation of its nucleic acids |
WO2012078863A2 (en) | 2010-12-09 | 2012-06-14 | Akonni Biosystems | Sample analysis system |
GB201104206D0 (en) | 2011-03-14 | 2011-04-27 | Ge Healthcare Uk Ltd | Biological sample holder and method of assembling same |
WO2013004366A1 (en) | 2011-07-01 | 2013-01-10 | Qiagen Gmbh | Filter module in biomolecule isolation |
US20130017545A1 (en) * | 2011-07-11 | 2013-01-17 | Agilent Technologies, Inc. | Apparatus and methods for acquiring analytes from a dried biological fluid sample |
US9399986B2 (en) | 2012-07-31 | 2016-07-26 | General Electric Company | Devices and systems for isolating biomolecules and associated methods thereof |
CN105164509B (en) * | 2012-08-28 | 2018-02-23 | 阿科尼生物系统公司 | Method and kit for purification of nucleic acid |
WO2014108882A1 (en) * | 2013-01-14 | 2014-07-17 | Sabio Innovative Solutions Pvt Ltd | Separation device, filtration device therefrom, and methods therefor |
CN105263627B (en) | 2013-01-18 | 2019-05-21 | 生米公司 | Analytical equipment |
JP6628954B2 (en) * | 2013-08-30 | 2020-01-15 | 学校法人東京理科大学 | Glass filter and cancer cell separation method |
CN111548903A (en) | 2013-11-01 | 2020-08-18 | 生米公司 | Sample extraction and preparation device |
JP6229471B2 (en) * | 2013-12-13 | 2017-11-15 | 東ソー株式会社 | Column for liquid chromatography having a convex filter |
WO2015195949A2 (en) | 2014-06-18 | 2015-12-23 | Clear Gene, Inc. | Methods, compositions, and devices for rapid analysis of biological markers |
JP6715190B2 (en) | 2014-06-19 | 2020-07-01 | アコーニ バイオシステムズ インコーポレイテッド | Molecular analysis system and method of using the same |
WO2017106790A1 (en) | 2015-12-18 | 2017-06-22 | Clear Gene, Inc. | Methods, compositions, kits and devices for rapid analysis of biological markers |
CN107130470A (en) * | 2016-02-29 | 2017-09-05 | 新材料与产业技术北京研究院 | A kind of composite filtering film and its preparation method and application |
US10845276B2 (en) | 2016-02-29 | 2020-11-24 | Hewlett-Packard Development, L.P. | Enzymatic sample purification |
US11161107B2 (en) | 2016-05-25 | 2021-11-02 | Integrated Micro-Chromatography Systems, Inc. | Dispersive pipette extraction system for purification of large biomolecules |
GB201704768D0 (en) * | 2017-01-05 | 2017-05-10 | Illumina Inc | Flow cell liquid degassing systema and method |
CN106701556B (en) * | 2017-01-06 | 2019-02-12 | 广东顺德永诺生物科技有限公司 | Digital pcr detection device and its liquid channel system |
CN108929841A (en) * | 2017-05-25 | 2018-12-04 | 菏泽学院 | The manual producing device of animal physiological experiment plasmid DNA samples |
US10739237B2 (en) * | 2017-08-02 | 2020-08-11 | Pocared Diagnostics Ltd. | Processor filter arrangement that includes method and apparatus to remove waste fluid through a filter |
CN108130325A (en) * | 2018-02-02 | 2018-06-08 | 苏州优卡新材料科技有限公司 | A kind of efficiently purifying column of ribonucleic acid extraction inorganic material filter core |
KR102136695B1 (en) * | 2018-08-22 | 2020-07-22 | 주식회사 인퓨전텍 | Method for pathogen enrichment and nucleic acid extract using device for point of care testing |
KR101996617B1 (en) * | 2018-10-11 | 2019-07-04 | 주식회사 엘지화학 | Integrated cartridge |
US10864483B2 (en) * | 2018-11-16 | 2020-12-15 | Integrated Protein Technologies, Snc. | Molecular weight filtration system and apparatus |
GB2581489B (en) * | 2019-02-15 | 2021-02-24 | Revolugen Ltd | Purification method |
US11591591B2 (en) | 2019-08-21 | 2023-02-28 | New England Biolabs, Inc. | Isolation of high molecular weight DNA using beads |
EP3821979A1 (en) * | 2019-11-18 | 2021-05-19 | Miltenyi Biotec B.V. & Co. KG | Enhanced process for sorting cells with a microfabricated valve |
CA3192080A1 (en) | 2020-09-18 | 2022-03-24 | Marc DEJOHN | Portable devices and methods for analyzing samples |
CN114618849B (en) * | 2020-12-11 | 2023-07-18 | 深圳市帝迈生物技术有限公司 | Cleaning device, cleaning method and sample analysis device for magnetic separation reaction cup |
CN112807825A (en) * | 2020-12-31 | 2021-05-18 | 上海金鑫生物科技有限公司 | High-flux automatic opening and closing type filtering suction head |
KR102562639B1 (en) * | 2021-04-29 | 2023-08-02 | 주식회사 한국과학 | Sample taking and diagnosing method for pcr(polymerase chain reaction) diagnosis and sample taking tool for pcr diagnosis |
CN116371259B (en) * | 2023-06-06 | 2023-08-01 | 南宫市喀秋莎润滑油科技有限公司 | Mixed blending machine for lubricating oil production |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4774058A (en) * | 1985-09-26 | 1988-09-27 | Mehl Ehrenfried L | Apparatus for, and methods of, operating upon a fluid |
US20090111193A1 (en) * | 2007-10-31 | 2009-04-30 | Cooney Christopher G | Sample preparation device |
US7759112B2 (en) * | 2007-10-31 | 2010-07-20 | Akonni Biosystems, Inc. | Apparatus, system, and method for purifying nucleic acids |
US20120149603A1 (en) * | 2010-12-09 | 2012-06-14 | Akonni Biosystems | Sample analysis system |
Family Cites Families (59)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US1117601A (en) * | 1913-05-09 | 1914-11-17 | Gen Filtration Company Inc | Filtering medium. |
US2114748A (en) * | 1934-05-09 | 1938-04-19 | Firm Jenaer Glaswerk Schott & | Method of making porous filter bodies of particles of glass |
US3414394A (en) * | 1965-06-21 | 1968-12-03 | Owens Illinois Inc | Sintered glass article and method of making |
DE3037898A1 (en) * | 1980-10-07 | 1982-05-06 | Bruker Analytische Meßtechnik GmbH, 7512 Rheinstetten | MIXING CHAMBER |
US4475818A (en) * | 1983-08-25 | 1984-10-09 | Bialkowski Wojciech L | Asphalt coating mix automatic limestone control |
DE3635598A1 (en) * | 1986-10-20 | 1988-05-05 | Eppendorf Geraetebau Netheler | PIPETTING DEVICE WITH A CLIP-ON CONE FOR A PIPETTE TIP AND PIPETTE TIP FOR SUCH A PIPETTING DEVICE |
US4765818A (en) | 1987-02-24 | 1988-08-23 | Hoechst Celanese Corporation | Porous glass monoliths |
US4810674A (en) | 1987-02-24 | 1989-03-07 | Hoechst Celanese Corp. | Porous glass monoliths |
US4912034A (en) | 1987-09-21 | 1990-03-27 | Biogenex Laboratories | Immunoassay test device and method |
US4923978A (en) | 1987-12-28 | 1990-05-08 | E. I. Du Pont De Nemours & Company | Process for purifying nucleic acids |
IT1240870B (en) * | 1990-02-14 | 1993-12-17 | Talent | PROCEDURE FOR THE EXTRACTION AND PURIFICATION OF HUMAN GENOMIC DNA |
DE4143639C2 (en) * | 1991-12-02 | 2002-10-24 | Qiagen Gmbh | Process for the isolation and purification of nucleic acids |
CA2105962A1 (en) | 1992-09-18 | 1994-03-19 | Margaret Patricia Raybuck | Device and method for affinity separation |
WO1994020831A1 (en) | 1993-03-08 | 1994-09-15 | Norman Wainwright | Aligned fiber diagnostic chromatography |
DE4321904B4 (en) * | 1993-07-01 | 2013-05-16 | Qiagen Gmbh | Method for chromatographic purification and separation of nucleic acid mixtures |
DE59502334D1 (en) * | 1994-01-07 | 1998-07-02 | Qiagen Gmbh | METHOD FOR CRUSHING HIGH MOLECULAR STRUCTURES |
DE4432654C2 (en) * | 1994-09-14 | 1998-03-26 | Qiagen Gmbh | Process for the isolation of nucleic acids from natural sources |
US5496523A (en) * | 1994-05-06 | 1996-03-05 | Sorenson Bioscience | Filtered micropipette tip for high/low volume pipettors |
DE19530132C2 (en) * | 1995-08-16 | 1998-07-16 | Max Planck Gesellschaft | Process for the purification, stabilization or isolation of nucleic acids from biological materials |
EP0912761A4 (en) * | 1996-05-29 | 2004-06-09 | Cornell Res Foundation Inc | Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions |
US6074827A (en) * | 1996-07-30 | 2000-06-13 | Aclara Biosciences, Inc. | Microfluidic method for nucleic acid purification and processing |
US6048457A (en) | 1997-02-26 | 2000-04-11 | Millipore Corporation | Cast membrane structures for sample preparation |
US6337214B1 (en) * | 1997-06-09 | 2002-01-08 | Acgt Medico, Inc. | Detection of DNA, RNA and proteins using a test column with two snares |
JPH11266864A (en) * | 1998-03-19 | 1999-10-05 | Hitachi Ltd | Purification of nucleic acid and device for purification |
AU6114199A (en) * | 1998-10-09 | 2000-05-01 | Whatman Bioscience Limited | Isolation method for nucleic acid and apparatus |
US6958392B2 (en) * | 1998-10-09 | 2005-10-25 | Whatman, Inc. | Methods for the isolation of nucleic acids and for quantitative DNA extraction and detection for leukocyte evaluation in blood products |
US6100084A (en) * | 1998-11-05 | 2000-08-08 | The Regents Of The University Of California | Micro-sonicator for spore lysis |
US6431476B1 (en) * | 1999-12-21 | 2002-08-13 | Cepheid | Apparatus and method for rapid ultrasonic disruption of cells or viruses |
AU2599800A (en) | 1999-01-06 | 2000-07-24 | Invitrogen Corporation | Methods and compositions for isolation of nucleic acid molecules |
WO2000073413A2 (en) * | 1999-05-28 | 2000-12-07 | Cepheid | Apparatus and method for cell disruption |
GB9913275D0 (en) * | 1999-06-08 | 1999-08-04 | Dna Research Instr Limited | Sample processing device |
DE19962577A1 (en) | 1999-12-23 | 2001-07-12 | Tittgen Biotechnologie Dr | Chromatography material and method using the same |
US6699713B2 (en) * | 2000-01-04 | 2004-03-02 | The Regents Of The University Of California | Polymerase chain reaction system |
GB0011443D0 (en) * | 2000-05-13 | 2000-06-28 | Dna Research Instr Limited | Separation device |
US6537502B1 (en) | 2000-07-25 | 2003-03-25 | Harvard Apparatus, Inc. | Surface coated housing for sample preparation |
JP3752417B2 (en) * | 2000-09-07 | 2006-03-08 | 株式会社日立製作所 | Nucleic acid purification method and purification apparatus |
US7157232B2 (en) * | 2000-12-13 | 2007-01-02 | The Regents Of The University Of California | Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change |
WO2002078847A1 (en) * | 2001-03-28 | 2002-10-10 | Hitachi, Ltd. | Instrument and method for recovering nucleic acid |
US20030138819A1 (en) * | 2001-10-26 | 2003-07-24 | Haiqing Gong | Method for detecting disease |
US20050092685A1 (en) * | 2002-01-17 | 2005-05-05 | Spark Holland B.V. | Set comprising a pipette and a cartridge, as well as a method for applying a sample to the cartridge and an analytical method |
US20040054160A1 (en) | 2002-09-16 | 2004-03-18 | Santona Pal | Nucleic-acid ink compositions for arraying onto a solid support |
US7595026B2 (en) * | 2003-05-29 | 2009-09-29 | Varian, Inc. | Solid phase extraction pipette |
US7722820B2 (en) * | 2004-11-19 | 2010-05-25 | Phynexus, Inc. | Method and device for sample preparation |
ATE531817T1 (en) * | 2003-07-15 | 2011-11-15 | Lukas Bestmann | SAMPLE PREPARATION UNIT |
US7541166B2 (en) * | 2003-09-19 | 2009-06-02 | Microfluidic Systems, Inc. | Sonication to selectively lyse different cell types |
US20050136477A1 (en) * | 2003-11-17 | 2005-06-23 | Hashem Akhavan-Tafti | Methods for isolating nucleic acids from biological and cellular materials |
JP4597870B2 (en) * | 2004-02-12 | 2010-12-15 | ジーエルサイエンス株式会社 | Mechanism for separation and purification of DNA, etc. |
NZ551646A (en) * | 2004-06-09 | 2010-11-26 | Pathogen Removal & Diagnostic Technologies Inc | Particles embedded in a porous substrate for removing target analyte from a sample |
CN101044248B (en) * | 2004-08-20 | 2010-05-05 | 旭化成株式会社 | Method for trapping nucleic acids using divalent metal ions |
DE102005005437A1 (en) * | 2005-02-05 | 2006-08-10 | Eppendorf Ag | Filter pipette tip |
JP2006246732A (en) * | 2005-03-09 | 2006-09-21 | Kanto Chem Co Inc | Nucleic acid purification supporter and method for purifying the same |
KR100657957B1 (en) * | 2005-04-12 | 2006-12-14 | 삼성전자주식회사 | 1 Method for isolating a nucleic acid using a material positively charged at the first pH and comprising an amino group and a carboxyl group and a solid material for a nucleic acid purification capable of being used for the method |
JP4791750B2 (en) * | 2005-04-15 | 2011-10-12 | ジーエルサイエンス株式会社 | Separation and purification method and separation and purification mechanism for DNA, etc. |
JP5240965B2 (en) * | 2005-09-02 | 2013-07-17 | 愛知県 | Method and apparatus for analyzing sulfated glycolipid |
JP4699868B2 (en) * | 2005-11-04 | 2011-06-15 | 株式会社日立ハイテクノロジーズ | Nucleic acid purification method and nucleic acid purification instrument |
JP2007155518A (en) * | 2005-12-06 | 2007-06-21 | Showa Denko Kk | Solid phase extraction cartridge |
DE102006002258B4 (en) * | 2006-01-17 | 2008-08-21 | Siemens Ag | Module for the preparation of a biological sample, biochip set and use of the module |
US8686129B2 (en) * | 2007-03-20 | 2014-04-01 | Agilent Technologies, Inc. | Methods for the separation of biological molecules using sulfolane |
US20080146789A1 (en) * | 2007-03-20 | 2008-06-19 | Braman Jeffrey C | Methods for the separation of biological molecules using dioxolane |
-
2007
- 2007-10-31 US US11/933,113 patent/US7759112B2/en active Active
-
2008
- 2008-03-11 WO PCT/US2008/056482 patent/WO2009058414A1/en active Application Filing
- 2008-03-11 JP JP2010531081A patent/JP5977921B2/en active Active
- 2008-03-11 CA CA2703950A patent/CA2703950C/en not_active Expired - Fee Related
- 2008-03-11 EP EP08731876.2A patent/EP2215103B1/en active Active
- 2008-03-11 CN CN200880116501.9A patent/CN101883776B/en active Active
- 2008-06-25 EP EP08780985.1A patent/EP2217344B1/en active Active
- 2008-06-25 CN CN2008801191314A patent/CN101883619B/en active Active
- 2008-06-25 WO PCT/US2008/068159 patent/WO2009058432A1/en active Application Filing
- 2008-06-25 CA CA2704771A patent/CA2704771C/en active Active
- 2008-06-25 JP JP2010531085A patent/JP5318110B2/en active Active
-
2010
- 2010-06-03 US US12/793,218 patent/US8236501B2/en active Active
- 2010-06-03 US US12/793,253 patent/US8236553B2/en active Active
-
2011
- 2011-04-28 HK HK11104271.9A patent/HK1150166A1/en not_active IP Right Cessation
-
2012
- 2012-07-03 US US13/541,353 patent/US20120283423A1/en not_active Abandoned
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4774058A (en) * | 1985-09-26 | 1988-09-27 | Mehl Ehrenfried L | Apparatus for, and methods of, operating upon a fluid |
US20090111193A1 (en) * | 2007-10-31 | 2009-04-30 | Cooney Christopher G | Sample preparation device |
US7759112B2 (en) * | 2007-10-31 | 2010-07-20 | Akonni Biosystems, Inc. | Apparatus, system, and method for purifying nucleic acids |
US8236553B2 (en) * | 2007-10-31 | 2012-08-07 | Akonni Biosystems, Inc. | Apparatus, system and method for purifying nucleic acids |
US20120149603A1 (en) * | 2010-12-09 | 2012-06-14 | Akonni Biosystems | Sample analysis system |
Non-Patent Citations (2)
Title |
---|
Whatman Catalog "Glass Microfiber Binder-Free" webpage downloaded 31 March 2010, pages 1-2. * |
Whatman Catalog "Glass Microfiber Filters" webpage dated 2007-2009. * |
Also Published As
Publication number | Publication date |
---|---|
US8236553B2 (en) | 2012-08-07 |
CN101883619A (en) | 2010-11-10 |
US20100240882A1 (en) | 2010-09-23 |
HK1150166A1 (en) | 2011-11-04 |
CA2703950A1 (en) | 2009-05-07 |
CA2704771C (en) | 2017-12-12 |
EP2217344A1 (en) | 2010-08-18 |
EP2217344A4 (en) | 2016-03-09 |
US7759112B2 (en) | 2010-07-20 |
EP2215103B1 (en) | 2019-08-07 |
JP2011505118A (en) | 2011-02-24 |
JP5977921B2 (en) | 2016-08-24 |
CN101883776A (en) | 2010-11-10 |
CN101883619B (en) | 2013-08-07 |
EP2215103A4 (en) | 2014-07-23 |
EP2215103A1 (en) | 2010-08-11 |
EP2217344B1 (en) | 2023-01-04 |
CA2703950C (en) | 2018-06-12 |
WO2009058414A1 (en) | 2009-05-07 |
CN101883776B (en) | 2014-04-02 |
US8236501B2 (en) | 2012-08-07 |
WO2009058432A1 (en) | 2009-05-07 |
CA2704771A1 (en) | 2009-05-07 |
JP5318110B2 (en) | 2013-10-16 |
US20100240123A1 (en) | 2010-09-23 |
JP2011502251A (en) | 2011-01-20 |
US20090107927A1 (en) | 2009-04-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8236553B2 (en) | Apparatus, system and method for purifying nucleic acids | |
US7988935B2 (en) | Handheld and portable microfluidic device to automatically prepare nucleic acids for analysis | |
US8574923B2 (en) | Sample preparation device | |
US9513196B2 (en) | Methods and systems for microfluidic DNA sample preparation | |
US20060166347A1 (en) | Sample preparation devices and methods | |
RU2380418C1 (en) | Replaceable microfluid module for automated recovery and purification of nucleic acids from biological samples and method for recovery and purification nucleic acids with using thereof | |
JP2007519917A5 (en) | ||
US20120071643A1 (en) | System and methods for purifying biological materials | |
JP2002502597A (en) | Integrated microfluidic device | |
KR20120044918A (en) | Nucleic acid purification | |
US20210008552A1 (en) | Nucleic acid extraction and purification cartridges | |
JP2010091568A (en) | Pipette tip part having separating material | |
AU2015215950B2 (en) | Nucleic Acid Purification | |
US11927600B2 (en) | Fluidic bridge device and sample processing methods | |
US20210220827A1 (en) | Systems and methods for nucleic acid purification using flow cells with actuated surface-attached structures | |
Malic et al. | Current state of intellectual property in microfluidic nucleic acid analysis | |
KR20240027088A (en) | Universal Assay Cartridges and Methods of Use | |
Verma et al. | Micro/nanofluidic devices for DNA/RNA detection and separation | |
RU2595374C2 (en) | Method for automated extraction with simultaneous purification of nucleic acids from several biological samples |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: AKONNI BIOSYSTEMS, MARYLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BELGRADER, PHILLIP;COONEY, CHRISTOPHER G.;REEL/FRAME:028485/0753 Effective date: 20120703 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |