EP2109499A2 - Installation améliorée de traitement biologique moléculaire - Google Patents

Installation améliorée de traitement biologique moléculaire

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Publication number
EP2109499A2
EP2109499A2 EP07857168A EP07857168A EP2109499A2 EP 2109499 A2 EP2109499 A2 EP 2109499A2 EP 07857168 A EP07857168 A EP 07857168A EP 07857168 A EP07857168 A EP 07857168A EP 2109499 A2 EP2109499 A2 EP 2109499A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
reaction
sequence
analyte
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07857168A
Other languages
German (de)
English (en)
Inventor
Peer STÄHLER
Markus Beier
Cord STÄHLER
Daniel Summerer
Mark Matzas
Sonja Vorwerk
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Febit Holding GmbH
Original Assignee
Febit Holding GmbH
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Filing date
Publication date
Priority claimed from DE102006062089A external-priority patent/DE102006062089A1/de
Priority claimed from DE102007018833A external-priority patent/DE102007018833A1/de
Application filed by Febit Holding GmbH filed Critical Febit Holding GmbH
Publication of EP2109499A2 publication Critical patent/EP2109499A2/fr
Withdrawn legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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Definitions

  • RNA functional biomolecules
  • proteins other classes of substances resulting from the activity of genes (eg products of enzymatic reactions such as certain metabolites) at a given time
  • the entire information for the functioning of a living being is coded in his DNA.
  • This in turn encodes RNA, which can encode proteins.
  • DNA and RNA are relatively easy to study for their stability and mating properties, which is why current analytical methods are aimed in particular at these two classes of molecules.
  • the focus is on the study of the DNA sequence of different organisms as well as different individuals of a species and their comparison with each other (eg genome sequencing, genotyping by analysis of sites of high genetic variability such as “single nucleotide polymorphisms", evolutionary biology, taxonomy.)
  • Another important analytical method is the characterization of the type and concentration of mRNA (expression profiling) in order to be able to make statements about the activity of genes under certain conditions, which have led to a wealth of new findings in recent years.
  • the human genome and the genome of some other complex organisms such as the mouse and a multitude of small organisms
  • Viruses, bacteria, etc. are explained as being from the sequence. However, the elucidation of the function of our and all other genomes is still in its infancy. Sequencing has revealed that humans have much fewer genes than previously thought, and comparison of sequenced organisms has shown that the number of genes, and thus the number of proteins, does not correlate with the complexity of an organism. And also the differences in the genes are minimal. Thus, the differences between the genes of two people in the per thousand range and the difference between humans and monkeys are just in the lower, single-digit percentage range. A very small genotypic difference compared to the obvious big difference in phenotype. In March 2005 issue of the "Spectrum of Science" describes the author John S.
  • introns as well as the remaining, non-coding DNA are considered by this model to be junk DNA or genetic waste. Overall, so 98.5% of the human genome would have no or no significant importance, and the large amount is explained by the long duration of evolution. This is precisely where Mattick comes in and postulates that at least the introns, possibly all the DNA that does not encode proteins, contains the information for using the genes. Accordingly, introns encode a large number of differently long RNAs. In fact, a variety of regulatory RNAs have now been discovered (e.g., microRNAs) encoded outside the exons.
  • microRNAs regulatory RNAs
  • CE capillary electrophoresis
  • Current is applied to capillaries filled with special gel, causing charged molecules to move. Smaller molecules get through the gel's network faster than large ones. If a cocktail of molecules of different sizes is added to a CE, these leave the capillaries sorted according to their size.
  • this method is used in conjunction with an enzymatic assay. This sample preparation assay generates the respective copy by primer extension from one end of the DNA to be sequenced. In this case, a small proportion of nucleotide analogues are introduced at each copied nucleotide position, causing chain termination and containing a nucleotide-specific fluorophore.
  • the identity and length of the resulting molecule are analyzed by the length and color of the resulting molecule at one position.
  • the colors are optically detected when exiting the capillary and the signals processed by computer and assembled into a sequence.
  • the PCR and related methods use particularly temperature-stable enzymes, which were taken from nature, the DNA or RNA comparable to the processes in one Cell to multiply. This is necessary because in most cases the DNA / RNA is present in the sample to be measured in such a low concentration that most measuring methods can not detect them.
  • the PCR method is a "one-pot-reaction" in which cyclically
  • Temperature is varied: from the denaturation step to near 100 ° C, addition steps at about 5O 0 C to 65 ° C to enzymatic steps at about 72 ° C. The result is depending on
  • Primer sequences can be defined as the area that will be duplicated, that is amplified, -will. This is in addition to the sample preparation for other detection methods, e.g. DNA arrays, but also used directly for detection.
  • the product of an amplification is colored and detected.
  • the mere presence of one or more successful amplifications in a given starting material allows detection.
  • the method is widely established and recognized and provides reliable results, especially in calibrated form as quantitative PCR.
  • the PCR method has two major weaknesses. One is the relatively high cost of a primer pair. This disadvantage is reduced by using a primer set for several reactions. A rule of thumb here are 100 reactions with a set.
  • the second disadvantage is that one does not obtain any information about the sequence between the two primers. Thus, any other sequence that contains the two primer sequences and that are close enough to one another may have been amplified so that an overlap could take place.
  • This disadvantage is addressed by the use of PCR as a method of analysis by several primer sets. The probability of successfully amplifying several very different primers in unknown material is correspondingly lower statistically.
  • RT-PCR real-time cycle-by-cycle
  • qPCR or qRT-PCR quantitative results
  • dNTPs fluorescently labeled dNTPs which contain a 3 'OH protecting group which prevents further extension after a single installation. After incorporation of a dNTP, the fluorescence present on the primer / template complex is detected and then the 3'-0H protecting group and the fluorophore cleaved so that a new cycle of incorporation, detection and cleavage can take place. All four dNTPs with different fluorophores can be offered in parallel or sequentially one single each; in this case, only a single color detection is necessary.
  • DNA arrays The most common method for expression profiling is DNA arrays. On these arrays, short DNA or RNA segments are spatially resolved in rows and columns or synthesized on site. For each gene whose expression is to be analyzed, one or more oligos are used. As with PCR, several oligos increase the statistical safety of the procedure. Further parameters for the quality of the array measurement are the oligo quality, length, sequence selection and the performance of the hybridization reaction. There are these arrays for all known genes of the human genome as well as for some other important model organisms. In addition, there are various topic arrays on which there are oligos, which code for genes that are assigned to a function or disease.
  • the sample material to be examined with an array must be amplified by means of PCR.
  • a generic PCR is used in which all genes expressed in the sample are amplified starting from the universal 3 'end. This overall method makes it possible to multiply a large number of genes with only one PCR reaction and then to detect gene-specifically with the DNA array.
  • the main problem with sample amplification is the use of arrays for genotyping. Since the positions to be examined lie in different regions of a genome, it is possible to amplify only one or a few measurement points with a PCR reaction. Since the cost of a primer set is significant, this greatly limits the use of genotyping arrays because very quickly the cost of upstream PCR reactions exceeds the cost of array analysis. Applied Biosystems therefore also addresses these segments with a parallel PCR system. In 2004, Roche and Affymetrix launched the first genotyping product to be approved for diagnostics. In the Amplichip, as many SNPs per PCR as possible are measured by means of a DNA array in order to make the product economically viable. However, a widespread use of this approach seems rather unlikely from a technical-economic point of view. The bottleneck remains the availability of the individual oligos as primers.
  • microarrays by the / ns / ta synthesis (array Arrangement in a matrix) known.
  • the most widespread is the zw-s / tw synthesis in an array arrangement of synthetic nucleic acids or oligonucleotides. This is carried out on a substrate that is loaded by the synthesis with a variety of different polymers.
  • the big advantage of the micro-array synthesis techniques is the provision of a large number of molecules of different and defined sequence at addressable locations on a common support.
  • the synthesis relies on a manageable set of starting materials (in DNA microarrays usually the 4 bases A, G, T and C) and builds on these arbitrary sequences of nucleic acid polymers.
  • the delimitation of the individual molecular species can be carried out by separate fluidic compartments during the addition of the synthetic ingredients, as described e.g. in the so-called / ns / ta spotting method or piezoelectric techniques based on the ink jet printing technique (A. Blanchard, in Genetic Engineering, Principles and Methods, Vol. 20, Ed. J. Sedlow, p. 111 -124, Plenum Press, AP Blanchard, RJ Kaiser, LE Hood, High-Density Oligonucleotide Arrays, Biosens. & Bioelectronics 11, p. 687, 1996).
  • An alternative method is the spatially-resolved activation of synthetic sites, e.g.
  • Examples of the methods known hitherto are photolithographic light-assisted synthesis [McGalley, G. et al; J. Amer. Chem. Soc. 119; 5081-5090; 1997], the projector-based light-assisted synthesis [PCT / EP99 / 06317], the fluidic synthesis by means of separation of the reaction spaces, the indirect projector-based light-triggered synthesis by means of photoacids and suitable reaction chambers in a microfluidic reaction support, the electronically induced synthesis by means of spatially resolved deprotection on individual electrodes on the Support and fluidic synthesis by means of spatially resolved deposition of the activated synthesis monomers.
  • MicroRNAs are RNA molecules about 22 nucleotides in length and make up the largest group of small RNAs in plants and animals.
  • miRNAs presumably regulate more than 30% of all human genes and, accordingly, their involvement in a wide range of different processes, such as the development of cancer via the control of transposon relocations, stem cell biology or muscle and brain development.
  • miRNAs are excised from primary transcripts (pri-miRNAs) by two steps of endoribonuclease III processing, first by Drosha, which produces hairpin-shaped pre-miRNA, then by Dicer, which produces siRNA-like double-stranded complexes. Mature miRNAs can then interfere with gene regulation through different mechanisms, such as by controlling mRN A digestion or binding to the UTR regions.
  • RNAse protection assay RPA
  • a primer extension reaction can be used.
  • a labeled primer is hybridized to the miRNA, extended with a polymerase and the reaction mixture gelelektrophoretisch separated and analyzed.
  • Much faster and more suitable methods for quantitative detection rely on PCR.
  • Reverse Trancription / PCR can be used when primers are inserted with a loop that introduce a universal sequence. This universal sequence is then used for the PCR.
  • Another approach for the detection and quantification of miRNAs is the use of microarrays.
  • Particular challenges arise from the short length of miRNAs for both probe design and labeling protocols.
  • Various methods for labeling are known to the person skilled in the art, both by direct labeling with biotin or fluorophores or indirectly during cDNA synthesis or amplification.
  • Both chemical and enzymatic methods are known for this purpose, for example based on cis-platinum compounds, periodate hydrazine labeling, T4 RNA ligase, poly-A polymerase or coupling to amino-modified RNAs.
  • Concerning the probe design special challenges arise from the short length of miRNAs, especially in terms of signal strength due to low duplex melting temperatures. For this purpose, modified nucleotides or the use of tandem probes or probes with more than 2 Binding sites for miRNAs are used.
  • Fluorescence in situ hybridization is an attractive method for the detection and identification of microorganisms directly from smears.
  • FISH Fluorescence in situ hybridization
  • Array-based methods can be used to analyze a large number of target molecules, but this usually requires amplification and labeling of the target molecules.
  • Taqman Probes Molecular Beacons, Scorpion Primers, Sunrise Primers, DNA Intercalators or Minor Groove Binder. These methods allow in particular the detection of rare nucleic acids in sample mixtures, for example for the detection of low virus concentrations.
  • Sequencing-based methods are also used, but are usually limited to common nucleic acids such as ribosomal RNA.
  • the invention is based on the object molecular biological processing of a
  • the invention relates to an improved molecular biology process plant.
  • the invention is therefore an improved molecular biology process plant and an improved method for processing biological samples.
  • This invention combines the provision of biologically functional molecules such as nucleic acids and peptides as well as derivatives or analogs of these two classes of molecules in miniaturized flow cells with the serial addition of reagents or fluids and serves to process biological samples such as proteins, nucleic acids, biogenic small molecules such as metabolites, Viruses or cells that are introduced into the miniaturized flow cells.
  • the material to be processed is held bound through several process steps in a substantially unchanged reaction space, whose upstream adaptation to specific samples by a spatially or / and time-resolved immobilization of biologically functional molecules such as nucleic acids, peptides and their derivatives or analogues in the miniaturized flow cells in an arrangement takes place as a microarray.
  • the molecular biology process plant of the invention enables a method for improved analysis of sequence, chemical or biochemical modification, and quantity of nucleotide sequences. This is achieved by combining selective spatially resolved binding of the analytes to an array of hybridizable probes synthesized in the miniaturized flow cells and optional sequence-unspecific or sequence-specific amplification, in particular DNA amplification.
  • the process uses one to several wash-separation steps and amplification steps.
  • the hybridizable probes synthesized in the miniaturized flow cell may be chemically or biochemically altered for this purpose. All steps of the method may optionally be optically monitored in a preferred embodiment, e.g. by a planar and thus parallel to the array or substantially parallel detector. While the reaction cycles are being run or thereafter, an optically detectable result, e.g. the location and quantity of optical markers, e.g. Fluorescence marker, to be detected.
  • the process plant according to the invention preferably has one or more heating elements which can increase the temperature in one or more flow cells, and preferably via one or more cooling elements, which can lower the temperature in one or more flow cells.
  • the various cyclic steps can be automated or partially automated.
  • the molecular biological process system according to the invention enables methods for improved analysis of sequence-specific binding and / or modification events between proteins and proteins the hybridizable probes synthesized in the miniaturized flow cell.
  • the hybridizable probes synthesized in the miniaturized flow cell may be chemically or biochemically altered for this purpose.
  • the process uses one to several wash-separation steps.
  • all steps of the method can optionally be monitored optically, for example by means of a planar and therefore substantially parallel detector. While the cycles are running or after, an optically detectable result, eg, the location and quantity of optical markers such as fluorescent markers, can be detected.
  • FIG. 1 shows an embodiment of the invention in which probe molecules 1 were synthesized on the reaction support, to which molecules 2b to be analyzed bind.
  • a polymerase 4b synthesizes the respective complementary strands of the molecules to be analyzed.
  • Figure 2 shows an embodiment of the invention in which on the reaction support
  • Probe molecules 1 were synthesized, to which a molecule 6 was attached.
  • An adapter molecule 7b was attached to the molecule 6.
  • a polymerase 4 from building blocks 9a synthesizes the strand complementary to molecule 6.
  • Blocks 9a carry a signaling group which may be removed during or after installation so that the formed string contains linked signaling group blocks 9b or associated blocks 9c with the remote signaling group removed.
  • FIG. 3 shows a tabular list of polymerases whose suitability for use in the process plant according to the invention and the processes according to the invention has been investigated.
  • FIG. 4 shows a fluorescence image of a reaction support on which DNA probe molecules were synthesized, which are attached to the surface via the 5 'end and have a free 3'-OH end.
  • the image was taken after hybridization of the reaction support with a PCR product and incubation with different polymerases, dNTPs and dNTPs with signaling groups in a suitable reaction buffer.
  • Polymerases used from left to right T7 DNA polymerase, Sequenase, Phi29, T4 DNA polymerase, Klenow fragment, Klenow fragment exo- and Bst DNA polymerase.
  • FIG. 5 shows a fluorescence image of a reaction support on which DNA probe molecules were synthesized, which are attached to the surface via the 5 'end and form a free 3'-OH reagent. Have end. The image was taken after hybridization of the reaction support with a PCR product and incubation with different polymerases, dNTPs and dNTPs with signaling groups in a suitable reaction buffer. Used polymerases from left to right: Taq DNA polymerase, 9 ° N, Vent DNA polymerase, Vent DNA polymerase, Pfu DNA polymerase, Therminator, Phusion Hotstart.
  • Figures 6A to 6F show fluorescence values (arbitrary units) of a reaction support with self-complementary hairpin probes synthesized on the surface after extension by various polymerases incorporating signaling groups and subsequent fluorescence detection.
  • inverse probes (5'-3 'synthesis) with a length between 27 and 30 nucleotides were designed, which pair with each other via a T-tetraloop (see FIG. 6G).
  • the DNA polymerases used are: Figure 6A Vent; 6B Vent; 6C, Pfu; 6D Therminator, 6E Phusion Hotstart, Figure 6F Klenow fragment E. coli DNA polymerase I.
  • FIG. 7 shows a fluorescence image of a reaction support on which DNA probe molecules were synthesized, which are attached to the surface via the 3 'end and to which the primers were hybridized.
  • the primers bind to the end of the probe molecule which is closer to the reaction support surface (i.e., proximal end).
  • the image was taken after incubation with different polymerases, dNTPs and dNTPs with signal-carrying groups in a suitable reaction buffer.
  • Polymerases used from left to right T7 DNA polymerase, Sequenase, Phi29, T4 DNA polymerase, Klenow fragment, Klenow fragment exo- and Bst DNA polymerase.
  • the dark shaded arrow indicates the direction of attachment of building blocks by the polymerase.
  • FIG. 8 shows the reaction support from FIG. 7 after washing with water.
  • FIG. 9 shows the reaction support from FIG. 8 after renewed hybridization of primers to the DNA probe molecules and incubation with different polymerases, dNTPs and dNTPs with signal-carrying groups in a suitable reaction buffer.
  • the polymerases used in this second copying procedure are from left to right: Taq DNA polymerase, 9 ° N, Vent DNA polymerase, Vent DNA polymerase, Pfu DNA polymerase, Therminator, Phusion Hotstart.
  • Figure 10 shows two variants of the preferred embodiment "oH-c / ⁇ -ligation.”
  • a linkage of two probe molecules occurs depending on a sample molecule to be analyzed:
  • the dark shaded arrow indicates the site of linkage, ie, ligation.
  • the ligation can be carried out, for example, enzymatically or chemically:
  • Figures I IA and I IB show two variants of the preferred embodiment "PCR-on-chip".
  • Common to both variants is the use of a locus-specific (and allele-specific) probe which has been synthesized on the reaction support and hybridizes with the sequence region of the sample molecule to be analyzed.
  • a so-called “universal tag” is covalently attached to the sample molecule to be analyzed and / or amplified .
  • a “universal primer” is used, which is complementary to this "universal tag.” Amplification takes place between the universal primer and the locus-specific (and allele-specific) probe
  • no "universal tag” is used.
  • the universal primer used here is a mixture of so-called random primers which bind at different sites of the sample molecule to be analyzed and / or amplified. The amplification takes place between the respective binding site of the universal primer and the locus-specific (and allele-specific) probe.
  • Figure 12 shows an embodiment in which reverse transcription and PCR are combined. Finally, the amplification takes place between the polyA region of the cDNA formed and a locus-specific probe which has been synthesized on the reaction support.
  • Figure 13 shows the preferred embodiment "microRNA capture-signal amplification” in which microRNA is bound by probe molecules, which can then be labeled with a universal sequence, eg an adenine strand (polyA tail) .
  • a universal sequence eg an adenine strand (polyA tail) .
  • This sequence can be used as a primer in order to copy a circular DNA with a primer-binding sequence in a reaction known to those skilled in the art as "rolling circle amplification”.
  • the resulting DNA can be labeled in different ways for detection.
  • FIG. 14 shows data from experiments relating to an embodiment of the invention for the analysis of microRNAs (miRNA).
  • FIG. 14A shows scatter plots in FIG Reproducibility of the signal intensities of the individual spots on used microarrays of two hybridizations of miRNAs from a human heart sample show (above), or the differences in the signal intensities between two hybridizations when comparing samples from different tissues (heart and brain, bottom).
  • Figure 14B shows two bar graphs showing the signal intensities of different microarray probes after hybridization with a human brain miRNA sample designed for the detection of a particular miRNA. The probes are either completely complementary to miRNA (PM) or carry one, two or three mismatches (MM, single, double, triple).
  • PM completely complementary to miRNA
  • MM single, double, triple
  • FIG. 15 shows a fluorescence image of a microarray of hybridizations of miRNAs from various tissues (heart and brain) which were hybridized under different conditions, as indicated in the table below.
  • 16 shows a fluorescence image of a microarray after hybridization with miRNAs and labeling with biotin / streptavidin-phycoerythrin (before signal amplification, recording time: 2780 ms).
  • FIG. 17 shows a fluorescence image of a microarray after hybridization with miRNAs and labeling with biotin / streptavidin-phycoerythrin (SAPE) and subsequent signal amplification by means of an antibody, which in turn is biotin-labeled and re-labeled with streptavidin-phycoerythrin (recording time: 1500 ms)
  • SAPE biotin / streptavidin-phycoerythrin
  • Figure 19 shows data and a scheme for the topic complex "Analysis of single nucleotide substitutions for SNP genotyping, resequencing or methylation analysis.”
  • a scheme is shown which illustrates the principle of the assay more or less efficient primer extension by a DNA polymerase on different primer molecules located on the surface instead.
  • On the left is a fluorescence image of a microarray after a primer extension as described. During the extension biotin was incorporated, which was subsequently labeled with streptavidin-phycoerythrin. In magnification, the signal differences for different nucleotide pairs in the primer can be clearly seen.
  • PCR-on-Chip 20 shows data on the topic complex "PCR-on-Chip.”
  • PCR reactions were carried out in the reaction support according to the two schemes with a PCR product of the GFP gene as template, wherein one of the primers in each case is immobilized on the surface Biotin was incorporated into the reaction and labeled with SAPE, fluorescence images of the arrays are shown to the right of the respective scheme, the data points labeled PCR are from a PCR reaction, the images titled PEX were the same incubations at the same Temperatures exposed, but not in a cyclical manner.
  • FIG. 21 shows data on the subject complex PCR-on-chip.
  • PCR reactions were carried out in the reaction support according to the scheme on the left with a PCR product of the GFP gene as a template, wherein within a reaction support both primers (GFPforwOl and GFPrevOl) are immobilized separately on the surface. Different primer lengths between 10 and 30 nucleotides were used.
  • biotin was incorporated and labeled via SAPE. Fluorescence images of the arrays are shown to the right of the scheme, respectively.
  • Identical PCR reactions were used in two identical reaction carriers, with GFPforwOl as the soluble primer and GFPrevOl alone in the other. Only at the positions where a PCR-capable, opposite primer pair is achieved, efficient signal generation by amplification is observed.
  • PCR-on-Chip 22 shows data on the topic complex "PCR-on-Chip.”
  • PCR reactions were carried out in the reaction support with a PCR product of the GFP gene as a template, wherein within a reaction support various primers were immobilized on the surface and in each case a primer ( As shown in the picture below, each 30 different immobilized primers were used in the sense and antisense direction, resulting in 30 different PCR products of different lengths in each array, whichever is more soluble Form is added only becomes observed for the sense or antisense primer product formation.
  • Figure 23 shows "on-chip primer extension" data for the copying of immobilized oligonucleotides synthesized in the reaction support
  • primers are hybridized to the oligonucleotides and extended by a polymerase be removed by washing from the reaction support and serve as a template in a PCR, which can be used for their propagation.
  • a scheme showing a so-called "Strand Displacement Amplification" in the reaction carrier is shown in Figure 24.
  • a hairpin probe is synthesized with a free 3 'nucleotide in the reaction support containing a double-stranded recognition sequence for a more distant nicking endonuclease Primer extension by a polymerase, the newly formed strand is cut by the nicking endonuclease and is available for re-primer extension, both enzymes, polymerase and nuclease can be present in the solution simultaneously and cause an isothermal, linear amplification.
  • FIG. 25 shows a scheme which shows a so-called "Strand Displacement Amplification" in the reaction carrier: A probe synthesized in the reaction carrier is hybridized with a primer so that a recognition sequence for a more remote cutting nicking endonuclease arises in the double strand region After primer extension by a polymerase, the newly formed strand is cut by the nicking endonuclease and is available for re-primer extension, both enzymes, polymerase and nuclease being simultaneously present in the solution, and a second isothermal, linear amplification Figure 26 shows a scheme in which an amplification on the surface of the
  • Reaction carrier takes place.
  • Two adjacent probe molecules of different sequence (primer A and primer B) can not be template-extended by a polymerase because they are too far apart to bind to each other (no formation of primer homo- or hetero-dimers known to those skilled in the art).
  • the primers can selectively bind to desired molecules from a complex mixture of molecules (eg DNA fragments from genomic DNA) and are extended by the polymerase. They then reach a length that allows binding of an adjacent primer, so that it can be extended by the polymerase.
  • the Reaction carrier stringently washed so that all non-covalently bound to the surface of the reaction carrier molecules and ions are removed from the reaction medium.
  • the reaction support is subjected to a temperature-time profile which enables a PCR reaction.
  • Figure 27 shows a scheme in which a Amplif ⁇ kation takes place on the surface of the reaction carrier.
  • Two adjacent probe molecules of different sequence primer A and primer B
  • primer A and primer B can not be template-extended by a polymerase because they are too far apart to bind to each other (no formation of primer homo- or hetero-dimers known to those skilled in the art). If soluble molecules not linked to the surface of the reaction support are added, the primers can bind.
  • a hybridization and wash profile is now performed that allows the selective binding of desired molecules from complex sample mixtures (e.g., fragments of genomic DNA). Undesired molecules are removed from the reaction carrier by washing.
  • the primers that have bound molecules are extended by the polymerase after addition of reagents necessary for a PCR.
  • reaction support After an initial extension step, the reaction support is optionally stringency washed, so that all non-covalently bound to the surface of the reaction support molecules and ions are removed from the reaction medium. After re-addition of reagents necessary for a PCR reaction known to those skilled in the art, the reaction support is subjected to a temperature-time profile which enables a PCR reaction.
  • FIG. 28 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture. Unwanted molecules are removed by washing. Optionally, a single-stranded (ssDNA) nuclease is added which processes all unbound, single-stranded probe molecules. The probe molecules bound to microRNAs are not processed. The bound microRNAs now function as primers and are extended by a polymerase, incorporating building blocks with signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 28 shows a principle for the detection of microRNAs.
  • microRNAs These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture. Unwanted molecules are removed by washing.
  • the microRNAs have been previously labeled with one or more signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 30 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture. Unwanted molecules are removed by washing.
  • the microRNAs have been previously labeled with one or more signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 31 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture.
  • the probe molecules contain several sites for the binding of a microRNA, preferably two, three, four or five. Unwanted molecules are removed by washing.
  • the microRNAs have been previously labeled with one or more signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 32 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture.
  • the probe molecules contain several sites for the binding of a microRNA, preferably two, three, four or five. Unwanted molecules are removed by washing.
  • the microRNAs have been previously labeled with one or more signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 33 shows a principle for the detection of microRNAs. These selectively bind in the
  • Reaction carrier synthesized probe molecules and can be known by those skilled in the art
  • Hybridization and washing steps selectively from a complex sample mixture out in
  • microRNAs are then purified by one or more enzymes, preferably
  • Polymerases and / or ligases labeled with one or more signaling groups or haptens After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 34 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture. Unwanted molecules are removed by washing.
  • the microRNAs are then labeled by one or more enzymes, preferably polymerases and / or ligases, with one or more signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 35 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture.
  • the probe molecules contain several sites for the binding of a microRNA, preferably two, three, four or five. Unwanted molecules are removed by washing.
  • the microRNAs are then labeled by one or more enzymes, preferably polymerases and / or ligases, with one or more signaling groups or haptens. After washing, these can be detected directly or after binding a hapten-specific ligand, which in turn contains one or more signaling groups.
  • FIG. 36 shows a principle for the detection of microRNAs. These selectively bind to probe molecules synthesized in the reaction support and can be selectively retained in the reaction support by hybridization and washing steps known to those skilled in the art from a complex sample mixture.
  • the probe molecules contain several sites for the binding of a microRNA, preferably two, three, four or five. Unwanted molecules are removed by washing.
  • the microRNAs are then labeled by one or more enzymes, preferably polymerases and / or ligases, with one or more signaling groups or haptens. After washing, these can be detected directly or after binding of a hapten-specific ligand, which in turn contains one or more signaling groups.
  • Figure 37 shows a workflow scheme for the detection and typing of viruses and other pathogens. After quantitative real-time PCR in the case of a positive test, the resulting PCR product is used directly, without re-PCR for hybridization in the reaction medium. This serves to characterize the detected virus or to discover new ones
  • Mutants, strains or types of a virus are Mutants, strains or types of a virus.
  • FIG. 38 shows an embodiment in which a probe molecule synthesized in the reaction carrier of the process plant according to the invention forms a hairpin structure.
  • a probe molecule synthesized in the reaction carrier of the process plant according to the invention forms a hairpin structure.
  • Figure 39 shows an embodiment in which a probe molecule forming a hairpin structure and having two sequences A and A * linked by a linker is used to sequence A ( Figure A) or A * ( Figure B) tie. This causes a change in the secondary structure of the probe molecule that can be detected.
  • FIG. 40 shows an embodiment as in FIG. 39, with the difference that a sequence X and Z are added to sequence A (FIG. A) and A * (FIG. B), wherein they do not mate with one another and have particular properties which are explained in more detail below ,
  • FIG. 41 shows an embodiment as in FIG. 38, with the difference that a fluorophore (FIG. A) or a quencher molecule (FIG. B) is added to sequence A and A *, which simplify detection of the change in the secondary structure in the probe molecule.
  • Figure 42 shows an embodiment as in Figure 39 which is used to both
  • target To bind strands of a double-stranded sample molecule (target).
  • FIG. 43 shows an embodiment in which a probe molecule synthesized in the reaction carrier of the process plant according to the invention forms a hairpin structure.
  • the probe molecule is not terminal, but internally linked to the surface of the reaction support.
  • the recognition sequence or sequences are in the loop and in type B is or are the recognition sequences in the star.
  • the recognition sequence is shown in dark hatching.
  • FIG. 44 shows a so-called RAKE assay for the detection of miRNA (miRNA RAKE assay).
  • RAKE assay (“RNA-primed, array-based Klenow enzyme assay") carried out using the Klenow fragment of DNA polymerase I an array-based elongation reaction starting from an RNA primer.
  • the miRNA to be detected binds to a DNA probe immobilized on the surface of the array or microarray.
  • NTP nucleotides
  • a part of the nucleotides can be replaced by labeled nucleotides, eg with biotin (bio) labeled nucleotides (bio-NTP), whereby the miRNA can be detected.
  • the DNA probe is immobilized with its 5 'end to the support surface and the 3' end of the DNA probe is free. After binding of the miRNA, the elongation reaction thus takes place in the direction of the carrier surface.
  • Advantages of the method shown are that the information contained in the analyte molecule (miRNA) is not copied to the chip, so that the chip is reusable. Furthermore, the stability of the duplex from probe and miRNA is increased by the elongation.
  • FIG. 45 shows further details of the embodiment of the miRNA-RAKE assay shown in FIG.
  • the DNA probe consists of two regions: the first region comprises a hybridization sequence (light gray) which is reverse complementary to the miRNA sequence to be detected; the second area comprises a marker sequence (mid-gray).
  • the hybridization sequence is located at the 3 'end of the DNA probe.
  • the tag sequence is located at the 5 'end of the DNA probe. This labeling sequence determines the incorporation of the labeled nucleotides, e.g. the incorporation of biotin-labeled uridine.
  • the various DNA probes immobilized on the support surface have identical tag sequences but different hybridization sequences.
  • Figure 46 shows a so-called "inverse" RAKE assay for detection of miRNA
  • the DNA probe is immobilized with its 3 'end to the support surface and the 5' end of the DNA probe
  • the hybridization sequence is located at the 3 'end of the DNA probe and is located at the 5' end of the DNA probe, so after binding of the miRNA, the elongation reaction will be in the direction away from the support surface 44 and 45 are also advantageous in that the information contained in the analyte molecule (miRNA) is not copied to the chip, so that the chip is reusable.Furthermore, the stability of the duplex of probe and miRNA is increased by the elongation.
  • miRNA analyte molecule
  • Figure 47 shows an inverse tandem miRNA RAKE assay.
  • the DNA probe comprises at least three regions: two hybridization regions which are immediately adjacent, ie in tandem, at the 3 'end of the DNA probe and a third region located at the 5 'end of the DNA probe comprising a tag sequence.
  • This DNA probe is immobilized with its 3 'end on the support surface, while the 5' end is free.
  • the two hybridization regions each have identical hybridization sequences.
  • the hybridization sequences are reverse-complementary to the miRNA to be detected.
  • two molecules of miRNA bind in close proximity, ie in tandem, to the DNA probe.
  • the DNA-RNA heteroduplex formed has increased stability compared to embodiments in which only one miRNA molecule can bind to the DNA probe.
  • an elongation reaction using Klenow enzyme and nucleotides is carried out starting from the 3 'end of a miRNA molecule hybridized to the DNA probe.
  • a part of the nucleotides can be replaced by labeled nucleotides, eg biotin-labeled nucleotides, whereby the miRNA can be detected.
  • the information contained in the analyte molecule is not copied to the chip, so that the chip is reusable.
  • the stability of the duplex from probe and miRNA molecules is additionally increased by the elongation.
  • FIG 48 shows a variant of the RAKE assay in which a ligation reaction is used.
  • This assay is referred to in the present application as a RALE assay ("RNA-primed, array-based Ugase enzyme assay”) .
  • the RALE assay immobilizes on the support surface a DNA probe comprising two hybridization regions: the first hybridization region on the The 3 'end of the DNA probe comprises a hybridization sequence that is reverse-complementary to the miRNA to be detected, and the second hybridization region at the 5' end of the DNA probe comprises a hybridization sequence that is reverse-complementary to a ligation probe.
  • the ligation probe has a free 5 '-phosphate group at its 5'-end and also has a label, eg a fluorescence label, which is preferably attached to the 3' end of the ligation probe.
  • an added ligase removes the 3'-end of the ligand miRNA is covalently linked to the 5 'end of the ligation probe.
  • the detection of miRNA takes place via the label present in the ligation probe.
  • the two hybridization regions on the DNA probe are reversed, ie the first hybridization region is located at the 5 'end of the DNA probe and the second Hybridization region is located at the 3 'end of the DNA probe.
  • the 5 'end of the miRNA is covalently linked to the 3' end of the ligation probe.
  • the label is preferably at the 5 'end of the ligation probe.
  • FIG. 49 shows a variant of the inverse tandem miRNA RAKE assay shown in FIG. 47.
  • a further process step in which the two miRNA molecules are covalently bound together by means of a ligase.
  • the additional ligation step leads to a further stabilization of the heteroduplex of DNA probe and miRNA molecules.
  • the information contained in the analyte molecule (miRNA) is not copied to the chip, so that the chip is reusable.
  • FIG. 50 shows an enzyme-free detection method for miRNA molecules.
  • immobilized on the support surface is a DNA probe comprising two hybridization regions: the first hybridization region of the DNA probe comprises a hybridization sequence that is reverse-complementary to the miRNA to be detected; the second hybridization region of the DNA probe comprises a hybridization sequence that is reverse-complementary to a so-called helper oligo.
  • This helper oligo is a short RNA oligonucleotide of 10 to 25 nucleotides in length, which has a label, eg biotin.
  • miRNA and helper oligo hybridize to the immobilized DNA probe.
  • an activated nucleotide is added which covalently links miRNA and helper oligo in a chemical reaction.
  • This chemical reaction is known as chemical ligation and has been described, for example, in International Patent Application WO 2006/063717 (the contents of this application are incorporated herein by reference in its entirety by reference to chemical ligation)
  • a stringent washing step excess non-ligated helper oligo is removed
  • the miRNA is detected via the marker in the helper oligo
  • Figures 50A and 5OB show two different embodiments which differ in the orientation of the DNA probe In the embodiment of Figure 50A, the DNA probe is immobilized to the support surface via its 3 'end and the 5' end is free: in the embodiment of Figure 50B, the DNA probe is immobilized to the support surface via its 5 'end and the 3'-end is free.
  • FIG. 51 shows a variant of the detection method for miRNA molecules shown in FIG.
  • a DNA probe is immobilized on the support surface comprising three hybridization regions in the following order: the first hybridization region of the DNA probe comprises a hybridization sequence which is reverse complementary to a first helper oligo; the second hybridization region of the DNA probe comprises a hybridization sequence that is reverse-complementary to the miRNA to be detected; the third hybridization region of the DNA probe comprises a hybridization sequence that is reverse-complementary to a third helper oligo.
  • the first helper oligo, the second helper oligo and the miRNA hybridize to the DNA probe.
  • the first and second helper oligo are RNA oligonucleotides of 10 to 25 bp in length.
  • activated nucleotides are added which covalently link the miRNA at one end to the first helper oligo and at the other end to the second helper oligo in a chemical reaction.
  • This chemical reaction is known as chemical ligation and has been described, for example, in International Patent Application WO 2006/063717.
  • the miRNA extended by the two helper oligos is separated from the DNA probe and amplified in an amplification reaction (eg PCR or "Whole Genome Amplification” (WGA))
  • Primer has the same sequence as the first helper oligo and the second primer has the sequence complementary to the second helper oligo
  • the first primer has the same sequence as the second helper oligo and the second primer has the
  • labels can be incorporated into the amplification products, eg by using labeled nucleotides such as bio-NTP
  • the amplification products are hybridized back to the microarray in a subsequent step The detection of the miRNAs can then over in the amplification reaction introduced mark done.
  • FIG. 52 shows a variant of the RAKE assay for the detection of miRNA.
  • two different DNA probes are immobilized on the carrier surface of a microarray.
  • the first of the two DNA probes contains at its 5 'end the recognition sequence for an RNA polymerase, for example the T7 promoter sequence, when the T7 RNA polymerase is used. At its 3 'end, this first DNA probe is reverse-complementary to a miRNA to be detected.
  • the second DNA probe on the support surface is reverse complementary to the first DNA probe.
  • NTP nucleotides
  • biotin-labeled uridine nucleotides can be used.
  • the detection of miRNA takes place via the incorporated labeled
  • extended miRNA molecules can be detached from the support surface by denaturation. To these single-stranded DNA
  • Amplification reaction is amplified to the DNA-RNA chimera reverse-complementary strand with about a 1000-fold amplification linear.
  • the amplification products are back-hybridized to the carrier surface of the microarray and hybridize with the above-mentioned second DNA probe.
  • Figure 53 shows probes with a cap group for use in the microarray systems of the invention.
  • the probe molecules immobilized on the carrier surface have a cap group at their 5 'end.
  • a target molecule e.g. a miRNA
  • the cap group interacts with the duplex and increases its thermal stability.
  • the chemical structure of an exemplary cap group is shown in the right part of the figure.
  • the interaction of the cap group with the duplex enhances differences in melting temperature between fully Watson-Crick paired duplexes and duplexes containing base mismatches. This is particularly useful for distinguishing miRNAs that differ only in one or a few nucleotides near the 3'-end or at the 3'-end, e.g. Members of the let-7 family.
  • the terms used herein have the meaning given to them in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kölbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • a "receptor" in the context of the present invention is any molecule that has a
  • the binding to the analyte is specific and selective.
  • the "receptor" is immobilized, preferably on a carrier body or carrier for short.
  • Preferred receptors of the invention include oligopeptides or polypeptides, also briefly summarized below by the term "peptide”. These oligopeptides or polypeptides may be composed of the known naturally occurring 20 amino acids, but may also contain naturally occurring or synthetic amino acid analogs and / or derivatives. These amino acids, amino acid analogs and / or derivatives may optionally carry labels such as dyes.
  • Oligopeptides typically consist of up to 30 amino acids, amino acid analogs and / or derivatives, while polypeptides consist of more than 30 amino acids, amino acid analogs and / or derivatives, with no sharp distinction between oligopeptides or polypeptides.
  • Oligopeptides or polypeptides used as receptor in the sense of the invention preferably comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 , 45, 50, 55 or 60 amino acids, amino acid analogs and / or derivatives.
  • receptors comprise oligonucleotides or polynucleotides, also referred to collectively below as nucleic acids.
  • These oligonucleotides or polynucleotides preferably consist of deoxyribonucleotides or of ribonucleotides or of mixtures thereof and may be single-stranded or double-stranded Nucleic acid analogs and / or derivatives, such as peptide nucleic acids (PNA), locked nucleic acids (LNA), etc.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • nucleobases of these deoxyribonucleotides, ribonucleotides, nucleotide analogues and nucleotide derivatives are selected from adenine (A), cytosine (C ), Guanine (G), thymine (T) and uracil (U), wherein deoxyribonucleotides typically contain nucleobases A, C, G or T and ribonucleotides typically contain nucleobases A, C, G or U.
  • the Receptors of the Erf also include variants and derivatives of these nucleobases, such as methylated nucleobases or those carrying covalently linked labels, such as dyes or haptens.
  • Oligonucleotides typically consist of up to 30 nucleotides, nucleotide analogs or derivatives, while polynucleotides consist of more than 30 nucleotides, nucleotide analogs or derivatives, with no sharp demarcation between oligonucleotides and polynucleotides.
  • oligonucleotides or polynucleotides used as receptor in the sense of the invention comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 , 45, 50, 55 or 60 nucleotides, nucleotide analogues or derivatives.
  • building blocks refers to the units linked in each case to receptors, which are usually individual amino acids or individual nucleotides or nucleotide analogues However, in certain embodiments, a building block may also consist of 2, 3, 4, 5, 6, 7, 8 , 9, 10 or more amino acids, nucleotides or Nukleotideanaloga.In this case, the synthesis time of the receptor, for example, when using blocks that consist of two nucleotides, halved.
  • the free blocks preferably have an activated or linkable group, via
  • activation is used here in the usual sense as a modification of a chemical group which enables this group to be attached to the support or to a building block previously attached to the support suitable conditions - ie those in microfluidic molecular biology achievable conditions - to form a covalent bond to another group.
  • activation is used here in the usual sense as a modification of a chemical group which enables this group to be attached to the support or to a building block previously attached to the support suitable conditions
  • asymmetric receptors refers to receptors consisting of at least 2 different types of receptor building blocks, ie containing more than 1, 2, 3, 4, 5, 6, 7, or 8 different types of receptor building blocks
  • a "type of receptor building blocks” or else a "set of receptor building blocks” each comprise a group of receptor building blocks which have a common structural feature but differ in another structural feature
  • a “set of receptor building blocks” includes all deoxyribonucleotides, regardless of which nucleobase the deoxyribonucleotide carries.
  • a second "set of receptor building blocks” encompasses all locked nucleic acids, ie, all LNA nucleotides, regardless of which nucleobase carries the particular LNA nucleotide, ie, an "asymmetric receptor” consisting of nucleic acids, at least two different nucleotide types, for example DNA + LN A or DNA + PNA or DNA + RNA.
  • the receptors of the invention may form one or more "secondary structures.”
  • a receptor of the invention may comprise one or more secondary structures in its entirety, or even in some subregions, in the case where the receptors of the invention are oligopeptides or polypeptides These secondary structures require a minimum length of the oligo- or polypeptide in question, for example in the case of ⁇ -helices at least 4 amino acids, in the case of ⁇ -sheets
  • These secondary structures preferably comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids
  • the receptor is a single-stranded oligo- or polynucleotide and that the secondary structure is a hairpin structure.
  • the hairpin structure is characterized by a stem region consisting of a self-complementary helix and a loop region consisting of a single-stranded, unpaired region.
  • the loop region has a length of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides.
  • the loop region has a length of at most 100, 90, 80, 70, 60, 50, nucleotides.
  • the stem region preferably has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more base pairs.
  • the stem region has a length of at most 40, 35, 30, or 25 base pairs.
  • the total length of the receptor forming a hairpin structure is preferably at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 32, 35, 40, 45, 50, 60, 80, 100 or more nucleotides.
  • an oligo- or polynucleotide can form a hairpin structure only if it has self-complementary regions.
  • the person skilled in the art is aware of methods, algorithms and computer programs for determining such self-complementary regions and for constructing oligo- or polynucleotides which have hairpin structures or other secondary structures.
  • the melting temperature of the hairpin structure is lower than the melting temperature of the hybridization product of receptor and specifically bindable analyte. In other words, in the presence of a specifically bindable analyte, the hairpin structure dissolves and the receptor and the specifically bindable analyte hybridize with each other.
  • a "light source matrix” in the sense of this invention is preferably a programmable light source matrix, for example selected from a light valve matrix, a mirror array, a UV laser array and a UV LED (diode) array
  • the light valve matrix may control a radiation source, which may preferably drive to predetermined positions
  • a radiation source which may preferably drive to predetermined positions
  • Such light matrices are disclosed, for example, in WO 00/13018 the light valve matrix selected from the group consisting of DLP, LCoS panels, and LCD panels, and the radiation source capable of driving predetermined positions is selected from an LED array and an OLED array
  • a "miniaturized flow cell" within the meaning of this invention is a three-dimensional
  • Microcavity which has at least one input and one output.
  • the interior space is designed to lead, like a single long channel, from one or more entrances to one or more exits, thus providing fast pressure-driven inflation (positive and / or negative pressure) with reagents and other media allowed.
  • This channel preferably has a diameter in the range of 10 to 10,000 .mu.m, particularly preferably from 50 to 250 .mu.m, and may in principle be configured in any desired form, for. B. with round, oval, square or rectangular cross-section.
  • the length of a flow cell can vary between 10 ⁇ m and 10 cm. For flow cell lengths that exceed the width or length of the carrier, this can also be attached meandering.
  • a “primer extension reaction” in the sense of the invention refers to any reaction in which a primer molecule which has hybridized to a template is extended as a function of the sequence of the template
  • the template can be a nucleic acid, ie DNA or RNA, or a nucleic acid analog.
  • the primer extension reaction may be accomplished by any suitable DNA-dependent polymerase known in the art
  • the DNA-dependent polymerase is a DNA polymerase, but suitable DNA-dependent RNA polymerases may also be used the "primer extension reactions" of the invention find use.
  • the primer extension reaction can be accomplished by any suitable RNA-dependent polymerase known to those skilled in the art.
  • the RNA-dependent polymerase is an RNA-dependent DNA polymerase.
  • Such RNA-dependent DNA polymerases are also known as "reverse transcriptases”.
  • an "amplification" of a nucleic acid means any generation of a new strand of nucleic acid from an existing nucleic acid strand.
  • the term “amplification” also includes the synthesis of a single complementary strand in a primer extension reaction.
  • the term “amplification” preferably also includes the duplication or further amplification of nucleic acid strands in processes such as polymerase chain reaction or multiple displacement amplification.
  • the invention relates to a molecular biological processing system comprising (a) a device for the ms // w synthesis of arrays of receptors, (b) one or more elements for the processing of fluidic steps, such as the addition of samples, reagent (C) a detection unit for the detection of an optical or electrical signal, (d) a programmable logic control unit, and (e) a programmable fluidics control, detection and control unit storage and management of measurement data.
  • the molecular biology Process plant characterized in that it comprises one or more miniaturized flow cells and that a fluidic step in said one or more miniaturized flow cells is 1 minute or less, more preferably 30 seconds or less, even more preferably 10 seconds or less, even more preferably 1 second or less, more preferably 0.1 sec or less, even more preferably 0.01 sec or less, even more preferably 0.001 sec or less, and most preferably 0.0001 sec or less.
  • the molecular biology process plant is characterized by having one or more miniaturized flow cells and the volume of fluid in said one or more miniaturized flow cells is 40% or less, more preferably 25% or less, even more preferably 10%.
  • the molecular biological process plant is characterized in that it has one or more miniaturized flow cells and that during the processing of the fluidic steps at least 2, more preferably at least 5, even more preferably at least 10, even more preferably at least 100, still more preferably at least 500 and most preferably at least 1000 different reagents are introduced into these one or more miniaturized flow cells.
  • these different reagents when processing the fluidic steps, will be in 10 minutes or less, preferably 1 minute or less, more preferably 30 seconds or less, even more preferably 10 seconds or less, even more preferably 1 second or less, more preferably in 0.1 second or less, even more preferably in 0.01 second or less, and most preferably in 0.001 second or less, into one or more miniaturized flow cells.
  • the invention in a second aspect, relates to a method of analyzing the nucleic acid sequence of a nucleic acid analyte comprising the steps of: (a) in situ synthesis of at least one oligonucleotide probe in at least one synthesis region in a miniaturized flow cell; (b) adding at least one single-stranded or double-stranded nucleic acid analyte to the miniaturized flow cells; (c) ligation or sequence-specific hybridization of the nucleic acid analyte to the oligonucleotide probe; and (d) at least one template-dependent nucleic acid synthesis step associated with a change in an optical or electrical signal.
  • the internal volume of the flow cell of step (a) is preferably 40% or less, more preferably 25% or less, even more preferably 10% or less, even more preferably 5% or less, even more preferably 1%. or less, more preferably 0.5% or less, even more preferably 0.1% or less, still more preferably 0.01% or less, still more preferably 0.001% or less, and most preferably 0.0001% or less the volume of the supply line to the fluid reservoir.
  • the flow cell of step (a) is characterized in that a fluidic step is preferably 1 minute or less, more preferably 30 seconds or less, even more preferably 10 seconds or less, even more preferably 1 second or less, even more preferably 0.1 sec or less, more preferably 0.01 sec or less, even more preferably 0.001 sec or less, and most preferably 0.0001 sec or less.
  • the invention in a third aspect, relates to a method of amplifying a target nucleic acid comprising the steps of: (a) ms / Yw synthesis of at least one oligonucleotide probe in at least one synthesis region in a miniaturized flow cell; (b) adding at least one single or double stranded nucleic acid analyte to the miniaturized flow cells; (c) ligation or sequence-specific hybridization of the nucleic acid analyte to the oligonucleotide probe; and (d) at least one round of nucleic acid amplification.
  • step (d) comprises the step of template-dependent nucleic acid synthesis and / or ligating an oligonucleotide primer or adapter nucleotide to the nucleic acid analyte and / or the step of digestion with a restriction endonuclease.
  • one or more of steps (a) through (d) is accompanied by a change in optical or electrical properties.
  • this change in optical property is a change in localization, emission, absorption, or amount of an optical marker.
  • the method additionally comprises the step of ms / tw synthesis of at least one oligonucleotide primer in the miniaturized flow cell detachably attached in its synthesis region.
  • the method additionally comprises releasing two or more oligonucleotide primers and hybridizing them to form a double-stranded adapter oligonucleotide.
  • the amplification method be selected from strand displacement amplification, PCR and ⁇ / Z / ng czVc / e amplification.
  • the amplification product is released from the surface of the miniaturized flow cell.
  • the amplification product be subjected to one or more further processing and / or analysis steps selected from PCR, gel electrophoresis, ligation, restriction digestion, phosphatase treatment, kinase treatment, m-vz ⁇ ro protein translation and m-vzvo -Proteintranslation.
  • the invention relates to a method of amplifying a target nucleic acid comprising the steps of: (a) / w-s / tw synthesis of at least one oligonucleotide probe in at least one synthesis region in a miniaturized flow cell; wherein the oligonucleotide probe has intramolecular hybridization regions; wherein one of the intramolecular hybridization regions is positioned at the 3 'end of the oligonucleotide probe; and wherein a recognition sequence for a nicking endonuclease (I) is present in the hybridization region at the 3 'end of the oligonucleotide probe, or (II) can be generated by a sequence-dependent extension of the hybridization region at the 3' end of the oligonucleotide probe, or (III) is partially present in the hybridization region at the 3 'end of the oligonucleotide probe and can be completed by a sequence-dependent extension
  • the invention relates to a method of amplifying a target nucleic acid comprising the steps of: (a) ms / tw synthesis of at least one oligonucleotide probe in at least one synthesis region in a miniaturized flow cell; (b) adding a primer molecule; wherein the primer is designed to have at least at its 3 'end a region complementary to the oligonucleotide probe; and wherein a recognition sequence for a nicking endonuclease (I) is present in the region of the primer complementary to the oligonucleotide probe, or (II) by a sequence-dependent Extension of the region complementary to the oligonucleotide probe can be generated, or (III) is partially present in the region of the primer complementary to the oligonucleotide probe and can be completed by a sequence-dependent extension of the region of the primer complementary to the oligonucleotide probe; (c) sequence-specific hybridization of the
  • the invention relates to a method of amplifying a target nucleic acid comprising the steps of: (a) / ss / Yw synthesis of a plurality of at least one first oligonucleotide probe in at least one synthesis region in a miniaturized flow cell; (b) in situ synthesis of a plurality of at least one second oligonucleotide probe in at least one synthesis region in a miniaturized flow cell; wherein the distance between any two oligonucleotide probes is selected so that they can not bind to each other; in each case associated first and second oligonucleotide probes are synthesized in the same synthesis region; (c) adding at least one single- or double-stranded nucleic acid analyte to the miniaturized flow cells; (d) ligating or sequence-specific hybridization of the nucleic acid analyte to a first oligonucleotide probe; (e) adding
  • the methods comprise one or more stringent washing steps, preferably a stringent washing step after step (d) and / or after step (f) of the sixth aspect.
  • the amount of newly synthesized nucleic acids is determined in real time.
  • the invention in a seventh aspect, relates to a method for the preparation of a carrier for the determination of nucleic acid analytes by hybridization, comprising the steps: (a) providing a carrier body and (b) constructing an array of several different receptors selected from nucleic acids step by step and nucleic acid analogs on the support by local or / and time-specific immobilization of receptor building blocks at respectively predetermined positions on or in the carrier body, wherein one uses several different sets of synthetic building blocks for the synthesis of the receptors to asymmetric, ie to obtain from several different types of receptor building blocks existing receptors.
  • the invention relates to a method of making a carrier for the determination of nucleic acid analytes by hybridization comprising the steps of: (a) providing a carrier and (b) constructing an array of several different receptors selected from nucleic acids stepwise and nucleic acid analogs on the carrier by local or / and time-specific immobilization of receptor building blocks at respectively predetermined positions on or in the carrier body, wherein in one or more of the predetermined positions, the nucleotide sequences of the receptors are selected such that the receptors are specifically bindable in the absence thereof Analytes at least partially present as a secondary structure.
  • the invention in a ninth aspect, relates to a method for the determination of analytes, comprising the steps of: (a) providing a carrier having a plurality of predetermined regions to each of which different receptors are immobilized selected from nucleic acids and nucleic acid analogs, wherein in one or more of (b) contacting the carrier with an analyte-containing sample, and (c) determining the analytes via their binding to the receptors immobilized on the carrier, the binding of an analyte to a specific one thereof bindable receptor leads to a detectable signal change.
  • the invention relates to a method of determination analyte comprising the steps of: (a) providing a carrier having a plurality of predetermined regions to each of which different receptors selected from nucleic acids and nucleic acid analogs are immobilized, wherein in one or more of the predetermined regions the receptors are at least partially absent in the absence of an analyte specifically capable of binding thereto (b) contacting the support with an analyte-containing sample, and (c) determining the analytes via their binding to the receptors immobilized on the support, wherein the binding of an analyte to a receptor capable of binding specifically with it proves the dissolution of the receptor Absence of the analyte present secondary structure includes.
  • the invention relates to a method for the determination of
  • An analyte comprising the steps of: (a) providing a support having a plurality of predetermined regions to each of which different receptors are immobilized selected from nucleic acids and nucleic acid analogs; wherein each individual receptor comprises at least one hybridization region to which an analyte can specifically hybridize; (b) contacting the carrier with a sample containing analyte; (c) performing a primer extension reaction; wherein the analyte acts as a primer; wherein in the primer extension reaction, building blocks are incorporated which carry one or more signaling groups and / or one or more haptens; and (d) determining the analyte via the incorporation of signal group-containing or hapten-containing building blocks.
  • the invention relates to a method for the determination of analytes, comprising the steps of: (a) providing a carrier having a plurality of predetermined regions, on each of which different receptors are immobilized selected from nucleic acids and nucleic acid analogs; wherein each individual receptor comprises at least one hybridization region to which an analyte can specifically hybridize; (b) contacting the carrier with a sample containing analyte; wherein the analytes in the sample have been linked before, during or after contacting with one or more signaling groups and / or with one or more haptens; (c) Determination of the analyte via the detection of the signaling group (s) or the hapten / haptene in the analyte.
  • the invention relates to a method for the determination of analytes, comprising the steps of: (a) providing a carrier having a plurality of predetermined regions, on each of which different receptors are immobilized selected from nucleic acids and nucle
  • Amplification of analytes comprising the steps of: (a) providing a support having a plurality of predetermined regions to each of which different receptors are immobilized selected from nucleic acids and nucleic acid analogs; each individual receptor having at its 3 'end a hybridization region to which an analyte is specific can hybridize; (b) contacting the carrier with a sample containing analyte; and (c) performing a primer extension reaction; wherein the different receptors function as primers, thereby obtaining a double-stranded nucleic acid consisting of analyte and extended receptor.
  • the method additionally comprises the following method steps which follow step (c): (d) thermal denaturation of the double-stranded nucleic acid obtained in step (c); (e) adjustment of reaction conditions allowing hybridization of analyte and non-extended receptors; (f) performing a primer extension reaction, wherein the different non-extended receptors function as primers; and (g) optionally repeating steps (d) to
  • the method additionally includes the following
  • Process step which is carried out during one of the steps (c) to (g) or after one of the steps (c) to (g): Determination of the analyte via the incorporation of the signal group-containing and / or hapten-containing building blocks.
  • the analyte is an RNA; wherein the different receptors additionally have a region with a primer sequence 1 positioned 5 'to the hybridization region, and wherein the process additionally comprises the following steps following step (c): (d) ligation of a nucleic acid cassette having a region a primer sequence 2, to the double-stranded nucleic acid obtained in step (c); (e) performing a second strand synthesis; (f) performing at least one cycle of an amplification reaction with the addition of a primer with primer sequence 1 and a primer with primer sequence 2.
  • building blocks which carry one or more signaling groups and / or one or more haptens will be incorporated in step (e) and / or in step (f).
  • the method additionally includes the following
  • Process step which is carried out during one of the steps (e) to (f) or after one of the steps (e) to (f): Determination of the analyte via the incorporation of the signal group-containing and / or hapten-containing components.
  • the invention relates to a method for Preparation of a carrier for nucleic acid analysis and / or synthesis, comprising the steps: (a) providing a carrier body and (b) constructing an array of several different receptors selected from nucleic acids and nucleic acid analogs on the carrier by site-specific or / and time-specific immobilization stepwise of receptor building blocks at respectively predetermined positions on or in the support body, wherein at least 2 different receptors are synthesized in at least one synthesis area by orthogonal chemical processes.
  • the invention relates to a reagent kit comprising a carrier body and at least two different sets of building blocks for the synthesis of receptors on the carrier body.
  • the invention relates to the use of the molecular biological process plant according to the first aspect for the detection or / and the isolation of nucleic acids; for sequencing; for point mutation analysis; for the analysis of genomes, genome variations, genome instabilities and / or chromosomes; for the typing of pathogens; for genotyping; for gene expression or transcriptome analysis; for the analysis of cDNA libraries; for the preparation of substrate-bound cDNA libraries or cRNA libraries; for generating arrays for the production of synthetic nucleic acids, nucleic acid double strands and / or synthetic genes; for the preparation of arrays of primers, ultra-longmers, probes for homogeneous assays, molecular beacons and / or hairpin probes; for generating arrays for the production, optimization and / or development of antisense molecules; for further processing of the analytes or target molecules for the logically subordinate analysis on the microarray, in a sequencing method, in an amplification method or for analysis in a
  • the analyte or nucleic acid analyte or the nucleic acid to be detected and / or to be isolated is selected from the group consisting of: a microRNA, a cDNA corresponding to a microRNA, a nucleic acid which acts pathogenically, and a pathogen Nucleic acid.
  • the specificity of the hybridization is integrated with the parallelism of a microarray and the amplification as in a conventional PCR amplification in the method according to the invention.
  • a microstructure with three-dimensional microcavities is used, each of which has at least one input and one output.
  • the interior space is designed such that it leads from an entrance to an exit, like a single long channel, and thus a fast pressure-driven filling
  • This reaction support is first prepared by in situ synthesis, e.g. equipped with oligonucleotides, oligonucleotide derivatives or Oligonukleotidanaloga, which are arranged in rows and columns of separate reaction fields.
  • the individual reaction fields preferably have dimensions of less than 100 ⁇ 100 ⁇ m.
  • Functional biological molecules are thus available in the reaction support, which can now selectively bind nucleic acids contained in an added sample via specific hybridization. These target molecules are thus bound depending on the sequences previously generated during the m-s / Yw synthesis. In the next step, all nucleic acids not bound to the desired extent are washed away. Only the specifically bound nucleic acids remain. However, the amount of these bound nucleic acids may be below the detection limit of a prior art confocal, e.g. on a scanning laser based, or parallel, e.g. based on CCD chips, optical detector lie.
  • a nonspecific or specific enzymatic amplification which is not dependent on additional specific primers.
  • Numerous methods are known to the person skilled in the art.
  • a cassette containing the necessary primer sequences can be ligated to the bound nucleic acids.
  • commercial kits such as e.g. recourse to the "GenomePlex Whole Genome Amplification WGA Kit” available from Rubicon Genomics, USA, or from Sigma-Aldrich, USA.
  • the nucleic acid material is allowed to hybridize again with the oligonucleotides of the array. It may be helpful to perform additional intermediate steps, such as a heating step to separate the strands. It is important in all steps that before each washing step or after a processing step Opportunity exists that those target molecules, which are to be further processed, can bind to the matrix of functional biological molecules, in this embodiment, to the microarray of oligonucleotides.
  • the hybridization step the material is washed again and all or some of the unspecific material is removed. Now, the detection can be carried out, which can be carried out as described above with various methods and apparatuses known to the person skilled in the art for the use of microarrays.
  • Examples include microscopes, optical scanners, laser scanners, confocal scanners, or parallel, for example, based on CCD chips, optical detectors that can record more than one measuring point at a time, or even record the entire reaction carrier in the whole, and mixed forms of devices described above, such as scanners with CCD lines.
  • Examples of signals which can be used in the analysis of the reaction results on the reaction support or array include the following signals well known in the art:
  • a further enrichment of the result-relevant material can first be achieved by repeating the wash-separation steps, in addition the signal-to-noise ratio can be improved.
  • Fluid or reagent changes In particular, systems with the possibility of rapid and automatable change of fluids or reagents are therefore the subject of this invention. Such systems are described in WO 00/13017 and in WO 00/13018, incorporated herein by reference
  • Oligonucleotides, oligonucleotide derivatives or oligonucleotide analogs are synthesized in the reaction support such that their 3 'OH end is extendible to a polymerase. This can e.g. can be realized by linking the 5 'end to the reaction carrier with free 3'-OH end.
  • the nucleic acid molecules to be copied by a polymerase are hybridized to the attached molecules and the 3'-OH end of the probes is extended by the polymerase by linking nucleotides or nucleotide analogues. During extension, but not necessarily, a copy of the hybridized molecule can be made.
  • the newly formed strand may belong to a different class of compound than the hybridized strand, for example, nucleic acid derivatives and analogues may be incorporated into the strand or attached.
  • the course of the reaction can optionally be monitored optically, e.g. by incorporation of modified nucleotides or presence of additional signaling substances, e.g. interact with DNA.
  • the hybridized, non-reaction-carrier molecule can function as a primer and be extended. Again, but not necessarily, a copy of the molecule made in the reaction support can be made.
  • An example of extension of the primer molecule, in which no copy of the hybridized strand is made are template-independent extension reactions, as known to those skilled in the art. This can e.g. the production of poly-A tails formed by certain polymerases.
  • Methods for the preparation or preparation of an analytical method can also be carried out directly in the reaction medium. These include, for example, the distance or conversion of interfering impurities (such as by enzymatic processing), attachment of signaling groups or their precursors and attachment of certain groups to bind ligands such as proteins, nucleic acids, signaling molecules or their precursors. These linkages can be carried out by chemical or, for example, also enzymatic methods known to the person skilled in the art.
  • the reaction carrier can furthermore be used for the purification of sample molecules, which is based on the affinity of the desired sample molecules from the biological sample mixture to probe molecules located on the surface of the reaction carrier. This affinity chromatography-like method is based on the binding of the sample molecules to said probe molecules and one or more washing steps, in which the temperature can be varied.
  • capture oligos in the reaction support are synthesized in such a way that they are specific with their sequence for all or a selection of genes for a region "downstream" of the poly-A tail, whereby it may be advantageous to close this region in the vicinity of the 5
  • transcripts derived from an mRNA preparation or an already processed mRNA population eg a cDNA library
  • the next step may be complementary in the synthesis of capture oligos with distal 3 'end Strands are synthesized to the isolated strand This is done with the addition of appropriate known in the art enzymes and other starting materials.
  • copies of the strand covalently linked to the solid phase can now be made.
  • all full-length sequences starting from the binding site of the capture oligos
  • This can be used for linear amplification with corresponding poly-A primers.
  • An advantage of such a linear amplification is the low distortion of the concentration ratios of individual transcripts to one another.
  • conservative primer sequences can also be inserted proximal to the carrier, which allow exponential amplification of the isolated strands.
  • Compartments containing individual process steps in series can be created by hydrophobic barriers, valves, separate reaction chambers or similar engineering details of the reaction support known from microreactor technology.
  • the method according to the invention can be used to carry out a "sequencing by synthesis.”
  • a microarray of oligonucleotides, oligonucleotide derivatives or oligonucleotide analogs (probes) is prepared in the reaction support and hybridized with a nucleic acid sample to be analyzed molecules produced contain free 3'-OH ends, so that - as known in the art - an extension of the ends by a polymerase is possible.
  • Several methods are known which allow attachment of only one nucleotide and the compound of the phosphate backbone, This blocking group can be cleaved off within the miniaturized reaction carrier so that a nucleotide which can be extended by a polymerase is formed.
  • the nucleotide can contain, for example, signal-emitting groups or their precursors can be split off within the miniaturized reaction medium (such as fluorophores).
  • the cleavable blocking group may be bound by a ligand linked to a signaling group or its precursor (eg, fluorescently labeled antibody).
  • test systems developed by 454 Life Sciences, Helicos or Solexa, which were described in more detail under 2.3 (Bennett ST, Barnes C, Cox A, Davies L, Brown C. Pharmacogenomics, 2005 Jun; 6 (4): 373-82 RL, Sutton GG, Jones SJ, Holt RA, Bioinformatics, 2006 Dec 8; Bentley DR. Curr Opin Genet Dev. 2006 Dec; 16 (6): 545-52, Bennett S. Pharmacogenomics, 2004 Jun; 5 (4): 433-8 Margulies, M. Eghold, M.
  • the gene sections to be examined are immobilized on surfaces without information about their identity in order to subsequently sequence them according to the method described.
  • the information on longer gene segments is then obtained bioinformatic by assembling the small single information.
  • the entire genome must always be analyzed and the number and length of the individual sequenced regions must exceed a critical size in order to allow assembly by sufficiently overlapping the sections at all. In many cases, however, interest only exists for the sequence of part of the genome.
  • the process plant according to the invention can be used in particular for a multi-step processing and analysis of sample material in the following manner:
  • probes in the reaction support of the process plant according to the invention which are specific for gene sections to be analyzed
  • first desired gene segments by binding to the Probes are selected.
  • a washing step may be performed to remove unwanted sample material from the reaction support. It may then be an amplification of the sample material, which can already provide information about the sequence of the bound.
  • Numerous methods are known to the person skilled in the art.
  • a sequencing of the bound and optionally amplified sample molecules can be carried out according to the method described.
  • Such sequential processing and analysis of sample material is considerably simplified by the design of the reaction support as a microfluidic unit and thus offers a substantial improvement over the prior art.
  • Examples of such signal amplification are known to those skilled in the art, including but not limited to: Rolling circle amplification, tyramide-mediated amplification, chemiluminescence and bioluminescence, phosphatase-induced amplification or decoration of the bound target molecules by one or more other oligonucleotides, which in turn are already labeled, e.g. using "branched DNA” or "bDNA” from Genospectra, USA (Collins M. L. et al., Nucleic Acids Res. 25 (15): 2979-2984, 1997).
  • conjugates of streptavidin and an oligonucleotide linked thereto via the 5 'end may be used.
  • reaction carriers For most embodiments of the processes according to the invention and molecular biological process plants, a plurality of reaction carriers can be used. Important is the targeted supply of reagents or fluids and the appropriate assembly of functional biological molecules by spatially or / and time-resolved immobilization.
  • the reaction carriers can in principle be flat glass slides, as they are used as microscope slides.
  • Known slide and microarray, wherein the surfaces can be prepared with one of the numerous known in the art configurations for the binding of molecules, such as by reactive or activatable functional groups (epoxy groups, amino groups, etc.).
  • a further layer such as a gel, a polyacrylamide or a porous coating, which can also increase the loading capacity of the reaction carrier.
  • the reaction supports may take the form of three-dimensional microstructures, such as e.g. in WO 00/13018, in WO 02/46091 and in WO 01/08799.
  • the reaction carriers may contain a plurality of small holes or pores that may be parallel or orthogonal to inlets and outlets.
  • organic and inorganic materials e.g. Silicon, plastic, plastic, polypropylene, resins, polycarbonate, cyclic olefin copolymers or mixtures of these materials.
  • Three-dimensional structures can be integrated directly into the system according to the invention with suitable connection techniques.
  • Flat or non-closed reaction carriers are introduced accordingly in a flow cell or another three-dimensional reaction space, so that the necessary change of reagents or fluids can take place.
  • These constructs can be permanent, so that no change of the actual flat or non-closed reaction carrier is provided for normal operation. This can be done by gluing, screwing, indirect mounting, clamping or clamping.
  • the reaction carriers are reversibly introduced into the three-dimensional reaction space.
  • the person skilled in methods for holding reaction carriers in flow cells and measuring devices are known.
  • the three-dimensional reaction spaces or closed structures are then provided with appropriate connections for the supply of fluids and reagents.
  • the molecules produced in the reaction carrier can act as a template and are written off. This can not only be used for analysis of the reaction support when signaling devices are incorporated in the write-off, but can be used to make a copy of the reaction support in the form of a mixture of soluble copies of the Reaction carriers synthesized molecules to generate.
  • the reaction carrier can then be reused eg for a new copy.
  • An example of such a process is the transcription of DNA molecules in the reaction support by a primer extension reaction by a polymerase.
  • an amplification can also take place if, for example, an excess of primer and suitable changes in the temperature during the reaction result in repeated binding and lengthening of the primers.
  • the resulting copies can then be isolated by washing from the reaction medium.
  • a primer extension reaction can also be used without washing in order to convert, for example, DNA strands synthesized in the reaction support into double strands. These can be used to analyze, for example, proteins that bind or modify double-stranded DNA.
  • DNA synthesized in the reaction carrier can be transcribed into RNA. This can be done, for example, by the described previous conversion of the DNA strands synthesized in the reaction support into double strands and subsequent transcription. Numerous methods are known to the person skilled in the art. It is also possible to incorporate or attach nucleic acid analogs or derivatives that are not natural DNA or RNA building blocks.
  • Figure 23 illustrates the described embodiment and shows data from experiments demonstrating successful copying of primer extension probes synthesized on the surface of the reaction support. The created copies can then be removed by washing from the reaction support and used successfully as a template in a PCR reaction, where they are propagated.
  • the molecules synthesized in the reaction support can belong to different classes of compounds. For example, DNA or RNA molecules but also peptides in the reaction carrier can be synthesized. It is also possible to synthesize various derivatives and / or analogs of these classes of compounds in the reaction carrier. These include peptide nucleic acids (PNA) Locked nucleic acids (LNA), various nucleobase-modified nucleic acid derivatives and analogs, such as nucleic acids with altered hybridization behavior or attached functional groups such as haptens, fluorescent dyes, luminescent groups or their precursors, photoreactive groups, inorganic particles, photoisomerizable groups or Groups with a desired certain binding or reaction behavior or a desired optical behavior.
  • PNA peptide nucleic acids
  • LNA Locked nucleic acids
  • nucleobase-modified nucleic acid derivatives and analogs such as nucleic acids with altered hybridization behavior or attached functional groups such as haptens, fluorescent dyes, luminescent groups or their precursors
  • These include but are not limited to gold nanoparticles, stilbenes, azobenzenes, nitrobenzyl compounds, biotin, digoxigenin, quantum dots, phosphate, phosphorothioates, groups that increase the stability of the molecule to, for example, enzymes, groups that are substrates for enzymes, etc.
  • Molecules may also include branches or dendritic structures. It is also possible to synthesize molecules in the reaction carrier, which belong to several classes of compounds or consist of different, linked parts, each of which belongs to different classes of compounds.
  • the linkage can be direct or via specific linker groups.
  • nucleic acids can be linked to peptides and / or proteins.
  • to produce and alter the desired molecules not only e.g. organic chemical methods are used, but also e.g. enzymatic methods.
  • the specific primers, aptamers, ribozymes, aptazymes or other oligonucleotide probes or functional oligonucleotides or polynucleotides can be prepared in the same reaction carrier and in some embodiments also on the same array and dissolved in one of the process steps. These can either be equipped with suitable labile linkers or as copies of on the
  • Reaction carrier generated oligonucleotide probes are produced.
  • known methods for preparing such arrays of nucleic acid polymers e.g. in the form of a so-called microarray, very many (typically more than 10) different
  • Nucleic acid polymers of at least more than 2, typically more than 10 bases in length, can be produced.
  • a portion of the microarray (s) immobilized thereon is used as copyable matrices for enzyme-based synthesis
  • the next step in the process according to the invention is now that on the solid Phase synthesized molecules using appropriate enzymes to copy.
  • Numerous enzyme systems are known and commercially available for this purpose. Examples include DNA polymerases, thermostable DNA polymerases, reverse transcriptases and RNA polymerases.
  • the reaction products are characterized by a large variety of sequence, which can be programmed arbitrarily indirectly via the template molecules during the upstream synthesis process.
  • a microarray from the genome instrument can synthesize 6,000 freely selectable oligonucleotides with a sequence of up to 30 nucleotides in a microarray arrangement as a reaction space in a microchannel array.
  • After the copying step there are correspondingly up to 6,000 freely programmable DNA 30mers or RNA 30mers in solution and can be made available as reactants for a next process step.
  • primer molecules which serve as the initiation point for polymerases.
  • These primers may consist of DNA, RNA, a hybrid of the two or modified bases.
  • nucleic acid analogs such as PNA or LNA molecules as an example, is contemplated in certain embodiments.
  • the distal end of the sequence synthesized on the support is self-complementary and can thus form a hybrid double strand, which is recognized by the polymerases as an initiation point.
  • nucleic acids into vectors or plasmids. All of these methods are the use of nucleic acids as hybridisierbarigem
  • nucleic acid polymers not or not exclusively via a hybridization reaction. These include aptamers, ribozymes and aptazyme.
  • markers include labeled nucleotides, e.g. labeled with haptens or optical markers such as fluorophores and luminescent markers
  • Primers or nucleic acid analogs having particular properties e.g. special melting temperature or accessibility for enzymes.
  • initiation on the template nucleic acids can be carried out by any means known to those skilled in the art for initiating an enzymatic copying of nucleic acids, e.g. from the applications Polymerase Chain Reaction, Strand Displacement and Strand Displacement Amplification, / n-v / 7ro-replication, transcription, reverse transcription or viral transcription (representatives of which are T7, T3 and SP6).
  • a Tl, T3 or an SP6 promoter is incorporated into a part or all of the nucleic acid polymers on the reaction support.
  • nucleic acid molecules which serve to bind microRNAs are synthesized in the reaction carrier.
  • the nucleic acid molecules may consist of DNA, but also of nucleic acid analogs which have a changed hybridization behavior.
  • nucleic acids which are bound to the molecules synthesized in the reaction support are linked by enzymatic methods with a universal group. This can be done by extension by template-independent polymerases, e.g. Poly A polymerase or telomerase.
  • a primer extension reaction is used to generate duplexes from single-stranded nucleic acid molecules synthesized in the reaction support. These can be used to analyze binding or modification events by eg Serve proteins that bind to the duplex. For this purpose, it may be expedient to incorporate general sequence sections in the molecules synthesized in the reaction support, which serve, for example, as a binding site for one or more primers. It is also possible to insert chemical groups which allow a covalent linking of the two strands. Numerous examples are known to the person skilled in the art, for example the use of psoralen.
  • the portion of the array of nucleic acids that is provided for these reaction products the initiation of an isothermal copying reaction.
  • One representative of this procedure is the beach displacement reaction.
  • a primer is chosen which binds to the template polymers at their distal end and can then be extended there in the 3 'direction. All or some of the nucleic acid polymers on the carrier contain this primer sequence distally.
  • an enzyme is added, for which the primer contains a recognition site, so that a single-strand break is induced.
  • the usual procedure involves the use of a restriction nuclease, e.g. N.BstNB I (available, for example, from New England Biolabs), which inherently introduces single-strand breaks (so-called nicks) because it can not form dimers.
  • double-stranded, circular nucleic acid fragments are provided, one strand anchored to the surface of the support and the other strand comprising a self-priming 3 'end so that elongation of the 3' end can occur.
  • the enzymatic synthesis in this variant of the method according to the invention comprises a replication analogous to the known for the replication of bacteriophages ito / Vmg-C / rc / e mechanism, wherein one strand of the annular nucleic acid fragments is anchored to the surface of the carrier and can be copied several times , If a double-stranded closed nucleic acid fragment is initially present, the second strand can first be opened by a single-strand break, forming a 3 'end, from which the elongation takes place. The cleavage of the elongated strand can be carried out enzymatically, for example.
  • nucleotide building blocks and a suitable enzyme By addition of nucleotide building blocks and a suitable enzyme, synthesis of the partial sequences complementary to the base sequences of the nucleic acid strands anchored on the surface of the support is then carried out.
  • single-stranded, circular DNA molecules are used to be copied in an i0 // mg OVc / e amplification.
  • primers nucleic acid molecules synthesized in the reaction support or nucleic acid molecules which interact with the molecules synthesized in the reaction support hybridize.
  • Preference is also given to using oligonucleotides which are linked to streptavidin as primers.
  • the streptavidin-biotin conjugate may have previously bound to biotin units previously attached to hybrids of probe molecules and sample molecules.
  • nucleic acid molecules hybridizing with the molecules synthesized in the reaction support may contain a universal group, such as a poly A tail. It may be expedient for this method to use universal binding sites in the circular DNA molecule for the binding of the primer. This creates long concatemers, in which signaling molecules are incorporated and can be used for analysis.
  • the resulting concatemers may serve as a template for other extendable molecules. These hybridize to the strand formed by the rolling C / rc / e amplification and are extended by a polymerase. Since several molecules can sequentially bind to each other, a molecule can reach a length by its extension, adjacent to the end of a molecule bound to the same strand.
  • the extension can be continued, if the hybridization of the second molecule is resolved by the progressive extension ("Strand displacement") and the hybridization region of the second molecule is copied again by the extension of the first molecule by the resolution of the hybridization of a molecule arise again
  • Single-stranded domains that can serve as a template for an extendable molecule give rise to complex, branched dendritic structures, and, in particular, signaling groups or their precursors or haptens can also be incorporated into the growing strand during elongation of the bound molecules may also contain signaling groups or their precursors or haptens, and the structures formed may be bound by substances that bind to the structures formed by the extension Change one or more of their optical properties experienced.
  • the products of the copying process can be given various labels, binding sites or markers that are desired for further processing or use in further assays or procedures.
  • markers and labels that allow for direct detection of the copies and are known to those skilled in the art from other methods of copying nucleic acids. Examples are fluorophores. Furthermore, binding sites may be provided for indirect detection or purification procedures. These include as examples haptens, such as biotin or digoxigenin.
  • the labels, binding sites or markers can be modified in a variant by Nucleotides are introduced. Another way is to use primers to initiate the copying process. The primers can already be introduced into the reaction with labels, binding sites or markers.
  • Label, binding sites or markers can subsequently be introduced by treating the reaction products of a subsequent labeling reaction with generic agents which react with the nucleic acids.
  • generic agents which react with the nucleic acids.
  • An example of this is cis-platinum reagents or nanogold particles, as e.g. offered by the company Aurogen, USA.
  • labels, binding sites, or markers may also be introduced by a further enzymatic reaction, such as, e.g. catalyzed by a terminal transferase.
  • the copies of the template nucleic acids in turn are used to react with the bound target nucleic acids. The initiation of their synthesis as copy products of nucleic acid probes can take place during or after the specific binding of the target molecules.
  • the non-specifically bound or unbound sample material is first washed away.
  • the sequences of the nucleic acid probes to be copied are selected such that the sequence to be analyzed later in a hybridization reaction only arises upon successful extension of the individual nucleic acid polymers copied in solution. These sections can then be detected by means of another area of the array.
  • Initiation of the copying process already carry a modification that supports the generation of the signal.
  • An example of such a modification is a primer carrying a branched DNA structure in its 5 'portion in a region that is not necessary for hybridization with the template (for bDNA see above).
  • Another variant provides that for each target sequence, e.g. a single gene or exon, two primers of opposite specificity are provided such that efficient exponential amplification occurs in a PCR or isothermal amplification.
  • An associated device as a preferred embodiment of the system according to the invention consists of a) a device for the in-situ synthesis of the arrays of template polymers and analytical nucleic acid probes, b) elements for the processing of fluidic steps, such as the sample addition, reagent addition, washing steps or / and sample extraction c) a detection unit for the detection of an optical or electrical signal, d) a programmable logic control unit, e) a programmable logic fluid control unit, detection and storage and management of measurement data.
  • the extended polymers are contacted with analytical nucleic acid probes, which in turn are useful for extension in the form of primer extension.
  • the arrangement of a primer extension expectorant is known from the specialist literature.
  • the signal of the primer extension to these analysis probes is then evaluated to determine the analysis result.
  • Such an analysis may e.g. the determination of single nucleotide polymorphisms (SNPs) in genomic DNA.
  • SNPs single nucleotide polymorphisms
  • only extensible primers are copied to template nucleic acids. The sequence is selected such that the SNPs to be examined are located in the 3 'region after the primer sequence on the target nucleic acid. In the next step, these primers are extended beyond the sequence of SNPs to be detected.
  • the reaction products of this extension are examined by primer extension or directly by hybridization and the results for the determination of the SNPs queried in the analysis are registered.
  • the data are processed in such a way that e.g. directly receive a report with the base positions and the found bases.
  • the big advantage of the invention lies in the fact that only a universal, generic sample preparation is necessary for such genotyping or SNP analysis assays. Primers and reagents specific to individual genotypes or SNPs are not needed since all sequence specificity is derived from the ms / tw synthesis of the underlying template array and the analysis array. In the embodiment with the combination of these two in a reaction carrier, the genotyping and SNP analysis is thereby maximally simplified. 6.11 Production of synthetic genes and other synthetic nucleic acid double strands using the method according to the invention by processing nucleic acids which have been prepared outside the reaction carrier
  • oligonucleotides which serve as building blocks of the synthetic gene, are prepared by synthesis in the reaction carrier.
  • carrier-bound libraries of nucleic acid probes is described for the synthesis of synthetic genes in PCT / EP00 / 01356.
  • the synthesis of oligonucleotides by copying carrier-bound nucleic acids e.g. for gene synthesis or for the preparation of reagents such as siRNAs or aptamers is described in DE 103 53 887.9.
  • oligonucleotides with a freely selectable sequence in a range from 10 to 100, possibly also up to 500 nucleotides are provided for the subsequent methods, such as the construction of synthetic genes.
  • oligonucleotides prepared outside the reaction carrier can be linked to the oligonucleotides synthesized in the reaction carrier by methods known to those skilled in the art.
  • high quality and freely programmable nucleic acids in the form of oligonucleotides are provided to produce synthetic double-stranded DNA (synthetic genes).
  • synthetic genes synthetic double-stranded DNA
  • the method according to the invention is used.
  • the oligonucleotides, which serve as building blocks of the synthetic gene, are prepared by synthesis and detachment by means of a labile linker or by synthesis via a copy of nucleic acid probes.
  • carrier-bound libraries of nucleic acid probes is described for the synthesis of synthetic genes in PCT / EP00 / 01356.
  • oligonucleotides by copying carrier-bound nucleic acids, for example for gene synthesis or for the preparation of reagents such as siRNAs or aptamers, is described in DE 103 53 887.9.
  • oligonucleotides with a freely selectable sequence in a range from 10 to 100, possibly also up to 500 nucleotides are provided for the subsequent methods, such as the construction of synthetic genes.
  • the subsequent methods such as the construction of synthetic genes.
  • the method according to the invention are more
  • Process steps which comprise the use, purification, modification or refinement of the oligonucleotides or the partial or complete construction of the target sequence, that is to say optionally of the finished synthetic gene, are carried out according to the process in an appropriate reaction carrier.
  • two strands are linked, one of which is a probe molecule synthesized in the reaction support.
  • the linkage is made possible by a template strand, which brings the two strands to be linked in spatial proximity.
  • the probe molecule synthesized in the reaction support can serve as a template, which brings two further strands in close proximity and thus allows a linkage.
  • the ligation may be e.g. be catalyzed by a ligase, but also by the skilled person known chemical coupling reactions.
  • the probe molecule synthesized in the reaction support can either be immobilized on the surface of the reaction support or have been detached before the attachment.
  • a transcription of the molecules of the reaction carrier before the linkage can be made and the linkage with the molecules that make up the copy done.
  • methods known to the person skilled in the art can be used, for example a primer extension reaction.
  • reaction support as a microfluidic system in combination with pumping systems is ideally suited for sequential modifications of molecules produced on the reaction support or those prepared on the reaction support Make molecules binding molecules. Since the molecules to be modified are immobilized on or bind to the reaction support, washing steps between the various modification events are greatly simplified over current methods.
  • a variety of molecular biological processes known to those skilled in the art include several sequential steps of particular modifications between which a purification step occurs. These are, for example, enzymatic modifications such as amplification, primer extension, ligation, phosphorylations or dephosphorylations, nuclease treatments, etc.
  • the purification steps are, for example, binding and washing of the sample with the aid of affinity columns, precipitation steps, gel electrophoretic methods, etc. In one embodiment, the invention is used to: consecutive
  • Modification events of molecules to perform The design of the reaction support, whose microfluidic channels can be flushed through with solutions and mixtures, can achieve a considerable simplification of such processes in comparison with methods of the prior art.
  • a washing step may be carried out in each case, in order to obtain e.g. Remove substances of a modification step that could interfere with a subsequent step.
  • asymmetric polymer probes are used in particular for the polymer probes. These allow the practice of the invention in a variant in which such probes, which are full-length products, are thermodynamically favored by other factors than solely by the fact that they are full-length products. This is achieved by making the probes individual building blocks with a particularly strong
  • binding behavior Including binding behavior. These particular building blocks are introduced asymmetrically or in a later step during polymer synthesis. This creates an asymmetry that gives the probes thermodynamic properties that affect the binding behavior.
  • nucleic acids In the case of nucleic acids, analogues are used which contribute to a stronger binding to the complementary bases. Alternatively, such building blocks are inserted distally on the probe molecules, which influence the binding behavior and contribute to stronger binding of these probes.
  • building blocks are inserted distally on the probe molecules, which influence the binding behavior and contribute to stronger binding of these probes.
  • peptide derivatives For example, those skilled in the art are aware of "minor groove binders" which are also used in the polymerase chain reaction.
  • the proportion of the full-length product can fall below a critical value after a certain number of synthesis cycles, so that the analysis result is not at all characterized by this full-length product.
  • the coupling rate of the individual addition steps is below 95% (Beier, M., Hoheisel, JD, Production by quantitative photolitogaphic synthesis of individually tested DNA microarrays, Vol. 28, No. 4, p. 6, 2000). With such methods, it makes sense to produce only DNA polymer probes up to a length of 25 bases.
  • the goal is to avoid the disadvantages described, which can result from a population of molecules of different lengths on the individual positions of a microarray, without accepting the disadvantages of an "off-chip" synthesis to have to.
  • This embodiment of the invention with asymmetrical probes thus describes one
  • This is achieved by an asymmetric configuration of the polymer probes.
  • modified building blocks are used, which in certain thermodynamic properties, such as. the binding stability, different from the previously used building blocks.
  • the same effect may be achieved by suitable modification of the distal end of the polymer probes, e.g. B. with a hybridization amplifier can be achieved.
  • Such a molecule is e.g.
  • Minor Groove Binder (Epoch Biosciences 2000 Annual Report, pages 4-5), which significantly increases the stability of binding to the last 4-5 bases of the polymer probe.
  • minor groove binders are some natural antibiotics having a shape that allows for folding into the minor groove of a DNA helix. This substitutes the lack of purification of the polymer probes in / w-s / ta syntheses prior to application in a polymer probe array. The quality disadvantage of / n-szYw synthesis methods is thus partially or fully compensated.
  • the method described as an embodiment of the invention improves the usability of in-situ synthesized polymer probe arrays in terms of the quality and validity of the analysis.
  • the method is further improved.
  • Modified synthetic building blocks are, for example, ribonucleoside analogs, such as locked nucleic acids (LNAs), modified purine or pyrimidine bases, such as superstabilizers
  • LNAs locked nucleic acids
  • pyrimidine bases such as superstabilizers
  • Adenosine analogues e.g., 2,4-diaminoadenosine
  • pyrazolopyrimidines e.g., PPG
  • Phosphate backbone analogs such as. As methylphosphonates, phosphorothionates, phosphoramidates, etc.
  • duplex stabilizers are building blocks which can lead to triple helix formation by a third nucleic acid or peptide strand, as well as stabilizing molecules, such as e.g. Intercalators intercalated between the base stacking of a DNA double strand.
  • Another aspect of the invention is the combination of the asymmetric probe design with m-s / tw purification methods in which the termination products of the probe synthesis are removed in situ.
  • the postsynthetic array optimization is enabled in this embodiment by the modified building blocks at the end of the last extended polymer probes. Shorter probes predominantly carry no such modified building blocks and can be obtained by suitable methods, such as e.g. a chemical and / or enzymatic digestion.
  • An object of the invention is therefore a method for producing a carrier for the determination of nucleic acid analytes by hybridization, comprising the steps: (a)
  • the different sets of building blocks are selected so that the individual building blocks with respect to the specificity for complementary nucleic acid building blocks are equal to the analyte, but have a different affinity for complementary nucleic acid building blocks from the analyte so that the preference for full length products of an in situ synthesized polymer probe array is achieved by targeted distribution of different types of building blocks along the polymer probes during synthesis.
  • the method according to the invention uses sets of synthesis building blocks that behave the same with regard to certain parameters, but in certain, e.g. thermodynamic, properties differ from each other.
  • the distribution of the building blocks along the growing polymer during the m-s / Y "synthesis is chosen so that the full-length products with the number of building blocks n or at least the synthesis products from the last addition steps of the polymer extension modified building blocks.
  • n can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.
  • a set of synthetic building blocks is used which has a higher affinity for complementary nucleic acid building blocks from the analyte than those previously used.
  • the set of synthesis building blocks used for the last step or steps in the construction of the receptors additionally a higher resistance to degradation reagents, eg. As enzymes, such as nucleases and / or chemical reagents, such as acids or bases, compared to the set used for the first steps of the construction of the receptors set of synthetic building blocks.
  • a targeted degradation step can be performed, with which the proportion of non-full-length products compared to the proportion of full-length products is reduced.
  • the incorporation of "degradable" building blocks and a subsequent degradation step can also take place one or more times during earlier steps of the receptor synthesis.
  • hybridization enhancers are site-specific, ie, increased hybridization affinity for complementary nucleotide building blocks from the analyte is provided for a predetermined number (ie, set) of individual building blocks from the receptor.
  • the hybridization enhancer is added to the distal end of the receptor, eg, modifying the last 3-5 bases of the receptor for hybridization affinity.
  • a nucleic acid array selected from DNA or RNA arrays, in particular a DNA array, constructed, wherein a first set of synthesis blocks, consisting of unmodified DNA or RNA synthesis blocks, which expediently in Form of suitable derivatives with phosphoramidites, H-phosphonates, etc., is used.
  • the second set of final or final steps of the receptor assembly is then a set of synthetic building blocks selected from N3'-P5'-phosphoramidate (NP) building blocks, Locked nucleic acid (LNA) building blocks, morpholinophosphordiamidate (MF) - Building blocks, 2'-O-methoxyethyl (MOE) building blocks, 2'-fluoro arabino-nucleic acid (FANA) building blocks, phosphorothioate (PS) building blocks, 2'-O-methyl (OMe) building blocks or peptide nucleic acid (PNA) building blocks.
  • NP N3'-P5'-phosphoramidate
  • LNA Locked nucleic acid
  • MF morpholinophosphordiamidate
  • MOE 2'-O-methoxyethyl
  • FANA 2'-fluoro arabino-nucleic acid
  • PS phosphorothioate
  • OMe 2'-O-methyl building blocks or
  • inventive method is also suitable for the construction of modified nucleic acid arrays, wherein a first modified block set is used as the first set of blocks and a second modified block set is used as a second set, wherein the two sets of blocks, as described above, with respect to Distinguish affinity for complementary nucleic acid components of the analyte and optionally additionally with respect to the resistance to degradation reagents.
  • This variant of the method according to the invention circumvents the cleaning problem for m-5 / Yw polymer probes by an asymmetrical configuration of the probes, which leads to an increased contribution of the full-length products to the binding energy in the double strand in the case of nucleic acids in a later application on the biochip.
  • This variant of the method according to the invention is suitable, in addition to the other applications described in this disclosure, for the detection or / and isolation of nucleic acids, eg. To perform Z) e "ovo-sequencing, re-sequencing and point mutation analyzes, e.g. B. SNP analyzes and the detection of new SNPs.
  • the method can be used for the analysis of genomes, genome variations, genome instabilities and chromosomes as well as gene expression or transcriptome analysis or for the analysis of cDNA libraries.
  • the method is also suitable for the production of substrate-bound cDNA libraries or cRNA libraries.
  • arrays can be generated for the production of synthetic nucleic acids, nucleic acid double strands and synthetic genes.
  • arrays of PCR primers, probes for homogeneous assays, molecular beacons and hairpin probes can also be prepared.
  • arrays can also be generated for the production, optimization or development of antisense molecules.
  • the inventive method is particularly suitable for the production of carrier bodies with channels, z. B. with closed channels.
  • the channels are in one embodiment microchannels with a cross section of z. B. 10-1000 microns.
  • suitable carrier bodies with channels are described in WO 00/13018.
  • a carrier body is used which is at least partially optically transparent and / or electrically conductive in the region of the positions to be equipped with receptors.
  • This variant of the method according to the invention is furthermore particularly suitable as integrated synthesis analysis method, i. the finished support is used in situ for the determination of analyte and then optionally for further synthesis-analysis cycles as described in WO 00/13018.
  • this variant of the method according to the invention also relates to a carrier for the determination of analytes containing a plurality, preferably at least 100 and more preferably at least 500, of different immobilized receptors, wherein the receptors from each of several different, eg. B. two or more
  • complementary nucleic acid building blocks from the analyte With respect to the specificity for complementary nucleic acid building blocks from the analyte are the same, but have a different affinity for complementary nucleic acid building blocks from the analyte.
  • this variant of the method according to the invention relates to a reagent kit comprising a carrier body and at least two different sets of building blocks for the synthesis of receptors on the carrier. Furthermore, the reagent kit may also contain reaction liquids.
  • This variant of the method according to the invention also relates to a device for integrated synthesis and analyte determination on a carrier, comprising a programmable light source matrix, optionally a detector matrix, a carrier preferably arranged between light source and detector matrix when using a detector matrix and means for supplying fluids in the carrier and for the discharge of fluids from the Carrier and optionally reservoirs for synthesis reagents and samples.
  • the programmable light source or exposure matrix may comprise a reflection matrix, a light valve matrix, e.g. Example, an LCD matrix, or a self-emitting exposure matrix. Such light matrices are disclosed, for example, in WO 00/13018.
  • the detector matrix, z. As an electronic CCD matrix, may be integrated as an option
  • the structure of the receptors on the support can include fluid-chemical synthesis steps, photochemical synthesis steps, electrochemical synthesis steps, or combinations of two or more of these steps.
  • An example of the electrochemical synthesis of receptors on a support is described in DE 101 20 663.1.
  • An example of a hybrid process comprising the combination of fluorochemical steps and photochemical steps is described in DE 101 22 357.9.
  • a DNA microarray is synthesized to a length of DNA probes of 25 building blocks.
  • an analogue with suitable properties is condensed on the probe.
  • This may be a locked nucleic acid (LN A) bridge, which is known to be able to be produced for all four bases of the DNA (and for a set of matching building blocks), and for all
  • LN A locked nucleic acid
  • DNA or other nucleic acid polymer probes are used which are all or partially capable of forming desired three-dimensional structures.
  • These three-dimensional structures may be hairpin structures or other structures known to those skilled in the art.
  • the invention encompasses arrays of nucleic acids immobilized on a support that are at least partially in the form of secondary structures, such as hairpin structures. Furthermore, methods for making such arrays and applications thereof are claimed.
  • a binding event between immobilized receptor and analyte is usually detected by detection of a label group bound to the analyte.
  • a carrier and method for analyte determination that allow integrated synthesis of receptors and analysis are known e.g. As described in WO 00/13018.
  • receptor arrays e.g. As DNA chips, complex biological issues (gene expression studies, target validation, sequencing by hybridization, re-sequencing) to be able to process, it is of fundamental importance that the hybridization between the receptor and target can be performed as error-free.
  • the detection system must therefore be able to distinguish between a so-called “filling match”, ie when the probe and target are completely complementary, and a "mismatch" when one or more erroneous base pairs are present.
  • a so-called “filling match” ie when the probe and target are completely complementary
  • a mismatch when one or more erroneous base pairs are present.
  • it is particularly difficult to distinguish between a single mismatch if only 1 base pair is faulty and the fill match.
  • terminal thermodynamic reasons particularly terminal (terminal) base mismatches are difficult or insufficient to detect. Mismatches in the middle of a sequence, however, are easier to detect for the same reasons.
  • Nucleic acid receptors are present in known single-stranded form on known DNA chips. In selecting the sequences for the receptors, care is therefore taken to avoid any formation of secondary structures.
  • the detection of base mismatches now takes place in that not only the actual query sequence, but also the corresponding sequence with a mismatch in the middle of the base sequence is applied as a negative control on the DNA chip. Whether it is a "filling match” or “mismatch” is detectable by the respective different signal intensities which result from hybridization of the sample (target) to the probe or its negative control sequence (mismatch sequence).
  • An object of this embodiment of the invention is therefore to provide a system ready make it possible to detect base mismatches very accurately. Furthermore, the system according to the invention should detect not only base mismatches in the middle of a sequence, but also at the end (terminal) on an array in high parallel.
  • This object is achieved by providing receptor arrays containing nucleic acid receptors that are at least partially in the form of hairpins.
  • Hairpins are a special form of secondary structures in nucleic acids, which are composed of two complementary sequence sections in the so-called star and a further sequence section in the so-called loop (FIG. 1a). In this case, there is a balance between the closed mold and the opened mold (FIG. 1b). Hairpin structures have already been used in solution for the label-free detection of hybridization events (Tyagi et al., Nature Biotechnology 1995, 14, 303-308). These hairpin structures ( Figure 2 type A) are characterized in that the recognition sequence is in the loop of the hairpin (Marras et al., Genetic Analysis, Biomolecular Engineering, 1999, 14, 151-156).
  • a quencher and a fluorophore molecule in the closed state are in close spatial proximity, so that the fluorescence is deleted. If a hybridization event occurs with the recognition sequence in the loop, the hairpin opens, whereby the fluorophore and the quencher are spatially separated. As a result, a fluorescence signal is observed.
  • poly-deoxyguanosine sequences can act as quenchers (M. Sauer, BioTec, 2000, 1, 30 ff). This has the advantage that the hairpin structure only has to be labeled with a fluorophore (M. Sauer et al., Anal. Chem. 1999, 71 (14), 2850 ff) that the incorporation of a quencher molecule is omitted.
  • An object of the invention in this embodiment is a method for the determination of analytes, comprising the steps of: a) providing a carrier with a plurality of predetermined regions, on each of which different receptors selected from nucleic acids and nucleic acid analogs are immobilized, wherein in one or more of the predetermined areas, the receptors in the absence of an analyte specifically bindable thereby at least partially present as a secondary structure.
  • this refers to each individual receptor in such a way that the presence of the respective analyte capable of binding specifically causes a change or abolition of the secondary structure in the receptor.
  • Another object of the invention is a device for the determination of analytes, comprising a) a light source matrix, b) a carrier having a plurality of predetermined positions, on each of which different receptors are immobilized on the carrier, c) means for supplying fluids to the carrier and the Discharging fluids from the carrier; and d) a detection matrix comprising a plurality of detectors associated with the predetermined positions on the carrier.
  • the hairpin structures according to the invention can surprisingly be used for a very precise discrimination of base mismatches on a solid phase, in particular on an array.
  • the hairpin structures according to the invention can be produced highly parallel both in situ on the solid phase, but can also, if prefabricated, be immobilized thereon.
  • the receptors are selected from nucleic acid biopolymers, e.g. As nucleic acids such as DNA and RNA or nucleic acid analogues such as peptide nucleic acids (PNA) and Locked nucleic acids (LNA) and combinations thereof. Particular preference is given to determining nucleic acids as analytes, where the binding of the analytes comprises hybridization. However, the method also allows the detection of other receptor-analyte-change effects, eg. B. the detection of nucleic acid-protein interactions.
  • nucleic acid biopolymers e.g. As nucleic acids such as DNA and RNA or nucleic acid analogues such as peptide nucleic acids (PNA) and Locked nucleic acids (LNA) and combinations thereof. Particular preference is given to determining nucleic acids as analytes, where the binding of the analytes comprises hybridization. However, the method also allows the detection of other receptor-analyte-change effects, e
  • This variant of the method according to the invention preferably comprises a parallel determination of a plurality of analytes, ie a carrier is provided which contains a plurality of different receptors which can react with respectively different analytes in a single sample.
  • the number of different receptors on a support is preferably at least 50, more preferably at least 100, even more preferably at least 200, even more preferably at least 500, even more preferably at least 1000, even more preferably at least 5000, even more preferably at least 10,000, even more preferably at least 50,000.
  • at least 50, preferably at least 100 and more preferably at least 200 analytes are determined in parallel.
  • Immobilization of the receptors to the support may be by covalent bonding, non-covalent self-assembly, charge interaction, or combinations thereof.
  • the covalent bond preferably comprises the provision of a carrier surface with a chemically reactive group, to which the starting components for receptor synthesis, preferably via a spacer or linker can be bound.
  • the non-covalent self-assembly can be carried out, for example, on a precious metal surface, e.g. As a gold surface, using thiol groups, preferably via a spacer or linker done.
  • the present invention is preferably characterized in that the detection system for analyte determination, a light source matrix, a microfluidic carrier and a detection matrix combined in an at least partially integrated structure.
  • This detection system can be used for integrated synthesis and analysis, in particular for the construction of complex carriers, for. As biochips, and for the analysis of complex samples, eg. For genomic, gene expression or proteome analysis.
  • the synthesis of the receptors takes place in situ on the support, for example by passing fluid with receptor synthesis units over the support, the building blocks being immobilized on respectively predetermined areas on the support in a location-specific or / and time-specific manner and these steps being repeated, until the desired receptors have been synthesized at the respective predetermined regions on the support.
  • This receptor synthesis preferably comprises at least one fluid chemical step, one photochemical step, one electrochemical step or a combination of such steps and one online process monitoring, for example using the detection matrix.
  • the light source matrix is preferably a programmable light source matrix, e.g. B. selected from a light valve matrix, a mirror array, a UV laser array and a UV LED (diode) array.
  • a programmable light source matrix e.g. B. selected from a light valve matrix, a mirror array, a UV laser array and a UV LED (diode) array.
  • the carrier is preferably a flow cell or a microfluidic cell, ie a microfluidic carrier with channels, preferably with closed channels, in which the predetermined positions with the respectively differently immobilized receptors are located.
  • the channels preferably have diameters in the range of 10 to 10,000 ⁇ m, more preferably from 50 to 250 microns and may in principle be configured in any form, for. B. with round, oval, square or rectangular cross-section.
  • the secondary structures in this embodiment of the invention preferably comprise a hairpin structure composed of a star and a loop.
  • the sequence of the receptor which is capable of binding with the analyte can be in the region of the loop of a hairpin. Binding the loop to the receptor opens the hairpin structure. This hairpin opening can in turn be detected by suitable means (eg see above).
  • suitable means eg see above.
  • the specific binding of the analyte sequence of the receptor is located in the star of the hairpin structure.
  • the binding of the analyte to the receptor causes a detectable dissolution of the hairpin structure.
  • the hairpin structures according to the invention having a recognition sequence in the star comprise complementary sequences A and A * in the star and a linker unit L in the loop.
  • base pairings eg polyethylene glycol, alkyl, polyethylene glycol phosphate or alkyl phosphate units
  • building blocks which can only accept weak base pairings eg a Tn loop
  • a sequence A located in the sample to be examined with the reference sequence A in the star of the hairpin around the sequence A * ( Figure 39A).
  • This competitive situation is used to increase the specificity of the hybridization. Is z.
  • the sequence A in the sample is not completely complementary to A * (ie, mismatches occur)
  • the pairing between the two sequences A and A * is more stable in the hairpin, with the result that the hybridization equilibrium is on the left side is displaced to the closed shape of the hairpin (Fig. 39A).
  • a labeled sample A is used for hybridization, this means that no or only a small signal is detectable, since the equilibrium lies on the side of the closed hairpin.
  • the hybridization equilibrium (and thus the stringency) of the reference probe or of the recognition sequence can be varied, inter alia, by virtue of the fact that these sequences contain building blocks of nucleic acid analogues which are characterized in that they bind more strongly with DNA than DNA with DNA ,
  • PNA or LNA building blocks or other components known to those skilled in the art with the described characteristics come into question.
  • the procedure described achieves that, in contrast to the usual procedure (using 1 Perfect Match + 1 single base mismatch probe), only a single probe is used for the discrimination between Perfect match and single base mismatch must, and thus parking spaces on the array can be saved or more information can be queried with a predetermined amount of parking spaces. Furthermore, this can also be queried terminal mismatches, since by the presence of the reference sequence in the same molecule higher stringency conditions can be set than when used for the discrimination of Perfect match and single-base mismatch 2 separate probes.
  • the hairpin structures comprise two complementary sequences (A, A *) and two non-complementary units (Z, X) in the star and one linker unit (L) in the loop ( Figure 40).
  • recognition sequences both the solid phase near sequence sequence AZ (FIG. 40A) and the solid phase remote sequence sequence A + -Z (FIG. 40B) can serve as recognition sequences.
  • X and Z do not mate with each other.
  • X is one or more nucleic acid building blocks which can be coupled and Z is one or more non-combinable building blocks.
  • Z may be, for example, an "abasie site” (DNA or RNA building block without heterobase) or a building block known to the person skilled in the art, which does not undergo base pairing but does not disturb the DNA structure.
  • Z may also be the mixture of the 4 bases adenosine, guanosine, cytidine and thymidine or uracil.
  • the hairpin structure includes an at least partially quenched label group in the closed state, e.g. B. a fluorophore.
  • a hairpin structure of the invention ( Figure 41) includes, for example, a quencher (Q) and a fluorophore (F) located at opposite ends of the nucleic acid sequence of the hairpin.
  • Q quencher
  • F fluorophore
  • the fluorescence in the closed hairpin is quenched by the spatial proximity of Q and F. When opened, fluorescence is detectable.
  • the hybridization can also be carried out with double-stranded targets (FIG. 42). If both strands are marked, the luminous intensity detectable for a parking space can thus be increased.
  • the carrier connection of the hairpin structures of both type A (recognition sequence in the loop) and of type B (recognition sequence in the star) can be carried out not only terminally but also internally (FIG. 43). It also discloses hairpin structures of type A bound internally to the wearer. Combinations of terminally and internally immobilized receptors on a support are possible.
  • the hairpin structure according to the invention comprising the recognition sequence in the stem.
  • the complementary sequence to the recognition sequence is used as a reference sequence for mismatch discrimination.
  • a target sequence present in the sample solution competes with the reference sequence (A *) for the probe sequence (A).
  • stringency can be further enhanced by incorporation of particular nucleic acid building blocks (PNA, LNA) in the reference strand. This means that only one hybridization will take place - ie the hairpin will change to the open form - if the target sequence present in the sample solution is exactly complementary to the probe sequence. If this is not the case, the hairpin built-in reference sequence ensures that the hairpin does not switch to the open form, and thus can not be hybridized with a target sequence.
  • PNA nucleic acid building blocks
  • the hairpin structure according to the invention allows the discrimination of terminal base mismatches. These are the hybridization of the target sequence to the
  • Position X possible.
  • Base pairings complementary to the terminal position X decide whether the hairpin increasingly changes to the open form and as a result a hybridization event can be detected.
  • a device for this purpose, comprising: a) a light source matrix, b) a microfluidic carrier having a plurality of predetermined positions, on each of which different receptors selected from nucleic acids and nucleic acid analogs are immobilized on the carrier, wherein in one or more of the predetermined regions the receptors c) means for supplying fluids to the carrier and for discharging fluids from the carrier, and d) a detection matrix comprising a plurality of detectors associated with the predetermined regions on the carrier.
  • the carrier is arranged between the light source matrix and the detection matrix.
  • Detectors of the detection matrix are preferably photodetectors and / or electronic detectors, eg. As electrodes selected.
  • the device according to the invention can be used for the controlled in situ synthesis of nucleic acids, eg.
  • DNA / RNA oligomers can be used as temporary protective groups photochemical, fluid chemical, and / or electronically cleavable protecting groups can be used.
  • the spatially or / and time-resolved receptor synthesis can be carried out by targeted control of electrodes in the detection matrix, targeted fluid supply to defined areas or area groups on the support and / or targeted exposure via the light source matrix.
  • All three-dimensional and fully or partially controlled or wholly or partially uncontrolled secondary and three-dimensional polymer probe structures can be used for binding studies and the multistep molecular biological processes of the invention such that the binding of proteins, peptides, cells, cell fragments, organelles, saccharides, Low molecular weight drugs, complex molecules, nanoparticles, synthetic organisms or molecules or cells from synthetic biology are analyzed and possibly optimized. From this, in one embodiment, the investigation of the binding of proteins from cell extracts or m-vz7ro production can be deduced whose binding pattern is examined on a set of sequence motifs. This set of sequence motifs can consist of a set of binding sites for transcription factors. Furthermore, this set may consist of hypothetical or empirically validated binding sites. A mixture of hypothetical or empirically validated binding sites may also be provided.
  • the three-dimensional and wholly or partially controlled or wholly or partially uncontrolled secondary and three-dimensional polymer probe structures for binding studies and the multistep molecular biological processes of the invention are used in such a way that thus the binding pattern of microRNA, other non-protein-coding RNA molecules or partially from RNA, partially consisting of proteins or peptides existing complexes with the double-stranded hairpin structures, other structures or derived from the hairpin structures double strands on the reaction carrier and possibly detected by markers on the analyte.
  • a FRET reaction can be generated and used for further analysis or information gathering. The FRET effect can be caused by a polymer probe and target interaction.
  • an acceptor or donor molecule is coupled to the assembled polymer probes. This can be done during the synthesis (by the building blocks) or after the synthesis, eg with reagents like Cis-Platin (see eg KREATECH ULS-Cy5).
  • the sample carries the corresponding other label to enable the FRET, eg the pairing Cy5 / Cy3 or phycoerythrin, or a quencher matching the donor. Energy transfer can only take place near the surface, so that autofluorescence of the solution or unbound, freely labeled sample material does not cause any disturbing background fluorescence.
  • the probe with free 3 'end can be the fluorescence acceptor, and by a polymerase reaction as described above (eg a primer extension) or another enzyme reaction (eg a ligation), an appropriate donor (EX) is attached to the bound complex or to the polymer probe itself, which enables the FRET.
  • a polymerase reaction as described above (eg a primer extension) or another enzyme reaction (eg a ligation)
  • EX enzyme reaction
  • the "big-dye” principle for "four-color sequencing” can be enabled in this way.
  • a donor exciter molecule fluorescein, etc.
  • a donor exciter molecule is excited and transfers its energy to the primer extension-attached dd nucleotide dye molecules.
  • the FRET reaction is made possible by incorporation of acceptor and donor molecule pairs in the sample after or during attachment to the polymer probe, eg, during or through a primer extension reaction.
  • this leads to high marking densities, so that many FRET transmissions can take place.
  • a particular advantage lies in an embodiment with a combination of FRET and CCD detection, since this allows the direct detection of the course of the reaction.
  • polymer probes are immobilized in or on a reaction support. This can be covalent or non-covalent. These polymer probes are constructed of nucleic acids or their analogs. On these polymer probes, the sample to be examined, which consists of at least 2 nucleic acid sequences, is immobilized by hybridization.
  • the specificity of the subsequent attachment of nucleic acids from the sample can be influenced by the sequence. For example, a reaction support can address the 3 'or 5' sequence motifs of all exons of a gene family or an entire genome. In the next step can still one Amplification carried out on the reaction carrier.
  • the sequence of the building blocks along the bound nucleic acids from the sample is determined by an enzymatic reaction. This can be done directly on the nucleic acid strands from the sample or through their amplicons or through the complementary sequence following primer extension on the polymer probes. Methods for the determination of the building blocks along the nucleic acid strands are known to the person skilled in the art, inter alia, under the term "sequencing by synthesis.” For this purpose, among others, polymerases, kinases and ligases are used as enzymes Technical embodiments have been presented by the companies Agencourt, 454 and Solexa.
  • a mixture of at least 2 short nucleic acid strands is added.
  • These short nucleic acid strands have a similar function as a primer molecule in the PCR reaction. They serve a second round of the different embodiments of the decryption reaction along the bound nucleic acids from the sample.
  • the sequence of short nucleic acid strands that perform a similar function as a primer molecule in the PCR reaction may have been determined prior to the first round of the decryption reaction along the bound nucleic acids. In a preferred alternative embodiment, this sequence is tuned to the results of the first round of the decryption reaction.
  • the skilled person is familiar in this connection from the conventional sequencing methods "primer walking.”
  • the method described here as a particularly preferred embodiment of the invention is a highly parallel primer walking on 2 or more nucleic acids from the sample by means of 2 or more short Nucleic acids having sequences which are selected on the basis of the sequence determined for the 2 or more nucleic acids from the sample It may be provided in one embodiment that the sequence determination of the second round initially comprises a short sequence motif which also already determined in the first round to derive a quality characteristic from it.
  • the short nucleic acids originate from a parallel synthesis process.
  • Such parallel synthesis methods are known to those skilled in the art of biochips and microarrays.
  • electrochemical, optically controlled and fluidically controlled processes for the production of 2 or more nucleic acid sequences on a carrier There are electrochemical, optically controlled and fluidically controlled processes for the production of 2 or more nucleic acid sequences on a carrier. Claimed here is the use according to the invention of two or more nucleic acid sequences from a parallel production process, in which the two or more nucleic acid sequences are either produced on a common carrier or prepared in a process in which at least one step comprises two or more reaction sites for the production of 2 or more nucleic acid sequences are simultaneously contacted with a reagent.
  • Examples of such parallel preparation methods for 2 or more nucleic acid sequences for the method according to the invention are DNA microarrays, which are produced by means of photolithography, projector technology, LED technology, emitting semiconductor components or LCOS projection.
  • examples are fabrication methods using direct photochemistry and fabrication methods using indirect photochemistry such as light-induced bases or acids.
  • Further examples are electrochemical processes.
  • fluidic methods which either apply the building blocks to a carrier (printing technology, printing technology) or selectively apply the reagents of the synthesis.
  • the immobilization of the nucleic acids from the sample can also take place directly on the solid phase, for example on beads or particles, on a reaction support, on a microscope slide or in a gel layer, followed by a first round the decryption reaction, followed by the second round described above, in between which further process steps may be taken.
  • two mating hybridisable molecules can be made at a location that bind together and form a hybrid.
  • These may be, for example, a DNA duplex or duplexes of nucleic acid analogs or derivatives.
  • Preferred is the synthesis of DNA oligonucleotides or derivatized DNA oligonucleotides or DNA oligonucleotide analogs having different sequences at a location. It can be synthesized 2, 3, 4, 5, or 6 different sequences per addressable for the synthesis location.
  • the synthesized molecules can be synthesized with different total surface concentrations or different individual surface concentrations.
  • the molar ratio of the different molecules is thereby variable.
  • first of all different building blocks with mutually orthogonal chemical protective groups can be applied, as known to the person skilled in the art.
  • the ratio of these components is arbitrary and is preferably 10/1, 9/1, 8/1, 7/1, 6/1, 5/1, 4/1, 3/1, 2/1, 1/1 or vice versa, but can also be between these values (1/1 - 10/1).
  • molecules are synthesized on the surface of the reaction support which are bound to the surface of the reaction support via a unit which can be cleaved under certain conditions (labile linker, labile spacer).
  • labile linker labile spacer
  • these units can now be cleaved and the molecules synthesized on the surface of the reaction support can be detached from the surface.
  • the cleavage can be triggered, for example, by changes in the temperature, the pH, by irradiation of light and / or by addition of chemicals such as acids, bases, nucleophiles, electrophiles, radicals, ions or catalysts, enzymes and others.
  • the speed of the cleavage reaction can be controlled and the Complete conversion of the cleavage reaction may take hours and / or days.
  • the cleavage reaction is preferably carried out so that it is carried out simultaneously with an analytical method in the reaction carrier.
  • an enzymatic reaction can be carried out in the reaction support, while a part of the molecules is slowly split off from the surface and only then the enzyme is available as a reaction or binding partner or as a substrate. It can be controlled during the reaction, the supply of molecules. This makes it possible, for example, to control the rate of the reaction or the amount of final product formed.
  • the cleavable molecule may preferably be a DNA oligonucleotide or derivatized DNA oligonucleotide or DNA oligonucleotide analog which may act as a primer in an enzyme reaction in the reaction support when cleaved off. It is also particularly preferred to use enzymes for cleavage.
  • enzymes for cleavage.
  • UNG, UDG uracil DNA glycosylase
  • reactions for an amplification in the reaction carrier are carried out, as are known to the person skilled in the art.
  • the methods described under 6.1, 6.5, 6.6, 6.9, 6.15, 6.16, 6.18, 6.19, 6.20 and 6.21 can be used.
  • a PCR is preferred in which DNA oligonucleotides or derivatized DNA oligonucleotides or DNA oligonucleotide analogs bound on the surface of the reaction support act as primers.
  • Figures 20-22 illustrate these applications and show data from successful PCR reactions on the surface of the reaction support. It is possible to use primers which, as described under 6.16, are present at the same addressable location for the synthesis.
  • primers per location are used.
  • These primers can be covalently linked to the surface of the reaction support in such a way that they can no longer bond to one another. Thus, no primer dimers known to those skilled in the art, neither homodimers nor heterodimers, can be formed during the PCR. If longer molecules are added to the reaction carrier which can bind to the primers and serve as a template in the PCR, the primers are extended by a polymerase and reach thereby a length that allows binding of a second primer in the opposite direction. Without the addition of longer molecules this primer extension does not take place.
  • a PCR is possible in which all primers and all but the originally added longer template molecules are covalently linked to the surface of the reaction support.
  • the only diffusible components of the reaction mixture in this case are enzymes, nucleotides and other buffer constituents known to the person skilled in the art, but not oligonucleotides or other nucleic acids which are contained in the mixture.
  • two primers with different sequences are located at one location, each binding specifically to particular sequences in complex sample mixtures.
  • Partially or completely purified or unpurified fragmented or unfragmented genomic DNA or partially or completely purified or untreated fragmented or unfragmented RNA extracts from sample material are preferably used as complex sample mixtures.
  • the primers are in opposite directions and after binding to the desired sample molecule are not more than 20000 nucleotides apart.
  • One primer binds to the sense strand of the sample molecules known to those skilled in the art, and the other to the antisense strand.
  • primer dimers can in fact no longer occur in such systems since the primers can no longer bind to one another by virtue of their covalent bonding to the surface.
  • all primers, template molecules, and PCR-generated products that can hybridize to each other, with primers or template molecules, are covalently bound to the surface and thereby isolated from each other.
  • reaction carrier simple systems of a few primers and covalently bound to the surface of the reaction carrier molecules that are formed by the extension of these primers and can serve as a template for other primers of the location. It is thus possible to carry many thousands or hundreds of thousands of individual PCRs isolated from one another in the reaction carrier without undesired cross-reactions occurring between the individual PCRs.
  • the only soluble and freely diffusing components in the reaction carrier are the components of a PCR reaction known to the person skilled in the art, such as buffer constituents, enzymes, building blocks such as triphosphates etc., but not specific nucleic acids or derivatives.
  • PCR reactions are carried out in the reaction support with participation of molecules which are synthesized on the surface of the reaction support and function as primers.
  • the progress of the reaction i. the amount of nucleic acid synthesized during the PCR reaction is monitored during the reaction by certain methods. For this, e.g. methods known to those skilled in the art, such as the use of molecular beacons, Scorpion primers, intercalators, minor groove binders, sunrise primers, and the like.
  • a signal is read out at different times during the PCR reaction, e.g. a fluorescent signal.
  • This signal can then be used to quantify the particular molecules contained in the sample mixture used.
  • the signals can be used to make statements about the sequence of the molecules contained in the sample. For example, it is possible to elucidate mutations such as SNPs, deletions or insertions known to the person skilled in the art. This method is particularly preferably used in combination with the PCR method described under 6.18. 6.20 Ultra-Longmers
  • molecules synthesized on the surface of the reaction support are used as primers. These are extended after specific binding to specific template molecules by a primer extension, so that very long, on the surface of the reaction carrier covalently bound molecules formed, so-called ultra-Longmers.
  • a PCR can also be carried out with a counterprimer or antisense primer known to the person skilled in the art, so that the length of the extended primer after a PCR can be defined by the position of this counterprimer.
  • a reaction carrier is then obtained which contains long, single-stranded probe molecules with known sequences and optionally defined lengths. For each addressable location on the surface of the reaction carrier, only one sequence of high purity is obtained.
  • the resulting probe molecules are then available to bind desired sample molecules from complex sample mixtures.
  • the length of the probe molecules is preferably 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, 5000 or 10000 nucleotides or Nucleotide derivatives or nucleotide analogs.
  • Particularly preferred is the described preparation of such very long probe molecules on the surface of the reaction carrier for probe molecules, which consist of DNA. These are preferably used to bind sequences of genomic DNA.
  • the result of these synthesis steps may be an array comprising a plurality of such ultralongamers.
  • these long fragments can be used to produce, for example, synthetic genes. If a copy of a sample material in the reaction carrier is prepared by the described primer extension and / or PCR, the reaction carrier can again be written off by a primer extension and the resulting noncovalently bound molecules to the reaction carrier be dissolved out of the reaction carrier. Alternatively, techniques with a labile linker molecule as described in 6.17 may be used, which allow detachment of the extended primer molecules from the reaction support.
  • reaction carriers can be used several times and represent a copy of the sample material used. This can be used, in particular, for example, to generate fingerprints based on the DNA or RNA sequence information of, for example, pathogens, biofuels or other organisms.
  • the long molecules covalently bound to the surface of the reaction support can, since they are a transcript of the sample material, be sequenced directly in the reaction support instead of the sample material and thus provide the same sequence information as a sequencing of the sample material itself.
  • methods known to those skilled in the art as well as those described in US Pat 2.3 and 6.5 described sequencing method can be used.
  • a sequencing simultaneously represents a quality assurance of the reaction carrier generated by the copying of the sample material, which can be used for a further use of the reaction carrier.
  • molecules synthesized on the surface of the reaction support are used as primers. It is possible to use so-called hairpin structures which have intramolecular hybridization regions which are completely or partially double-stranded and have a free 3 'end which can be extended by a polymerase.
  • primer molecules can be hybridized to the molecules synthesized on the surface of the reaction support and extended by a polymerase.
  • the primers may be previously covalently linked (crosslinked) to the molecules synthesized on the surface of the reaction support by certain methods. For example, psoralen can be used.
  • the probe molecules synthesized on the surface of the reaction support also contain a recognition sequence for nicking endonucleases known to those skilled in the art.
  • a recognition sequence for nicking endonucleases known to those skilled in the art.
  • the strand can also be displaced by a temperature change. This process can be repeated, which results in an increase in the molecules synthesized by the polymerase and cut off by the nicking endonuclease.
  • Figures 24 and 25 illustrate this preferred embodiment.
  • mixtures of polymerases and nicking endonucleases can be used, whereby re-synthesis and truncation of the strands to be propagated can proceed in a mixture in a reaction carrier, without having to exchange mixtures. It can also be used thermostable enzymes. Enzymes to be used are, for example, the Klenow fragment of E.
  • the newly formed molecules are preferably DNA oligonucleotides or derivatized DNA oligonucleotides or DNA oligonucleotide analogs.
  • other molecules are bound to and covalently linked to the molecules synthesized on the surface of the reaction support.
  • Different methods known to those skilled in the art for crosslinking biomolecules are used.
  • Nucleic acids are preferably linked to one another, ie the molecules synthesized on the surface of the reaction support are oligonucleotides or derivatives or analogs of DNA or RNA. These are hybridized with specific oligonucleotides or derivatives or analogs of DNA or RNA introduced sequence-specific in the reaction carrier and linked together.
  • linking methods known to those skilled in the art which are based on, for example, a psoralen unit, or on aldehydes or ketones or radical or carbene or nitrene-forming chemical groups can be used for this purpose. 6.23 Assembly of short probe molecules to longer molecules directly in the reaction support
  • molecules synthesized in the reaction support are specifically linked together directly in the reaction support to form longer molecules.
  • the molecules to be linked may have been chemically synthesized on the surface of the reaction carrier or prepared by certain enzymatic methods. Preferably, these are present directly after the synthesis or by a cleavage in soluble form and are no longer bound to the surface. Preference is given to using methods such as PCR, primer extension or the methods described under 6.21.
  • Different molecules can be linked together in a defined sequence. This can be done for example by complementary, contained in the molecules, specific binding sites. These binding sites cause a specific, initially non-covalent assembly of the molecules. By using certain enzymes such as ligases or polymerases, these molecules can now be covalently linked together.
  • the length of the molecules to be linked can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.
  • molecules synthesized in the reaction support are specifically linked together directly in the reaction support to form longer molecules.
  • the linkage as under 6.23 is controlled by certain properties of the reaction carrier.
  • the amount of substance of individual types of molecules relative to the molar amount of other molecules may be altered in a manner that favors a particular positioning at specific locations in the later, covalently linked target molecule.
  • the sequence of the linkage and the position of the individual molecules in the later, covalently linked target molecule can be controlled by the individual Molecules at addressable locations on the surface of the reaction support are synthesized in such a way that their spatial position on the surface favors each other a directed, specific linkage.
  • molecules which are to be linked directly to one another in the later, covalently linked target molecule and thus should lie next to one another can also be synthesized at adjacent locations of the reaction carrier.
  • physical effects such as diffusion of the individual molecules to one another at different speeds, for example controlled by different path lengths, it is also possible to control the time of the collisions and thereby the linking of individual molecules.
  • individual populations can be synthesized very close to each other in island-like groups and, after peeling or after creating soluble, non-surface-associated copies, preferentially be linked to one another by the diffusion paths being short and the molecules colliding rapidly.
  • linked molecules are formed, which are composed of relatively few single molecules, whereby the complexity of the assembly and linking remains small.
  • the receptors synthesized on the surface of the reaction support are used to detect microRNAs (also called “miRNAs") and other small RNAs in sample mixtures
  • the receptors synthesized on the reaction support may preferably be linked to the surface via the 5 'or 3' end, so that the free end of the receptor is preferably either a 3 'or a 5' end
  • the linkage may be direct or via a linker
  • the receptors preferably contain one or more binding sites that specifically bind, ie hybridize with, certain microRNAs or other small RNAs while 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 binding sites, more preferably 1, 2 or 3 binding sites.
  • the binding sites can be directly adjacent to each other or separated by small intermediate areas of predetermined length. These intermediate regions may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.
  • the molecules synthesized on the surface of the reaction support are preferably oligonucleotides or derivatives or analogs of DNA or RNA with a length of preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150,
  • microRNAs or other small RNAs can thus be specifically bound and thereby detected in complex sample mixtures by detection methods, as known to those skilled in microarray technology.
  • the molecules to be detected can be linked with signaling groups or haptens prior to introduction into the reaction support or else directly into the reaction support before, during or after binding to the molecules synthesized on the surface of the reaction support, which enable their detection, preferably by an optical signal.
  • signaling groups or haptens prior to introduction into the reaction support or else directly into the reaction support before, during or after binding to the molecules synthesized on the surface of the reaction support, which enable their detection, preferably by an optical signal.
  • a variety of labeling methods known to those skilled in the art are available for this, e.g. by direct labeling with biotin or fluorophores or indirectly during cDNA synthesis or amplification. Both chemical and enzymatic methods are known for this, e.g.
  • the signal-emitting groups may be in particular fluorescence-active groups or fluorophores or FRET quencher or FRET acceptor groups or luminescent groups known to the person skilled in the art. These may be introduced directly or as groups with other chemical moieties such as e.g. Linked to dendrimers or with ligands that previously bind to the RNA-linked hapten groups.
  • Figures 28-36 and 44-53 illustrate these preferred embodiments of the present invention.
  • signal amplification may also be used, such as the introduction of a hapten such as biotin, the subsequent binding of a conjugate of streptavidin and a fluorophore or multiple fluorophores.
  • a another ligand bound which in turn is linked to one or more haptens or fluorophores, so that a larger number of haptens or fluorophores binds. It may then bind another ligand, which in turn is linked to one or more haptens or fluorophores, so that a larger number of haptens or fluorophores bind. This process can be repeated several times, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.
  • the ligands used are preferably antibodies; as haptens preferably biotin or digoxigenin.
  • oligonucleotides can also be used as primers in a known to those skilled i o // mg c / rc / e amplification, which are linked to streptavidin.
  • the streptavidin-biotin conjugate may have previously bound to biotin units previously attached to hybrids of probe molecules and sample molecules.
  • FIGS 16 and 17 show data from successful experiments for this preferred embodiment.
  • microRNAs or other analytes are amplified on the surface of the reaction carrier. This amplification may be a single strand amplification or a double strand amplification.
  • the microRNA or another analyte is detected before, during or after the amplification as described above by the incorporation of labeled building blocks.
  • the microRNA or other analyte is detected by DNA intercalators, molecular beacons, Taqman probes, and other methods known to those skilled in the art.
  • the molecules synthesized for the binding of certain miRNAs or other small RNAs synthesized on the surface of the reaction support can be completely or only partially complementary to the sequence of the RNA to be bound with regard to their desired binding site or binding sites.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 individual bases may be non-complementary to the sequence of the RNA to be bound.
  • Figure 14 shows data from successful experiments in which microRNAs were detected in complex samples of different tissues in the manner described. Molecules having 1 or 2 binding sites were used for the binding of the microRNAs which were directly adjacent or separated by intermediate regions and were either completely complementary to the sequence of the RNA to be bound or at 1, 2 or 3 nucleotide positions not complementary to the sequence of binding RNA.
  • FIG. 15 illustrates detailed optimization results regarding the temperature and buffer conditions for the specific detection of a multiplicity of microRNAs from a complex sample mixture.
  • the type of sample mixture may be different, uncleaned or wholly or partially purified extracts of cells or tissues may be used. These can be, for example, total nucleic acid purification, total RNA purification or special purification, which allow enrichment of small RNAs such as microRNAs. Numerous methods are known to the person skilled in the art.
  • Figure 18 shows data from successful experiments in which microRNAs from different tissues were detected in the manner described. The complexity of the sample mixtures was different, total RNA extracts were used or small RNAs were previously enriched using a special purification procedure.
  • RNAs to be detected can also be enzymatically processed directly in the reaction carrier before detection in a specific manner.
  • This can preferably be done by the incorporation of signaling groups or haptens modified nucleotides by a polymerase.
  • the type and number of nucleotides modified with signaling groups or haptens can be determined by the composition of the probe molecule. This can preferably be done by a template sequence that genetically encodes the presence of certain nucleotides type and number of built-in building blocks.
  • Figure 28 illustrates this preferred embodiment of the invention.
  • desired microRNAs present in the sample mixture to be examined and other small RNAs are specifically amplified. Due to the ability to generate a large number of different sequences in the reaction carrier and to use them as primers, a multiplicity of amplification reactions individually adapted to the respective RNAs to be amplified can be carried out in parallel.
  • RNAs to be examined can either function as a template or as a primer or both and / or be linked to universal sequences before the amplification.
  • FIGS. 44 and 45 show a preferred embodiment in which the receptors are linked to the surface of the carrier via the 5 'end. The hybridization region with the microRNA is positioned at the free 3 'end of the receptor.
  • the hybridization region is preferably arranged such that after the hybridization of the microRNA the receptor is at least one, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25 or more unhybridized receptor building blocks at the 5 'end.
  • This non-hybridized 5 'region of the receptor preferably has 1, 2, 3, 4, 5, 6, 7, or more building blocks, which can serve as template for signal-containing building blocks, such as adenines using biotin-labeled uridines as hapten group-containing building blocks.
  • the free 3 'end of the hybridized microRNA serves as a primer for subsequent amplification.
  • the amplification may consist only in the single extension, preferably by a DNA-dependent DNA polymerase, eg Klenow fragment.
  • signal-group-containing or hapten-containing building blocks preferably nucleotide building blocks, are incorporated in this amplification.
  • the amplification preferably comprises the covalent linking of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more building blocks.
  • the result of this amplification is an RNA-DNA hybrid when using deoxynucleotide building blocks.
  • the receptor can be linked to the surface of the support via the 3 'end.
  • the hybridization region with the microRNA is preferably at the 3 'end of the receptor and is preferably arranged such that after hybridization of the microRNA the receptor is at least one, preferably 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more non-hybridized receptor building blocks at the 5 'end.
  • the amplification is preferably carried out from the 3 'end of the microRNA to the end of the receptor.
  • the unhybridized 5 'region of the receptor is preferably selected as previously described.
  • the receptor may have 2, 3, 4, 5, 6, 7, or more preferably 2 hybridization regions that are reverse-complementary to the microRNAs to be detected. Preferably, these regions are arranged such that there are no unhybridized receptor building blocks between the hybridized microRNAs.
  • the receptors may be linked to the surface of the support via the 3 'end (see FIGS. 47 and 49) or may be linked to the surface of the support by the 5' end.
  • the two, three, four or more are attached to the receptor hybridized microRNAs are covalently linked by a suitable ligase, for example RNA ligase or T4 DNA ligase (see FIG. 49).
  • the amplification is preferably carried out after the ligation of the microRNAs by a suitable polymerase, preferably a DNA-dependent DNA polymerase such as Klenow fragment.
  • a suitable polymerase preferably a DNA-dependent DNA polymerase such as Klenow fragment.
  • the receptor is linked either to the surface of the carrier via the 5 'or 3' end, in addition to the analyte to be detected, eg the microRNA, one or two ligation probes, either together with the analyte or before or after the Hybridization of the analyte hybridized to the receptor (see Figure 48).
  • the ligation probe (s) is, for example, added to the sample to be analyzed and mixed with it.
  • the ligation probe (s) has a sequence that allows it to hybridize 3 'and / or 5' of the microRNA to be detected to the receptor.
  • the receptor sequence and the sequence of the ligation probes are chosen so that after hybridization of the microRNA and the ligation probe (s) between the respective free 3 'and 5' ends no unhybridized receptor building block are arranged.
  • the ligation probe has at least one signal-generating group or detectable group, such as, for example, a hapten and at least one group which can be linked by a ligase to a free 3 'OH group or 5'-phosphate group of the microRNA, for example an OH group. Group or a monophosphate group.
  • the ligation is preferably carried out in a separate step following hybridization with a suitable ligase, for example RNA ligase or T 4 DNA ligase.
  • a suitable ligase for example RNA ligase or T 4 DNA ligase.
  • the hybridization and the ligation can also be done in one step, whereby an acceleration of the detection reaction can be achieved.
  • the detection of the group (s) contained in the ligation probe is preferably carried out after washing the surface of the support, with a stringency that is preferably chosen so that hybridized unligated microRNAs are washed away from the surface of the support.
  • a chemical linkage via suitable activated nucleotides is achieved.
  • Suitable chemical linkages have been described, for example, in International Patent Application WO 2006/063717 (the content of this application is incorporated herein by reference in its entirety by reference to chemical ligation). This linkage can take place both at the 3 'and at the 5' end of the analyte, for example the microRNA.
  • either only one activated nucleotide or an oligonucleotide, at the 3 'or 5' end of which an activated nucleotide is arranged, can be used (see FIGS. 50 and 51).
  • a activated oligonucleotide is linked to the analyte, in particular a microRNA
  • the sequence is selected to be complementary to the receptor sequence located 5 'and / or 3' adjacent to the receptor sequence to which the analyte is hybridized. In this case, first a sequence-specific hybridization of the oligonucleotide takes place followed by a chemical linkage with the hybridized microRNA.
  • the oligonucleotide and / or the activated nucleotide comprises a signaling and / or a detectable group, such as, for example, hapten-containing groups, in particular biotin.
  • a detectable group such as, for example, hapten-containing groups, in particular biotin.
  • one or two short helper oligonucleotides are hybridized to the receptor before, after or together with the analyte, in particular the microRNA.
  • the helper oligonucleotide is preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and has a sequence that satisfies the art Helper oligonucleotides allow 3 'and / or 5' in addition to the analyte, in particular to hybridize the microRNA. Following hybridization of the one or two helper oligonucleotides and the analyte, chemical ligation is performed using an activated nucleotide or oligonucleotide.
  • the sequence of the helper oligonucleotide is chosen so that after hybridization, an unhybridized receptor nucleotide is located between the respective ends of the helper oligonucleotide and the analyte.
  • the sequences of the helper oligonucleotide and the receptor are chosen so that after hybridization of the analyte and the helper oligonucleotide to the receptor, the number of unhybridized receptor nucleotides between the 3 'and 5' ends of the helper oligonucleotide and the 5 ' or 3 'end of the analyte correspond to the number of nucleotide units of the activated oligonucleotide.
  • this may include signaling and / or detectable groups, such as hapten-containing groups, in particular biotin.
  • the activated oligonucleotide may additionally contain a signaling and / or detectable group.
  • helper oligonucleotide and activated nucleotide or activated oligonucleotide may each contain the groups of a FRET pair, so that only after the chemical linkage of the helper oligos to the analyte a FRET pair is present.
  • the detection step follows. Preferably, prior to detection, the surface of the support is washed with a stringency, which is preferably selected to wash off hybridized unligated helper oligonucleotides from the surface of the support.
  • one or more additional ones may be added to the first amplification step or ligation step in the previously described methods Connect amplification steps.
  • the first amplificate see FIGS.
  • Ligation product can be amplified linearly (using a primer) or exponentially (using two primers).
  • the sequence of the primer (s) is preferably chosen to match that in the first amplification or by the ligation
  • the primer (s) are selected to hybridize to both the analyte and the attached portion or ligated portion in the first amplification. This can ensure a higher specificity for amplificates of certain analytes. If, at the same time, the first amplicons or ligation products of different receptors are to be amplified, it is preferred that the primer or primers hybridize exclusively to the sequences which have been added by ligation or the first amplification to the different analytes, since thereby an amplification reaction all different amplified first amplicons or ligation products.
  • the primers and / or the nucleotide building blocks may be labeled with signal-donating or detectable groups.
  • amplification may be followed by backhybridization to the receptors on the surface of the support. The detection of the amplificates takes place, if appropriate, after a stringent washing step on the surface of the support.
  • a cap group is preferably located at the free end of the receptor at the free 5 'end of the receptor (see FIG. 53).
  • an analyte preferably a microRNA
  • cap groups in the sense of the invention interact and stabilize with the duplex formed by the hybridization
  • Suitable stabilizing cap groups include, for example, substituted or unsubstituted bicyclic or tricyclic aromatics or heteroaromatics and stilbenes, in particular trans-stilbene (eg, trans-1,2,3-trimethoxy-5-stilbene.)
  • Particularly preferred cap groups are capable of intercalating into the formed duplex, and by intercalation, stabilize the duplex, ie, particularly preferred cap groups are DNA -Interkalatoren.
  • the utilization of the described molecular biological process plant represents is a significant improvement over the prior art for the detection, quantification and characterization of microRNAs and other small RNAs. Since the number of naturally occurring small RNAs such as microRNAs is still unknown and new RNAs are being discovered in very short periods of time an adaptation of analytical methods to the conditions found in each case, ie the number and type of RNAs to be examined is absolutely necessary. Due to the possibility of synthesizing and testing a large number of different probe molecules on the surface of the reaction support in a very short time, it is possible with the described process equipment to react very rapidly to changes in the types of molecules to be investigated and to adapt the analysis methods.
  • the molecules synthesized on the surface of the reaction support are used to detect and characterize microorganisms and / or pathogens.
  • certain nucleic acids characteristic of a particular pathogen or a microorganism are selectively bound by the molecules synthesized on the surface of the reaction support.
  • Genes, mRNAs, genomic RNA or DNA or other RNAs of the pathogen are preferably used as nucleic acids.
  • molecules synthesized on the surface of the reaction support are used to analyze the identity of individual nucleotides in these nucleic acids. Thus, mutations such as nucleotide substitutions, deletions or insertions can be elucidated.
  • nucleic acids to be examined are bound and / or used as a substrate, wherein the efficiency of the reaction catalyzed by the particular enzyme then changes when a mutation is present in the nucleic acid to be examined.
  • this efficiency may also change if, by binding the nucleic acid to be investigated to the molecules synthesized on the surface of the reaction support, certain nucleotide pairs are formed which differ, for example, in that they form pairs known to the person skilled in the art as Watson-Crick base pairs or others couples.
  • APEX allele-specific hybridization
  • allele-specific primer extension allele-specific PCR
  • ASA Taqman assays
  • molecular beacons minisequencing
  • SSCP-PCR mismatch cleavage
  • OLA branch migration assay
  • BMI branch migration assay
  • pyrosequencing dynamic allele specific Hybridization
  • DASH Dynamic allele specific Hybridization
  • MAA Multiplex Automated Primer Extension Assay
  • Figure 19 illustrates such an assay and shows data from successful identifications of individual nucleotide positions in a DNA sample.
  • These methods are particularly preferably used to distinguish pathogen strains from each other and to allow typing of pathogens. All pathogen species occur in nature with different genotypes, which often differ only by a few mutations, but have different properties, such as infection potential, resistance to certain drugs and many more.
  • desired existing in the sample to be examined mixture of pathogen-originating or self-pathogens representing nucleic acids are specifically amplified. Due to the ability to generate a large number of different sequences in the reaction carrier and to use them as primers, a multiplicity of amplification reactions individually adapted to the respective RNAs to be amplified can be carried out in parallel.
  • the nucleic acids to be examined can either function as a template or as a primer or both and be linked before the amplification with universal sequences.
  • Molecular diagnostics is used today in many areas to detect microorganisms and viruses and to quantify them in samples.
  • quantitative real-time PCR is used.
  • samples in individual PCRs or multiplex PCRs are assayed for the content of a particular pathogen. If the test is positive, a large number of further studies based on quantitative real-time PCR will follow to elucidate the exact genotype of the pathogen species. This may be necessary, for example, to decide on the type of chemotherapy or to conduct epidemiological investigations or to monitor the populations of certain strains or to detect the appearance of new strains.
  • the products of the first real-time PCR which are used for the detection of a pathogen, are used directly in the Reaction carrier to elucidate an elucidation of the exact genotype of the pathogen species.
  • the reaction carrier can be designed in such a way that both known and new genotypes can be detected.
  • Figure 37 illustrates this preferred embodiment of the invention.
  • the process plant described is used to empirically test probe molecules and thus rapidly provide a large number of suitable probe molecules for certain applications. It is known that the binding properties of probe molecules such as oligonucleotides or derivatives or analogs of DNA or RNA can not be predicted very accurately. There are no bioinformatic methods to predict the binding strength and / or specificity of such probe molecules with respect to desired sample molecules. This results in a need for empirical testing of probe molecules.
  • probes can then be selected in a "rapid prototyping process" according to certain quality criteria, which fulfill the desired conditions and will be used in later experiments. If it is desired to examine a sample for which no suitable or limited For example, if suitable probe molecules are known, a number of probes can be generated, tested and / or optimized very quickly in model experiments, which can then be used in later experiments for this type of sample Most systems known to those skilled in the art for producing a variety of different probe molecules are either very slow or can not afford this at an economically acceptable cost In contrast, dining equipment offers the opportunity to produce a very large number of products in a very short time To generate, test and / or optimize probe molecules at a low price, so that it is possible to react quickly to changes in the type of sample molecules to be examined, ie the analysis method can be adapted individually.
  • DNA probes of 21-23 nucleotides in length were synthesized analogously to published methods in a micro fluidic reaction support (Baum, M. et al., Nucleic Acids Research, 2003, 31, el 51 and references cited therein). It was an inverse synthesis chemistry was used so that the probes were bound to the 5 'end of the surface of the reaction carrier and had a free 3' end. For the experiments, an external reaction unit was used, which allows the filling and temperature control of the reaction carrier.
  • the reaction support was reacted with a mixture of 200 nM PCR product and 33 to 200 ⁇ M of each of the four dNTPs (of which 33% of the TTP was replaced by biotin-16-dUTP (Roche)) in the reaction buffer provided by the manufacturer for each DNA polymerase heated, heated to 80 0 C for 5 min and cooled to room temperature over 20 min.
  • the reaction medium was heated for mesophilic DNA polymerases at 37 0 C and for thermostable DNA polymerases to 72 0 C, the mixture was removed and the reaction carrier with the same mixture, containing 1 .mu.l of enzyme per 15 .mu.l filled. After a reaction time of 10 minutes, the reaction carrier was washed with 500 ⁇ L of water.
  • the reaction carrier was incubated within the molecular biological process plant with a streptavidin-phycoerythrin-containing buffer solution, washed and analyzed by fluorescence measurement.
  • DNA probes were prepared as above containing a self-complementary sequence and thereby forming a hairpin structure having a paired 3 'end that can be used as a primer by a DNA polymerase.
  • the probes had a length of 27 and 30 nucleotides, the length of the self-complementary region varied between 4-7 nucleotides, the loop region between the self-complementary regions was in each case a TTTT sequence.
  • the experiments were carried out analogously to the experiments described above with reference to FIGS. 4 and 5.
  • RNA was fractionated and purified by means of the flashP AGE kit known to those skilled in the art; or fragmented and used directly; or used directly; and marked by the mirVana kit known to those skilled in the art according to the manufacturer's protocol.
  • the purified, labeled RNA samples were introduced into the reaction support and incubated at certain temperatures under buffer conditions as described in FIG. After the incubation, the solution was removed from the reaction medium.
  • Reaction carrier was incubated within the molecular biological process plant with a streptavidin-phycoerythrin-containing buffer solution, washed and analyzed by fluorescence measurement.
  • signal amplification was performed as described in the figure description of Figs. 16 and 17 (Figs. 16 and 17).
  • DNA probes of between 21-50 nucleotides in length were synthesized analogously to published methods in a microfluidic reaction support (Baum, M. et al., Nucleic Acids Research, 2003, 31, el51 and references cited therein).
  • An inverse synthesis chemistry was used for the data shown in FIG.
  • FIG. 19 The reaction carrier was incubated with a mixture of 200 nM PCR product and
  • FIG. 23 The reaction support was filled with a mixture of 13 .mu.M of a primer and 33 .mu.M of each of the four dNTPs (of which 33% of the TTP was replaced by biotin-16-dUTP (Roche)) in the reaction buffer "2" from NEB, 5 The reaction mixture was heated to 37 ° C., the mixture was removed and the reaction mixture was removed with the same mixture containing 1 ⁇ L of Klenow fragment (3'-5'-exo).
  • reaction support was washed with 500 ⁇ l of water, the reaction support was incubated with a streptavidin-phycoerythrin-containing buffer solution within the molecular biological process plant, washed and analyzed by fluorescence measurement The same reaction was repeated, this time without biotin-16-dUTP
  • the reaction carrier was washed with 25% ammonia solution , the eluate is dried, used as a template in a PCR reaction, and analyzed by gel electrophoresis.
  • DNA probes were synthesized as for the data shown in Fig. 19.
  • an external reaction unit was used, which allows infilling and tempering of the reaction medium.
  • the reaction carrier was incubated within the molecular biological process plant with a streptavidin-phycoerythrin-containing buffer solution, washed and analyzed by fluorescence measurement.

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Abstract

L'invention concerne une installation améliorée de traitement biologique moléculaire, et un procédé amélioré de traitement d'échantillons biologiques. L'invention combine la mise à disposition dans des cellules d'écoulement miniaturisées de molécules biologiquement fonctionnelles - comme des acides nucléiques et des peptides ainsi que des dérivés ou analogues de ces deux classes de molécules - avec l'addition en série de réactifs ou de fluides; elle sert au traitement d'échantillons biologiques comme des protéines, des acides nucléiques, de petites molécules biogènes comme par exemple des métabolites, des virus ou des cellules, qui sont introduits à cet effet dans les cellules d'écoulement miniaturisées. L'invention concerne en outre des procédés et l'utilisation de l'installation de traitement biologique moléculaire selon l'invention pour déceler et/ou isoler des acides nucléiques; pour le séquencement; pour l'analyse de mutation ponctuelle; pour l'analyse de génomes et/ou de chromosomes; pour l'obtention d'acides nucléiques synthétiques; pour l'obtention de réseaux d'amorces, de sondes et/ou de molécules antisens; ainsi que d'autres processus d'analyse et de synthèse.
EP07857168A 2006-12-29 2007-12-31 Installation améliorée de traitement biologique moléculaire Withdrawn EP2109499A2 (fr)

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US20100184045A1 (en) * 2008-09-23 2010-07-22 Helicos Biosciences Corporation Methods for sequencing degraded or modified nucleic acids
WO2010043418A2 (fr) * 2008-10-17 2010-04-22 Febit Holding Gmbh Amplification intégrée, traitement et analyse de biomolécules dans un support de réaction microfluidique
DE102009029831A1 (de) 2009-06-17 2011-01-13 W.O.M. World Of Medicine Ag Vorrichtung und Verfahren für die Mehr-Photonen-Fluoreszenzmikroskopie zur Gewinnung von Informationen aus biologischem Gewebe
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US10407460B2 (en) * 2015-05-07 2019-09-10 The Regents Of The University Of Califonia Solid phase sequence-independent nucleic acid assembly
WO2019014185A1 (fr) * 2017-07-10 2019-01-17 The Charles Stark Draper Laboratory, Inc. Appareil permettant un stockage d'informations de haute densité dans des chaînes moléculaires
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