WO2001053817A2 - Methods of and apparatus for separating and detecting nucleic acid - Google Patents
Methods of and apparatus for separating and detecting nucleic acid Download PDFInfo
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- WO2001053817A2 WO2001053817A2 PCT/US2001/001963 US0101963W WO0153817A2 WO 2001053817 A2 WO2001053817 A2 WO 2001053817A2 US 0101963 W US0101963 W US 0101963W WO 0153817 A2 WO0153817 A2 WO 0153817A2
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- gel
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- holder
- sample
- electrode
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
Definitions
- Nucleic acids include DNA, RNA, and analogues thereof.
- Different species of microbes such as bacteria, viruses, fungi, and/or parasites have unique genetic (DNA and/or RNA) sequences that can be used to detect the presence or absence of a particular microbe.
- Common sequences of nucleic acids are also recognized within a group of microbes such as in bacteria, which can be used to detect the presence or absence of members of that particular group.
- DNA or RNA sequences are modified, such as by single nucleotide polymorphisms (SNPs) or mutation, disease or dysfunction can result.
- SNPs single nucleotide polymorphisms
- Detection of polymorphisms or mutations can facilitate early intervention during the life of a patient in the form of medication or education to improve quality of life. For example, when genetic analysis indicates that a child will be born with cystic fibrous, early medical intervention can be provided that can minimize complications of the disease.
- a biological sample can be composed of a heterogeneous mixture of components, for example, proteins and nucleic acids.
- a target molecule e.g., a particular DNA, RNA or protein
- the ability to rapidly separate the components and identify a target molecule, e.g., a particular DNA, RNA or protein, in a sample can be a limiting factor in developing a useful diagnostic test.
- a target molecule e.g., a particular DNA, RNA or protein
- the present invention relates to a system for detecting, separating, purifying and/or concentrating a component or target molecule, e.g., an analyte, in a biological sample by allowing an electric field to transport the component into the vicinity of a capture probe, wherein the component specifically binds to or hybridizes with a capture probe.
- the system can include a housing, e.g., an electrophoresis cassette for receiving at least two electrodes; one or more structures for receiving a thin gel matrix that includes a capture probe; a source of ions to support an electric field, e.g., a buffered salt solution.
- the capture probe is, optionally contained within a gel matrix, for example agarose.
- the apparatus further includes structures for holding the thin gel or for holding a reinforced thin gel; structures for introducing a sample into a sample compartment in the system, wherein the sample potentially contains a component that binds to, or hybridizes with, the capture probe, e.g., a nucleic acid sequence which is substantially complementary to the capture probe.
- the system can further include an electrical power source, or can include structures for connecting the system, or for connecting a part of portion of the system to an electrical power source.
- the system of the present invention can be used for determining the presence or absence of a component or target molecule.
- a sample can be transferred to the electrophoresis cassette, e.g., the sample is introduced into a sample compartment of the cassette and an electrical force, e.g., a voltage gradient is applied across the electrodes of the cassette resulting in one or more components of the sample to pass from the sample compartment through the thin gel matrix.
- One or more capture probes are associated with the gel.
- a capture probe may be associated with a gel by affixing, immobilizing, entrapping, and/or by chemically, ionically, or covalently binding the capture probe to the gel.
- the capture probe can also be referred to herein as a ligand.
- the target molecule e.g., the analyte to be detected is a nucleic acid having a nucleotide sequence that is substantially complementary to the nucleotide sequence of the capture probe
- the nucleic acid is retained in the thin gel, while nucleic acids lacking a complementary sequences move through the gel.
- binding of the target molecule to the capture probe can be detected by one or more chemical or physical properties exhibited by the capture probe-target complex, which property will be known to those skilled in the art.
- the capture probe-target complex includes a detectable label.
- a system for capturing an analyte includes an electrophoresis cassette.
- the electrophoresis cassette has a housing, e.g., a base having a pair of electrode channels, a barrier interposed between the electrode channels, a barrier having at least one migration channel extending between the electrode channels, and a pair of enlarged slots bounded and opening into the migration channel.
- a first electrode extends in a first electrode channel and a second electrode extends in a second electrode channel.
- a capture gel holder is receivable in an enlarged slot.
- the capture gel holder can have an opening aligned with a migration channel.
- a thin gel is positioned in an opening of a capture gel holder.
- the capture gel holder is a comb.
- the thin gel has a matrix which can be a polymeric mesh; optionally the matrix may be non- conductive.
- a cassette in one embodiment, includes an evaporation cover positioned to overlie the base of the cassette.
- the evaporation cover can have at least one opening for a capture gel holder, at least one opening for venting of gas, and/or at least one opening to allow a sample well-forming comb to pass into the migration channel.
- the cassette can include a pair of wash well sets for receiving the teeth of the capture gel holder.
- the electrodes can have a pair of terminals extending through the evaporation cover. The terminals can be flush with the top of the evaporation cover.
- a capture gel holder has a handle and a plurality of teeth projecting from the handle.
- One or more of the teeth can have a bore through the tooth.
- a polymeric material e.g., a non-conductive material, can occupy space within the bore of the tooth, so as to be stretch over or within the bore of that tooth.
- the material can support a gel, e.g., a gel matrix.
- the bore can be bordered by, preferably surrounded by, a recessed central region, and can further be bordered by, or surrounded by, a flange on the bore so that the recessed central region and the flange facilitate release of gas.
- At least one tooth has a shape that is adapted to fit in the cassette in a particular or specified direction, for example a keyed shape.
- a thin gel is used for separating and detecting a target molecule, (e.g., an analyte), for example, a nucleic acid sequence, a nucleic acid analogue, where such analogue is for example, a peptide nucleic acid (PNA), a modified nucleic acid sequence, or a polypeptide having a particular binding affinity, a particular structure, or a particular amino acid sequence, in a sample.
- PNA peptide nucleic acid
- the present invention provides a polymeric gel matrix, optionally reinforced to facilitate removal from an apparatus, e.g., an electrophoretic cassette, wherein the polymeric gel matrix includes capture probes capable of specifically hybridizing with, or binding to, a target molecule.
- the gel is "thin".
- the term “thin” as used herein refers to the thick mass of the get matrix.
- the gel matrix of the present invention has the attributes of either a filter or a sieve, so as to retain one or more sample components on the gel matrix while permitting other components to pass through. It is further desirable that the gel matrix of the present invention be capable of manipulation, so that it can be placed into and removed from e.g., the electrophoretic cassette. It can be appreciated by one of skill in the art, that by reducing the thickness of the gel matrix greater assay speeds may be attained. However, the term “thin” can also refer to characteristics of selective permeability and ease of handling so as to decrease the amount of time required to achieve desired detection, separation, or concentration. Importantly, using the methods and apparatuses described herein, no prior amplification of sample analytes is required, yet the sensitivity of this rapid assay is comparable to more complex and time consuming methods of analysis.
- the gel can comprise any material that can be used in an electrophoretic assay system for detecting, separation, or concentrating an analyte, such as a form of polyacrylamide, agarose, starch, or dextran.
- the gel may be formulated so that it is stable enough to be relocated, moved, or physically transported by manual or mechanical means.
- the gel matrix can be provided with reinforcement that acts to reinforce the gel thereby facilitating handling. When the thin gel matrix is cast on a reinforcing material, the thin gel matrix is called a reinforced thin gel.
- the thin gel reinforcement includes a non-conductive porous material such as a mesh, a web, a mat, or a felt.
- the thin gel includes a polymeric additive, cross-linking agent, or other chemical agent that can be incorporated into the gel so as to structurally reinforce the gel matrix.
- the reinforcement increases the strength and durability of the gel matrix thereby rendering it resistant to tearing or stretching.
- the gel can be used with a holder that facilitates transfer and processing of the gel within a detector or detection station to detect binding of the target molecule to a cassette probe contained in the gel.
- a highly sensitive, non-amplified, rapid assay for the detection of a target molecule in a biological sample is provided.
- a sample e.g., sample suspected to be contaminated with an undesirable species, e.g., a target molecule such as a microbial species, e.g., bacteria, fungus, or virus
- a target molecule such as a microbial species, e.g., bacteria, fungus, or virus
- a target molecule such as a microbial species, e.g., bacteria, fungus, or virus
- render a nucleic acid characterized as present in that species preferably nucleic acid known to be unique to that species, available to hybridize to a capture probe associated with the gel matrix on the capture gel holder.
- the hybridized nucleic acid is thereby captured on the gel, and can then be detected and reported.
- a biological sample is first diluted with an appropriate diluent, and then centrifuged at high speed to pellet any target bacteria present in the sample.
- the pelleted bacteria is treated in a manner to solubilize membrane components, and render target bacterial nucleic acids suitable for hybridization with specific nucleic acid probes.
- Buffer containing alkaline phosphatase-conjugated reporter probes is added to the sample and the sample is heated to temperatures sufficient for unfolding of target nucleic acids and hybridization of reporter probes.
- the samples are cooled, and an aliquot of each sample is loaded into a pre-warmed electrophoresis cassette as described herein, and a voltage is applied.
- the samples migrate past capture combs (also referred to herein as capture gel holder) containing capture probes specific to the target bacterial nucleic acids.
- the capture probes are immobilized on a thin gel.
- the combs are then dip-washed and moved to a slot in the cassette which is upstream of the original electrophoretic path.
- a voltage is again applied for a brief time to electrophoretically wash the combs.
- the combs are placed in a conditioning buffer for a set time, then removed and are placed flat in a reader tray.
- Enzyme substrate is added in a quantity sufficient to react with the reporter probes hybridized to target immobilized in the gel.
- the reader trays are then scanned by the reader, and any signal present is detected and reported. The detection of signal is indicative of the presence of the target bacteria in the sample.
- a sample suspected of bacterial contamination can be cultured to grow up any colonies of bacteria present in the sample and the culture is transferred to the thin gel matrix by blotting the sample with an agarose gel encapsulated polyester or nylon membrane.
- the transferred bacterial colonies can be exposed to conditions that result in lysis of the bacteria and release of the analyte or analytes, e.g., nucleic acids contained therein.
- the nylon membrane can then be sandwiched against a thin gel matrix comprising capture probes, specific for the bacteria to be detected and any air bubbles between the two are eliminated, e.g., by gentle pressure.
- the sandwich can then be placed in the electrophoretic apparatus and, under suitable conditions, an electrical current is applied and the bacterial components comprising the analyte or analytes to be detected migrate (i.e., move) into contact with the immobilized capture probes. If the bacterial analyte to be detected is present within the sample, the analyte can hybridize, or bind to, the immobilized probe and remain immobilized (i.e., does not further migrate) in the gel. Bound analyte can then be detected, e.g., by hybridization of the analyte to a reporter probe and reported as described herein. In addition, the methods and apparatuses described herein permit rapid capture and purification of hybridized nucleic acids.
- target nucleic acids can be rapidly isolated from a complex sample and specifically captured by the immobilized capture probes of the gel on the capture comb.
- the capture comb can be removed to a different set of slots in the same electrophoretic cassette, a separate container, or another apparatus where the immobilized target/probe complex is subjected to conditions which release the target from the probe, thus the target molecule is recovered from the sample and can be used in further procedures such as for polymerase chain reaction.
- confirmatory testing for the presence of a specific polymerase chain reaction (PCR) product can be performed using the methods and apparatus of the present invention.
- a complex PCR reaction mixture can be contacted with immobilized capture probes on a capture comb and the specific PCR product can be captured and detected to confirm the accurate performance of the PCR reaction.
- FIG. 1 is a top perspective view of an electrophoresis cassette
- FIG. 2 is a top perspective view of the electrophoresis cassette of FIG.1 with the cover;
- FIG. 3 is a top view of the electrophoresis cassette
- FIG. 4A is a top perspective view of the evaporation cover
- FIG. 4B is a top view of the evaporation cover
- FIG. 5 is a front perspective view of a capture gel holder
- FIG. 6A is a front view of the capture gel holder
- FIG. 6B is a side view of the capture gel holder
- FIG. 7 A is an enlarged front view of a portion of the capture gel holder of FIG. 6A;
- FIG. 7B is an enlarged front view of a portion of the capture gel holder of FIG. 6A;
- FIG. 7C is a sectional view taken along the line 7C-7C in FIG. 6A
- FIG. 7D is a sectional view taken along the line 7D-7D in FIG. 6A
- FIG. 7E is a sectional view taken along the line 7E-7E in FIG. 6A
- FIG. 7F is a sectional view taken along the line 7F-7F in FIG. 6A;
- FIG. 7G is a sectional view taken along the line 7G-7G in FIG. 6A;
- FIG. 8 A is a top perspective of a sample well-forming comb for making sample wells in the electrophoresis cassette
- FIG. 8B is a top perspective view of a molding comb
- FIG. 8C is a top perspective view of an alternative molding comb
- FIG. 9 A is a schematic of an exemplary method of determining if bacteria is present in a biological sample
- FIG. 9B is a second schematic of an exemplary method of determining if bacteria is present in a biological sample
- FIG. 9C shows SEQ ID Nos. 1-17, RNA sequences from a panel of target organisms along with the complements of the probe sequences.
- the lines enclosed by the thin boxes indicates the DNA sequence that is complementary to the probes.
- the white dotted lines underline the sequences corresponding to the capture probes that are linked to the thin gel membrane.
- the solid white lines underline the sequences corresponding to the reporter probes.
- FIG. 10 is a schematic of a method for rapid assay of an analyte in a sample
- FIG. 11 A illustrates a front perspective view of a capture gel reinforced with a non-conductive mesh
- FIG. 1 IB is a cross-sectional view of the capture gel taken along the line
- FIG. 12A illustrates a side view of an alternative embodiment of the capture gel having an encapsulating enmeshed glass fibers
- FIG. 12B is a cross-sectional view of the capture gel taken along the line 12B-12D ofFIG. 12 A;
- FIG. 13 is a side perspective view of an electrophoresis machine with a side removed;
- FIG. 14 is a side perspective view of the electrophoresis machine and overlying incubator unit;
- FIG. 15 A is a top view of a tray for receiving a pair of capture gel holders for use in a detection device such as a luminescence reader;
- FIG. 15B is a sectional view of the tray taken along the line 15B-15B in FIG. 15 A;
- FIG. 16 is a schematic representation of an embodiment of the electrophoretic cassette and a detection station in which a target detection is accomplished by an enzyme stimulated chemiluminescence reaction;
- FIG. 17 is a schematic outline of a method of purification
- FIG. 18 A is a front view of a capture gel holder
- FIG. 18B is a perspective view of the capture gel holder of FIG. 18 A;
- FIG. 19A is a top view of an alternative embodiment of an electrophoresis cassette
- FIG. 19B is a sectional view of the electrophoresis cassette taken along the line 19B-19B of FIG. 19 A;
- FIG. 19C is a side perspective view of the electrophoresis cassette
- FIG. 20 is a side perspective of a comb for making sample wells in the electrophoresis cassettes.
- FIG. 21 A illustrates in a top view of an alternative embodiment of the electrophoretic parallel channel device
- FIG. 2 IB is a cross-sectional view of the electrophoresis cassette of FIG. 21B;
- FIG. 21C illustrative a gel capture holder having thin gel or reinforced thin gel;
- FIG. 22 illustrates a graph of results obtained using the assay method disclosed
- FIG. 23 is a graph showing a dose response for in vitro target transcripts spiked into the assay after the centrifugation step
- FIG. 24 is a graph showing a similar dose response of FIG. 23, but using E. coli spiked into sterile platelet concentrates.
- FIG. 25 depicts the time estimates for running up to 8 samples in the assay method described herein. DETAJXED DESCRIPTION OF THE INVENTION
- the present invention relates to the discovery that target molecule detection can be rapidly achieved by utilizing a thin gel matrix comprising immobilized capture probes. Capture and subsequent detection of the target molecule depends upon the density of the capture probe in the gel rather than upon the distance traveled by the target molecule through the thin gel matrix. Further, incorporation of a reinforcement into the thin gel matrix facilitates its handling and its removal from the device, thereby permitting improved procedures for removing background signals and detection.
- the reinforcement can be provided by a separate material such as a non-conductive mesh or fibrous mat. Alternatively, reinforcement can be a molecular reinforcement for example a cross-linking molecule that is incorporated into the thin gel matrix that strengthens and/or reinforces the matrix.
- the present invention comprises a thin gel, an electrophoretic system for use with the thin gel, and a method of using the electrophoretic system with the thin gel. More specifically, the present invention comprises a reinforced thin gel, an electrophoretic apparatus or device, a sample comb or capture gel holder for use with the electrophoretic device, and a method of using the electrophoretic device and the capture gel holder (sample comb) for separating and subsequently detecting an analyte from a sample.
- An electrophoresis cassette 190 according to the invention is shown in FIGS.
- the cassette 190 has a base 192 and an evaporative cover 194, shown in FIG. 2.
- the base 192 of the electrophoresis cassette 190 has a pair of electrode channels 92 and 94.
- each electrode channel 92 and 94 there are a plurality of ribs 198 that break each of the channels into portions.
- Each rib 198 has a slot 200 through which an electrode 202, such as seen in FIG. 3, extends.
- the slot 200 of the rib 198 is used to ensure the electrode 202 is properly positioned as explained in detail below.
- the electrode 202 extends the length of the respective electrode channel 92 and 94.
- the electrodes 202 each include a pair of posts 204 and a stainless steel or platinum strip 206 extends between the posts 204 as seen in FIG. 3.
- a portion of the post 204 is shown for illustrative purposes in FIG. 3, in that the post 204 is not installed in the base 192 until after the evaporative cover 194 is installed.
- Each post 204 has a cylindrical rod 208, shown in section in FIG. 3, which is received in a bore 210 in the base 192 and an integral circular disk or head 212 which sits on top of the evaporative cover 194, as seen in FIG. 2.
- the posts 204 are formed of stainless steel in one embodiment.
- the base 192 has a plurality of channels, the migration channel 96 as seen in FIG. 1, which extend between the electrode channels 92 and 94. Within each migration channel 96 there is a pair of enlarged slots 98 adapted to receive a tooth 218 of a capture gel holder 220.
- the capture gel holder 220 is further described below with reference FIGS. 5-7G.
- An alternative embodiment of the capture gel holder 66 is described briefly below. Whether a capture gel holder 66 can be used with a particular electrophoresis cassette 190 is dependent on numerous factors including spacing and dimension of teeth and any keying features.
- the base 192 of the electrophoresis cassette 190 has a pair of sets of wash wells 222 and 224 FIG. 1.
- Each set of wash wells 222 and 224 has a wash well 226 associated with and spaced similarly to the migration channel 96.
- Each wash well 226 is adapted to receive one of the teeth 218 of the capture gel holder.
- Each of the wash wells 226 of each set of wash wells 222 and 224 has a rib or support 228 with a slot 230 for receiving a tooth 70 or 218 of the capture gel holder 60 or 220.
- the rib 228 with the slot 230 is offset so that it does not align with the migration channel 96 therein holding the capture gel holder 220 aligned and upright while not interfering with the capture gel 40, as seen in FIG. 5, ensuring that the bore 76 in each tooth 218 which holds the capture gel 40 is accessible by the solution in the wash well.
- the wash wells 226 of the set of wash wells 224 have a larger capacity or volume than those of the other set of wash wells 222. The purpose of the difference in size is to allow the capture gel 40 to react with different amounts and concentrations of solutions or reagents.
- the set of wash wells 224 is referred to as a set of conditioner wells and the other set of wash wells 222 is referred to as the set of wash wells.
- the evaporative cover 194 has a plurality of vent holes or slots 234 for overlying the electrodes 202.
- the evaporative cover 194 in addition has a plurality of generally square openings 236.
- the square openings 236 allow a plurality of teeth 382, as seen in FIG. 8A, of a sample well- forming comb 380, to pass into the migration channel 96.
- the evaporative cover 194 has a plurality of slots 238 for receiving the teeth 218 of the capture gel holder 220 and which overlies the enlarged slot 98 of the migration channel 96, as seen in FIG. 1, of the base 192.
- the slots 238 have a rounded side edge 240 and a squared side edge 242 as best seen in FIG. 4B.
- the slots 238 are shaped so that the capture gel holder 220 can only pass through the slots 238 in a certain orientation, as explained in further detail below with reference FIGS. 5-7G.
- the evaporation cover 194 is attached to the base 192 by ultrasonic welding. The evaporative cover 194 does not cover the sets of wash wells 222 and
- the circular disk 212 of the post 204 at the electrode 202 overlies the evaporative cover 194.
- the evaporative cover 194 has a hole 246 through which the cylindrical rod of the post 204 extends.
- the evaporative cover 194 has a plurality of tabs 248 to be received in complimentary notches 250 in the base 192 of the electrophoresis cassette 190, as seen in FIGS. 1 and 2.
- One of these tabs, tab 252, of the tabs 248 is positioned slightly inward of the position of the other tabs 248 as seen in FIGS. 4A and 4B such that the evaporative cover 194 can only be placed on the base 192 in one direction.
- both the evaporative cover 194 and the base 192 have markings 256 such as the letters "A,” “B,” “C,” and “D” adjacent to the slots for the teeth 238 and the sets of washing wells 222 and 224 to indicate the order of receiving (i.e. , the steps) the teeth 218 of the capture gel holder 220 that are inserted into the respective components. The steps are described in greater detail below.
- the electrophoresis cassette 190 has a handle portion 258 on the base 192 for assisting in the movement of the electrophoresis cassette 190.
- the electrophoresis cassette 190 is used in conjunction with the capture gel holder in the method of capture of target molecule.
- the capture gel holder 220 as illustrated in FIG. 5 has a plurality of teeth 218 and a handle portion 260.
- the handle portion 260 has a recess 262 to assist in grasping and a second recess or label receiving portion 264.
- each of the teeth 218 has a thin central region 266 in which is located a bore 268 which extends through the tooth 218.
- the thinning of the central region 266 helps to prevent entrapment of bubbles on the face of the capture gel 40 and tooth 218 when inserting the capture gel holder into the slot 98 and 238 of the assembled and filled cassette.
- the bore 268 has a larger opening 270 and a smaller opening 272 creating a shoulder 274 as best seen in FIG. 7D.
- the smaller opening 272 has a taper 276.
- a capture gel 40 covers or overlies the bore 268 for capture of the target molecules as explained below.
- the teeth 218 are tapered so that each of the teeth is both narrower and shorter at the bottom as compared to the top of the tooth near the handle.
- the capture gel holder 220 has a length of slightly larger than 4 inches and has six teeth 218.
- the center lines of adjacent bores 268 are 0.709 inches apart with the spaces between center lines of the first and last bores 268 being 3.543 inches.
- the teeth 218 are tapered with the bottom having a length of 0.520 inches and a width in the thin central region 266 of 0.08 inches and the top of the tooth, near the handle portion, having a length of 0.545 inches and width in the central region of 0.138 inches.
- the bore 268, in one embodiment, has a diameter of 0.260 inches.
- the top of the bore 268 being 0.381 inches above the bottom of the tooth.
- migration channel 96 has a width of 0.295 inches which is larger than to the diameter of the bore 268 in the tooth 238.
- the teeth 218 of the capture gel holder 220 have a rounded side edge 280 and a square side edge 282 as best seen in FIGS. 7B, 7F, and 7G, and 7C.
- some of the teeth 218 can contain an additional protrusion 284 on a square side edge 282 to assure that the capture gel holder 220 is placed into the electrophoresis cassette 190 in the proper orientation.
- the capture gel holder 220 has a flat optical detector surface 288 on both sides of the part. These optical detector surfaces 288 are used in conjunction with an electrophoresis machine 292 described in conjunction to FIG. 13. While the capture gel holder 220 can only be placed in the cassette 190 in one position, optical detector surfaces 288 are located on both edges of the capture gel holder because of the two locations in the electrophoresis machine 292 that receives electrophoresis cassette 190.
- a sample well forming comb 380 as • shown in FIG. 8 A and a molding comb 386 as shown in FIG. 8B are used with the electrophoresis cassette 190.
- the sample well forming comb 380 as shown in FIG. 8A has a plurality of teeth 382 which pass through the square openings 236 in the evaporative cover 194 to create sample wells in agarose or electrophoretic matrix 120 as described below.
- the molding comb 386 as shown in FIG. 8B is received in the enlarged slots 98 during the pouring of the agarose or electrophoretic matrix 120 as described below.
- the method and apparatus allows rapid detection of target molecules in a biological sample.
- electrophoresis cassette 190 described and prior to a more detailed description of the capture gel 40 and of the electrophoresis cassette 190, a methods of detection using these items will be described.
- a biological sample believed to contain a target nucleic acid is obtained and processed in a manner (e.g., cells are lysed) to release the nucleic acids and render them suitable for detection.
- the released nucleic acids are contacted with a reporter probe, under conditions suitable for the reporter probe to hybridize to the target nucleic acids in the sample resulting in labeled (or tagged, e.g., detectably-labeled) sample nucleic acids.
- an adapter probe can also be contacted with the sample nucleic acids under suitable conditions wherein the adapter probe also hybridizes to the sample nucleic acids.
- the sample now comprises a mixture of labeled nucleic acids, unlabeled nucleic acids, and other components.
- This sample mixture is then subjected to electrophoresis in the electrophoresis cassette described herein, where the mixture is brought into contact with the capture probe.
- the capture probe is specific for the target molecule contained in the sample (e.g., the target bacterial nucleic acid). If the sample contains the target molecule, e.g., the nucleic acid has a sequence that is complementary to that of the covalently bound capture probe sequence, the target molecule is immobilized to the capture probe in the thin gel of the gel holder. Due to the continued application of electrophoretic current, the unbound components of the sample continue to move through the thin gel and are separated from the bound components.
- the gel holder which contains the immobilized target/capture probe complex can be transferred to a different slot in the electrophoresis cassette and the gel can be washed with buffer by briefly applying voltage which results in the unbound (e.g., unhybridized) components being removed from the gel. After separation, the bound components can be detected by a number of known methods. In one embodiment, detection of the bound target molecule is facilitated by moving the thin gel from the electrophoretic device to a detection device, such as a fluorescence or luminescence reader.
- FIG. 9A shows the flow of molecules in the assay.
- FIG. 10 outlines schematically the steps for manipulating the sample and the reagent. Referring to text block 130 in FIGS.
- the sample which is suspected of containing the target analyte is prepared.
- the molecule to be detected is a target nucleic acid which is contained in a cell, bacterium, or virus.
- conserved bacterial nucleic acid sequences can be used to detect a variety of bacteria present in a biological sample.
- two probes can be used for each target RNA. One is the reporter probe that is used to label the target RNA. The other is the capture probe which is linked to the thin gel membrane.
- FIG. 9C shows the SRP RNA sequences (SEQ ID Nos. 1-17) from a panel of target organisms along with the complements of the probe sequences.
- the lines enclosed by the thin boxes indicates the DNA sequence that is complementary to the probes. (Sequence complements are shown so that the mispaired regions can be more easily identified.)
- the white dotted lines underline the sequences corresponding to the capture probes that are linked to the thin gel membrane.
- the solid white lines underline the sequences corresponding to the reporter probes.
- the capture probes are synthesized as 2'-O-methyl RNA ohgonucleotides with a 5'-Acrydite (tm) modification (Acrydite (tm) is available commercially from Mosaic Technologies , Waltham, MA).
- the reporter probes are synthesized as 2'-O-methyl RNA ohgonucleotides with a 5 '-amino modification and are conjugated to an alkaline phosphatase enzyme label.
- One of the first steps of sample processing is to lyse the cells to render the nucleic acids available for hybridization to a capture probe.
- the sample is placed into a tube with a buffer or diluent and spun in a centrifuge at a speed and for a time sufficient for the cellular matter (e.g., platelets, cells, bacteria, viruses) to pellet in the tube.
- the cellular matter e.g., platelets, cells, bacteria, viruses
- the supernatant is poured off the sample in the tube and the sample is resuspended in the rinse buffer and then spun down again as before. After spinning, the supernatant is poured off.
- a lysis buffer is then pipetted into the tube with the sample as represented by block 132 in FIG. 9A.
- Lysis buffer comprises a buffered solution comprising components sufficient for lysing the cellular matter and preserving the target nucleic acids in an undegraded state.
- the lysis buffer can optionally comprise detergent or chaotrophic agents to facilitate lysis.
- Such buffers and components are well-known to those skilled in the art.
- the sample is heated to about, for example, greater than about 100°C, more specifically, approximately 124°C ⁇ 4°C, for a time sufficient for the cells to lyse, e.g., for approximately five minutes, and then cooled to a specific temperature of less than 50°C, such as about 45°C for about 2 to 6 minutes.
- the sample is heated in the incubator unit 302 in FIG. 14.
- a reporter probe mixture is then introduced into the lysed sample.
- the reporter probes comprise ohgonucleotides which are conjugated to flourescent, phosphorescent chemiluminescent or enzymatic labels.
- enzymatic labels include horseradish peroxidase or alkaline phosphatase. In general, enzymatic labels producing flourescent or chemiluminescent signals are used.
- the reporter probe is contacted with the lysed sample, as illustrated by block 134 in FIGS. 9A and 9B and block 154 in FIG. 10, under conditions sufficient for the reporter probe to hybridize to the nucleic acids in the sample, e.g., about 45°C ⁇ 2°C for approximately 10 minutes, e.g., 9-11 minutes.
- one reporter probe well-known to those of skill in the art is an alkaline phosphatase reporter probe.
- the sample hybridizes to the reporter probe as represented by block 136 in FIGS. 9A and 9B and 156 in FIG. 10, resulting in a labeled sample.
- the labeled sample is transferred into the sample wells of the electrophoresis cassette 190 shown in FIGS. 1-3.
- the voltage is applied to the electrophoresis cassette 190 cause the labeled sample to migrate through the electrophoresis matrix 120 from the location where it was introduced in the migration channel 96 away from one electrode 100 and toward the other electrode 100, as illustrated by block 140 in FIG. 9 A and block 160 in FIG. 10.
- the cassette is pre-warmed to a temperature of approximately 27-35°C prior to electrophoresis.
- the electrophoresis temperature is approximately 31 -35°C.
- voltage is adjusted from about 24- about 40 volts.
- the capture probes/ligands may all have the same sequence or may have different sequences. It is recognized that each sequence in a thin gel can be localized in a specific area, for example of a specific tooth, or a spatially defined area within a single tooth. Therefore, each tooth of the capture gel holder can have a different capture probe/ligand for a different target molecule, or each tooth can contain multiple species of capture probes. In the case where multiple probes are present, each probe can be present in a unique spatially delineated area of the gel. Alternatively, the capture probes can be mixed together and the mixture dispersed throughout the capture gel.
- the voltage is turned off to the electrophoresis cassette 190 and the capture gel holder 220 is moved to the other enlarged segment 98, sometimes referred to as a wash slot.
- the voltage is turned on again and electrophoresis is continued for an additional time period, such as five minutes.
- the current is in the same direction during the second or wash period.
- the capture gel holder 220 such as the comb shown in FIG. 5, is removed from the electrophoresis cassette 190 with the capture gel 40 with the labeled target nucleic acid hybridized to the capture probes of the capture gel matrix.
- the capture gel holder 220 is then placed in a buffer solution and the substrate specific for the reporter probe of the labeled sample is contacted with the sample for a time and at a temperature sufficient for the reporter probe to interact with the substrate to generate a detectable signal.
- the capture gel holder 220 is placed in a reader as represented by block 164 in FIG. 10 so that the signal, such as chemiluminescence, is detected.
- Presence of a detectable signal is indicative of the presence of the target molecule, e.g. nucleic acid, in the sample.
- the detectable signal is produced by the label of the reporter probe, which is the probe shown in block 134 of Fig. 9 A or block 154 of Fig. 10.
- Any biological sample can be analyzed using the methods and devices described herein.
- the methods of the present invention are applicable to analysis of any chemical entity that can be electrophoresed (e.g., a charged molecule that has detectable mobility when placed in an electrophoretic field) and that binds to, or is bound by, nucleic acids.
- Such entities include, for example, DNA or RNA samples, nucleic acid binding proteins, and aptamer binding partners (aptamers are nucleic acids that are selected to bind to specific binding partners such as peptides, proteins, drugs, polysaccharides and small organic molecules, e.g., theophylline and caffeine (Jenison, et al., Science, 263:1425-1429 (1994)).
- methods described herein can be used for analysis and purification of target nucleic acids using immobilized capture probes, where specific binding involves base pairing interactions between sample nucleic acids and the capture probe, as in nucleic acid hybridization.
- the methods described herein are also useful for purification of sequence-specific nucleic acid binding proteins, since synthetic nucleic acids of defined sequence can be immobilized in matrices commonly used for protein electrophoresis.
- the test sample can be from any source and can contain any molecule that can form a binding complex with a capture probe.
- samples from biological sources containing cells, or platelets obtained using known techniques, from body tissue (e.g., skin, hair, internal organs), or body fluids (e.g., blood, plasma, urine, semen, sweat).
- body tissue e.g., skin, hair, internal organs
- body fluids e.g., blood, plasma, urine, semen, sweat.
- microbiological samples such as viruses, yeasts and bacteria; plasmids, isolated nucleic acids and agricultural sources, such as recombinant plants.
- the test sample is treated in such a manner, known to those of skill in the art, so as to render the target molecules contained in the test sample available for binding or hybridizing.
- the target molecule is a nucleic acid present in a cell
- a cell lysate is prepared, and a crude cell lysate (e.g., containing the target nucleic acid as well as other cellular components such as proteins and lipids) can be analyzed.
- the target nucleic acids can be isolated (rendering the target nucleic acids substantially free from other cellular components) prior to analysis. Isolation can be accomplished using known laboratory techniques.
- the target nucleic acid can also be amplified (e.g., by polymerase chain reaction or ligase chain reaction techniques) prior to analysis. Probes
- Capture probes are ligands, or molecular sequences complimentary to a predicted analyte immobilized in an electrophoretic gel matrix by covalent attachment to a polymer suitable for use as an electrophoretic medium.
- a variety of capture probes can be used in the methods of the present invention.
- the capture probes of the present invention comprise a nucleic acid with a nucleotide sequence substantially complementary to the target molecule wherein the target molecule hybridizes to the capture probe.
- the complementarity of nucleic acid capture probes need only be sufficient to specifically bind the target molecule and demonstrate the presence or absence of the target molecule.
- Probes suitable for use in the present invention comprise RNA, DNA, nucleic acid analogues, and chimeric probes of mixed class comprising a nucleic acid with another organic component, e.g., peptide nucleic acids. Capture probes can be single-stranded or double-stranded nucleic acids.
- nucleic acid includes DNA or RNA.
- Nucleic acids referred to herein as “isolated” are nucleic acids separated away from the components of their source of origin (e.g., as it exists in cells, or a mixture of nucleic acids such as a library) and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods known to those skilled in the art. These isolated nucleic acids include substantially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated.
- substantially complementary means that the nucleotide sequence of the capture probe need not reflect the exact nucleotide sequence of the target molecule, but must be sufficiently similar in identity of sequence to hybridize with the target molecule under specified conditions.
- non-complementary bases, or additional nucleotides can be interspersed in sequences provided that the sequences have sufficient complementary bases to hybridize therewith.
- Specified conditions of hybridization can be determined empirically by those of skill in the art. For example, conditions of stringency should be chosen that significantly decrease non-specific hybridization reactions. Stringency conditions for nucleic acid hybridizations are explained in Current Protocols in Molecular Biology, Ausubel, F.M., et al., eds., Vol.
- nucleic acid hybrids Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Stringent conditions, e.g., moderate, or high stringency, can be determined empirically, depending in part on the characteristics of the probe and target molecule.
- the length of a capture probe will be at least 5 nucleotides in length, more typically between 5 and 50 nucleotides, and can be as long as several thousand bases in length.
- Probes comprising modified nucleotides may also be useful.
- nucleotides containing deazaguanine and uracil bases may be used in place of guanine and thymine-containing nucleotides to decrease the thermal stability of hybridized probes (Wetmur, Critical Reviews in Biochemistry and Molecular Biology, vol. 26, pp. 227-259, 1991).
- 5-methylcytosine can be substituted for cytosine if hybrids of increased thermal stability are desired (Wetmur, Critical Reviews in Biochemistry and Molecular Biology, vol. 26, pp. 227-259, 1991).
- Modifications to the ribose sugar group can reduce the nuclease susceptibility of immobilized RNA probes (Wagner, et. al. Nature, 372: 333-335, (1994)). Modifications that remove negative charge from the phosphodiester backbone can increase the thermal stability of hybrids (Moody, et. al, Nucleic Acids Res., 17: 4769-4782 (1989); Iyer et. al, J. Biol Chem., 270: 14712-14717 (1995)). Nucleic acid analogues can also be useful as immobilized probes.
- PNA peptide nucleic acid
- standard DNA bases are attached to a modified peptide backbone comprised of repeating N-(2-aminoethyl)glycine units (Nielsen, et. al, Science, 254: 1497-1500, (1991)).
- the peptide backbone is capable of holding the bases at the proper distance to base pair with standard DNA and RNA single strands.
- PNA-DNA hybrid duplexes are much stronger than equivalent DNA-DNA duplexes, probably due to the fact that there are no negatively charged phosphodiester linkages in the PNA strand.
- PNAs are very resistant to nuclease degradation.
- PNA nucleic acid analogues are useful for immobilized probe assays. It will be apparent to those skilled in the art that similar design strategies can be used to construct other nucleic acid analogues that will have useful properties for immobilized probe assays.
- label is any means for distinguishing a capture probe having an analyte bound thereto.
- the label may be a molecule complementary to the capture probe - an analyte complex where the label has been provided with a radio-isotope, a fluorescent, a chemiluminescent, or similar chemical tag such as used in a sandwich hybridization assay.
- the label may be an enzyme capable of interacting with a substrate to produce a detectable signal.
- a signal probe can be used to provide a label on the capture probe-analyte complex.
- each capture probe in a thin gel or polymeric gel is localized in a specific area.
- a plurality of thin gels having differing capture probes/ligands can be arranged to form arrays so all the probes in a particular area have a known sequence.
- the thin gel or polymeric gel is further described below.
- Capture probes can also be covalently attached to a matrix using a thiol attachment chemistry as described in U.S. Provisional Patent Application No. 60/151,267 filed August 27, 1999, U.S. Provisional Patent Application No. 60/177,844 filed January 25, 2000, and U.S. Patent Application No. 09/649,637 titled "Methods of Immobilizing Ligands on Solid Supports and Apparatus and Methods of Use Therefor" by
- Capture probes are attached to a polymeric material that provides a sieving function in order to increase contact with the capture probe.
- Thin gel matrices may also include spacer molecules and matrix strengthening molecules.
- Nucleic acids, modified nucleic acids and nucleic acid analogs can also be coupled to agarose, dextrans, cellulose and starch polymers using cyanogen bromide or cyanuaric chloride activation. Polymers containing carboxyl groups can be coupled to capture probes provided with a primary amine group using carboiimide coupling.
- Polymers having primary amine groups incorporated therein can be coupled to amine-containing capture probes with glutaraldehyde or cyanuric chloride. Many polymers can be modified with thiol-reactive groups which can be coupled to thiol-containing synthetic capture probes. Representative protocols and other examples can be found in Cass and Ligler (eds.), "Immobilized Biomolecules in Analysis: A Practical Approach,” 1998, Oxford University Press, Oxford, UK, and Hermanson, Mallia, and Smith, "Immobilized Affinity Ligand Techniques,
- Adaptor probes/molecules can also be used in the methods of the present invention. Such probes are described in U.S. Patent Application No. 09/285,380 and PCT/US00/08529, the teachings of which are herein incorporated by reference.
- Gel Matrix One of the elements of the invention is the capture gel 40, such as a polymeric gel or a thin gel. This capture gel matrix is used in conjunction with the probe or capture probe, to capture the target molecule (i.e., desired component).
- the capture gel refers to the gel framework to which complementary sequences are attached. Examples of materials suitable for use in forming the gel matrix include both gel-forming and non-gel forming polymers. Any gel matrix suitable for electrophoresis can be used for preparation of the gels of the present invention.
- Suitable matrices include acrylamide and agarose. It is recognized that other materials can be used as well for the capture gel. Examples include chemically modified acrylamides, starch, dextrans and cellulose-based polymers. Additional examples include modified acrylamides and acrylate esters (for example see Polysciences, Inc. Polymer & Monomer catalog, 1996-1997, Warrington, PA), starch (Smithies, Biochem. J., 71 :585 (1959); product number S5651, Sigma Chemical Co., St. Louis, MO), dextrans (for examples see Polysciences, Inc.
- a composite matrix contains a mixture of two or more matrix forming materials, such as for example acrylamide-agarose composite gels. These gels typically contain 2-5% acrylamide and 0.5%-l% agarose. hi these gels, the acrylamide concentration determines the functional pore size. Agarose provides mechanical strength without significantly altering the gel pore size of the acrylamide.
- the capture gel 40 has non-conductive mesh 42 encapsulated in a polymer gel 44.
- a plurality of capture probe molecules are covalently bound to the polymer gel 44 throughout the three dimensional capture gel, which fills the spaces 48 of the fibers 50 of the mesh 42, as best seen in FIG. 1 IB.
- the non-conductive mesh 42 is seen to be substantially entrapped in bubble-free capture (thin) gel 40.
- a top surface 52 and a bottom surface 54 of the capture gel (reinforced thin gel) 40 are shown substantially flat.
- the top surface 52 and the bottom surface 54 are illustrated as substantially flush with the top surface and the bottom surface of the non-conductive mesh 42, respectively, either or both of the top surface 52 and the bottom surface 54 of the capture gel 40 can be distanced from their respective surfaces of the mesh 42.
- the polymer gel 44 is an acrylamide gel.
- the type of capture probe molecules are dependent on what is desired to be capture.
- the capture probe molecules can be an oligonucleotide carrying reactive ethylene groups.
- the capture gel 40 with the non-conductive mesh 42 can be formed by various methods.
- One method of forming the capture gel 40 is by capillary action. Porous material, a non-conductive mesh, is placed between two glass plates so that the material extends from between the two plates while being supported by one of the plates. Gel-forming solution is applied to an edge of the material that extends from under the top glass plate. The gel-forming polymer solution is drawn into the material by capillary action and between the two glass plates. Plate spacers may be used between the glass plates to allow an increase in the thickness of the layer when gelled.
- a releasing agent is applied to the mold, be it glass or some other material, in order to facilitate release of the molded and reinforced thin gel.
- porous material to be placed in a mold and a gel-forming polymer solution, having ligand covalently bound to the polymer providing sieving properties to the gel, can be added to the mold and allowed to solidify. Air bubbles within the gel matrix should be avoided, for example by forming the thin gel under pressure or by vibrating the mold.
- FIG. 12A and FIG. 12B an alternative embodiment of a reinforced capture (thin) gel 58 having a polymer gel 44 encased within a fibrous glass fiber infra-structure 60 is illustrated.
- the glass fiber reinforcement 60 is seen to extend above and below the surfaces of the polymer gel 44.
- Capture probe molecules are covalently bound to polymers 44 throughout the three dimensional capture gel 58.
- the capture gel 40 of the present invention has sufficient mechanical strength to allow its removal from the electrophoresis device for washing and detection procedures, such as described below.
- the capture gel can be reinforced either by mechanical or molecular means.
- Examples of an internal structural reinforcement for such a thin gel matrix include a variety of non-conductive materials having pores.
- Such materials include for example mesh, screen or honeycomb material; non-woven fibrous material such as glass fiber and mat; and woven or knitted material such as fabric.
- materials having pores comprise mesh, woven, and non-woven materials.
- Mesh materials include polyester silk screening material such as is available from Sefar America, Inc., Briancliff Manor, NJ. Corp., polyamide resin mesh, nylon mesh, and polyethylene or polypropylene honeycomb material.
- Woven materials include loosely woven cloth such as for example J-ClothTM available from Johnson & Johnson Ltd.
- Non-woven materials include membranous materials comprising cellulose esters such as cellulose acetate butyrate, nitrocellulose, cellulose propionate such as described in United States Patent No. 4,006,069 (Hiratsuka , et.al.), the disclosure of which is incorporated herein by reference.
- the reinforcement is to be internal to the thin gel matrix and there is to be a layer of gel on both sides of the reinforcing mesh or cloth so that the reinforcement material is encased by, and enmeshed with, the gel
- the thickness of the gel is preferably not greater than that which can be supported by the mesh or cloth.
- the surface of the reinforcement material is either rough, irregular, porous or otherwise capable of allowing the gel to interpenetrate the material.
- three-dimensionally stable reinforcement materials are porous materials such as porous particles made by a method as disclosed in British Patent No. 1421 531 (UKAEA), the disclosure of which is incorporated herein by reference in its entirety.
- Other examples of reinforcement materials include thinly sectioned sponges.
- other contemplated reinforcements include porous materials that are coated to enhance the adhesion of the gel matrix to the porous material such as coated polyester, as described in United States Patent No. 5,672,416 (Radola), the disclosure of which is incorporated herein by reference in its entirety.
- the capture gel 40 can also include spacer molecules and matrix strengthening molecules.
- a "spacer molecule" is a structure capable of polymerizing with or binding to at least one of the gel matrix forming polymer molecules and functions to distance a moiety bound to the spacer from the gel matrix forming polymer backbone.
- the spacer molecule is capable of binding with any two of the gel matrix forming polymeric components and can thereby affect the density and/or spatial orientation of the capture probes bound to a gel matrix forming polymeric component in any particular region of the thin gel matrix by distancing one polymer from another polymer in the matrix.
- spacer molecules may provide reactive sites for binding to a moiety or molecule other than the matrix forming polymers.
- the spacer molecule may provide binding sites for polyethylene glycol or a detergent.
- matrix strengthening polymers are polymers useful for cross-linking, stiffening, or otherwise modifying the gel matrix to make it more durable during handling, without adversely affecting the desired sieving and/or analyte binding properties of the resulting thin gel matrix.
- Matrix strengthening polymers toughen the thin gel matrix increasing its mechanical strength thus allowing it to be handled without additional external reinforcement.
- Exemplary polymers include agarose.
- this polymer component may also provide additional sites for coupling gel modifying constituents. For example, chemical groups such as methyl or hydroxyl groups may be added to modify the hydrophobic/hydrophilic nature of the gel. Sulfate or quaternary amine groups may be added to introduce ionic groups into the thin gel.
- the stainless steel or platinum strips 206 are placed the electrode channels 92 and 94 seen in FIG. 1 of the base 192.
- the strips 206 have curved ends to receive the posts 204 as seen in FIG. 3 as explained below and the slots 200 in the ribs 198 ensure the strips are installed properly.
- the evaporative cover 194 As seen in FIGs. 4A and 4B is positioned on the base 192. As indicated above, the cover 194 and the base 192 have tabs 248 and notches 250 to ensure proper installation. In one embodiment, the evaporative cover 194 and the base 192 are welded together by an ultrasonic process.
- the four posts 204 associated with the two electrodes 202 are each inserted into one of the holes 246 in the evaporative cover 194.
- the bore 210 in the base 192, as seen in FIG. 1 receives a portion of the cylindrical rod portion of the post 204 with the rod portion passing through the curved end of the strips 206.
- the circular disk portion 212 of the post 204 overlies the top of the evaporative cover 194.
- the teeth of a pair of molding combs are inserted through the slots 238 in the evaporative cover 194 and into the enlarged slots 98, as seen in FIG. 1.
- One of the identical molding combs 386 is seen in FIG. 8B. Both sets of enlarged slots 98 receive teeth of the molding combs.
- a single molding comb 390 having two parallel sets of teeth such as seen in FIG. 8C, can be used.
- an electrophoretic matrix 120 is dispensed in the base 192 through the sample openings and fills a portion of the migration channel 96 to a predetermined height.
- the electrophoretic matrix 120 can be formed from gel forming polymers such as agarose or starch.
- the agarose is formed by using a molten solution of 1% agarose in 0.1 X TrisBP. The following is mixed 100 ml 0.1X TrisBP buffer with 1 g agarose powder (Sigma A9539 General Use Molecular Biology Grade).
- the procedure is repeated for all the migration channels 96, and a sample well comb 380 is inserted into the cassette.
- the sample well comb is shown in FIG. 8A. Molten agarose solution is dispensed into channels 96 through sample opening 236 in evaporative cover, taking care to reserve enough empty space to allow insertion of sample well comb.
- the agarose or electrophoretic matrix 120 is shown in FIG. 1 filling one migration channel 96.
- a sample well 174 is shown in the agarose 120.
- the evaporative cover 194 is welded to the bore 192 prior to the dispensing of the agarose 120 into the channels 96.
- the electrophoresis cassette 190 can be foiled sealed for shipping in one embodiment.
- the capture gel holder 220 is sealed separately and contains the desired capture probes in one embodiment.
- the cassettes of the present invention can be disposable (i.e., used once and discarded) or reused after a suitable cleaning process. For instance, in embodiment utilizing alkaline phosphate (AP) as a label, the following treatment can be used to remove trace amounts of AP.
- AP alkaline phosphate
- the parts of the electrophoresis cassette 190 are soaked in a solution of 1% hydrochloric acid and 1 millimolar (mM) EDTA (ethylenediaminetetraacetic acid) for at least 30 minutes to remove residual reagents. The parts or components are then washed thoroughly with water and air-dried on a clean surface.
- mM millimolar
- EDTA ethylenediaminetetraacetic acid
- the electrophoresis cassette 190 and capture gel holder 220 are unsealed if sealing step had occurred.
- a sample is also prepared as described with reference FIGS. 9A, 9B and 10.
- a hybridization mixture is loaded into the sample wells 174 in the electrophoresis cassette 190 through the square openings 236 in the evaporative cover 194.
- the capture gel holder 220 which contains the capture gel 40 with the desired capture probes is inserted in the electrophoresis cassette so that the teeth 218 enter the first set of enlarged slots 98 in the migration channel 90.
- the first set of slots 98 is that set closest to the (+) electrode.
- the electrophoresis cassette 190 is placed in an electrophoresis machine 292.
- the electrophoresis machine 292 for receiving the electrophoresis cassettes 190 is shown.
- the electrophoresis machine 292 has a tray 294 which slides out of the machine and receives the electrophoresis cassettes 190.
- the machine 292 has electrical connections in the center of the electrophoresis machine 292 in a bar 296 such that when the tray 294 slid in to the machine 292, the bar 296 in the center engages the cylindrical disc 212 of the post 204 of the electrodes 202.
- each of the electrodes 202 Only one side of each of the electrodes 202 is contacted.
- the purpose for having a pair of posts 204 for each electrode 202 is such that the electrophoresis cassettes 190 may be placed in either spot on the tray 294 in the electrophoresis machine 292.
- the electrophoresis machine 292 has timing features and temperature sensors to ensure that the electrophoresis cassette 190 does not exceed a certain temperature.
- the optical detector surface 288 is used in conjunction with the electrophoresis machine 292 such that machine 292 does not operate without a capture gel holder 220.
- the electrophoresis machine 292 is shown with an incubator unit 302 overlying it.
- the incubator unit 302 has a pair of warming blocks 304 for receiving the electrophoresis cassettes 190.
- the blocks 304 are used for prewarming cassettes 190 to proper running temperature prior to electrophoresis.
- the electrodes 100 of the electrophoresis cassette 190 of FIG. 2 are connected to a DC power supply in the electrophoresis machine 292 and a constant voltage is applied for a set time period. Temperature will increase during the run, but should not be allowed to increase above a temperature which will impair hybridization of the probes with the target nucleic acid. In a preferred embodiment the cassette temperature is controlled within the electrophoresis machine 292.
- the power is turned off.
- the capture gel holder 66 is moved from the enlarged segment 98 in the migration channel 96 to the other enlarged segment 98 in the same electrophoresis cassette 86 which is proximal to the (-) electrode.
- the teeth of the capture gel holder 220 are placed into the set of wash wells 222 that contains the wash buffer.
- the buffer is the wash buffer of example 1 and the capture gel 40 on the teeth 218 are in the buffer for 5 seconds.
- the electrodes 100 are connected to a DC power supply and a constant voltage is applied for a set time period. Once again, care is taken not to allow the temperature of the cassette to exceed a temperature which will impair hybridization of the probes. As indicated above, when discussing the method in relation to FIG. 10, the current is in the same direction during the second or wash period.
- the propagation of the residual sample which may be trapped in the channel 96 is away from the capture gel 40 of the capture gel holder 220 and thus any sample on the capture gel holder 220 that will not be captured by the capture probe will pass through and no new sample will come in contact with the capture gel 40.
- the capture gel 40 of the capture gel holder 220 is accomplished.
- the capture gel holder 220 is removed from the wash or second set of slots 98 of the electrophoresis cassette.
- the teeth 218 with the capture gel 40 are placed in a conditioner buffer in the second set of wash wells 224.
- the capture gel 40 is soaked in the solution and incubated at room temperature for 5-10 minutes on the incubator unit of FIG.14.
- the detection tray 178 for receiving the capture gel holders 220 is placed into a detector 172 such as a luminometer of FIG. 16.
- the detection tray 178 has a recess 312 for receiving a pair of capture gel holders 220.
- the recess 312 is shaped to fit the capture gel holder 220 described above.
- the detection tray 178 is sized in one embodiment to be approximately the size of a 96- well type tray plate in length and width. In this embodiment, the detection tray 178 is not as tall as a conventional 96-well type tray plate. Therefore, it has a length of approximately 5.03 inches and a width of 3.365 inches.
- the capture gel holder 220 is then placed in a detection tray 178, as shown in FIGS. 15A & 15B.
- the capture gel holder 66 and the detection tray 178 are placed in a microplate luminometer 172 such as a Walla Victor 2 sold by E.G.&G Walla of Turku, Finland, as represented in FIG. 16.
- target nucleic acids are purified by contact with the capture probe covalently attached to a thin gel matrix.
- the bound target nucleic acid can be released from the probe by changes in conditions (e.g., increased temperature) and the released target nucleic acids can be collected into a clean receptacle for further processing.
- Such purification can be performed using capture probes covalently bound to thin gel membranes where the gel membranes are contained in any suitable holder or support device, e.g. a comb such as described herein or micro liter plates, such as the 96-well micro liter well plates well-known to those of skill in the art.
- the purification steps are similar to the method shown in FIG. 10 and discussed above until the detection step, block 162 and 164 in FIG. 10
- the capture gel holder 220 is removed from the electrophoresis cassette 190
- the capture gel holder 220 is subjected to conditions sufficient to break the bonds between the capture probe and the target molecule, thereby releasing the target molecule from the probe.
- the thin gel membrane is removed from the holder and placed in a tube with elution buffer. The tube is heated to elute the hybridized target from the capture probes.
- the released target molecules can be recovered using techniques well- known to those skilled in the art and then used in further procedures, such as, for example, PCR amplification.
- FIGS. 18A and 18B An alternative capture gel holder 66 is seen in FIGS. 18A and 18B.
- the capture gel holder also sometimes referred to as a comb or sample comb, 66 has a handle 68 and a plurality of teeth 70.
- Each of the teeth 70 has a thinned central region 74 in which is located a bore 76.
- the bore 76 extends through the tooth 70.
- the thinned central region 74 facilitates bubble removal as explained above with respect to the first embodiment.
- the non-conductive mesh 42 of the capture gel 40 overlies the bore 76.
- the capture gel 40 can be affixed to the capture gel holder 66 by many methods including but not limited to ultrasonic welding, heat staking, and gluing.
- the polymer gel 44 component of the capture gel 40 can be cast onto the non- conductive mesh 42 before or after attaching the mesh to the holder 66. In one embodiment, the capture gel 40 placed on non-conductive mesh 42 before the mesh is attached to the holder 66.
- FIGS. 19A-19C An alternative electrophoresis cassette 86 that is used with the capture gel holder 66 is seen in FIGS. 19A-19C is used.
- the electrophoresis cassette 86 has a base 88 and an evaporative cover 90.
- the evaporative cover 90 is transparent in one embodiment but is not limited to such.
- the base 88 of the electrophoresis cassette 86 has a pair of electrode channels 92 and 94, as seen in FIG. 19B.
- the base 88 has a plurality of migration channels 96 which extend between the electrode channels 92 and 94. Within each migration channel 96, there is a pair of enlarged slots 98 adapted to receive a tooth 70 of the capture gel holder 66.
- Each electrode channel 92 and 94 has an electrode 100 which extends the length of the respective electrode channel 92 and 94.
- the electrodes 100 are each a pair of stainless steel pins 108, and a stainless steel strip 110 extends between the pins 108.
- the evaporation cover 90 has a strip 102 which overlies the interface between the electrode channels 92 and 94 and the migration channel 96. Strip 102 restricts the flow of bubbles generated by buffer hydrolysis at the electrodes thereby facilitating their removal through vents 104 in the cover.
- the evaporative cover 90 has a plurality of vent holes 104 for overlying the electrodes 100.
- the evaporative cover 90 has holes 106 for receiving the teeth 70 of the capture gel holder 66 which overlies the enlarged slot 98 of the migration channel 96 of the base 88.
- the evaporative cover 90 in addition has a plurality of generally square openings 114.
- the square openings 114 allow a plurality of teeth 116, as seen in FIG. 20, of a sample well-forming comb 118, to pass into the migration channel 96 as explained below.
- a second alternative electrophoretic cassettes 150 for use in the method of electrophoretically introducing a sample into a reinforced thin gel (or a thin gel) having capture probes covalently bound to the thin gel matrix is exemplified.
- the cassette 450 comprises a base 452, a first electrode 454, a second electrode 456, at least one holder 458 for holding reinforced thin gel 406, and an electrophoretic matrix 550.
- the electrophoretic matrix is buffer filled and substantially occupies the cassette base as a layer having a predetermined height.
- the cassette is provided with at least one well 460 in the electrophoretic matrix for receiving a liquid sample.
- the electrophoretic matrix can be formed from gel forming polymers such as for example agarose or starch.
- the electrophoretic matrix can be formed from a porous solid such as a polymeric sponge described in Harrington, et.al., US Patent No. 5,637,202, the disclosure of which is incorporated herein in its entirety by reference.
- the reinforced thin gel is held in the proper orientation within the cassette by mean of the holder 458.
- the holder allows easy removal and transportation of the reinforced thin gel to a detection station for reading of the reinforced thin gel and detection of the target molecule when present in the sample.
- the holder a gel capture holder, has at least one tooth 459 that extends down from a handle. The tooth has an aperture for receiving the reinforced thin gel.
- the holder is oriented so that the teeth of the holder extend down into the electrophoretic matrix when the holder is seated n the electrophoretic system.
- the holder positions the reinforced thin gel so that a target molecule in the sample in the well will migrate through the reinforced thin gel when a potential gradient is applied across the electrodes 454 and 456.
- the holder 458 fits into slots on the left and right sides of the base 452 to position the holder within the cassette.
- the electrodes can be formed from many conductive materials including wire, carbon rods, metal strips, metal-plated fabric, conductive plastics, and the like.
- the conductive electrode materials can be formed as wires, thin strips, pins, rods, coatings deposited on the ends of the base 452, or conductive adhesive strips.
- a preferred method for preparing the cassette of FIG. 21 A is described below. First the electrodes and base are assembled. Then the holder 458 with attached reinforced thin gel 406 is placed into the base. The teeth of holder 458 are immersed in the matrix 550 up to the depth indicated by dashed line 601 in FIG. 21C. Then a comb with solid teeth is placed adjacent to, and parallel with the holder 158.
- FIG. 8B An illustrative example of a comb 180 having a comb handle 182 and a tooth 184 is provided in FIG. 8B.
- the base 452 the base is filled with molten agarose in electrophoresis buffer. The filled cassette is allowed to cool and the agarose solidifies. After cooling, the sample well-forming comb is removed.
- a finished cassette is exemplified in FIG. 21 A.
- any holder or manifold capable of supporting the gel matrix is suitable for use in the methods described herein.
- a key characteristic of the holder is its ability to stable support the matrix (e.g. weld the mesh coated gel to the holder) in a manner suitable for electrophoresis.
- many devices and manifolds commonly used for filtration or vacuum filtrations can be adapted for use as capture gel holders.
- sample material e.g., platelet concentrate
- sample vials(s) containing 0.5mL Sample Diluent Cap the tubes and invert 3 to 5 times to mix. Centrifuge the tubes in a balanced microcentrifuge at 10,000 ⁇ 1,000 x g for 1 ⁇ 0.25 minute. The control tubes are not centrifuged. Carefully remove the tubes from the rotor and remove the caps. Decant the supernatant by pouring into a BioHazard waste container, and briefly holding the lip of the tubes to a clean absorbent material. The tube should not be tapped or shaken.
- sample material e.g., platelet concentrate
- a new pipet tip should be used for each sample. Turn on the voltage to 40 volts, and run for 4 minutes, then reduce the voltage to 24 volts for another 16 minutes. Turn off the voltage, and move the membrane comb to the wash buffer wells , held at room temperature or in the warmed cassette, for ⁇ 5 seconds. Move the comb to the electrophoretic wash slot, and turn on the voltage to 40 volts for 5 ⁇ 0.5 minutes. Turn off the voltage, and move the membrane comb to the Conditioning Buffer, hold at room temperature or in the warmed cassette, for 5-10 minutes. Detection Remove the comb from the cassette, and blot the sides of the comb with clean absorbent material held in the blotting fixture for 10 seconds. Place the comb into a reader tray, and place the reader tray into the reader. Add 25 ⁇ 3 ⁇ l room temperature substrate to each well and read.
- Sample Diluent Buffer NaH 2 PO 4 .monohydrate 15mM Na 2 HPO 4 .heptahydrate 15mM
- Example 2 Assay using a thin gel membrane.
- cassettes similar to those of FIG. 19 were used with the following exceptions: 1) carbon rod electrodes were used in place of metal ones; 2) individual mesh reinforced thin gel membranes were used instead of using a multi- toothed capture gel holder. The individual thin gel membranes were manipulated with forceps. Part I. Preparation of thin gel membrane for capture of target nucleic acid
- the mesh is cut into rectangles (0.48 inch x 0.52 inch) and washed by gentle shaking in ethanol (one wash of 5 minutes), deionized water (three washes of 5 minutes), and aqueous nonionic detergent (0.1 % Tween 20, one wash for 5 minutes.)
- the mesh is air dried on clean filter paper.
- TrisBP buffer is 4 parts of 900 rnM Tris borate buffer (TrisBp) pH8.3 mixed with 1 part 900 mM Tris phosphate buffer pH 8.3)
- TrisBp Tris borate buffer
- 1 micro liter 20% TWEEN 20 and 3 microliters 10% TEMED (5 microliters in 45 microliters H2O)
- Sandwich mesh rectangles between clean silanized glass plates Stagger the plates slightly at one end so that on one end of the sandwich, the mesh lying against the bottom plate is exposed.
- the mesh When the mesh is saturated, slide the top plate so that the mesh is completely sandwiched between the two plates. Allow polymerization for one hour at room temperature.
- cassette base used for this example was similar to that shown in FIGS. 19A-19C.
- Other cassette components include: one sample well-forming comb for forming sample wells in channel 301; and two carbon rod electrodes, wrapped with platinum wire (platinum wire is present for connection of carbon rods to the power supply). These cassette components are soaked in a solution of 1% hydrochloric acid and 1 millimolar (mM) EDTA (ethylenediaminetetraacetic acid) for at least 30 minutes to remove residual alkaline phosphatase activity. The components are then washed thoroughly with water and air dried on a clean surface.
- mM millimolar
- cassette components is cleaned and dried including: a cassette base as shown in FIG. 19; two carbon rod electrodes, wrapped with platinum wire (platinum wire is present for connection of carbon rods to the power supply); and one sample well-forming comb (as shown in FIG. 20) for forming wells to hold the thin membrane within the cassette slots 98 during capture and washing.
- This second set will be used for electrophoretic washing of the thin gel membrane after target capture.
- a molten solution of 1% agarose in 0.1 X TrisBP is prepared in a 250 ml glass Ehrlenmeyer flask as follows. Mix: 100 ml 0.1X TrisBP buffer and lg agarose powder (Sigma A9539 General use Molecular Biology Grade). Stopper top of flask loosely with clean lab tissues (Kimwipes) and heat to boiling in a commercial microwave oven. Cover flask with aluminum foil and equilibrate to 60 ° C in a water bath.
- Example 3 Rapid format for detection of bacteria using non-amplified nucleic acid probes with immobilized capture chemistry
- SRP bacterial signal recognition particle
- RNA:RP complex is captured during electrophoresis by hybridization to capture probes (CP) which are immobilized with AcryditeTM chemistry in a polyacrylamide gel on a thin membrane.
- Membranes are washed and exposed to chemiluminescent substrate for detection.
- the whole assay, including sample processing, can be conducted in about 60 minutes, with the following Signal-to-Noise values for E. coli:
- Bacteria was spiked into a platelet concentrate and enriched by centrifugation.
- the sample is lysised in the presence of sodium dodecyl sulphate at high temperature.
- the sample is hybridized with a high concentration of alkaline phosphatase-conjugated reporter probes.
- the selection of probes was based on published sequence information. (See, e.g., http://psyche.uthct.edu/dbs/srpdb/s ⁇ db.html)
- AcryditeTM attachment chemistry U.S. Patent No. 5,932,711
- Capture probe layers were supported on a thin membrane and inserted into an agarose- filled electrophoresis cassette 190, and subjected to electrophoresis of target molecules through the probe layer for rapid capture of targets and removal of excess unbound reporter probe.
- the capture gel holder 220, capture membrane comb, was removed from the electrophoresis cassette 90, and inserted into a tray, the detection tray 78 of FIGS. 15 A and 15B, designed to fit in a standard 96-well micro liter plate footprint. Chemiluminescent substrate was pipetted into these horizontal wells and light output was read in the luminometer over several minutes.
- FIG. 23 shows a dose response for in vitro target transcripts spiked into the assay after the centrifugation step.
- FIG. 24 shows a similar dose response, but using E. coli spiked into sterile platelet concentrates.
- Table 1 shows a list of the bacterial species which have been detected with the current probe set using the thin gel described herein.
- FIG. 25 depicts the time estimates for running up to 8 samples. (4 samples plus a negative and positive control per cassette, 2 cassettes run in parallel.)
- rectangles are each embedded in a matrix forming gel solution having a unique probe.
- the rectangles, each provided with a specific probe or known probe set, are arranged in a known pattern between two clean silanized glass plates that are separated by spacers and sufficient acrylamide gel matrix is wicked in to form a thin gel surrounding each rectangle, joining the rectangles into a sheet.
- a sample was prepared for detection of E. coli contamination in platelet concentrates.
- log phase E. coli were added to a platelet sample to provide a simulation of a contaminated platelet sample.
- the nucleic acid probes, the probes 46 on the capture gel 40, used are directed toward detection of a target, E. coli 4.5S RNA, also known as the signal recognition particle RNA.
- the alkaline phosphatase conjugated detection probe hybridizes directly to the 4.5S RNA target.
- the adaptor probe (block 154 of FIG. 10) also hybridizes directly to the 4.5 S RNA target at a location distinct from that occupied by the detection probe.
- One portion of the adaptor probe is not complementary to the target and forms a single stranded tail when hybridized to the target 4.5S RNA.
- This single stranded region of the adaptor is complementary to the capture probe which is immobilized in the reinforced thin gel.
- lysis buffer is 100 mM sodium phosphate buffer pH 7.0, 3 mM EDTA, 5% SDS, 0.001% phenol red
- the pellet is resuspend by vortex mixing or by pipetting up and down with a micropipette.
- Each tube was capped tightly and placed into a pre-warmed 135°C heating block for 5 minutes as illustrated by block 152 in FIG. 10. After heating, the tubes are moved to a room temperature rack for at least 90 seconds. In one embodiment, the tubes are placed in an incubation unit 304, such as shown in FIG. 14, which both heats and cools. Twenty (20) ⁇ l of 5X DHB+AP is added to ea. tube with
- Eppendorf repeater (5XDHB+AP is 525 mM NaCl, 0.5 mM MgCl 2 , 0.05 mM ZnCl 2 , 125 mM Tris HCl pH 8.3, 5% Ficoll 400, 0.05% wt/vol xylene cyanole, 125 nanomolar (nM) alkaline phosphate conjugated oligondeoxynucleotide probe (3-CTTCC GTCTA CTGCG CACAC GG- alkaline phosphatase-5') (SEQ ID NO: 19), 2.5 mM aurin tricarboxylic acid, 125 nM adaptor oligonucleotide probe (3'- TCCGG GCCCT TGCAT AAGTG AGTCC AGGCC TTCCT TCGTC G-5') (SEQ ID NO: 20), 0.05%) sodium azide).
- Each tube is manually shaken several times.
- the tubes are incubated at 52°C for 10 minutes to hybridize the reporter probe and adaptor probe to the 4.5S RNA from the bacteria.
- Twenty (20) ul of the hybridization mixture is loaded into the sample wells 174 in cassette 86 of FIG. 19B..
- the electrodes 100 are connected to a DC power supply and apply constant voltage of 50V for 10 minutes. Temperature will increase during the run, but should not be allowed to increase above 35 degrees centigrade (C). If necessary, the cassette 86 can be placed on a surface cooled by a recirculating water bath to prevent overheating. Referring to block 158 of FIG. 10, after a set period of time of migration of the sample towards the capture gel 40 in the capture gel holder 66, the power is turned off. The capture gel holder 66 is moved from the enlarged segment 98 in the flow channel 96 to the other enlarged segment 98 in the same electrophoresis cassette 86. The removal of the membranes from the sample electrophoresis cassette is facilitated by rocking the membrane back and forth before pulling upward.
- the capture gel holder is moved from the first set of enlarged slots 98 of the electrophoresis cassette 86 and transferred to the second set of enlarged slots, the wash segment, 98 of the electrophoresis cassette 86.
- the teeth 70 of the capture gel holder 66 are placed in the buffer- filled slot created by the enlarged slot 98.
- the electrodes 100 are connected to a DC power supply and a constant voltage of 50 V for 5 minutes is applied. Once again, care is taken not to allow the temperature of the cassette to exceed 35°C.
- the current is in the same direction during the second or wash period.
- the prorogation of the sample is away from the capture gel 40 of the capture gel holder 66 and thus any sample on the capture gel holder 66 that will not be captured will pass through and no new sample will come in contact with the capture gel 40.
- the detection of captured nucleic acid target-alkaline phosphatase conjugated probe complex on thin gel membranes, the capture gel 40 of the capture gel holder 66 is accomplished.
- the capture gel holder 66 is removed from the wash or second set of slots 98 of the electrophoresis cassette.
- the teeth 70 with the capture gel 40 are placed in Tropix reagent (CDP-star with Emerald II, Tropix catalog MS100RY).
- the capture gel 40 is soaked in the solution and incubated at room temperature with gentle rocking or shaking for 15 minutes, as illustrated in FIG. 8, by reference numeral 170.
- the capture gel holder 66 is then placed in a detection tray 178, as shown in FIGS. 15A & 15B.
- the capture gel holder 66 and the detection tray 178 are placed in a microplate luminometer 172 such as a Walla Victor 2, E.G.&G Walla, Turku, Finland.
- FIG. 22 illustrates the results of three different experiments (assays 1 through 3); results are displayed as a signal to noise ratio (S/N). The overall mean S/N ratio is also displayed along with its standard deviation.
- the samples tested comprise human platelet concentrates spiked with the indicated number of log-phase E. coli bacteria (expressed as the number of colony forming units (cfu) loaded per lane).
- the expression "1.6E+5" is an abbreviation for the value "1.6 X I 0 5 ".
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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JP2001554055A JP2003520961A (en) | 2000-01-19 | 2001-01-18 | Methods and devices for separating and detecting nucleic acids |
CA002397883A CA2397883A1 (en) | 2000-01-19 | 2001-01-18 | Methods of and apparatus for separating and detecting nucleic acid |
AU31028/01A AU3102801A (en) | 2000-01-19 | 2001-01-18 | Methods of and apparatus for separating and detecting nucleic acid |
EP01903178A EP1255983A2 (en) | 2000-01-19 | 2001-01-18 | Methods of and apparatus for separating and detecting nucleic acid |
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US17683900P | 2000-01-19 | 2000-01-19 | |
US60/176,839 | 2000-01-19 |
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WO2001053817A2 true WO2001053817A2 (en) | 2001-07-26 |
WO2001053817A3 WO2001053817A3 (en) | 2002-02-07 |
WO2001053817A9 WO2001053817A9 (en) | 2003-01-09 |
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PCT/US2001/001963 WO2001053817A2 (en) | 2000-01-19 | 2001-01-18 | Methods of and apparatus for separating and detecting nucleic acid |
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EP (1) | EP1255983A2 (en) |
JP (1) | JP2003520961A (en) |
AU (1) | AU3102801A (en) |
CA (1) | CA2397883A1 (en) |
WO (1) | WO2001053817A2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003101591A1 (en) * | 2002-06-01 | 2003-12-11 | Novartis Ag | Separation of molecules |
US6911308B2 (en) | 2001-01-05 | 2005-06-28 | Exact Sciences Corporation | Methods for detecting, grading or monitoring an H. pylori infection |
US7452668B2 (en) | 1997-05-16 | 2008-11-18 | Exact Sciences Corporation | Electrophoretic analysis of molecules using immobilized probes |
EP2856136A4 (en) * | 2012-05-31 | 2016-02-17 | Ge Healthcare Bio Sciences Ab | An electrophoresis system and a separation and identification method |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP4261546B2 (en) * | 2003-09-26 | 2009-04-30 | 三菱レイヨン株式会社 | Electrophoresis apparatus and electrophoresis method |
JP5544759B2 (en) * | 2009-05-21 | 2014-07-09 | 株式会社島津製作所 | Sample analysis method using mass spectrometry |
US20220099619A1 (en) * | 2020-09-30 | 2022-03-31 | Icare Diagnostics International Co. Ltd. | Electrode and application thereof |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US4164464A (en) * | 1977-03-25 | 1979-08-14 | Instrumentation Specialties Company | Sample concentrator |
WO1993013412A1 (en) * | 1991-12-31 | 1993-07-08 | E.I. Du Pont De Nemours And Company | Apparatus for direct blotting and automated electrophoresis, transfer and detection and processes utilizing the apparatus thereof |
WO1994009185A1 (en) * | 1992-10-14 | 1994-04-28 | Labintelligence, Inc. | Electrophoretic quantitation of specific binding complexes |
US5348633A (en) * | 1993-01-22 | 1994-09-20 | Northeastern University | Method for quantitating trace amounts of an analyte in a sample by affinity capillary electrophoresis |
US5538614A (en) * | 1994-08-17 | 1996-07-23 | Han; Dawn D. | Macromolecule recovery cassette |
-
2001
- 2001-01-18 EP EP01903178A patent/EP1255983A2/en not_active Withdrawn
- 2001-01-18 WO PCT/US2001/001963 patent/WO2001053817A2/en not_active Application Discontinuation
- 2001-01-18 CA CA002397883A patent/CA2397883A1/en not_active Abandoned
- 2001-01-18 AU AU31028/01A patent/AU3102801A/en not_active Abandoned
- 2001-01-18 JP JP2001554055A patent/JP2003520961A/en not_active Withdrawn
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4164464A (en) * | 1977-03-25 | 1979-08-14 | Instrumentation Specialties Company | Sample concentrator |
US4164464B1 (en) * | 1977-03-25 | 1992-05-12 | Instrumentation Specialties Co | |
WO1993013412A1 (en) * | 1991-12-31 | 1993-07-08 | E.I. Du Pont De Nemours And Company | Apparatus for direct blotting and automated electrophoresis, transfer and detection and processes utilizing the apparatus thereof |
WO1994009185A1 (en) * | 1992-10-14 | 1994-04-28 | Labintelligence, Inc. | Electrophoretic quantitation of specific binding complexes |
US5348633A (en) * | 1993-01-22 | 1994-09-20 | Northeastern University | Method for quantitating trace amounts of an analyte in a sample by affinity capillary electrophoresis |
US5538614A (en) * | 1994-08-17 | 1996-07-23 | Han; Dawn D. | Macromolecule recovery cassette |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7452668B2 (en) | 1997-05-16 | 2008-11-18 | Exact Sciences Corporation | Electrophoretic analysis of molecules using immobilized probes |
US6911308B2 (en) | 2001-01-05 | 2005-06-28 | Exact Sciences Corporation | Methods for detecting, grading or monitoring an H. pylori infection |
WO2003101591A1 (en) * | 2002-06-01 | 2003-12-11 | Novartis Ag | Separation of molecules |
EP2856136A4 (en) * | 2012-05-31 | 2016-02-17 | Ge Healthcare Bio Sciences Ab | An electrophoresis system and a separation and identification method |
Also Published As
Publication number | Publication date |
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AU3102801A (en) | 2001-07-31 |
EP1255983A2 (en) | 2002-11-13 |
WO2001053817A9 (en) | 2003-01-09 |
CA2397883A1 (en) | 2001-07-26 |
WO2001053817A3 (en) | 2002-02-07 |
JP2003520961A (en) | 2003-07-08 |
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