CA2407652A1 - Device and method for hybridizing double-stranded dna samples on oligomer arrays - Google Patents
Device and method for hybridizing double-stranded dna samples on oligomer arrays Download PDFInfo
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- CA2407652A1 CA2407652A1 CA002407652A CA2407652A CA2407652A1 CA 2407652 A1 CA2407652 A1 CA 2407652A1 CA 002407652 A CA002407652 A CA 002407652A CA 2407652 A CA2407652 A CA 2407652A CA 2407652 A1 CA2407652 A1 CA 2407652A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J19/0006—Controlling or regulating processes
- B01J19/0013—Controlling the temperature of the process
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00054—Controlling or regulating the heat exchange system
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00074—Controlling the temperature by indirect heating or cooling employing heat exchange fluids
- B01J2219/00087—Controlling the temperature by indirect heating or cooling employing heat exchange fluids with heat exchange elements outside the reactor
- B01J2219/00094—Jackets
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00074—Controlling the temperature by indirect heating or cooling employing heat exchange fluids
- B01J2219/00087—Controlling the temperature by indirect heating or cooling employing heat exchange fluids with heat exchange elements outside the reactor
- B01J2219/00103—Controlling the temperature by indirect heating or cooling employing heat exchange fluids with heat exchange elements outside the reactor in a heat exchanger separate from the reactor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00132—Controlling the temperature using electric heating or cooling elements
- B01J2219/00137—Peltier cooling elements
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00351—Means for dispensing and evacuation of reagents
- B01J2219/00353—Pumps
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00495—Means for heating or cooling the reaction vessels
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/00527—Sheets
- B01J2219/00529—DNA chips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
- B01J2219/00608—DNA chips
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/0068—Means for controlling the apparatus of the process
- B01J2219/00686—Automatic
- B01J2219/00689—Automatic using computers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
- B01J2219/00722—Nucleotides
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
- C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
Abstract
The invention relates to a device for hybridizing double-stranded DNA sample s on oligomer arrays, comprising at least one pump (4, 14), which has two conveying directions, a closed hybridization chamber (1, 11), a cooling element (2, 12) and a heating element (3, 13). The individual components are interconnected in the above order by lines (5, 15) in which liquids are conveyed.
Description
Device and method for hybridizing double-stranded DNA samples to oligomer arrays.
The invention concerns a device and a method for hybridizing double-stranded DNA samples to oligomer arrays.
Hybridization of sample DNA on oligomer chips, for example, oligonucleotide arrays, is conducted for detecting specific sequences in sample DNA. One possible approach, "sequencing by hybridization (SBH)p, in fact determines the complete sequence of the sample DNA or at least large portions thereof. Allele-specific hybridizations, however, are also conducted in order to detect specific changes in the sample DNA, e.g. point mutations. The sample DNA, however, usually is present in double-stranded form, since it has previously been amplified for the most part by means of PCR, and thus it is no longer available essentially for hybridizing with the oligomers. The present invention describes a device, which serves for the efficient hybridizing of double-stranded DNA to oligomer arrays.
A number of hybridizing chambers are known from the prior art. Thus, US-A-5,100,775; US-A-5,360,741; or US-A-5,466,603 describe hybridization chambers, which are adapted to the most varied objectives and requirements. Such hybridization chambers have in the meantime become available commercially in many forms, but generally they cannot be separately temperature-controlled. Hybridization chambers that are suitable for uptake of specimen carriers or slides are also known. In addition, there are foils, which are self adhering at the edges and which can form hybridization chambers by gluing onto the slide. A
pneumatically controllable and temperature-controllable hybridization chamber is also known, in which the hybridization properties will be improved by movement of the hybridization fluid.
All of these devices, however, require that the double-stranded sample DNA is either thermally denatured or one strand is selectively separated prior to hybridization (e.g., a primer in PCR can be labeled with biotin and one strand can be removed selectively from the solution by binding to streptavidin in the following step) or one strand is produced in excess by enzymatic means, and thus the segments hybridizing to the oligomer an-ay are not blocked by complementary strands.
These methods represent not only an additional step, but they are also expensive, particularly in the case of biotin, and are often poorly reproducible in the case of enzymatic reactions. If both strands are to be detected, however, by the oligomer array, then only thermal denaturing is considered, without anything further. The problem, however, is so-called reannealing, that is, the complementary strands hybridize again with one another after the denaturation, which can occur more rapidly than hybridization with oligomers on the chip. This problem is also not solved by one-time denaturation. In contrast, if denaturation occurs in the chamber, then DNA
fragments already hybridized to the oligomer array will again be removed.
The object of the present invention is thus to aeate a device, which overcomes the disadvantages of the prior art and makes possible an effective hybridization of double-stranded DNA samples.
The object is solved by the features of Gaim 1. Advantageous embodiments are characterized in the dependent sub-Gaims.
The object is thus solved by a device for hybridizing double-stranded DNA
samples to oligomer arrays (oligomer chips), which comprises at least one pump transporting in two directions, a closed hybridization chamber, a cooling element and a heating element, whereby the individual components are joined with one another in the above-named sequence, by pathways for transporting liquids.
The device described here makes possible a periodic denaturation of the DNA
sample, without removal of DNA that has already hybridized to oligomers and thus overcomes the problems mentioned in the prior art. Due to the fact that the DNA sample is denatured thermally prior to introduction onto the chip and then is suddenly cooled, it is present in single-stranded form for the most part when it contacts the chip. Thus, a large part of the otherwise double-stranded DNA sample is available for hybridizing with the oligomer array. By pumping the sample fluid back and forth, the device assures that this process is repeated frequently until a sufficient portion of the double-stranded DNA sample has hybridized to the oligomers of the chip. Also, during the hybridization phase, a mixing occurs due to this process.
It is preferred according to the invention that the pump is a peristaltic pump, a hose pump or a piston pump. The pump will be able to transport small quantities of liquid precisely in [both] the aspiration and pressure directions. This can also be done according to the invention by means of valves, such as multiple-way valves, which are known to the person of average skill in the art, and these can be controlled andlor regulated in tum again externally.
It is particularly preferred that the pump is programmable or is controlled by a computer. Such controls and/or computer programs are known in and of themselves to the person of average skill in the art.
The invention concerns a device and a method for hybridizing double-stranded DNA samples to oligomer arrays.
Hybridization of sample DNA on oligomer chips, for example, oligonucleotide arrays, is conducted for detecting specific sequences in sample DNA. One possible approach, "sequencing by hybridization (SBH)p, in fact determines the complete sequence of the sample DNA or at least large portions thereof. Allele-specific hybridizations, however, are also conducted in order to detect specific changes in the sample DNA, e.g. point mutations. The sample DNA, however, usually is present in double-stranded form, since it has previously been amplified for the most part by means of PCR, and thus it is no longer available essentially for hybridizing with the oligomers. The present invention describes a device, which serves for the efficient hybridizing of double-stranded DNA to oligomer arrays.
A number of hybridizing chambers are known from the prior art. Thus, US-A-5,100,775; US-A-5,360,741; or US-A-5,466,603 describe hybridization chambers, which are adapted to the most varied objectives and requirements. Such hybridization chambers have in the meantime become available commercially in many forms, but generally they cannot be separately temperature-controlled. Hybridization chambers that are suitable for uptake of specimen carriers or slides are also known. In addition, there are foils, which are self adhering at the edges and which can form hybridization chambers by gluing onto the slide. A
pneumatically controllable and temperature-controllable hybridization chamber is also known, in which the hybridization properties will be improved by movement of the hybridization fluid.
All of these devices, however, require that the double-stranded sample DNA is either thermally denatured or one strand is selectively separated prior to hybridization (e.g., a primer in PCR can be labeled with biotin and one strand can be removed selectively from the solution by binding to streptavidin in the following step) or one strand is produced in excess by enzymatic means, and thus the segments hybridizing to the oligomer an-ay are not blocked by complementary strands.
These methods represent not only an additional step, but they are also expensive, particularly in the case of biotin, and are often poorly reproducible in the case of enzymatic reactions. If both strands are to be detected, however, by the oligomer array, then only thermal denaturing is considered, without anything further. The problem, however, is so-called reannealing, that is, the complementary strands hybridize again with one another after the denaturation, which can occur more rapidly than hybridization with oligomers on the chip. This problem is also not solved by one-time denaturation. In contrast, if denaturation occurs in the chamber, then DNA
fragments already hybridized to the oligomer array will again be removed.
The object of the present invention is thus to aeate a device, which overcomes the disadvantages of the prior art and makes possible an effective hybridization of double-stranded DNA samples.
The object is solved by the features of Gaim 1. Advantageous embodiments are characterized in the dependent sub-Gaims.
The object is thus solved by a device for hybridizing double-stranded DNA
samples to oligomer arrays (oligomer chips), which comprises at least one pump transporting in two directions, a closed hybridization chamber, a cooling element and a heating element, whereby the individual components are joined with one another in the above-named sequence, by pathways for transporting liquids.
The device described here makes possible a periodic denaturation of the DNA
sample, without removal of DNA that has already hybridized to oligomers and thus overcomes the problems mentioned in the prior art. Due to the fact that the DNA sample is denatured thermally prior to introduction onto the chip and then is suddenly cooled, it is present in single-stranded form for the most part when it contacts the chip. Thus, a large part of the otherwise double-stranded DNA sample is available for hybridizing with the oligomer array. By pumping the sample fluid back and forth, the device assures that this process is repeated frequently until a sufficient portion of the double-stranded DNA sample has hybridized to the oligomers of the chip. Also, during the hybridization phase, a mixing occurs due to this process.
It is preferred according to the invention that the pump is a peristaltic pump, a hose pump or a piston pump. The pump will be able to transport small quantities of liquid precisely in [both] the aspiration and pressure directions. This can also be done according to the invention by means of valves, such as multiple-way valves, which are known to the person of average skill in the art, and these can be controlled andlor regulated in tum again externally.
It is particularly preferred that the pump is programmable or is controlled by a computer. Such controls and/or computer programs are known in and of themselves to the person of average skill in the art.
Also, according to the invention, it is preferred that the hybridization chamber comprises at least one cover, with input/output channels passing through it and a tempering block, with an oligomer array that can be applied or fastened thereon.
it is particularly prefer-ed that a cooling unit is also present, on which the tempering block is arranged.
According to the invention, a device is also preferred, wherein the volume of the hybridization chamber amounts to less than 200 . I when the oligomer chip is inserted.
It is particularly advantageous that the hybridization chamber is equipped for the uptake of commercially available slides or microscope slides.
it is also preferred according to the invention that the cooling element tightly surrounds the pathway.
It is also preferred that the heating element tightly surrounds the pathway and that the pathway projects from the heating element via its open end. Alternatively, it is preferred that the heating element surrounds a sample vessel at least partially and that the pathway is immersed by its open end into the sample solution present in the sample vessel, and that this pathway is optionally conducted doom to the bottom of the sample vessel on the inside.
It is also preferred that the pathways are tubings and preferably comprised of an inert material, silicone rubbers, polytetrafluoroethylene, polyvinyl chloride, polyethylene and/or special steel.
it is particularly prefer-ed that a cooling unit is also present, on which the tempering block is arranged.
According to the invention, a device is also preferred, wherein the volume of the hybridization chamber amounts to less than 200 . I when the oligomer chip is inserted.
It is particularly advantageous that the hybridization chamber is equipped for the uptake of commercially available slides or microscope slides.
it is also preferred according to the invention that the cooling element tightly surrounds the pathway.
It is also preferred that the heating element tightly surrounds the pathway and that the pathway projects from the heating element via its open end. Alternatively, it is preferred that the heating element surrounds a sample vessel at least partially and that the pathway is immersed by its open end into the sample solution present in the sample vessel, and that this pathway is optionally conducted doom to the bottom of the sample vessel on the inside.
It is also preferred that the pathways are tubings and preferably comprised of an inert material, silicone rubbers, polytetrafluoroethylene, polyvinyl chloride, polyethylene and/or special steel.
Other inert materials are also considered and are known to the person with average skill in the art.
It is most particularly preferred that the hybridization chamber, the cooling element, the heating element and the tempering block can be temperature-controlled independent of one another.
It is also preferred that the tempering block of the hybridization chamber is simultaneously configured as a cooling element.
It is also preferred that the volume of the pathways between the heating element and the inlet channel of the hybridization chamber is smaller than the volume of the hybridization chamber itself.
The present invention will be explained in more detail on the basis of the attached illustrations.
Here:
Fig. 1a shows a schematic representation of a device according to the invention in a first example of embodiment, Fig. 1 b shows a schematic representation of a device according to the invention in a second example of embodiment, and Fig. 2 shows a perspective view of a form of embodiment of a hybridization chamber according to the invention.
It is most particularly preferred that the hybridization chamber, the cooling element, the heating element and the tempering block can be temperature-controlled independent of one another.
It is also preferred that the tempering block of the hybridization chamber is simultaneously configured as a cooling element.
It is also preferred that the volume of the pathways between the heating element and the inlet channel of the hybridization chamber is smaller than the volume of the hybridization chamber itself.
The present invention will be explained in more detail on the basis of the attached illustrations.
Here:
Fig. 1a shows a schematic representation of a device according to the invention in a first example of embodiment, Fig. 1 b shows a schematic representation of a device according to the invention in a second example of embodiment, and Fig. 2 shows a perspective view of a form of embodiment of a hybridization chamber according to the invention.
The subject of the present invention is a device for hybridizing double-stranded DNA samples on oligomer arrays (oiigomer chips) as schematized in Figure 1 alb. It is comprised of a closed hybridization chamber 1, 11 that can be temperature-controlled, a pump 4, 14, a heating element 3, 13 and a cooling element 2, 12, which are joined together each time by pathways 5, 50, preferably plastic tubings, for transporting liquid.
Hybridization chamber 1, 11 (Figure 2) is preferably comprised of two parts, a dish for uptake of the oligomer array 23 and a cover 21, which preferably can be pressed together by a hinge mechanism. Preferably a recess is found in the cover for a sealing ring, which forms the side walls of the chamber. The cover also contains the ducts 22 for the tubing connections 5, 15 or other transport channels for liquids. The chamber can preferably be temperature-controlled by a Pettier element.
The pump preferably involves a tubing hose operating according to the peristaltic principle or a piston pump, which can be programmed for automatically conducting the method by itself, or can be controlled preferably by means of a PC. The sample is moved cyclically by means of the pump and is first denatured in the heating element, then cooled in the cooling element and subsequently hybridized in the hybridization chamber. After this, it is again pumped into the heating block and denatured. This process is cyclically repeated and the device can preferably conduct however many, but at least two, such cycles automatically, one after the other.
The heating element as well as the cooling element are preferably comprised of a metal block, whose temperature is controlled most preferably by a Pettier element. !n a preferred variant both the heating element and the cooling element each surround a tubing, through which the sample solution is transported. Alternatively, the heating element can take up a vessel, preferably comprised of plastic (e.g., "Eppendorf cup"). In this case, the tubing or another channel extends down to the bottom of this vessel in order to aspirate the sample fluid therein.
The DNA sample can thus be transported to the cooling element and to the hybridization chamber. In another variant of the invention, a cooling element is not used, but rather the sample heated in the heating element is rapidly cooled by contact with the temperature-controlled hybridization chamber.
The hybridization chamber can take up oligomer chips, on which oligonucleotides and/or PNA
oligomers (peptide nucleic acids) oligomer are immobilized. In a particular prefer-ed variant, the hybridization chamber takes up commercially available slides, such as are also used in microscopy. The volume which the hybridization chamber holds when the oligomer chip is inserted most preferably amounts to less than 200. I.
A subject of the present invention is also a method for hybridizing double-stranded DNA
samples to oligomer arrays with the use of a device according to the invention.
Another subject of the present invention is thus a device for hybridizing double-stranded DNA
samples to oligomer arrays (oligomer chips), wherein a device according to the invention as described above is used and wherein the DNA sample is moved cyclically via the pump and is first denatured in the heating element, then cooled in the cooling element, and subsequently hybridized in the hybridization chamber, and is then again denatured in the heating element, whereby at least two such cycles are automatically conducted, one after the other.
Hybridization chamber 1, 11 (Figure 2) is preferably comprised of two parts, a dish for uptake of the oligomer array 23 and a cover 21, which preferably can be pressed together by a hinge mechanism. Preferably a recess is found in the cover for a sealing ring, which forms the side walls of the chamber. The cover also contains the ducts 22 for the tubing connections 5, 15 or other transport channels for liquids. The chamber can preferably be temperature-controlled by a Pettier element.
The pump preferably involves a tubing hose operating according to the peristaltic principle or a piston pump, which can be programmed for automatically conducting the method by itself, or can be controlled preferably by means of a PC. The sample is moved cyclically by means of the pump and is first denatured in the heating element, then cooled in the cooling element and subsequently hybridized in the hybridization chamber. After this, it is again pumped into the heating block and denatured. This process is cyclically repeated and the device can preferably conduct however many, but at least two, such cycles automatically, one after the other.
The heating element as well as the cooling element are preferably comprised of a metal block, whose temperature is controlled most preferably by a Pettier element. !n a preferred variant both the heating element and the cooling element each surround a tubing, through which the sample solution is transported. Alternatively, the heating element can take up a vessel, preferably comprised of plastic (e.g., "Eppendorf cup"). In this case, the tubing or another channel extends down to the bottom of this vessel in order to aspirate the sample fluid therein.
The DNA sample can thus be transported to the cooling element and to the hybridization chamber. In another variant of the invention, a cooling element is not used, but rather the sample heated in the heating element is rapidly cooled by contact with the temperature-controlled hybridization chamber.
The hybridization chamber can take up oligomer chips, on which oligonucleotides and/or PNA
oligomers (peptide nucleic acids) oligomer are immobilized. In a particular prefer-ed variant, the hybridization chamber takes up commercially available slides, such as are also used in microscopy. The volume which the hybridization chamber holds when the oligomer chip is inserted most preferably amounts to less than 200. I.
A subject of the present invention is also a method for hybridizing double-stranded DNA
samples to oligomer arrays with the use of a device according to the invention.
Another subject of the present invention is thus a device for hybridizing double-stranded DNA
samples to oligomer arrays (oligomer chips), wherein a device according to the invention as described above is used and wherein the DNA sample is moved cyclically via the pump and is first denatured in the heating element, then cooled in the cooling element, and subsequently hybridized in the hybridization chamber, and is then again denatured in the heating element, whereby at least two such cycles are automatically conducted, one after the other.
Another subject of the present invention is a kit, comprising a device as described above for hybridizing double-stranded DNA samples to oligonucleotide arrays and one or more oligomer arrays or biochips and/or documentation for using the device and/or buffer solutions for conducting the hybridizations.
Reference list 1, 11 hybridization chamber 2, 12 cooling element 3, 13 heating element 4, pump 5, 15 pathway 6 sample vessel 21 cover 22 inlet/outlet channels 23 oligomer array 24 tempering block 25 cooling unit
Claims (15)
1. A device for hybridizing double-stranded DNA samples to oligomer arrays (oligomer chips) comprising at least one pump (4, 14) transporting in two directions, a closed hybridization chamber (1, 11), a cooling element (2, 12) and a heating element (3, 13), whereby the individual components are connected in the above-named sequence each time with one another by pathways (5, 15) for transporting fluids.
2. The device according to claim 1, further characterized in that pump (4, 14) is a peristaltic pump, a hose pump or a piston pump.
3. The device according to one of the preceding claims, further characterized in that the pump (4, 14) is programmable or is controlled by a computer.
4. The device according to one of the preceding claims, further characterized in that hybridization chamber (1, 11) comprises at least one cover (21) with inlet/outlet channels (22) passing through this cover, and a tempering block (24) with an oligomer array (23) that can be applied or fixed thereon.
5. The device according to claim 4, further characterized in that a cooling unit (25) is also present, on which the tempering block (24) is arranged.
6. The device according to one of the preceding claims, further characterized in that the volume of hybridization chamber (1, 11) amounts to less than 200. I with an inserted oligomer chip.
7. The device according to one of the preceding claims, further characterized in that the hybridization chamber (1, 11) is equipped for taking up conventional specimen carriers or microscope slides.
8. The device according to one of the preceding claims, further characterized in that the cooling element (2, 12) tightly surrounds the pathway (5, 15).
9. The device according to one of the preceding claims, further characterized in that heating element (3, 13) tightly surrounds pathway (5, 15) and that pathway (5, 15) projects from the heating element by its open end.
10. The device according to one of claims 1 to 8, further characterized in that the heating element (3, 13) at least partially surrounds a sample vessel (6) and that pathway (5, 15) by its open end is immersed in the sample solution present in the sample vessel (6) and that this pathway (5, 15) is optionally guided down to the bottom of the sample vessel (6) on the inside.
11. The device according to one of the preceding claims, further characterized in that pathways (5, 15) are tubings and preferably comprised of an inert material, silicone rubbers, polytetrafluoroethylene, polyvinyl chloride, polyethylene, and/or special steel.
12. The device according to one of the preceding claims, further characterized in that the hybridization chamber (1, 11), the cooling element (2, 12), the heating element (3, 13) and the tempering block (24) can be temperature-controlled independent of one another.
13. The device according to one of the preceding claims, further characterized in that tempering block (24) of hybridization chamber (1, 11), is formed simultaneously as cooling element (2, 12).
14. The device according to one of the preceding claims, further characterized in that the volume of pathways (5, 15) between the heating element (3, 13) and the inlet channel (22) to the hybridizaiton chamber (1, 11) is smaller than the volume of the hybridization chamber (1, 11) itself.
15. A method for hybridizing double-stranded DNA samples to oligomer arrays (oligomer chips), whereby a device according to one of the preceding claims is used and whereby the DNA sample is moved cyclically via the pump and is first denatured in the heating element, then cooled in the cooling element, and subsequently hybridized in the hybridization chamber and is then again denatured in the heating element, whereby at least two such cycles are conducted automatically one after the other.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19952723A DE19952723C2 (en) | 1999-10-26 | 1999-10-26 | Device and method for hybridizing double-stranded DNA samples on oligomer arrays |
| DE19952723.7 | 1999-10-26 | ||
| PCT/DE2000/003771 WO2001030489A2 (en) | 1999-10-26 | 2000-10-18 | Device and method for hybridizing double-stranded dna samples on oligomer arrays |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2407652A1 true CA2407652A1 (en) | 2001-05-03 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002407652A Abandoned CA2407652A1 (en) | 1999-10-26 | 2000-10-18 | Device and method for hybridizing double-stranded dna samples on oligomer arrays |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1230014B1 (en) |
| JP (1) | JP2003533969A (en) |
| AT (1) | ATE335539T1 (en) |
| AU (1) | AU774249B2 (en) |
| CA (1) | CA2407652A1 (en) |
| DE (2) | DE19952723C2 (en) |
| WO (1) | WO2001030489A2 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7776273B2 (en) | 2000-04-26 | 2010-08-17 | Life Technologies Corporation | Laser capture microdissection (LCM) extraction device and device carrier, and method for post-LCM fluid processing |
| DE10050943B4 (en) * | 2000-10-10 | 2005-08-25 | Epigenomics Ag | Device for hybridizing samples to arrays of biological substances |
| DE10156329A1 (en) * | 2001-07-17 | 2003-02-06 | Frieder Breitling | Method and arrangement for attaching substances immobilized in transport means as well as monomer particles |
| DE10149684B4 (en) * | 2001-10-09 | 2005-02-17 | Clondiag Chip Technologies Gmbh | Device for holding a substance library carrier |
| DE10160983B4 (en) * | 2001-12-05 | 2004-12-09 | Epigenomics Ag | Method and integrated device for the detection of cytosine methylation |
| CA2492491A1 (en) * | 2002-06-13 | 2003-12-24 | Millenium Biologix Ag | Reaction chamber |
| DE10233212B4 (en) * | 2002-07-22 | 2006-07-06 | Siemens Ag | Measuring device with a biochip arrangement and use of the device for a high-throughput analysis method |
| US6913931B2 (en) | 2002-10-03 | 2005-07-05 | 3M Innovative Properties Company | Devices, methods and systems for low volume microarray processing |
| DE10251338B4 (en) * | 2002-11-05 | 2008-05-21 | Intavis Bioanalytical Instruments Ag | Device for carrying out staining and hybridization reactions |
| DE10316723A1 (en) * | 2003-04-09 | 2004-11-18 | Siemens Ag | Test slide with sample wells, forming sealed reaction chamber with casing, also includes bonded seal forming resting surface for casing |
| DE10318219A1 (en) * | 2003-04-22 | 2004-11-11 | Febit Ag | Plastics housing, to handle and protect a biochip for synthesis and analysis applications, has a recess in the base body to accommodate the biochip with a frame to define its position |
| DE10319712A1 (en) * | 2003-05-02 | 2004-11-25 | Sirs-Lab Gmbh | Apparatus for duplicating reactions of samples, of biological molecules in microbiology, has a reaction vessel clamped over the sample holding zone wholly covered by the lower vessel openings |
| DE10320957A1 (en) * | 2003-05-09 | 2004-12-09 | Evotec Technologies Gmbh | Docking device for a fluidic microsystem |
| DE102006022511B3 (en) * | 2006-05-11 | 2007-08-16 | Fachhochschule Jena | Mounting for a planar microreaction chamber for optical analysis of liquids or gases comprises lower and upper parts held together by permanent magnets |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5100775A (en) * | 1988-03-16 | 1992-03-31 | Smyczek Peter J | Method for conducting nucleic acid hybridization in chamber with precise fluid delivery |
| US5360741A (en) * | 1992-09-29 | 1994-11-01 | Triangle Biomedical Sciences, Inc. | DNA hybridization incubator |
| US5840573A (en) * | 1994-02-01 | 1998-11-24 | Fields; Robert E. | Molecular analyzer and method of use |
| US5466603A (en) * | 1994-02-15 | 1995-11-14 | Meehan; Brian W. | Temperature regulated hybridization chamber |
| GB9506312D0 (en) * | 1995-03-28 | 1995-05-17 | Medical Res Council | Improvements in or relating to sample processing |
| US5856174A (en) * | 1995-06-29 | 1999-01-05 | Affymetrix, Inc. | Integrated nucleic acid diagnostic device |
| US6132580A (en) * | 1995-09-28 | 2000-10-17 | The Regents Of The University Of California | Miniature reaction chamber and devices incorporating same |
| US5741647A (en) * | 1996-02-16 | 1998-04-21 | Tam; Joseph Wing On | Flow through nucleic acid hybridisation uses thereof and a device thereof |
| AUPO427996A0 (en) * | 1996-12-20 | 1997-01-23 | Co-Operative Research Centre For Diagnostic Technologies | Method for detecting a nucleotide at a specific location within a polynucleotide sequence and apparatus therefor |
| US6558901B1 (en) * | 1997-05-02 | 2003-05-06 | Biomerieux Vitek | Nucleic acid assays |
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1999
- 1999-10-26 DE DE19952723A patent/DE19952723C2/en not_active Expired - Fee Related
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2000
- 2000-10-18 WO PCT/DE2000/003771 patent/WO2001030489A2/en active IP Right Grant
- 2000-10-18 DE DE50013311T patent/DE50013311D1/en not_active Expired - Fee Related
- 2000-10-18 CA CA002407652A patent/CA2407652A1/en not_active Abandoned
- 2000-10-18 AT AT00984864T patent/ATE335539T1/en not_active IP Right Cessation
- 2000-10-18 AU AU21492/01A patent/AU774249B2/en not_active Ceased
- 2000-10-18 EP EP00984864A patent/EP1230014B1/en not_active Expired - Lifetime
- 2000-10-18 JP JP2001532895A patent/JP2003533969A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| AU774249B2 (en) | 2004-06-24 |
| DE19952723C2 (en) | 2002-10-31 |
| EP1230014B1 (en) | 2006-08-09 |
| AU2149201A (en) | 2001-05-08 |
| WO2001030489A2 (en) | 2001-05-03 |
| JP2003533969A (en) | 2003-11-18 |
| DE50013311D1 (en) | 2006-09-21 |
| DE19952723A1 (en) | 2001-05-10 |
| EP1230014A2 (en) | 2002-08-14 |
| ATE335539T1 (en) | 2006-09-15 |
| WO2001030489A3 (en) | 2001-10-25 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |