EP1192007B1 - Microchip matrix device for duplicating and characterizing nucleic acids - Google Patents

Abstract

The aim of the invention is to provide a device for duplicating and characterizing nucleic acids almost simultaneously and with a high sample throughput rate. The device consists of a chamber body with a recess whose edge sealingly holds an optically transparent chip. Said chip holds nucleic acids in individual spots on a detection surface. The chamber body is placed on an optically transparent chamber support with a bearing surface, in such a way that a capillary gap, which can be filled with a liquid sample, is formed between the detection surface of the chip facing towards the chamber support and said chamber support. The chamber body is provided with an inlet and an outlet, which are spatially separate from each other, and has a space, which laterally encompasses the chip and which has a gas reservoir. The chamber support is provided with heating elements.

Description

The invention relates to a device for reproduction and Characterization of nucleic acids.

It has been known for decades that the amplification (replication) of deoxyribonucleic acid (DNA), the molecules that encode the genome (genetic information) of organisms, takes place in vivo (in the cell) by transcription and in vitro (outside the cell) by the method of polymerase chain reaction (PCR) can be operated.
It has now become the laboratory standard to amplify nucleic acids by PCR, to clone the PCR products (to insert them into a carrier molecule and to insert them into a microorganism), to amplify the cloned PCR products in microorganisms and to isolate the amplified PCR products ( Sambrook, J; Fritsch, EF and Maniatis, T., 1989, Molecular cloning: a laboratory manual 2nd edn. Cold Spring Harbor, NY, Cold Spring Habor Laboratory). This routine two-stage duplication enables an enormously large number of identical molecules to be produced from a few starting nucleic acid molecules, but has the disadvantage that it is very laborious and time-consuming, has a low sample throughput (the number of processed nucleic acids in a unit of time) and therefore very much is expensive.
The one-step duplication by PCR, on the other hand, is relatively fast, enables a high sample throughput in small batch volumes due to miniaturized processes and is not so labor intensive due to automation.
Characterization of nucleic acids by duplication alone is not possible. Rather, after duplication, it is necessary to use analysis methods, such as nucleic acid sequence determinations or electrophoretic analyzes of the PCR products or their enzymatically produced individual fragments, to characterize the PCR products.

From the documents US 5,716,842; DE 195 19 015 A1; WO 94/05414; US 5,587,128; US 5,498,392; WO 91/16966; WO 92/13967; F 90 09894 and the publications by S. Poser, T. Schulz, U. Dillner, V. Baier, JM Köhler, D. Schimkat, G. Mayer, A. Siebert (Chip elements for fast thermocycling, Sensors and Actuators A, 1997 : 62 672-675) and M. U. Kopp, AJ de Mello, A. Manz (Chemical amplification: Continuous-flow PCR on a chip, Science, 1998: 280 1046-1048), various miniaturizable or miniaturized methods and devices (thermocyclers) for carrying out the PCR are known.
In DE 195 19 015 A1; WO 94/05414; US 5,587,128; US 5,498,392 and the publication by S. Poser, T. Schulz, U. Dillner, V. Baier, JM Köhler, D. Schimkat, G. Mayer, A. Siebert (Chip elements for fast thermocycling, Sensors and Actuators A, 1997: 62 672-675) thermal cyclers are described which consist of capped chambers which hold the samples.
The in US 5,716,842; DE 195 19 015 A1, WO 91/16966; WO 92/13967; F 90 09894 and the publication MU Kopp, AJ de Mello, A. Manz (Chemical amplification: Continuous-flow PCR on a chip, Science, 1998: 280 1046-1048) presented miniaturizable or miniaturized thermocyclers work on the principle that with them the sample liquid is pumped continuously across three temperature zones.
The disadvantage of all the above-mentioned solutions is that only the information as to whether nucleic acid has been amplified and, if necessary, how much nucleic acid has been amplified can be obtained in the online detection. A further characterization of the amplification products is not possible.
US Pat. No. 5,856,174 discloses a system with which it is possible to pump sample liquids back and forth between, for example, three miniaturized chambers. The PCR is carried out in one chamber of this system, a workup reaction is carried out in the next and the reaction products are detected in the third, for example with a DNA chip. The PCR chamber is a standard tube, as described in the literature (S. Poser, T. Schulz, U. Dillner, V. Baier, JM Köhler, D. Schimkat, G. Mayer, A. Siebert; Chip elements for fast thermocycling, Sensors and Actuators A, 1997: 62, 672-67). The disadvantage of this system is that a complicated, fault-prone and control-technically complex system of a pressure-driven fluidics has to be set up in order to convey the sample liquid from the PCR chamber into the detection chamber. In addition, the separation of amplification and detection leads to an increase in the total analysis time.

The genetic characterizations, e.g. for identification and Taxonomic classification of microorganisms is currently taking place based on DNA-DNA hybridization studies, rRNA gene sequence comparison (e.g. by means of the 16S or 23S rRNA gene segments) after sequencing these sections and using Restriction fragment length polymorphism (RFLP) studies or PCR tests using specific primers gel electrophoretic separation and detection of restriction or PCR products (T. A. Brown, 1996, genetic engineering for beginners, Spectrum Academic Publishing House Heidelberg, Berlin Oxford).

The known RFLP studies are based on an individual-specific distribution of restriction endonuclease interfaces, which relates to DNA sequence differences in the area of genomic DNA, which has a high degree of homology to a labeled DNA probe used for hybridization (TA Brown, 1996, Genotechnologie für Beginners, Spectrum Academic Publishing House Heidelberg, Berlin Oxford).
The RFLP examination, which is used, for example, in HLA diagnostics (human leukocyte antigen) in immunology prior to transplantation or transfusion (cf. Cesbron A., Moreau P., Milpied N., Muller JY., Harousseau JL ., Bignon JD., "Influence of HLA-DP mismatches on primary MLR responses in unrelated HLA-A, B, DR, DQ, Dw identical pairs in allogeneic bone marrow transplantation" Bone Marrow Transplant 1990, Nov 6: 5, 337- 40 or Martell RW., Oudshoom M., May RM., Du Toit ED., "Restriction fragment length polymorphism of HLA-DRw53 detected in South African blacks and individuals of mixed ancestry" Hum. Immunol. 1989, Dec 26: 4, 237-44), comprises the isolation of genomic DNA, the restriction endonuclease cleavage of the DNA, a separation of the DNA fragments, a transfer and immobilization of the DNA fragments, the preparation and labeling of the hybridization probes, the hybridization, the detection and the correlation and interpretation. The disadvantage of this previously non-automatable examination is that such an analysis is very labor-intensive and time-consuming (it takes 5 to 10 working days) and has a low sample throughput (a worker only typed up to 50 samples in parallel), so that it is very cost-intensive ,

The characterization of genome sections which can be carried out using DNA molecules or ribonucleic acid molecules (RNA molecules) by hybridization with specific gene probes (Leitch, AR, Schwarzacher, T., Jackson, D. and Leitch IJ, 1994, in situ hybridization , Spektrum Akademischer Verlag Heidelberg, Berlin Oxford), has been routinely carried out for several years. Gene probes are single-stranded nucleic acid molecules of a known nucleotide base sequence with an optimal length of 100 to 300 bases, which specifically lead to a double-stranded nucleic acid pairing with single-stranded nucleic acid sections, e.g. a gene, and mostly with a non-radioactive or radioactive reporter element (marker), e.g. a radionucleotide dye. are provided, which serve the detection of the gene probes. A distinction is made between double-stranded DNA probes, single-stranded RNA probes, tailor-made synthetic oligonucleotide probes with a length of 10 to 50 bases, genome probes and DNA probes produced by PCR (Leitch, AR, Schwarzacher, T., Jackson, D. and Leitch IJ, 1994, In situ hybridization, Spektrum Akademischer Verlag Heidelberg, Berlin Oxford).
In hybridization, a distinction is made between the hybridization of probes with isolated single-stranded nucleic acid (DNA or RNA) and the so-called in situ hybridization (on-site hybridization in tissues, cells, cell nuclei and chromosomes), in which the gene probe spreads to one in the cell (Single-stranded) nucleic acid (DNA or RNA) couples (Leitch, AR, Schwarzacher, T., Jackson, D. and Leitch IJ, 1994, in situ hybridization, Spektrum Akademischer Verlag Heidelberg, Berlin Oxford). With this in situ hybridization it is particularly important that the target sequence and the tissue morphology are preserved and that the preserved tissue is permeable to the probe and the detection reagents. This permeability is not always present, which is a disadvantage of this method.
The hybridization of probes with isolated and spread chromosomes, which is also referred to as in situ hybridization, circumvents the disadvantage of the permeability barrier, since the chromosomes are freely accessible, for example fixed on a support. (TA Brown, 1996, genetic engineering for beginners, Spektrum Akademischer Verlag Heidelberg, Berlin Oxford).
Essential for the hybridization is the presence of single-stranded nucleic acid target and nucleic acid probe molecules, which usually occurs through heat denaturation, as well as the selected optimal stringency (setting of the parameters: temperature, ionic strength, concentration of helix-destabilizing molecules), which ensures that only probes with almost perfectly complementary ones (corresponding) sequences remain paired with the target sequence (Leitch, AR, Schwarzacher, T., Jackson, D. and Leitch IJ, 1994, in situ hybridization, Spektrum Akademischer Verlag Heidelberg, Berlin Oxford).
Classic applications of probe technology that enable the identification of unknown organisms or the detection of certain organisms in a mixture of organisms are, for example, phylogenetic studies or the detection of germs in medical diagnostics. In both areas, the detection of organisms is often based on the analysis of the genes for ribosomal RNA (rRNA, rDNA), which are particularly suitable for this purpose because of their ubiquitous distribution and the existence of variable, species-specific sequence segments. In addition to these properties, the rDNA contains flanking sequence sections that are highly conserved within the respective organism kingdom. Primer sequences directed against these sections can be used for species-independent amplification of the rDNA (G. Van Camp, S. Chapelle, R. De Wachter; Amplification and Sequencing of Variable Regions in Bacterial 23S Ribosomal RNA Genes with conserved Primer Sequences. Current Microbiology, 1993, 27: 147-151 and WG Weisburg, SM Barns, DA Pelletier, DJ Lane; 16S ribosomal DNA Amplification for Phylogenetic studies. J. Bactertiol, 1991, 173: 697-703), which significantly increases the sensitivity of downstream detection methods.
Depending on the specific objective, various established methods for rDNS-based identification of organisms are available.
For the identification of unknown organisms, the entire (usually 16S) rDNA is usually amplified with two universal primers by PCR and then sequenced. In this way, extensive rDNA databases have been created which currently contain sequences from several 1000 organisms (e.g. RDP / Ribosomal Database Project II, Michigan state University, http://www.cme.msu.edu/RDP/) and the phylogenetic assignment allow new sequences. In principle, this method allows the detection of any organism, but is very time-consuming and therefore not suitable for diagnostic applications. The process also has a number of sources of error (F. Wintzingerrode, UB Göbel, E. Stackebrand; Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiology Reviews, 1997, 21: 213-229) , in particular recombination processes and point mutations during the PCR amplification lead to incorrect results.
A number of alternative techniques have been developed for diagnostic applications. Mattsson and Johansson (JG Mattsson, KE Johansson; Oligonucleotide probes complementary to 16S rRNA for rapid detection of mycoplasma contamination in cell cultures. FEMS Microbiiol Lett., 1993: 107 139-144) describe a method in which ribosomal RNA is isolated from mycoplasma, immobilized on filters and detected by hybridization of three different specific oligonucleotides. This procedure is relatively fast, but the number of organisms to be identified and the sensitivity of the detection is limited.
McCabe and co-workers (KM McCabe, YH Zhang, BL Huang, EA Wagar, E. McCabe; Bacterial species identification after DNA amplification with a universal Pprimer pair. Mol. Genet. Metab, 1999; 66: 205-211) describe a method in which rDNA is amplified from clinical bacterial isolates lysed on filter spots by using universal primers and then identified by hybridization with specific probes. This process is sensitive; however, the number of species to be detected is also relatively narrow.
In a method used by Oyarzabal and co-workers (OA Oyarzabal, IV Wesley, KM Harmon, L. Schroeder-Tucker, JM Barbaree, LH Lauerman, S. Backert, DE Conner; Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16S rDNA. Vet Microbiol, 1997, 58: 61-71), in which 16S rDNA of a Campylobacter species is amplified using specific probes and the size of the product is determined, only a yes / no response can be generated for a single specific germ.

Document WO-A-9 533 846 describes a device that a nucleic acid bearing Chip in a reaction chamber having the chip opposite chamber wall from an optically transparent Material is.

The invention has for its object a device for Specify the duplication and characterization of nucleic acids, which has an almost simultaneous duplication and characterization allows a high sample throughput, and thus the disadvantages of Bypasses state of the art.

The task is characterized by the distinctive features of the first Claim resolved. Advantageous refinements are due to the subordinate claims recorded.

The essence of the invention is that the device spatially combines the PCR and the parallel hybridization against chip-bound nucleic acid in a temperature and flow controllable cell (chamber). The inside of the chamber has a chip that generates a capillary gap between the chamber bottom and the detection surface of the chip, which receives the sample liquid, the sample liquid being mixed by an induced electroosmotic flow.
The chamber around the capillary gap and the chip advantageously forms a gas reservoir through which a gas reservoir leads to the capillary gap, which separates an inlet from an outlet, so that the samples can be injected via the inlet, due to the capillary forces from the inlet into the capillary gap arrive and can be removed from it via the outlet. When the capillary gap is filled, an air gap is generated as a ring around the chip stored in the chamber and the capillary gap (which serves as a sample reservoir) due to surface tension effects, so that the chip and the capillary gap are thermally insulated from the chamber body, which leads to the fact that the Samples in the chamber gap can be quickly heated and cooled by heating and cooling elements, which, together with temperature sensors and electrodes, are placed on a chamber support that holds the chamber and is in heat-conducting contact with it over the chamber floor. Because the capillary gap serves as a sample reservoir, the evaporation rate of the sample liquid is greatly reduced even at temperatures near the boiling point of the sample liquid, since the sample can only evaporate over the edge of the capillary gap.
The capillary gap (the sample reservoir) is the location of the nucleic acid amplification in the sample liquid by PCR with specific primers and the genetic characterization of the sample. The labeled PCR products are fished out of the sample liquid by the immobilized specific probes, which are bound on the nucleic acid chip. The chamber and the chip are optically transparent and, due to their design, enable the on-line detection of the marking signal of the PCR products bound to the probes.
The device according to the invention has the advantage over the previously used methods that a maximum genetic typing using specific probes can be automated in a minimal diagnosis time with minimal sample volumes and is possible with a high sample throughput in a temperature and flow controllable cell, with PCR being used to highlight the diagnosis relevant gene structures against a sequence background and the almost simultaneous, parallel hybridization of the PCR products against the chip-bound nucleic acid results in a specific detection.

The device according to the invention finds e.g. for the simultaneous Detection of various microbial pathogens (e.g. based on 16S or 23S rRNA analysis), the screening for Resistance of individual pathogenic microorganisms or a genomic typing of diagnostically relevant allele structures of Eukaryotic cells use, the parallel recognition by the chip with its different, for the different Target sequences specific probes is made possible.

Fig. 1:

eine prinzipielle Darstellung einer möglichen Ausführungsform einer erfindungsgemäßen Vorrichtung zur Vervielfältigung und Charakterisierung von Nukleinsäuren,

Fig. 2:

einen Querschnitt entlang der Ebene A-A gemäß der Fig. 1,

Fig. 3:

eine Draufsicht auf die Vorrichtung gemäß der Fig. 1,

Fig. 4:

eine schematische Darstellung der Ansicht der Unterseite der Vorrichtung gemäß Fig. 1,

Fig. 5.

einen Querschnitt entlang der Ebene B-B gemäß der Fig. 4,

Fig. 6:

eine schematische Darstellung der Draufsicht auf den Kammerträger der Vorrichtung gemäß der Fig. 1,

Fig. 7:

einen Querschnitt entlang der Ebene C-C gemäß der Fig. 6,

Fig. 8:

eine Schematische Darstellung einer möglichen Quadrupolanordnung auf dem Kammerträger der Vorrichtung gemäß der Fig. 1,

Fig. 9:

einen Querschnitt entlang der Ebene D-D gemäß der Fig.8 ,

Fig. 10a:

eine schematische Darstellung einer möglichen Positionierung einer Probenflüssigkeit innerhalb der Vorrichtung gemäß Fig. 1,

Fig. 10b:

einen Querschnitt entlang der Ebene E-E gemäß der Fig. 10a,

Fig. 11:

eine schematische Blockdarstellung eines möglichen Einbaus der Vorrichtung gemäß Fig. 1 in ein Analysensystem,

Fig. 12a:

eine Angabe der Abmessungen einer Vorrichtung gemäß der Fig. 1 in Millimetern,

Fig. 12b:

eine Angabe der Abmessungen einer Vorrichtung gemäß der Fig. 2 in Millimetern,

Fig. 12c:

eine Angabe der Abmessungen einer Vorrichtung gemäß der Fig. 3 in Millimetern,

Fig. 13:

eine schematische Darstellung des optischen Strahlengangs durch die Vorrichtung gemäß Fig. 1,

Fig. 14:

eine schematische Darstellung einer Ausführungsform eines Chips der Vorrichtung gemäß Fig. 1 und

Fig. 15:

eine schematische Darstellung sekundärer und tertiärer Amplifikationsprodukte des Chips gemäß Fig. 14.

The invention will be explained below with reference to the schematic drawings and the application examples. Show it:

Fig. 1:

2 shows a basic illustration of a possible embodiment of a device according to the invention for the duplication and characterization of nucleic acids,

Fig. 2:

2 shows a cross section along the plane AA according to FIG. 1,

Fig. 3:

2 shows a plan view of the device according to FIG. 1,

Fig. 4:

2 shows a schematic representation of the view of the underside of the device according to FIG. 1,

Fig. 5.

3 shows a cross section along the plane BB according to FIG. 4,

Fig. 6:

2 shows a schematic illustration of the top view of the chamber support of the device according to FIG. 1,

Fig. 7:

6 shows a cross section along the plane CC according to FIG. 6,

Fig. 8:

2 shows a schematic representation of a possible quadrupole arrangement on the chamber support of the device according to FIG. 1,

Fig. 9:

3 shows a cross section along the plane DD according to FIG. 8,

10a:

1 shows a schematic illustration of a possible positioning of a sample liquid within the device according to FIG. 1,

10b:

10 shows a cross section along the plane EE according to FIG. 10a,

Fig. 11:

2 shows a schematic block diagram of a possible installation of the device according to FIG. 1 in an analysis system,

Fig. 12a:

an indication of the dimensions of a device according to FIG. 1 in millimeters,

Fig. 12b:

an indication of the dimensions of a device according to FIG. 2 in millimeters,

Fig. 12c:

an indication of the dimensions of a device according to FIG. 3 in millimeters,

Fig. 13:

2 shows a schematic illustration of the optical beam path through the device according to FIG. 1,

Fig. 14:

is a schematic representation of an embodiment of a chip of the device according to FIGS. 1 and

Fig. 15:

14 shows a schematic illustration of secondary and tertiary amplification products of the chip according to FIG. 14.

The device 20 shown in FIG. 1 for reproduction and

Characterization of nucleic acids consists of a chamber body 1 and a chamber carrier 5. The chamber body 1 is provided with a bearing surface 4, via which it is sealingly connected to the chamber carrier 5, so that a sample chamber 3 is formed. This sample chamber 3 consists of a gas reservoir 6 and a capillary gap 7 and is provided with at least one inlet 81 and at least one outlet 82. The inlet 81 and the outlet 82 lead into the sample chamber 3 and are spaced apart by an intermediate gas reservoir nose 9 of the gas reservoir 6. The chamber body 1, which, for example, is inseparably sealed by a bond or weld to a chamber carrier 5 (not shown in detail), holds a chip 2, for example a nucleic acid chip. This chip 2, which carries detection surfaces 12 in the form of spots 13, is held in the chamber body 1 in such a way that the detection surfaces 12 in the form of spots 13 face the surface of the chamber carrier 5 and through the edge 42 of the chamber body 1 from the chamber carrier 5 are positioned evenly spaced so that the chip 2 and the chamber carrier 5, as shown in FIG. 2, generate the capillary gap 7, which serves as a sample reservoir. This capillary gap 7 receives the sample liquid 19.
The chamber body 1 consists, for example, of optically transparent plastic or glass, the sample chamber 3, which represents a space for filling the sample liquid 19, by milling, and the inlet 81 and the outlet 82, which are routes for the sample liquid, by drilling into the Chamber body 1 can be introduced.
As is known, the nucleic acid chip 2 consists of an optically transparent support, the material of which can be, for example, silicon or glass, and of nucleic acid molecules of a specific sequence (for example probes) immobilized on this support.
The sample chamber 3 comprises the gas reservoir 6 and the capillary gap 7, gas and air bubbles collecting in the gas reservoir 6 when filling the sample liquid 19 due to surface tension effects, so that the chip 2 and the capillary gap 7 are thermally insulated from the chamber body 1. The capillary gap 7, which forms the sample reservoir (for example with a volume of 1.8 μl), ensures that the detection surface 12 is completely wetted with the sample liquid 19.
The inlet 81 and the outlet 82 serve to direct the sample liquid 19, which enables filling and emptying of the sample chamber 3, and thus also filling and emptying of the capillary gap 7 as a result of the acting capillary forces.
The inlet 81 and the outlet 82, which can for example run side by side as shown in FIG. 1, are spatially separated from one another by a gas reservoir 9, so that the sample liquid 19 is prevented from flowing from the inlet 81 to the outlet 82 without entering the Capillary gap 7 to arrive.
The chamber support 5, which is optically transparent and has good thermal conductivity, is made, for example, of glass and, as shown in FIGS. 4, 6 and 8, is provided with means for applying temperature 17, for example in the form of miniaturized heaters, and with miniaturized temperature sensors 16 and electrodes of a quadropole 18 so that temperature control of the sample liquid 19 and mixing of the sample liquid 19 by an induced electroosmotic flow is made possible. In another embodiment of the device 20, not shown in detail, the chamber body 1 can be provided with the means for applying temperature 17 and the miniaturized temperature sensors 16 and the electrodes of the quadropole 18.

The temperature sensors 16 can be designed, for example, as nickel-chrome thick-film resistance temperature sensors. The length of the temperature sensor 16 is, for example, in the case that the chamber support 1 has an area of 8 x 8 mm and the chip 2 has an area of 3 x 3 mm or less, 10.4 mm and the width of the temperature sensor 16 in this example 50 µm, so that the resistance at 20 ° C is 4 kOhm and the temperature coefficient TK _R at 0 ° C is 1500 ppm. Alternatively, the temperature sensors 16 can also be designed as optically transparent thin layers.
The means for applying temperature 17 can, for example, be designed as a nickel-chrome thick-film resistance heater. In the dimensions of the previous example, the means for applying temperature 17 have a length of 2.6 mm and a width of eight individual webs, each 50 μm wide, so that the resistance at 20 ° C. is 300 ohms. As an alternative to this, the means for applying temperature 17 can also be designed as optically transparent thin layers.
The quadrupole 18 can be designed, for example, as gold-titanium electrodes. In the dimensions of the previous example, these electrodes have a length of 2.2 mm and a width of 0.5 mm. The quadropole serves to induce an electroosmotic flow, which leads to the mixing of the sample liquid 19 in the sample chamber 1. Alternatively, the quadruple 18 can also be designed as an optically transparent thin layer.

Fig. 2 shows the chamber body 1, which over the bearing surface 4 with the chamber support 5 is in rigid, non-detachable connection. This Connection can be made, for example, by gluing. alternative there is, for example, the possibility that the chamber support 5 and the chamber body 1 is connected to one another by fusing or are made in one piece. Between the chamber support 5 and through the chamber body 1 over the edge 42 held chip 2 there is the capillary gap 7 (which serves as a sample reservoir), which Due to its capillary action it is able to extract sample liquid from the Record sample chamber 3.

Through the chamber body 1, the inlet 81 and the outlet 82 lead in the gas reservoir 6 of the sample chamber 3, so that through the inlet 81st Sample liquid 19 via the gas reservoir 6 into the capillary gap 7 can be filled and discharged via the outlet 82. The chip 2 is like the chamber support 1 made of optically transparent or transparent Material such as Glass, so a conical opening in the Chamber body 1, the continuous cone forming a viewing cone Recess 11, optical and photometric evaluations, such as. Fluorescence measurements from which the detection surface 12 are possible.

Fig. 3 shows the inlet 81 and the outlet 82 and the Recess 11 through which the detection surface 12 with spots 13 of the Chips 2 are optically accessible. This visual accessibility enables the above optical and photometric evaluations of the Signals emanating from the detection surface 12, which are not in the example shown fluorescence signals.

4 are those on the underside of the transparent Chamber support 5 located means for temperature 17 including interconnects 1517 and pads 1417 shown. The In the example, means for applying temperature 17 consist of eight individual microstructured in parallel Resistance heating lines 171 through which the under the chamber body 1st located chamber support 5 and with it the filled Sample liquid 19 in the capillary gap 7 is homogeneously enicable. The Resistance line 171 of the means for applying temperature 17, those with a different, definable temperature can be acted upon have dimensions such that the above optical Accessibility of the detection surfaces 12 of the chip 2 is ensured.

5 shows the positioning of the means for Temperature exposure 17 on the chamber body 1 facing away Side of the chamber support 5, which the chamber body 1 with the supported chip 2 carries.

6 is on the top of the transparent chamber support 5 stored temperature sensor 16 including conductor tracks 1516 and Pads 1416 shown. The temperature sensor 16 is around the detection surface 12 of the chip 2 is mounted, so that said optical accessibility of the detection surface 12 is ensured. The Temperatufühler 16 are by a not shown in the figure Passivation layer electrically opposite subsequent elements of the Device 20 and opposite the sample liquid 19 electrically isolated.

Fig. 7 shows the positioning of the temperature sensor 16 on the Chamber body 1 facing surface side of the chamber support 5, which is also the surface side of the chamber support 5 with the chip 2 held by the chamber body 1 has the capillary gap 7 generated.

Fig. 8 shows one on the, not shown in detail Passivation layer of the temperature sensor 16 applied Quadrupole 18 including assigned conductor tracks 1518 and Pads 1418. Quadrupole 18 is electrically conductive Contact with the sample liquid 19 so that the alternating Applying a voltage of +1 V to two electrodes 181 of the Quadrupole 18 an inducible by the electroosmotic flow Swirling in the capillary gap 7 filled with sample liquid 19 can be caused. Another pair of electrodes 181 of quadrupole 181 put under tension, so change Turbulence conditions. By constantly alternating the pairs electrodes 181, which are energized, are made effective mixing of the sample liquid 19. The applied Low voltage of only one volt prevents the Sample liquid 19 in the capillary gap 7 electrochemical Changes are subject to and gas bubbles form, for example. The Quadrupole 18 is designed, as shown in this figure, that the optical accessibility of the detection surface 12 ensures is. Alternatively, the quadrupole 18 can also be an optically transparent one Thin layer.

FIG. 9 shows the positioning of the quadrupole 18 on the surface side of the chamber carrier 5 facing the chamber body 1. FIGS. 10a and b schematically show the sample liquid 19 stored in the capillary gap 7 between the chamber body 1 and the chamber carrier 5.
Due to the size of the gas reservoir 6, driven by the minimization of the interfacial energy, any air bubbles, not shown in detail, can be discharged from the capillary gap 7 into the gas reservoir 6 of the sample chamber 3. This forms an air ring around the sample liquid 19, which thermally insulates it and the chip 2 from the chamber body 1, so that the sample liquid 19 can be quickly heated and cooled in the capillary gap 7 with low energy consumption. At the same time, the evaporation rate of the sample liquid 19 is greatly reduced even at temperatures near the boiling point, since the sample liquid 19 can only evaporate over the edge of the capillary gap 7. In addition, the need for sample liquid 19 is low (in the .mu.l range) sample reservoir 7, since the capillary gap 7 forms only a small volume, which means that the required sample volumes are very small.
Due to the described good thermal insulation of the chip 2 and the sample liquid 19 from the chamber body 1 and the small volume of the sample liquid 19, the heating and cooling rates customary for microthermal cyclers described by Posner and others can be achieved (S. Poser, T. Schulz, U. Dillner, V. Baier, JM Köhler, D. Schimkat, G. Mayer, A. Siebert; Chip elements for fast thermocycling, Sensors and Actuators A, 1997, 62: 672-675). At the same time, the temperature homogeneity of the sample liquid 19 and the heat input into the sample liquid 19, which is positioned in the capillary gap 7 between the chip 2 and the temperature-controllable chamber support 5, are to a large extent ensured due to the large heating surface-to-sample volume ratio.

FIG. 11 shows the installation of the device 20 for the duplication and characterization of nucleic acids in an analysis system 200. The analysis system 200 consists of a temperature controller 21, a mixing control 22, electrical lines 23, 24, 33, 34, a total inlet 25, a waste container 26 , a conditioner 27, valves / pumps 28, storage containers 29, connecting hoses 30, a conditioner control 31, an automatic control 32, a control computer 35, a computer bus 36 and an automatic pipetting device 37. The device 20 is directly connected via the inlet 81 and the outlet 82 , Conditioner 27 and the waste vessel 26 and via the electrical lines 23 and 24 directly to the temperature controller 21 and the mixing control 22 in connection, the temperature controller being coupled to the temperature sensors 16 and the means for applying temperature 17 and the mixing control to the Quatrupol 18.
In the device 20 built into the analysis system 200 for the duplication and characterization of nucleic acids, the sample liquid 19 can be pipetted into the total inlet 25 via the automatic pipetting device 37 from microplates not shown in detail. Through the valves and pumps 28, which is in liquid-conducting connection with the total inlet 25, the sample liquid 19 can be conducted through the connecting hoses 30 into the conditioner 27, the conditioner 27 being used for processing the sample liquid 19 (for example pH adjustment and Filter out interfering substances). The buffer liquids and reaction solutions for this workup can be supplied from the storage containers 29, which are in a liquid-conducting connection with the conditioner 29. The automatic pipetting device 37 and the conditioner 29 are connected to the conditioner control 31 and the machine control 32 via the electrical lines 33 and serve to control and regulate them. The inlet 81 and the outlet 82 of the chamber body 1, which lead into the gas reservoir 6, serve the liquid line from the conditioner 29 via the capillary gap 7 to the waste 26.
In the device 20, the sample liquid 19 can be tempered and mixed in the area of the capillary gap 7 by means of the temperature controller 21 and the mixing control 22. The capillary gap 7 is therefore the site of the amplification and characterization of a nucleic acid, in the example the target DNA.

Figures 12a to c show an example of an embodiment of the device 20 that the chamber body 1 has a length and width of 8 mm and a height of 3 mm, the gas reservoir length and width of 5.4 mm and a height of 0.5 to 0.8 mm, the chamber support 5 has a thickness of 0.9 mm, the recess 11 on its side facing the chip 2 has a diameter of 2.8 mm and the inlet 81 and the outlet 82 have a diameter of 0.5 mm have, the inlet 81 and the outlet 82, and the recess 11 with respect to the chamber support 5 have an inclination of 70.
FIG. 13 shows the optical beam path through a further embodiment of the device 20, in which the support surface 4 is detachably and sealingly connected to the chamber support 5 via an additional sealing surface 43, for the dark field fluorescence image of the detection surface 12 chips 2. As shown, the excitation light is directed by the dark field mirror 38 onto the detection surface 12 along the excitation light beam path 39. The fluorescent light emanating from the detection surface 12 is directed along the detection light beam path 40 onto a microscope objective 41. In the example, the distance between the dark field mirror 38 and the detection surface 12 is approximately 4.6 mm and the distance between the detection surface 12 and the microscope objective 41 is approximately 22.0 mm.

The optical readout of the interaction signal between the target DNA 50 shown in FIG. 14 and the probe DNA 56, 57, 58, 59 on the surface of the chip 2 can take place online due to the construction of the device 20.
The chip 2 is held in the chamber body 1 in such a way that it can be irradiated by light in a wide solid angle, so that the hybridization can be tracked online or in situ by means of the marked probes 56, 57, 58, 59, for example fluorescence measurements. The arrangement and size of the temperature sensor 16 and the quadrupole 19 is designed in such a way that the beam path for the online detection or the subsequent in situ detection is not disturbed and the detection of the interactions on the spots 13 by all forms of optical detection or spectroscopy ( For example, photometry, differential photometry, confocal fluorescence measurement, dark field fluorescence measurement, transmitted light fluorescence measurement, incident light fluorescence measurement, etc.) can be evaluated, whereby the label 60 and measurement method must be coordinated.

Fig. 14 shows the schematic representation of the chip 2, which bears the primers 54 (A) and 53 (B '), these showing the specific sequence region of the target DNA 50, ie the sequences A, X, S1, X, B and B ', X, S1', X, A '. The sequences A and B or A 'and B' define the region of the target DNA 50, which is identical for all species, or of the single-stranded AB target DNA 51 and A'B 'target DNS 52 Example immobilized via spacer 55 probes 56, 57, 58 and 59, which carry sequences which are specific for the target DNA 50 of a specific origin, ie in the example shown only the probes 56 and 57 hybridize with the sequences S1 and S1 ' to the amplification products of the target DNA 50 (shown in Fig. 15). In contrast, no hybridization takes place at probes 58 and 59 with sequences S2 and S2 '.
The primers 53 and 54 carry, for example, a fluorescent label 60 which can be incorporated into the secondary amplification products 61 and 62 by the amplification process, as a result of which the hybridization to the probes 56 and 57 can be detected during amplification by fluorescence measurement, so that the decision is made possible whether the target DNA 50 has the sequence S1 or S1 'and / or the sequence S2 or S2' between the sequence areas A and B or A 'and B'.
Since the probe sequences can be specific for a particular species, for example, this method can be used to provide evidence of the presence of a particular species in a sample.

Figure 15 shows the schematic representation of the secondary and tertiary Amplification products 61, 62 and 63, which are generated by the device 20 The amount of secondary amplification product 61 and 62 is from the second reaction cycle within the Capillary gap 7 almost doubled with each cycle, so that the Concentration of secondary amplification product after a few Cycles sufficient to attach to probes 56, 57 attached to spots 13 are immobilized to hybridize, extending the Probes 56, 57 complementary to the secondary amplification product 61, 62 takes place. This tertiary amplification product 63 out Probes 56, 57 and secondary amplification product 61, 62 can, for example. via a label 60, which is coupled to the primers 53, 54 used, be detected by means of fluorescence detection.

In a first application example, the specific detection of individual microorganism species will be described:
The chip 2 of the device 20 is a DNA chip in this example and serves, during or after the DNA amplification, for the detection of the amplification products and possibly also for the provision of solid phase coupled DNA primers (Figs. 14 and 15). For example. a sequence S1 which is specific for a species (for example Escherichia coli ) is copied out of a large number of possible targets by the thermal amplification process (for example PCR) so often that the reliable detection of this sequence by hybridization to the probes 56, 57, 58 and 59 and fluorescence measurement on the detection surface 12 is possible. If several sequences are known, each of which is specific for a species, a strain or a disease and which are all between two conserved areas that are identical in all cases, immobilization of the corresponding probes on chip 2 can be used to identify all species, strains and Detect diseases in parallel with only one thermal amplification reaction in the device 20. The range of applications can be expanded by using several pairs of primers 53, 54. In the simplest case, the fluorescence detection of the tertiary amplification products 63 is carried out by means of fluorescent labeling 60 of the primers 53, 54. Other types of labeling, such as intercalators, radioisotopes, FRET systems, fluorescence-labeled nucleotides, etc., are not thereby excluded.
The molecular biological process taking place in the device 20 will be described below with reference to FIGS. 14 and 15.
The target DNA 50 originating from a biological sample is added to the sample reservoir (the capillary gap) 7 together with primers 53, 54, which can be labeled 60. The spots 13 of the chip 2 on the detection surface 12 carry, via spacers 55, probe DNA with sequences S1, S1 ', S2, S2' etc., which are characterized in that they can be complementary to those in the target DNA 50 occur. In the example shown in Fig. 14, the target DNA contains 50 sequences that are complementary to probes 56 and 57. Each sequence S1, S1 'and S2, S2', etc. of the probes (56, 57, 58, 59) was chosen in such a way that it is specific for a specific problem. If, for example, certain pathogens are to be detected by means of the device 20, S1 and S1 'are specific for the Bacillus cereus pathogen , S2 and S2' for the Campylobacter jejuni pathogen etc. If there is only the Bacillus cereus pathogen in a stool sample, so After the sample has been properly processed, there will be a target DNA 50 in the sample liquid which only contains the sequences S1 and S1 '. In order to make them detectably hybridize on the detection surface 12, the number of copies of target DNA 50 must generally be increased significantly. A noise-suppressing, specific DNA amplification method is therefore carried out in the sample reservoir (capillary gap) 7. For this purpose, two primers 53, 54 with sequences A and B ', which are the same for all pathogens, are selected, which frame all possible pathogen-specific probe sequences (S1, S2, S3 ...) (as in Fig. 14) Sequences S1 and S1 'are framed by sequences A and B'). Then, as in the PCR, the target DNA 50 is denatured at approx. 90 ° C, the primers 53, 54 aneal at approx. 65 ° C at B or A 'and it becomes a primer at approx. 70 ° C -Extensions reaction performed, which makes the target DNA 51, 52 double-stranded. The product obtained is then the primary amplification product with the sequence A, X, S1, X, B, Y or B ', X, S1', X, A ', Y. The cycle of denaturation, anal and extension is repeated, whereupon the secondary amplification product 61.62 (see FIG. 15) is obtained. By repeating the amplification cycle several times, the number of secondary amplification products 61, 62 is almost doubled in each case. As a result, the concentration of DNA which contains the sequences S1 and S1 'increases in such a way that reliable detection of the hybridization on the probes 56, 57 is possible. DNA which is not specifically bound to the spots and which is still in the sample liquid is not detected by the amplification process, as a result of which the selectivity of the overall method is greatly increased.
In this way, Bacillus cereus is detected in a highly specific and highly sensitive manner. Instead of the PCR protocol, other amplification methods can also be used.
By installing the device 20 for the duplication and characterization of nucleic acids in the analysis system 200 (FIG. 11), it is possible to carry out the processes of processing samples automatically and continuously.

A second application example describes a parallel detection of bacterial pathogens in stool samples:
In this example, the chip 2 of the device 20 is a DNA chip and serves for the parallel detection of several bacterial pathogens in human or animal stool samples.
The total DNA from each stool sample is isolated using standard techniques (eg using the Qiagen kit provided for this). The DNA is taken up in a volume of a standardized, optionally commercially available buffer system suitable for use in the device 20, in which a PCR amplification can be carried out. In addition to the buffer component, this contains at least one thermostable polymerase, an optionally isomolar mixture of the four natural deoxynucleotide triphosphates, a divalent salt, optionally components to increase the effectiveness of the PCR, and building blocks for labeling the PCR products (e.g. fluorescence-biotin or similarly labeled deoxynucleotide triphosphates ).
For the detection of the organisms, a chip 2 is used, on the surface of which oligonucleotide probes 56, 57, 58, 59 are immobilized, which are complementary to one or more variable regions of the 16S rRNA genes and / or the 23S rRNA genes and / or the internal genes Ranges between 16S and 23S rRNA genes of different organisms to be detected are. The probes 56, 57, 58, 59 are directed, for example, against one or more of the corresponding sequences from Aeromonas spec. and / or Bacillus cereus and / or Campylobacter jejuni and / or Clostridium difficile and / or Clostridium perfringens and / or Plesiomonas shigelloides and / or Salmonella spec. and / or Shigella spec. and / or Staphylococcus aureus and / or Tropheryma whippelii and / or Vibrio cholerae and / or Vibrio parahaemolyticus and / or Yersinia enterocolitica .
The oligonucleotide probes 56, 57, 58, 59 are arranged in spots 13, so that each individual spot 13 contains a multiplicity of oligonucleotide probes (for example the probe 56) of the same sequence. The probes 56, 57, 58, 59 are immobilized either at their 3 'end or at the 5' end or at an internal position, the 3 'end of the probes 56, 57, 58, 59 possibly blocking, for example by amination is so that it can not serve as a substrate for DNA polymerases.
The selection of the probes 56, 57, 58, 59 takes place in such a way that on the one hand each of the probes has a high sequence specificity for the organism to be detected and on the other hand there are motifs in the genomes of the germs at a short distance from the binding site of the specific probes all or for groups of the organisms to be detected have the same sequence.
Against these motifs, universal primers 53, 54 are directed, which are suitable for PCR amplification of a sequence section, which contains the binding site of the probes immobilized on chip 2, in all organisms to be detected. This
Primers 53, 54 are added to the DNA isolated from the stool sample and taken up in the amplification solution (sample liquid 19). Optionally, the primer 53, 54, which specifies the synthesis of the strand which contains the sequence complementary to the sample immobilized on the chip 2 during the subsequent PCR amplification, can be added as a labeled component.
The amplification mixture is filled into the device 20 provided with a chip 2 as described. The solution in the device 20 is subjected to a cyclic temperature regime, so that the target DNA 50 is amplified according to a typical PCR mechanism and, if necessary, simultaneously labeled. After sufficient amplification, there is a hybridization step in which the target sequences amplified with the universal primers 53, 54 hybridize with the specific probes 56, 57, 58, 59 immobilized on the chip 2.
After the reaction has ended, a rinsing step follows in which DNA molecules which are not linked to the chip and are bound non-specifically are removed.
Subsequently, the marking remaining on the chip 2 is detected. Organisms present in the stool sample are identified by marking the sample spots 13 specific to them on the chip 2.

To sample liquids 19, for example, from stool samples or tissue a large number of processing steps are required. It cells have to be broken down, proteins, lipids and solids are separated and the DNA is worked up and cleaned. The enzymes, primers and necessary for the use of the device other substances must also be in the sample liquid 19 are fed. These steps can be done by installing the Device 20 for the duplication and characterization of Nucleic acids in the analysis system 200 that i.a. from pumps and Valves 28 that move and control the liquids, from filters and Reaction chambers (conditioner 27) in which the individual Process steps are carried out sequentially and from Storage containers 29, which supply the chemicals required for this, exists (shown in Fig. 11), automatically and continuously. The samples are not made from one by a pipetting robot 37 Standard delivery system shown in detail in the Total inlet 25 filled for conditioning. The through that Analysis system 200 processed samples pass through inlet 81 in the device 20, so that a duplication and Automated characterization of sample nucleic acids can be carried out. The whole process is done by one Control computer 35 monitors the over a computer bus 36 with electronic controllers and control devices 21, 22, 31, 32 is connected.

All in the description, the following claims and the Features shown in the drawing can be used both individually and any combination with each other be essential to the invention.

LIST OF REFERENCE NUMBERS

1 -

chamber body

2 -

chip

3 -

sample chamber

4 -

bearing surface

5 -

chamber support

6 -

gas reservoir

7 -

capillary

81 -

Inlet

82 -

outlet

9 -

Gas reservoir nose

11 -

recess

12 -

detection area

13 -

commercial

14 -

lands

15 -

conductor path

16 -

temperature sensor

17 -

Means for applying temperature

171 -

resistance lines

18 -

quadrupole

181 -

electrodes

19 -

sample liquid

20 -

contraption

21 -

thermostat

22 -

mixing control

23 -

electrical cables for temperature control

24 -

electrical cables for quadrupole control

25 -

total intake

26 -

waste

27 -

conditioner

28 -

Pumps / valves

29 -

reservoir

30 -

connecting hoses

31 -

Konditionierersteuerung

32 -

The machine control

33 -

electrical cables for conditioner control

34 -

electrical lines for automatic control.

35 -

tax calculator

36 -

computer bus

37 -

Automatic pipetting device (pipetting robot)

38 -

Darkfield mirror

39 -

Anregungslichtstrahiengang

40 -

Detection light beam path

41

microscope objective

42

boundary

43 -

sealing surface

50 -

Target DNA

51 -

AB Target DNS

52 -

A'B 'target DNS

53 -

Primer B '

54 -

Primer A

55 -

spacer

56 -

Probe S1

57 -

Probe S1 '

58 -

Probe S2

59 -

Probe S2 '

60 -

label

61 -

secondary amplification product

62 -

secondary amplification product

63 -

tertiary amplification product

200 -

analysis system

1416 -

Connection surfaces of the temperature sensor

1417 -

Pads of the heater

1418 -

Quadrupole pads

1516 -

Conductor path of the temperature sensor

1517 -

Conductor heater

1518 -

Quadrupols trace

A-A -

cutting plane

B-B -

cutting plane

C-C -

cutting plane

D-D -

cutting plane

E-E -

cutting plane

Claims (19)

1. Device for amplifying and characterizing nucleic acids in a reaction chamber,
   characterized in that an optically transparent chamber body (1), which contains an optically transparent chip (2) having nucleic acids bound thereto and a detection area (12), is sealingly placed on an optically transparent chamber support (5), so that a sample chamber (3) having a capillary gap (7) is formed between the chamber support (5) and the detection area (12) of the chip (2), the chamber being temperature-adjustable and flow-controllable.
2. Device according to claim 1,
   characterized in that the temperature adjustment means are connected with the chamber support (5) and enable a rapid heating and/or cooling of the sample chamber (3) having the capillary gap (7).
3. Device according to claim 2,
   characterized in that the temperature adjustment means are situated on the side of the chamber support (5) facing towards the chamber body (1).
4. Device according to any one of the preceding claims,
   characterized in that the temperature adjustment means (16, 17) are configured in the form of optically transparent thin films and/or are so finely structured that the optical transparency of the chip (2) remains unaffected at least in the area of the nucleic acids bound in spots on the detection area (12).
5. Device according to claim 4,
   characterized in that the temperature adjustment means comprise micro-structured heating elements (17), preferably nickel-chromium-thick film-resistance heaters and/or micro-structured temperature sensors (16), preferably nickel-chromium-thick film-resistance sensors.
6. Device according to any one of the preceding claims,
   characterized in that the chamber support (5) comprises systems for thoroughly mixing the sample liquid, which are configured in the form of optically transparent thin films and/or are so finely structured that the optical transparency of the chip (2) remains unaffected at least in the area of the nucleic acids bound in spots on the detection area (12), whereby preferably a quadrupole system for inducing an electro-osmotic flow is concerned.
7. Device according to claim 6,
   characterized in that the quadrupole system is realized as gold-titanium electrodes.
8. Device according to any one of the preceding claims,
   characterized in that the chamber support (5) and the chamber body (1) preferably consist of glass and/or an optically transparent synthetic material, particularly preferably of polycarbonate and/or polymethane ethyl acrylate.
9. Device according to any one of the preceding claims,
   characterized in that the chamber support (5) consists of a thermally conducting material.
10. Device according to any one of the preceding claims,
    characterized in that the chip consists of glass and/or silicon.
11. Device according to any one of the preceding claims,
    characterized in that the chamber body (1) comprises at least in the area of the chip (2) an optically transparent conical recess.
12. Device according to any one of the preceding claims,
    characterized in that the chamber body has an inlet (81) and an outlet (82) spatially separate from each other, for charging the sample chamber (3) and the capillary gap (7).
13. Device according to claim 12,
    characterized in that the inlet (81) and the outlet (82) are arranged unilaterally to the chip (2) and are separated by a gas reservoir nose (9).
14. Device according to any one of the preceding claims,
    characterized in that the chamber body (1) is sealingly and unreleasably connected with the chamber support (5) by an adhesive and/or weld connection, or is releasably connected through an additional sealing surface (43).
15. Device according to any one of the preceding claims,
    characterized in that the detection area (12) is configured in the form of spots, to which probes (56, 57, 58, 59) in the form of nucleic acid molecules are immobilized, said nucleic acid molecules preferably being DNA molecules and/or RNA molecules.
16. Device according to claim 15,
    characterized in that the probes (56, 57, 58, 59) are immobilized through spacers (55).
17. Device according to any one of the preceding claims,
    characterized in that the evaluation of the chip-based characterization of nucleic acids may ensue by forms of the optical detection and/or spectroscopy, particularly preferably by transmission light fluorescence measurement, dark field fluorescence measurement, confocal fluorescence measurement, reflective light fluorescence measurement, photometry and/or differential photometry.
18. Use of a device according to any one of the preceding claims for an almost simultaneous amplification and chip-based characterization of nucleic acids.
19. Use of a device according to any one of the claims 1 to 17 for an almost simultaneous amplification by PCR and for a chip-based characterization of nucleic acids.

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