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WO2003018834A2 - Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique - Google Patents

Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique Download PDF

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Publication number
WO2003018834A2
WO2003018834A2 PCT/DE2002/003122 DE0203122W WO03018834A2 WO 2003018834 A2 WO2003018834 A2 WO 2003018834A2 DE 0203122 W DE0203122 W DE 0203122W WO 03018834 A2 WO03018834 A2 WO 03018834A2
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid oligomer
ligate
signal
ligands
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PCT/DE2002/003122
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German (de)
English (en)
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WO2003018834A3 (fr
Inventor
Gerhard Hartwich
Peter Frischmann
Ute Haker
Herbert Wieder
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Friz Biochem Gmbh
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Priority claimed from DE10141691A external-priority patent/DE10141691A1/de
Application filed by Friz Biochem Gmbh filed Critical Friz Biochem Gmbh
Priority to US10/487,816 priority Critical patent/US20060228703A1/en
Priority to EP02762252A priority patent/EP1554396B1/fr
Priority to AT02762252T priority patent/ATE513058T1/de
Publication of WO2003018834A2 publication Critical patent/WO2003018834A2/fr
Publication of WO2003018834A3 publication Critical patent/WO2003018834A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present invention relates to a method for the detection of nucleic acid oligomer hybridization events.
  • Immunoassays and increasingly sequence analysis of DNA and RNA are used in disease diagnosis, in toxicological test procedures, in genetic research and development, as well as in the agricultural and pharmaceutical sectors.
  • serial methods with autoradiographic or optical detection parallel detection methods using array technology using so-called DNA or protein chips are increasingly being used.
  • parallel detection is based either on optical, radiographic, mass spectrometric or electrochemical methods.
  • oligonucleotide or DNA chips can also be used for SNP (single nucleotide polymorphism) or gene expression analysis, since they allow the activity level of a large number of individuals to be more active
  • probe oligonucleotides For gene analysis on a chip, a library of known DNA sequences (“probe oligonucleotides”) is fixed in an ordered grid on one surface, so that the position of each individual DNA sequence is known. If there are fragments of active genes ("target oligonucleotides”) in the test solution, the sequences of which are complementary to certain probe oligonucleotides on the chip, the target oligonucleotides can be identified (read) by detecting the corresponding hybridization events on the chip.
  • probe oligonucleotides fragments of active genes
  • Protein chips whose test sites carry certain antigen (or antibody) probes instead of probe oligonucleotides, can be used in proteome analysis or in the parallelization of diagnostics.
  • radioactive labels in DNA / RNA sequencing has several disadvantages, e.g. the elaborate, legally prescribed safety precautions when handling radioactive materials.
  • the costs of the equipment are very high for fluorescence and mass spectrometric detection.
  • the disadvantages of labeling with radioactive elements or fluorescent dyes can partly. are avoided if association events are detected on the basis of the associated change in the electrochemical properties (cf. WO 97/46568, WO 99/51778, WO 00/31101, WO 00/42217).
  • the object of the present invention is therefore to create a method for the detection of nucleic acid oligomer hybridization events which does not have the disadvantages of the prior art.
  • DNA deoxyribonucleic acid RNA ribonucleic acid PNA peptide nucleic acid synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid.
  • synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid.
  • the sugar-phosphate unit is replaced by the -NH- (CH 2 ) 2 - N (COCH 2 base) -CH 2 CO unit hybridizes PNA with DNA.
  • Nucleic acid At least two covalently linked nucleotides or at least two covalently linked pyrimidine (eg cytosine, thymine or uracil) or purine bases (eg adenine or guanine).
  • the term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or Purine bases, such as on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous structures (eg phosphoramide, thio-phosphate or dithio-phosphate backbone).
  • An essential feature of a nucleic acid in the sense of the present invention is that it can bind naturally occurring cDNA or RNA in a sequence-specific manner.
  • Nucleic acid oligomer Nucleic acid of unspecified base length (e.g. nucleic acid octamer: A nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bonded to one another.). ns oligomer Nucleic acid oligomer
  • Oligomer equivalent to nucleic acid oligomer Oligomer equivalent to nucleic acid oligomer.
  • Oligonucleotide equivalent to oligomer or nucleic acid oligomer e.g. a DNA, PNA or RNA fragment of unspecified base length.
  • Oligo Abbreviation for oligonucleotide Oligo Abbreviation for oligonucleotide.
  • nucleic acid oligomer a nucleic acid oligomer to associate the formation of the Watson-Crick structure of double-stranded nucleic acid oligomers, i.e. hybridize, the two single strands, the nucleotide sequence of one strand being complementary to the nucleotide sequence of the other strand, so that base A (or C) of one strand forms hydrogen bonds with base T (or G) of the other strand (in the case of RNA T is replaced by uracil).
  • the two single strands hybridize in such a way that the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (in RNA, T is replaced by uracil ). Any other Base pairing within the hybrid does not form hydrogen bonds, distorts the structure and is referred to as a "mismatch".
  • Reducing agent Chemical compound (chemical substance) that reduces this other chemical compound (chemical substance) by donating electrons to another chemical compound (chemical substance).
  • redox active Designates the property of a unit under certain external circumstances to release electrons to a suitable oxidizing agent or to take up electrons from a suitable reducing agent.
  • Linker Molecular connection between two molecules or between a surface atom, surface molecule or a surface molecule group and another molecule.
  • linkers can be purchased as alkyl, alkenyl, alkynyl, hetero-alkyl, hetero-alkenyl or hetero-alkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partner.
  • the reactive groups can also be photoactivatable, ie the reactive groups are only activated by light of certain or any wavelength.
  • Preferred linkers are those of chain length 1-20, in particular chain length 1-14, the chain length here being the shortest continuous connection between the structures to be connected, i.e. between the two molecules or between a surface atom, surface molecule or a surface molecule group and another molecule , represents.
  • mice Mica muscovite platelets, carrier material for thin application
  • the terminal phosphate group of the oligonucleotide is esterified at the 3 'end with (HO- (CH 2 ) 2 -S) 2 to PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH, the SS bond being cleaved homolytically and this causes an Au-SR bond.
  • Au-S-fCHJ ⁇ -ss-oligo spacer hybridizes with the oligonucleotide complementary to ss-oligo.
  • Cyclic voltammetry Recording a current / voltage curve.
  • the potential of a stationary working electrode is changed linearly as a function of time, starting from a potential at which no electrooxidation or reduction takes place up to a potential at which a dissolved or adsorbed species is oxidized or reduced (i.e. current flows).
  • the direction of the potential feed is reversed. The behavior of the products of electrooxidation or reduction is then recorded in the return.
  • Amperometry Recording a current / time curve.
  • the potential of a stationary working electrode is e.g. by a potential jump to a potential at which the electro-oxidation or reduction of a dissolved or adsorbed species takes place and the flowing current is recorded as a function of time.
  • Chronocoulometry Recording a charge / time curve.
  • the potential of a stationary working electrode is e.g. is set to a potential at which the electrooxidation or reduction of a dissolved or adsorbed species takes place and the transferred charge is recorded as a function of time. Chronocoulometry can therefore be understood as an integral of amperometry.
  • the present invention provides a method for the detection of nucleic acid oligomer hybridization events which comprises the steps of providing a modified surface, the modification consisting in the attachment of at least one type of ligate nucleic acid oligomer, providing signal nucleic acid oligomer ligands, providing a sample with nucleic acid oligomer Ligands, bringing a defined amount of the signal nucleic acid oligomer ligands into contact with the modified surface and bringing the sample and the nucleic acid oligomer ligands contained therein into contact with the modified surface, detection of the signal Nucleic acid oligomer ligands, and comparison of the values obtained in the detection of the signal nucleic acid oligomer ligands with reference values.
  • a defined amount of the signal nucleic acid oligomer ligands can be brought into contact with the modified surface and the sample can be brought into contact with the modified surface at the same time, but preferably the signal nucleic acid oligomer ligands and the sample are brought into contact separately.
  • the reference values can be determined anew before the sample is brought into contact with the modified surface .
  • a detection of the signal nucleic acid oligomer ligands is carried out and only then is the sample brought into contact with the modified surface.
  • the signal nucleic acid oligomer ligands are then detected a second time and the values determined in the second detection are compared with the reference values determined in the first detection.
  • the modified surface is washed after the first detection of the signal nucleic acid oligomer ligands and, after the sample has been brought into contact with the modified surface, the same defined amount of signal nucleic acid oligomer ligands as before the first Detection (reference measurement) brought into contact with the modified surface. Only then is the second detection of the signal nucleic acid oligomer ligands carried out.
  • conditions are set or measures are taken after the first detection of the signal nucleic acid oligomer ligands, before and / or during washing of the modified surface, or measures are taken which at least predominantly dissociate ligate nucleic acid oligomers and signal nucleic acid oligomer Lead ligands.
  • measures are taken which at least predominantly dissociate ligate nucleic acid oligomers and signal nucleic acid oligomer Lead ligands.
  • the temperature can be increased above the melting temperature of the double-stranded oligonucleotides consisting of ligate nucleic acid oligomer and signal nucleic acid oligomer ligand, chaotropic salts can be added or one can Potential that is above the electrostringent potential.
  • the reference values can be determined according to a preferred embodiment of the present invention after simultaneous or separate contacting of the modified surface with nucleic acid oligomer ligands and signal nucleic acid oligomer ligands and subsequent detection of the signal nucleic acid oligomer ligands.
  • conditions are first set or measures are taken which at least predominantly dissociate ligate nucleic acid oligomers and
  • Nucleic acid oligomer ligands and lead to at least predominant dissociation of ligate nucleic acid oligomers and signal nucleic acid oligomer ligands, then the modified surface is washed, then signal nucleic acid oligomer ligands are brought into contact with the modified surface, the same defined amount of signal Nucleic acid oligomer ligands are used as in the first addition, and finally the signal nucleic acid oligomer ligands are detected and thus the reference values are determined. This method of determining the reference value is particularly preferred when the sample is brought into contact with the modified surface before a defined amount of the signal nucleic acid oligomer ligands is brought into contact with the modified surface.
  • An increase in temperature above the melting temperature of the double-stranded oligonucleotides consisting of ligate nucleic acid oligomers and nucleic acid oligomer ligands and above the melting temperature of the double-stranded oligonucleotides consisting of ligate nucleic acid oligomers and signal nucleic acid oligomer ligands also leads to an increased dissociation of the double-stranded oligomers.
  • the temperature is preferably raised to at least 5 ° C. above the melting temperature.
  • a further measure is the application of a potential which is above the electrostringent potential, in particular an increase in the (negative) potential by at least 10 mV above the electrostringent potential, and also a treatment with NaOH.
  • the melting temperature of the double strand of ligate nucleic acid oligomers and signal nucleic acid oligomer ligands or of the double strand of ligate nucleic acid oligomers and nucleic acid oligomer ligands can either be determined under the corresponding external conditions, that is to say at a given concentration of the oligonucleotides, a certain salt content, a certain salt , in the presence of dissociation-promoting organic solvents such as formamide, DMF etc. or even in a wide range can be calculated relatively accurately (see for example the oligo analyzer "www.idtdna.com/program/main/home.asp" available on the Internet).
  • the electrode potential can be used in electrode-immobilized ligate nucleic acid oligomers with an associated counter strand to displace the polyanionic counter strand from the electrode.
  • negative to slightly positive potentials E ⁇ 0.2 V against silver wire
  • both single and double-stranded nucleic acid oligomers bound to a conductive surface are in a rather elongated conformation due to the repulsion of the negatively charged DNA backbone. If the potential is shifted further into the negative, the surface becomes more negatively charged and less strongly bound nucleic acid oligomer ligands and signal nucleic acid oligomer ligands, i.e. those which have one or more base mismatches in the binding region, are displaced from the modified surface.
  • this so-called electrostringent potential can also be determined experimentally for certain external parameters.
  • Anions of chaotropic salts for the dissociation of double-stranded oligonucleotides are, for example, CCI 3 COO “ , CNS “ , CF 3 COO “ , CIO 4 “ , I “ , (see also: Robinson and Grant, Journal of Biological Chemistry, 241, 1966, p 1329ff and Kessler et al. US 5,753,433)
  • the melting temperature is reduced by chaotropic salts.
  • a correction measured value can be determined which corresponds to the background signal of the modified surface used.
  • the correction measurement value is generally subtracted from the detection and reference signal in order to improve the quantification of the detection.
  • the determination of a correction measurement value for modified surfaces not associated with nucleic acid oligomer ligands and signal nucleic acid oligomer ligands can in principle take place at three different times: a) after the sample and the nucleic acid oligomer ligands contained therein have been brought into contact with the modified surface but before the addition of the signal nucleic acid oligomer ligands; b) before contacting the sample and the nucleic acid oligomer ligands contained therein with the modified surface and before adding the signal nucleic acid oligomer ligands; c) after dissociation of the double-stranded hybrids from ligate-nucleic acid oligomer and nucleic acid oligomer ligand or from ligate-nucleic acid oligomer and signal-nucleic acid oligomer ligand, after washing the modified surface in the absence of signal-nucleic acid oligomer ligands.
  • the correction measured values can also be determined several times at the same time or at the different times described above when the method according to the invention is carried out.
  • the measured value used in each case is then determined by forming the arithmetic mean from the various measurements for the respective area.
  • the separate implementation of a reference measurement can be avoided.
  • there are reference sites on the modified surface applied which can be assigned a very specific degree of association after adding the sample.
  • the signal obtained during the detection is then characteristic of this particular degree of association and can be used to normalize the signals from the test sites.
  • All of the methods described in the context of the present invention can namely also be carried out using a modified surface, the surface being modified by binding at least two types of ligate nucleic acid oligomers.
  • the different types of ligate nucleic acid oligomers are bound to the surface in essentially spatially separated areas. "Substantially separated areas” are understood to mean areas of the surface that are largely modified by binding a certain type of ligate. Only in areas in which two such essentially separated areas adjoin one another can spatial mixing of different types of ligate nucleic acid oligomers occur.
  • a type of nucleic acid oligomer ligand is added to the sample, the nucleic acid oligomer ligand being a binding partner with a high association constant with a specific type of ligate nucleic acid oligomer that is bound to the surface in a certain area (test site T 100 ).
  • This added type of nucleic acid oligomer ligand does not associate with the other types of ligate nucleic acid oligomers bound to the surface, or only very slightly.
  • the nucleic acid oligomer ligand is added to the sample in an amount that is greater than the amount of nucleic acid oligomer ligands that is necessary to fully associate the ligate nucleic acid oligomers of the T 10 o test sites.
  • the last step of this process - after bringing the sample into contact with the modified surface, the sample containing the type of nucleic acid oligomer ligand with a high association constant with the ligate nucleic acid oligomers bound in the region T 10 o, optional stringent hybridization and after contacting the signal Nucleic acid oligomer ligands with the modified surface - the comparison of the values obtained in the detection of the signal nucleic acid oligomer ligands with the value obtained for the region T 100 .
  • the value obtained for the area T 10 o thus corresponds to the value when the association is complete (100%).
  • a modified surface is used which has been modified by binding at least three types of ligate nucleic acid oligomers.
  • the different types of ligate nucleic acid oligomers are bound to the surface in essentially spatially separated areas. At least one type of ligate nucleic acid oligomer is bound to the surface in a certain area (test site T 0 ), from which it is known that there is no binding partner with a high association constant in the sample, i.e. the complementary nucleic acid oligomer ligand is not in the sample occurs.
  • one type of nucleic acid oligomer ligand is added to the sample before the sample is brought into contact with the modified surface, the added type of nucleic acid oligomer ligand being a binding partner with a high association constant with a certain type of ligate nucleic acid oligomer is present in a certain area (test site T 10 o) bound to the surface.
  • This added type of nucleic acid oligomer ligand does not associate with the other types of ligate nucleic acid oligomers bound to the surface, or only very slightly.
  • the nucleic acid oligomer ligand is added to the sample in an amount that is greater than the amount of nucleic acid oligomer ligands that is necessary to fully associate the ligate nucleic acid oligomers of the T 10 o test sites.
  • the last step of this process is - after bringing the sample into contact with the modified surface, the sample containing the type of nucleic acid oligomer ligand with a high association constant with the ligate nucleic acid oligomers bound in the region T ⁇ 0 o, optional stringent hybridization and after contacting the signal Nucleic acid oligomer ligands with the modified surface - the comparison of the values obtained in the detection of the signal nucleic acid oligomer ligands with the value obtained for the area T 100 and with the value obtained for the area T 0 .
  • the value obtained for the area T 0 thus corresponds to the value in the absence of association (0%).
  • At least one further type of nucleic acid oligomer ligand is added to the sample before contacting the sample with the modified surface, it being known that this nucleic acid oligomer ligand is not included in the original sample.
  • This further type of nucleic acid oligomer ligand has an association constant> 0 to a type of ligate nucleic acid oligomer which is bound to the surface in a certain region (test site T n ).
  • the Nucleic acid oligomer ligand is added to the sample in such an amount that after contacting the sample with the modified surface, n% of the ligate nucleic acid oligomers of the test site T n are in an associated form.
  • the last step of this process is - after the sample has been brought into contact with the modified surface, the sample containing the types of nucleic acid oligomer ligands which associate with the ligated nucleic acid oligomers bound in the T 10o and T n regions, optional stringent hybridization and after being brought into contact the signal nucleic acid oligomer ligand with the modified surface - the comparison of the values obtained in the detection of the signal nucleic acid oligomer ligands with the value obtained for the area T 100 , with the value obtained for the area T 0 and with that for the areas T n values obtained.
  • the value obtained for a specific test site T n thus corresponds to the value in the presence of n% ligate-ligand associates
  • the amount of nucleic acid oligomer ligand that has to be brought into contact with the modified surface in order to bring about an n% association at the test site T n can be determined by the person skilled in the art by simple routine tests. For this purpose, for example after detection of the values for T 0 and T 100, a calibrated measurement is carried out, in which the signal intensity is determined by (different) detection labels with which the ligate nucleic acid oligomer and the nucleic acid oligomer ligand are equipped. The ratio of ligand label signal to ligate label signal corresponds to n%.
  • T n mixtures are used as ligate nucleic acid oligomers for modifying the measuring ranges from n% ligate nucleic acid oligomers required for the test site T 100 are used, and (100-n)% ligate nucleic acid oligomers, which are used for the test site T 0 .
  • the determination of suitable T n - nucleic acid oligomer ligands and their addition to the sample before contacting the sample with the modified surface can be dispensed with if sufficient T 100 - nucleic acid oligomer ligand is added to the sample, so that not only the test Site T 100 can fully associate with T 10 o-nucleic acid oligomer ligand, but can also associate the respective n% of the T ⁇ test sites with the tio-nucleic acid oligomer ligand.
  • a sufficient number of reference sites T n will be available applied to the modified surface, a reference curve can be recorded with high accuracy. Standardizing the measurements of the actual test sites using With the help of this reference curve, the reproducibility of the analysis using chip technology is significantly improved.
  • the measurement values for the areas T 0 , T n and T 10 o can also be determined several times at the same time or at the different times described above when the method according to the invention is carried out. The measured value used in each case is then determined by forming the arithmetic mean from the various measurements for the respective area. As described above, the measured values for the areas T 0 , T n and T 10 o can be improved by determining correction measurement values or arithmetically averaged correction measurement values.
  • test area M it is advantageous to also improve the measured values of all other areas of the modified surface (test area M) by means of correction measured values, whereby mathematical determination methods are preferred which are used to determine the corrected measured values for the areas T 0 , T n and T 10 o and the actual measuring range M are identical (same standardization).
  • nucleic acid oligomer ligand that is not contained in the sample presents no problems, since even the most extensive genomes still offer a sufficient selection of sequences that are not available.
  • the hybridization step must be carried out under stringent conditions.
  • preference is given to using sequences which differ significantly, that is to say in several bases, from the sequences present in the sample. Particularly good results are achieved if oligonucleotides with the same or at least a similar number of bases are used for the test sites and for the reference sites.
  • the ligate nucleic acid oligomers of the present invention consist of n nucleotides which are present in a specific nucleotide sequence (sequence).
  • the ligate nucleic acid oligomers are complementary or at least predominantly complementary to a sequence region of n nucleotides of the nucleic acid oligomer ligands.
  • the nucleic acid oligomer ligands, the presence of which is to be detected on the basis of the present invention, can have further sequence regions in addition to the contiguous sequence region which is n nucleotides long and whose sequence is complementary to the n nucleotide sequence of the ligates.
  • ligate nucleic acid oligomers are used, each type of ligate nucleic acid oligomer consisting of a number of n nucleotides of a specific sequence.
  • the ligate nucleic acid oligomers of one type are complementary to a sequence region of n nucleotides of nucleic acid oligomer ligands of one type, wherein the ligate nucleic acid oligomer can form a perfect match with this n nucleotide-contiguous sequence region of the nucleic acid oligomer ligand, ie in this n nucleotide long region After association (hybridization), the ligate and the appropriate ligand form a double helix with n only suitable base pairings.
  • the signal nucleic acid oligomer ligands associated with this type of ligate nucleic acid oligomer have sequence regions which are only partially complementary to the n nucleotide sequence of the ligate; in particular, the signal nucleic acid oligomer ligands have only those sequence segments that, after association with the n nucleotide long sequence of the ligate nucleic acid oligomer are at most n-1, n-2, n-3, n-4 or n-5 nucleotides complementary to one another.
  • n stands for an integer from 3 to 80, in particular from 5 to 50, particularly preferably from 15 to 35 or from 8 to 25.
  • Signal nucleic acid oligomer ligands which meet these conditions are also referred to below as SNP-ID ligands.
  • the signal nucleic acid oligomer ligands which are used as SNP-ID ligands consist of less than n nucleotides, all nucleotides of the signal nucleic acid oligomer ligand being sequence-specifically complementary to the n-nucleotide sequence of the ligate nucleic acid oligomer are.
  • signal nucleic acid oligomer ligands can also be used which consist of n or more nucleotides, but only have regions of n nucleotides whose sequence in fewer than n nucleotides, in particular in n-1 to n-5 nucleotides, for the sequence of n nucleotide long ligate nucleic acid oligomer is complementary.
  • signal nucleic acid oligomer ligands can also be used which consist of n or more nucleotides, but which only have partial sequences such that, after association of two or more partial sequences of the signal nucleic acid oligomer ligand to the ligate nucleic acid oligomer, fewer than n nucleotides of the hybrid from ligate and signal nucleic acid oligomer ligand, in particular only n-1 to n-5 nucleotides, can be present complementarily hybridized to one another.
  • These coordinated complementary regions of ligate nucleic acid oligomers and signal nucleic acid oligomer ligands bring with them a particular advantage in the detection of nucleic acid oligomer hybridization events.
  • nucleic acid oligomer ligand hybridizes to the ligate nucleic acid oligomer, the nucleic acid oligomer ligand having a coherent nucleotide sequence which is complementary to the ligate nucleic acid oligomer in all bases, with which a so-called perfect match can form.
  • mismatches that is to say to suppress or detect as negative hybridization events in which the associate (hybrid) of ligate-nucleic acid oligomer and nucleic acid oligomer ligand have one or more non-complementary base pairs
  • association constant K SIG of the hybrid of ligate-nucleic acid oligomer and signal-nucleic acid oligomer ligand must namely be greater than the association constant K MM of a mismatch, because in this case the nucleic acid oligomer ligands whose association has at least one mismatch with the ligate-nucleic acid oligomers by the signal nucleic acid oligomer ligands are displaced or largely displaced from their binding to the ligate nucleic acid oligomers and conversely signal nucleic acid oligomers associated with the ligate nucleic acid oligomer can no longer or only very little be displaced from the associate by mismatch nucleic acid oligomer ligands.
  • association constant K SIG of the hybrid of ligate-nucleic acid oligomer and signal-nucleic acid oligomer ligand must be smaller than the association constant K M of a perfect match, since in this case the nucleic acid oligomer ligands, whose association with the ligate-nucleic acid oligomers form a perfect match , by which signal nucleic acid oligomer ligands are not displaced from their binding to the ligate nucleic acid oligomers and conversely signal nucleic acid oligomers already associated with the ligate nucleic acid oligomer are displaced from the associate by a nucleic acid oligomer ligand which enables a perfect match.
  • SNP-ID ligands Signal nucleic acid oligomer ligands that meet these conditions are referred to as SNP-ID ligands. These SNP-ID ligands only have such sequence sections that, after association with the ligate nucleic acid oligomer, a maximum of n-1, n-2, n-3, n-4 or n-5 nucleotides to one another are complementary.
  • the correlation K M > K SIG ⁇ K then applies to the association constants of the possible hybrids - with a suitable choice of the signal nucleic acid oligomer ligand, in particular when using signal nucleic acid oligomer ligands which consist of less than n nucleotides in total, all nucleotides of the signal-nucleic acid oligomer ligand are sequence-specifically complementary to the n-nucleotide sequence of the ligate-nucleic acid oligomer, the association constant of the hybrid of ligate-nucleic acid oligomer and signal-nucleic acid oligomer ligand is somewhat larger than the association constant of a mismatch because the non-complementary bases Mismatches are usually located inside the nucleic acid chain.
  • the hybrid is more disturbed than by a missing base at the chain end, which means that the association constant of the hybrid consisting of ligate-nucleic acid oligomer and shortened signal-nucleic acid oligomer ligand is somewhat larger than the association constant of a mismatch and a correlation K M > K SIG KM can thus be established can be set.
  • association constants when using ligate nucleic acid oligomers and signal nucleic acid oligomer ligands with coordinated lengths are transferred to displacement assays using, for example, ligate antibodies, signal antigen ligands and antigen ligands can.
  • the nucleotide regions for sequence-specific hybridization and the associated association constants K M , K SIG and K MM instead of the nucleotide regions for sequence-specific hybridization and the associated association constants K M , K SIG and K MM , the differ in this case Association constants of the signal antigen / ligate antibody and antigen ligand / ligate antibody associates, for example, by an order of magnitude.
  • a molar ratio of signal nucleic acid oligomer ligands to ligate nucleic acid oligomers is between 0.01 and 1000, preferably between 0.1 and 100, particularly preferably between 1 and 10 used. This avoids displacement of the nucleic acid oligomers from the perfect match by the signal nucleic acid oligomer ligands.
  • a molar ratio of signal nucleic acid oligomer ligands to nucleic acid oligomer ligands is between 0.01 and 1000, preferably between 0.1 and 100 , particularly preferably used between 1 and 10.
  • the expected amount of nucleic acid oligomer ligands must be known at least to an order of magnitude. If it is not known, a series of experiments with varying molar ratios of signal nucleic acid oligomer ligands to ligate nucleic acid oligomers in the range between 0.01 and 1000 can also be carried out.
  • the resulting measured value curve from measured values versus added sample portions shows a significant increase in measured value when fewer sample portions are added if perfectly hybridizing nucleic acid oligomer ligands were present in the sample, while nucleic acid oligomer ligands that form mismatches during hybridization only with a significantly increased addition of Lead to an increase in the measured values and these two measured value ranges - if there are matches and mismatches in the sample - are separated from one another by a plateau of the measured values.
  • the modified surface is washed before the first detection of the signal nucleic acid oligomer ligands. This removes the supernatant solution with the SNP-ID ligands contained therein that are not associated with ligate nucleic acid oligomers.
  • a signal is measured which originates exclusively from the SNP-ID ligands bound to the ligate nucleic acid oligomers. This prevents the measurement values from being overlapped by unbound signal nucleic acid oligomer ligands, which means that detection is achieved with greater accuracy.
  • the type of SNP-ID ligands which are suitable for each type of ligate nucleic acid oligomer are adapted such that they only have those sequence segments which, after association with the n ml nucleotide sequence of the ligate nucleic acid oligomer in the Hybrid maximum n Ml -1, n Ml -2, n Ml -3, n M ⁇ -4 or n Ml -5 have mutually complementary nucleotides.
  • the variation of the nucleotide length of the ligate nucleic acid oligomers with parallel detection of different nucleic acid oligomer ligands allows a more targeted and facilitated selection of ligate nucleic acid oligomers which enable a perfect match with the nucleic acid oligomer ligands if the nucleic acid oligomer ligands of the sample to be examined have a large pool of potentially present nucleic acid oligomer ligands, as z. B. is the case with SNP studies of the entire human genome.
  • the adapted SNP-ID ligands enable indirect stringent hybridization, which is carried out simultaneously for all test sites, but is individually adapted to each test site.
  • ligate PNA oligomers are used as ligate nucleic acid oligomers and signal PNA oligomer ligands are used as signal nucleic acid oligomer ligands.
  • the advantage of using ligate PNA oligomers and signal PNA oligomer ligands lies in the fact that the methods of the present invention without additional modification of ligate nucleic acid oligomers, nucleic acid oligomer ligands or signal nucleic acid oligomer ligand with a detectable label can be carried out.
  • PNA is known to differ from DNA in that it lacks an electrical charge.
  • the charge of the nucleic acid oligomer ligand can be regarded as a "label" when using ligate PNA oligomers and signal PNA oligomer ligands, which can be detected by suitable measurements, in particular by electrochemical measurements such as electrochemical impedance measurements or by SECM.
  • the present invention also relates to a kit for carrying out a method for the detection of nucleic acid oligomer hybridization events.
  • the kit comprises a modified surface, the modification consisting in the attachment of at least one type of ligate nucleic acid oligomer, and an effective amount of signal nucleic acid oligomer ligands.
  • a double detection of the Siganl nucleic acid oligomer ligands can be carried out, ie a determination of the Reference values and the second detection of the signal nucleic acid oligomer ligands and a measurement of correction values.
  • the reference values are already included in the kit, so that the signal nucleic acid oligomer ligands only have to be detected once by the end user. The values obtained in this detection then only need to be compared with the existing reference values.
  • the reference values and correction values are already included in the kit, so that the signal nucleic acid oligomer ligands only have to be detected once by the end user.
  • the values obtained in this detection then only need to be compared with the existing reference values and standardized to the correction values.
  • the kit comprises a modified surface which has at least one area T 0 and at least one area T 100 .
  • the modified surface additionally comprises at least one region T n are particularly preferred.
  • the present invention also relates to a kit for carrying out a method for the detection of SNP examinations.
  • the kit includes a modified surface, the modification being the attachment of at least one type of ligate nucleic acid oligomer, and an effective amount of SNP-ID ligand.
  • a double detection of the SNP-ID ligands can be carried out, ie a determination of the reference values and the second detection of the signal nucleic acid oligomer ligands as well as a measurement of correction values.
  • the reference values and correction values are already included in the kit, so that the SNP-ID ligands only have to be detected once by the end user.
  • the values obtained during this detection then only need to be compared with the already existing threshold values, which were determined from reference values and correction values, in order to recognize the absence or presence of an SNP and its composition (homo- or heterozygous).
  • the present invention thus provides a method for the detection of sequence-specific nucleic acid oligomer hybridization events using a displacement assay.
  • nucleic acid oligomers such as DNA / RNA / PNA oligomer single strands as an association matrix (probe) for the detection of targets, ie for the detection of oligonucleotides or DNA fragments.
  • association matrix ligate nucleic acid oligomers are contacted with a solution of signal nucleic acid oligomer ligands, whereby some of the signal nucleic acid oligomer ligands are hybridized to the surface-immobilized ligate nucleic acid oligomers and the remaining signal nucleic acid oligomer ligands remain in the supernatant solution .
  • the modified surface can be washed before the detection of the signal nucleic acid oligomer ligands.
  • the signal nucleic acid oligomer ligands are selected so that the surface associates of ligate nucleic acid oligomer and signal nucleic acid oligomer ligand have an association constant that is lower than the association constant between ligate nucleic acid oligomer and nucleic acid oligomer ligand.
  • the signal nucleic acid oligomer ligands or the nucleic acid oligomer ligands either themselves function as signal-generating substances for detection or they are labeled with a detectable signal-generating substance.
  • the surface-immobilized signal nucleic acid oligomer ligands are detected in the absence of nucleic acid oligomer ligands - either before adding the ligand nucleic acid oligomers or after dissociation of the ligand oligonucleotides - using a suitable measurement method. If there are signal-nucleic acid oligomer ligands in the supernatant solution during the reference measurement, a suitable surface-sensitive measurement method (e.g.
  • total infernal reflection fluorescence or electrochemical methods such as chronocoulometry is selected as the measurement method, which allows between surface-immobilized signal nucleic acid oligomer ligands and discriminate signal nucleic acid oligomer ligands in the bulk phase.
  • an unknown type of nucleic acid oligomer ligand is therefore determined by detecting a third compound (signal nucleic acid oligomer ligand), which, like the unknown type of nucleic acid oligomer ligand, associates with a probe molecule (ligate nucleic acid oligomer).
  • signal nucleic acid oligomer ligand a third compound
  • nucleic acid oligomer ligand a probe molecule
  • nucleic acid oligomer ligand there are associations of nucleic acid oligomer ligand and ligate nucleic acid oligomer, it is of course also possible to displace part of the nucleic acid oligomer ligands from the ligand-ligate complex by adding signal nucleic acid oligomer ligands.
  • nucleic acid oligomer ligand with ligate nucleic acid oligomer, since an equilibrium will in any case be established which is determined by the ratio of the association constants of nucleic acid oligomer ligand / ligate nucleic acid oligomer to signal nucleic acid oligomer ligand / Ligate nucleic acid oligomer and the concentrations used of nucleic acid oligomer ligand and signal nucleic acid oligomer ligand is determined.
  • the displacement assay thus comprises a complexation event between a ligate nucleic acid oligomer and a signal nucleic acid oligomer ligand, which is in competition with a further complexation event from the actual target (nucleic acid oligomer ligand) and ligate nucleic acid oligomer.
  • the displacement assay comprises in particular a complexation event between a ligate nucleic acid oligomer and a signal nucleic acid oligomer ligand, which is followed by a further complexation event after addition of the actual target (nucleic acid oligomer ligand), which takes place with displacement of the signal nucleic acid oligomer ligand.
  • the displacement assay includes in particular a complexation event between a ligate nucleic acid oligomer and a target (nucleic acid oligomer ligand), which is followed by a further complexation event after addition of signal nucleic acid oligomer ligands, which can take place with displacement of at least some of the nucleic acid oligomer ligands.
  • the term “surface” denotes any carrier material which is suitable for covalently binding derivatized or non-derivatized ligate nucleic acid oligomers directly or after corresponding chemical modification or via other specific interactions.
  • the solid support can consist of conductive or non-conductive material.
  • conductive surface means any carrier with an electrically conductive surface of any thickness, in particular surfaces made of platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead, aluminum and manganese.
  • any doped or undoped semiconductor surfaces of any thickness can also be used. All semiconductors can be used as pure substances or as mixtures. As non-limiting examples, carbon, silicon, germanium, ⁇ -tin, Cu (l) and Ag (l) halides of any crystal structure may be mentioned here. Also suitable are all binary compounds of any composition and structure from the elements of groups 14 and 16, the elements of groups 13 and 15, and the elements of groups 15 and 16. In addition, ternary compounds of any composition and structure from the elements of Groups 11, 13 and 16 or the elements of groups 12, 13 and 16 can be used. The names of the groups in the Periodic Table of the Elements refer to the 1985 IUPAC recommendation.
  • glass and modified glass are preferred as the material.
  • the modification can be carried out, for example, by silanization and leads in all cases to functional groups which are suitable for binding appropriately functionalized ligate nucleic acid oligomers in coupling reactions.
  • This modification includes layered structures on the glass surface using polymers such as Dextran polymers, which allow a variation of the layer thickness and surface quality with.
  • Further possibilities of derivatization of the glass for the final attachment of the ligate nucleic acid oligomers are, for example, the application of a thin (approx. 10-200 nm) metallization layer, in particular a gold metallization layer, which can additionally be coated with (thiol-functionalized) polymers, especially dextrans.
  • the glass can also be functionalized with biotin after the silanization (e.g. amino-functionalized glass surface after the silanization and coupling of the carboxylic acid biotin via EDC and NHS or via a biotin active ester such as biotin-N-succinimidyl ester) or alternatively with immobilized on dextran-lysine or dextran Biotin can be coated.
  • biotinylated glass surfaces produced in this way are then treated with avidin or streptavidin and can then be used to bind biotinylated ligate nucleic acid oligomers.
  • nucleic acid oligomers bound to a surface are referred to as ligate nucleic acid oligomers.
  • Methods for immobilizing nucleic acid oligomers on a surface are known to the person skilled in the art.
  • the ligate nucleic acid oligomers can, for example, be covalently bonded to the surface via hydroxyl, epoxy, amino or carboxy groups of the support material with thiol, hydroxyl, amino or carboxyl groups present on the nucleic acid oligomer or by derivatization on the ligate nucleic acid oligomer.
  • the ligate nucleic acid oligomer can be bound directly or via a linker / spacer to the surface atoms or molecules of a surface.
  • the ligate nucleic acid oligomer can be anchored by the methods customary in immunoassays, for example by using biotinylated ligate nucleic acid oligomers for non-covalent immobilization on avidin or streptavidin-modified surfaces.
  • the chemical modification of the ligate nucleic acid oligomers with a surface anchor group can already be introduced in the course of automated solid-phase synthesis or in separate reaction steps.
  • the nucleic acid oligomer is also linked directly or via a linker / spacer to the surface atoms or molecules of a surface of the type described above. This binding can be carried out in various ways (see, for example, WO 00/42217). Ligate nucleic acid oligomers / nucleic acid oligomer ligands / signal nucleic acid oligomer ligands
  • the ligate nucleic acid oligomers of the present invention consist of n nucleotides which are present in a specific nucleotide sequence (sequence).
  • the ligate nucleic acid oligomers are complementary or at least predominantly complementary to a sequence region of n nucleotides of the nucleic acid oligomer ligands.
  • nucleic acid oligomer ligands Molecules that specifically interact with the ligate nucleic acid oligomer immobilized on a surface to form a double-stranded hybrid are referred to as nucleic acid oligomer ligands.
  • nucleic acid oligomer ligands are therefore nucleic acid oligomers which act as complex binding partners of the complementary nucleic acid oligomer.
  • the nucleic acid oligomer ligands, the presence of which is to be detected on the basis of the present invention, can have further sequence regions in addition to the contiguous sequence region which is n nucleotides long and whose sequence is complementary to the n nucleotide sequence of the ligates.
  • the ligate nucleic acid oligomers of one type are complementary to a sequence region of n nucleotides of nucleic acid oligomer ligands of one type, wherein the ligate nucleic acid oligomer can form a perfect match with this n nucleotide-contiguous sequence region of the nucleic acid oligomer ligand, i.e. in this n-nucleotide-long region, the ligate and matching ligand form a double helix with n exclusively matching base pairs after the association (hybridization).
  • the signal nucleic acid oligomer ligands associated with a type of ligate nucleic acid oligomer have only partially complementary sequence regions to the n nucleotide long sequence of the ligate.
  • Nucleic acid oligomer ligands only such sequence sections that, after association with the n nucleotide long sequence of the ligate nucleic acid oligomer, a maximum of n-1, n-2, n-3, n-4 or n-5 nucleotides are complementary to one another.
  • n for an integer from 3 to 80, in particular from 5 to 50, particularly preferably from 15 to 35 or from 8 to 25.
  • Signal nucleic acid oligomer ligands which satisfy these conditions are also called SNP-ID ligands - Nucleic acid oligomer ligands can be determined by suitable detection methods themselves or after appropriate modification with a detection label.
  • the signal nucleic acid oligomer ligands can be detected by a surface-sensitive detection method, in particular by a spectroscopic, an electrochemical or an electrochemiluminescent method.
  • Spectroscopic detection is the detection of fluorescence, in particular total infernal reflection fluorescence (TIRF) of the signal nucleic acid oligomer ligands, whereas electrochemical detection can be carried out using amperometry, chronocoulometry, impedance measurement or scanning electrochemical microscopy (SECM).
  • TIRF total infernal reflection fluorescence
  • SECM scanning electrochemical microscopy
  • ligate PNA oligomers are used as ligate nucleic acid oligomers and signal PNA oligomer ligands are used as signal nucleic acid oligomer ligands.
  • the advantage of using ligate PNA oligomers and signal PNA oligomer ligands lies in the fact that any embodiment of the displacement assay of the present invention can be used without additional modification of ligate nucleic acid oligomer, nucleic acid oligomer ligand or signal nucleic acid oligomer ligand can be carried out with a detectable label.
  • PNA is known to differ from DNA in that it lacks an electrical charge.
  • the charge of the nucleic acid oligomer ligand can be regarded as a "label" which can be detected by suitable measurements, in particular by electrochemical measurements such as electrochemical impedance measurements or by SECM.
  • SNP-ID ligands are used as signal nucleic acid oligomer ligands.
  • the modified surface is washed before the detection of the signal nucleic acid oligomer ligands. This removes the supernatant solution with the SNP-ID ligands contained therein that are not associated with ligate nucleic acid oligomers.
  • a signal is measured that is unique to the the ligate nucleic acid oligomer bound SNP-ID ligand is derived. This prevents the measurement values from being superimposed by unbound signal nucleic acid oligomer ligands, as a result of which detection is achieved with greater accuracy and any method that is sensitive to the detector label used can be used as the measurement method.
  • the signal nucleic acid oligomer ligands have a lower tendency to form complexes with the ligate nucleic acid oligomers than the actual nucleic acid oligomer ligands (targets), i.e. the association constant between ligand and ligate is greater than the association constant between signal ligand and ligate.
  • the signal oligonucleotide therefore only has nucleic acid regions in which less than 20 consecutive bases of the signal oligonucleotide are complementary to the 20 bases of the ligate oligonucleotide.
  • an arbitrarily long signal oligonucleotide labeled with a detection label can be used, as long as the signal oligonucleotide only has nucleic acid regions in which fewer than 20 consecutive bases of the signal oligonucleotide are complementary to the 20 bases of the ligate oligonucleotide.
  • the signal oligonucleotide can be a 20 nt oligo labeled with at least one detection label or contain a 20 nt oligo sequence that is complementary to the 20 nt ligate oligonucleotide and one or more base pair mismatches in the complexing between signal oligonucleotide and ligate oligonucleotide. It is also possible to use an oligonucleotide labeled with at least one detection label which is less than 20 nt and is completely complementary to the ligate oligonucleotide.
  • a nucleic acid oligomer is a compound of at least two covalently linked nucleotides or of at least two covalently linked pyrimidine (for example cytosine, thymine or uracil) or purine bases (for example adenine or guanine), preferably a DNA, RNA or PNA fragment used.
  • pyrimidine for example cytosine, thymine or uracil
  • purine bases for example adenine or guanine
  • nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as, for example, the sugar-phosphate backbone of DNA, cDNA or RNA, a peptide backbone of PNA or analogous backbone structures, such as a thio-phosphate, a dithio-phosphate or a phosphoramide backbone.
  • Essential feature of a nucleic acid in the sense of the present invention is the sequence-specific binding of naturally occurring DNA or RNA or structures derived therefrom (transcribed or amplified) such as cDNA or amplified cDNA or amplified RNA (aRNA).
  • Signal nucleic acid oligomer ligands that cannot be detected themselves are provided with one or more detectable labels by derivatization.
  • This label enables the detection of the complexation events between the signal nucleic acid oligomer ligand and the surface-bound ligate nucleic acid oligomer.
  • the label can deliver a detection signal directly or, as in the case of enzyme-catalyzed reactions, indirectly.
  • Preferred detection labels are fluorophores and redox-active substances.
  • nucleic acid oligomer ligands When using ligate PNA oligomers as ligate nucleic acid oligomers and signal PNA oligomer ligands as signal nucleic acid oligomer ligands, it is possible to work with a detectable label without additional modification of ligate nucleic acid oligomer, nucleic acid oligomer ligands or signal nucleic acid oligomer ligands.
  • the charge of the nucleic acid oligomer ligand is used as a "label", which can be detected by suitable measurements, in particular by electrochemical measurements such as electrochemical impedance measurements or by SECM.
  • fluorophores commercially available fluorescent dyes such as Texas red, rhodamine dyes, fluorescein etc.
  • redox molecules are used as labels.
  • Transition metal complexes in particular those of copper, iron, ruthenium, osmium or titanium with ligands such as pyridine, 4,7-dimethylphenanthroline, 9,10-phenanthrenquinone diimine, porphyrins and substituted porphyrin derivatives can be used as redox labels.
  • riboflavin of quinones such as pyrrolloquinoline quinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone or derivatives thereof, of metallocenes and metallocene derivatives such as ferrocenes and ferrocene derivatives, cobaltocenes and cobaltocene derivatives, of porphyrins, methylene blue, daaminomin derivative derivatives
  • Derivatives para or ortho dihydroxy benzene derivatives, para or ortho dihydroxy anthraquinone derivatives, para or ortho dihydroxy naphthoquinone derivatives
  • Surface sensitive detection methods are possible.
  • Electrochemical, spectroscopic and electrochemiluminescent methods are suitable as the detection method.
  • the kinetics of the electrochemical processes can in principle be used to distinguish between redox-active detection labels adsorbed on a surface and dissolved in the supernatant.
  • Surface-adsorbed detection labels are generally converted electrochemically faster (e.g. oxidized or reduced) than redox-active detection labels from the bulk phase, since the latter first have to diffuse to the (electrode) surface before the electrochemical conversion.
  • Examples of electrochemical surface-sensitive methods are amperometry and chronocoulometry.
  • the chronocoulometry method allows to distinguish near-surface redox-active components from (identical) redox-active components in the volume phase and is e.g. in Steel, A.B., Herne, T.M. and Tarlov M.J .: Electrochemical Quantitation of DNA Immobilized on Gold, Analytical Chemistry, 1998, Vol. 70, 4670-4677 and references cited therein.
  • the measurement signal of the chronocoulometry (transferred charge Q depending on the
  • Time t is composed of three components: (i) a diffusive part, which is caused by the redox-active components in the volume phase and
  • n number of electrons per molecule for the reduction
  • C 0 * concentration [mol / cm 2 ]
  • Q d ⁇ capacitive charge
  • C nFAF 0 charges which are converted during the electrochemical conversion of the adsorbed redox-active detection label, where F 0 [mol / cm 2 ] represents the surface coverage density of the detection label.
  • F 0 therefore stands for the amount of detection label on the electrode surface.
  • a chronocoulometrically detected displacement assay in the sense of the present invention will be explained using the example of a 20 nt ligate nucleic acid oligomer.
  • the (working) electrode modified with 20 nt ligate oligonucleotides is made with a defined amount of signal nucleic acid oligomer ligands, for example a 12 nt signal nucleic acid oligomer ligand that carries one or more redox labels and becomes a region of the ligate that is as close to the surface as possible -Oligonucleotide is complementary, brought into contact so that an association between ligate oligonucleotide and redox-labeled ss-nucleic acid oligomer complexing agent can take place.
  • the (working) electrode is initially set to a potential Ei at which little to no electrolysis (electrochemical change in the redox state) of the redox marking can take place (e.g. with ferrocene-modified ligate oligonucleotide approx. 0.1 V against Ag / AgCI ( sat.KCI)).
  • the working electrode is set to a potential E 2 by means of a potential jump, at which the electrolysis of the redox marking takes place in the diffusion-limited limit case (for example in the case of ferrocene-modified SS Nucleic acid oligomer complexing agent approx. 0.5 V against Ag / AgCI (sat. KCI)).
  • the transferred charges are recorded as a function of time.
  • the sample solution is added, which should (can) contain the ligand nucleic acid oligomer (target), which has an nt sequence which is complementary to the 20 nt of the ligate oligonucleotides in a region.
  • target the ligand nucleic acid oligomer
  • a second electrochemical measurement is carried out.
  • the change in the instantaneous charge signal is proportional to the number of signal oligonucleotide ligands displaced and is therefore proportional to the number of target oligonucleotides present in the test solution.
  • This change in the instantaneous charge signal depends on the length of the ligand oligonucleotides, that is to say on the number of nucleotides of the ligand oligonucleotides. If the ligand oligonucleotides have a length which corresponds approximately to or is shorter than the length of the ligate oligonucleotides, a decrease in the instantaneous charge signal is observed, since signal oligonucleotides are generated by the ligand oligonucleotides from the association with the ligate oligonucleotides and thus be displaced from the vicinity of the modified surface.
  • the ligand oligonucleotides have a greater length than the ligate oligonucleotides, only a part of the nucleotides of the ligand oligonucleotides can bind to the nucleotides of the ligate oligonucleotides and a remaining part of the ligand oligonucleotide with freely accessible nucleotides remains.
  • the signal oligonucleotides will then bind to these free bases of the ligand oligonucleotides.
  • the protruding part of the ligand oligonucleotides is in the vicinity of the surface, an increase in the instantaneous charge signal can occur, since the number of signal oligonucleotides that are in the vicinity of the surface can be found by adding the long-chain ligand oligonucleotides and binding of the signal oligonucleotides to these ligand oligonucleotides increased.
  • Total internal reflection fluorescence (TIRF, cf. Sutheriand and Dahne, 1987, J. Immunol. Meth., 74, 253-265) can serve as the optical measurement method for the detection of fluorescence-labeled signal nucleic acid oligomer ligands.
  • Fluorescence molecules which are located in the vicinity of the interface between a solid waveguide medium, typically glass, and a liquid medium or are immobilized on the surface of the waveguide medium facing the liquid, can be excited by the evanescent field that protrudes from the waveguide and emit detectable fluorescent light.
  • Displaced or dissolved fluorescent-labeled complexing agents are not detected by the evanescent field (or only insofar as they are in the area of the penetration depth of the evanescent field) and therefore make (almost) no contribution to the measured signal.
  • the penetration depth of the evanescent field is typically 100 to 200 nm, but can be increased to several 100 nm by means of a thin metallization layer (approx. 10 to 200 nm), in particular a gold metallization layer.
  • the layer thickness of the ligate-modified carrier surface is adapted to the penetration depth of the evanescent field, e.g.
  • Biotin active esters such as biotin-N-succinimidyl ester) on amino-derivatized surfaces and coupling of avidin or streptavidin to the biotinylated so produced
  • 10-200 nm) metallization layer in particular a gold metallization layer, which is coated with a (thiol functionalized) polymer, in particular dextrans or polylysine, can be occupied, which in turn is used to bind the ligate nucleic acid oligomer or (b) by applying a polymer layer composed of polylysine, dextran-lysine or dextran-immobilized biotin and coupling of avidin or Streptavidin to the biotinylated surfaces thus generated with subsequent attachment of biotinylated ligate nucleic acid oligomers.
  • a polymer layer composed of polylysine, dextran-lysine or dextran-immobilized biotin and coupling of avidin or Streptavidin to the biotinylated surfaces thus generated with subsequent attachment of biotinylated ligate nucleic acid oligomers.
  • SNP-ID ligands are used as signal nucleic acid oligomer ligands.
  • the modified surface is washed before the detection of the signal nucleic acid oligomer ligands. This removes the supernatant solution with the SNP-ID ligands contained therein that are not associated with ligate nucleic acid oligomers. During the subsequent detection of the signal nucleic acid oligomer ligands, a signal is measured which originates exclusively from the SNP-ID ligands bound to the ligate nucleic acid oligomers.
  • Show it 1 shows a schematic representation of the detection of nucleic acid oligomer hybridization events by means of displacement assay
  • FIG. 3 shows a chronocoulometric measurement of the sequence-specific hybridization of a 20mer ligate nucleic acid oligomer with a complementary counter strand (nucleic acid oligomer ligand) by detection of the ferrocene-labeled tetramer signal nucleic acid oligomer ligands displaced by the hybridization.
  • D Array with specific complexation of a test site with target
  • E Array with specific complexation of a ligate nucleic acid oligomer with target and with signal nucleic acid oligomer ligands associated with a ligate nucleic acid oligomer
  • nucleic acid oligomer 1 101 ligate nucleic acid oligomer 1 102 ligate nucleic acid oligomer 2 103 signal nucleic acid oligomer ligand 104 detection label, eg ferrocene 105 surface, eg gold 106 nucleic acid oligomer ligand and: addition of a signal nucleic acid oligomer ligand ⁇ : hybridization @: dissociation @: dissociation and removal of the signal Nucleic acid oligomer ligand and subsequent addition of the nucleic acid oligomer ligand ⁇ : Addition of the nucleic acid oligomer ligand
  • the curve labeled "1” represents the chronocoulometric measurement after hybridization of the ferrocene-labeled tetramer signal nucleic acid oligomer ligands
  • the curve labeled “2” shows the chronocoulometric measurement after addition of the complementary nucleic acid oligomer ligand and (partial) displacement of the redox-labeled tetramers from the ligate surface.
  • the DNA chip can also be derivatized with a single ligate oligonucleotide.
  • the ligate nucleic acid oligomers are nucleic acid oligomers (for example DNA, RNA or PNA fragments) with a base length of 3 to 80 or 3 to 70 or 3 to 50, preferably with a length of 5 to 50 or 8 to 50 or 5 to 30, particularly preferably the Length 15 to 35 or 10 to 30 or 8 to 25 used.
  • the surface thus provided with immobilized ligate oligonucleotides is incubated with a solution of a certain amount of signal nucleic acid oligomer ligands, for example nucleic acid oligomers labeled with redox labels, which can only be hybridized to a certain sequence section of the ligate oligonucleotide, but not to the entire sequence of the ligate oligonucleotide. This leads to the formation of hybrids from ligate nucleic acid oligomer and the signal nucleic acid oligomer ligands in the area of complementary sequences.
  • signal nucleic acid oligomer ligands for example nucleic acid oligomers labeled with redox labels
  • the surface-immobilized portion of the signal nucleic acid oligomer ligands is determined in a reference measurement (e.g. by a first chronocoulometric measurement, see section "Surface-sensitive detection methods").
  • the (as concentrated as possible) test solution with ligand oligonucleotide (s) is added to the surface with immobilized ligate oligonucleotides, associated signal nucleic acid oligomer ligands and excess solution (with free, non-surface-adsorbed signal nucleic acid oligomer ligands).
  • Hybridization only occurs in the case in which the solution contains ligand-nucleic acid oligomer strands which are complementary to the ligate-nucleic acid oligomers bound to the surface, or at least complementary in wide areas (or further areas than the signal oligonucleotide) are.
  • the originally associated signal oligonucleotides are, at least partially, displaced.
  • the proportion of the remaining surface-immobilized signal nucleic acid oligomer ligands is determined in a second measurement (e.g. a second chronocoulometric measurement, see section "Surface-sensitive detection methods").
  • the difference between the reference measurement and the second measurement per test site is proportional to the number of complementary (or in many areas complementary) ligand oligonucleotides originally present in the test solution for the respective test site (cf. FIG. 1, process route with the steps ⁇ , ⁇ , ⁇ ).
  • the associates of ligate nucleic acid oligomer and signal nucleic acid oligomer ligand can be dissociated on the surface, for example by increasing the temperature, and all signal nucleic acid oligomer ligands or only the signal nucleic acid oligomer ligands in the supernatant Solution are removed by washing, so that after the reference measurement, the surface originally used with immobilized ligate oligonucleotides is available.
  • the removal of the signal nucleic acid oligomer ligands from the associates with the ligate nucleic acid oligomers is generally carried out by removing the modified surface from the solution containing the signal nucleic acid oligomer ligands and then washing the modified surface. Dehybridizing conditions can be set when washing the modified surface and / or before removing the modified surface from the solution containing the signal nucleic acid oligomer ligands.
  • the sample solution with ligand oligonucleotide (s) is then added to the surface with immobilized ligate oligonucleotides and the ligand oligonucleotides which may be present can be hybridized to the ligate oligonucleotides under any stringency conditions known to the person skilled in the art.
  • stringency conditions it can be achieved that only complementary ligand oligonucleotides remain hybridized to the ligate oligonucleotides, whereas "ligand oligonucleotides" which have one or more mismatches dehybridize.
  • the reference measurement can be omitted if the size of the reference signal is known beforehand (for example, from previous measurements etc.).
  • step ⁇ hybridization with nucleic acid oligomer ligands and then step ⁇ are carried out (cf. FIG. 1, process route with step ⁇ ).
  • the sample solution with ligand oligonucleotides is first added to the surface with immobilized ligate oligonucleotides and optionally hybridized under stringent conditions.
  • a solution is then added which contains a certain amount of signal nucleic acid oligomer ligands.
  • the proportion of signal nucleic acid oligomer ligands which is associated with the ligate oligonucleotides is then determined with the aid of a measurement, and the values obtained are compared with the known reference signal.
  • n nucleotide (nt) long ligate nucleic acid (DNA, RNA or PNA, for example a 20 nucleotide long oligo) (Fig. 1A, 101 or 102) is close to one of its ends (3 'or 5' end ) directly or via any spacer with a reactive group for covalent anchoring to the surface, eg as a 3'-thiol-modified ligate oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold electrodes.
  • a reactive group for covalent anchoring to the surface eg as a 3'-thiol-modified ligate oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold electrodes.
  • Other covalent anchoring options arise e.g.
  • ligate nucleic acid oligomer modified in this way becomes
  • a monofunctional linker in buffer (e.g. 100 mM
  • Alkanethiols or ⁇ -hydroxyalkanethiols in phosphate buffer / EtOH mixtures in the case of thiol-modified ligate oligonucleotides are brought into contact, the monofunctional linker binding via its reactive group to the surface, which may be correspondingly derivatized (see section "The Surface”).
  • FIG. 1A The surface modified in this way (FIG. 1A), after corresponding washing steps, is brought into contact with redox-labeled signal nucleic acid oligomer ligands (FIGS. 1B, 104) composed of fewer than n complementary nucleotides (FIG. 1B, 103).
  • signal nucleic acid oligomer ligands labeled with redox labels e.g.
  • one or more ferrocenecarboxylic acid-modified nucleic acid tetramers are used, the sequence of which is complementary to partial tetramer sequences of the ligate oligonucleotides, or SSB (single stranded DNA binding protein) modified with redox labels (eg ferrocene derivatives) ) are used. Care is taken to ensure that significantly more labeled signal nucleic acid oligomer ligands (at least a 1.1-fold molar excess) are added than can be bound to the surface via the ligate nucleic acid oligomers.
  • the detection label on the signal nucleic acid oligomer ligand is detected by a suitable method, e.g. by chronocoulometry in the case of the signal oligonucleotides labeled with ferrocene redox labels.
  • the dissolved ligand is then added and the measurement for detecting the detection label is repeated using the appropriate method (e.g. renewed chronocoulometric measurement in the case of the signal oligonucleotides labeled with ferrocene redox labels).
  • the modified surface is removed from the solution containing signal nucleic acid oligomer ligands, optionally washed as described above and then brought into contact with the solution containing the nucleic acid oligomer ligands.
  • the hybridization can be carried out under suitable conditions known to the person skilled in the art (arbitrary, freely selectable stringency conditions of the parameters potential / temperature / salt / chaotropic salts etc. for the hybridization).
  • the modified surface is brought into contact with signal nucleic acid oligomer ligands (in the same concentration as in the previous measurement) and a measurement for detecting the detection label is carried out using a suitable method.
  • the difference in the measurement signal is proportional to the number of hybridization events between the ligate nucleic acid oligomer on the surface and the appropriate nucleic acid oligomer ligand in the test solution (see Example 6).
  • a decrease in the almost instantaneous signal portion of the excess surface of redox labels is detected, cf. "Surface sensitive detection methods”.
  • the complexes of surface-bound ligate-nucleic acid oligomers and labeled signal-nucleic acid oligomer ligands are released after the first detection (reference measurement) (for example by increasing the temperature) and the free labeled signal-nucleic acid oligomer ligands are washed out the supernatant removed (Fig. 1, ®).
  • the first detection for example by increasing the temperature
  • the free labeled signal-nucleic acid oligomer ligands are washed out the supernatant removed (Fig. 1, ®).
  • hybridization FIG. 1D
  • adding the marked signal nucleic acid oligomer ligands FIG. 1, (D)
  • a second chronocoulometric measurement is carried out.
  • the method can be for a ligand type, that is to say a specific ligand oligonucleotide type with a known sequence, on one electrode or for several ligand types, that is to say different ligand oligonucleotide types, on individually addressable electrodes of an electrode array which is used in more complex arrays eg can be controlled and read out via CMOS technology.
  • nucleotide long ligate nucleic acid (DNA, RNA or PNA) (FIGS. 1A, 101 or 102, for example a 20 nucleotide long oligo) is direct near one of its ends (3 'or 5' end) or via a (any) spacer with a reactive group for covalent
  • anchoring on the surface for example a carboxy-modified ligate oligonucleotide for binding to amino-modified silanized glass (for example to (3-aminopropyl) -triethoxysilane-modified glass). More covalent
  • Anchoring options arise e.g. from amino-modified ligate oligonucleotide, which is used for anchoring to dextran polymers derivatized with carboxylic acid and immobilized on glass, in this embodiment the thickness of the layer of dextran polymer with attached ligate oligonucleotides using methods known to the person skilled in the art via the dextran polymer composition, dextran anchor groups on the glass surface, Dextrans anchor groups for immobilization on the glass, incubation time on the glass etc. can be varied.
  • the thickness of the dextran / ligate oligonucleotide layer is selected such that it approximately corresponds to the depth of penetration of the evanescent field of light for exciting the fluorophores (approximately 50 nm to approximately 500 nm, depending on whether or not there is a metallization layer on the glass to increase the penetration depth of the evanescent field).
  • FIG. 1A the surface modified in this way (FIG. 1A) is brought into contact with fluorophore-labeled signal nucleic acid oligomer ligands (FIG. 1B, 104) composed of less than n complementary nucleotides (FIG. 1B, 103).
  • fluorophore-labeled signal nucleic acid oligomer ligands e.g. Fluorescein-modified nucleic acid dodecamers are used, the sequences of which are complementary to partial dodecamer sequences of the ligate oligonucleotides, or SSB (single stranded DNA binding protein) modified with one or more fluoresceins (e.g.
  • FITC derivatives can be used. Care is taken to ensure that significantly more labeled signal nucleic acid oligomer ligands (at least a 1.1-fold molar excess) are added than can be bound to the surface via the ligate nucleic acid oligomers.
  • the detection label on the signal ligand oligonucleotide is detected by a suitable method, for example by Total Infernal Reflection Fluorescence (TIRF) in the case of the Fluorophore label labeled signal nucleic acid oligomer ligands.
  • TIRF Total Infernal Reflection Fluorescence
  • the signal nucleic acid oligomer ligand in the supernatant solution is removed by washing, the test solution is added and potential hybridization events are made possible under suitable conditions known to the person skilled in the art (arbitrary, freely selectable stringency conditions of the parameters potential / temperature / salt / chaotropic salts etc. for the hybridization).
  • the amount of signal ligand originally used for the reference measurement is then added again and the measurement for the detection of the detection label is repeated using the appropriate method (eg renewed TIRF measurement in the case of the signal nucleic acid oligomer ligands labeled with fluorescein).
  • the difference in the measurement signal is proportional to the number of hybridization events between the ligate nucleic acid oligomer on the surface and the appropriate nucleic acid oligomer ligand in the test solution (see Example 6).
  • a decrease in the fluorescence signal is expected.
  • the complexes of surface-bound ligate nucleic acid oligomer and labeled signal nucleic acid oligomer ligands are released after the first detection (reference measurement) and the free labeled oligonucleotides are removed from the supernatant by washing (FIG. 1, ® ). After adding the nucleic acid oligomer ligands, hybridization (FIG. 1 D) and adding the labeled signal nucleic acid oligomer ligands (FIG. 1, ⁇ ), a second measurement is then carried out (FIG. 1 E).
  • the method can be used for a ligand type, ie a specific ligand oligonucleotide type with a known sequence, on a glass fiber or for several ligand types, ie different ligand oligonucleotide types, on individually addressable glass fibers of a glass fiber bundle.
  • 1A, 101 or 102 is close to one of its ends (3 'or 5' end) directly or via any (any) Provide spacers with a reactive group for covalent anchoring to the surface, for example as a 3'-thiol-modified ligate oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold electrodes.
  • the ligate nucleic acid oligomer modified in this way becomes
  • the amount of SNP-ID ligand is adjusted so that the molar ratio of SNP-ID ligand and ligate nucleic acid oligomer is 2-5 and the concentration of the SNP-ID ligand is as high as possible.
  • the detection label on the SNP-ID ligand is detected by a suitable method, e.g. by cyclic voltammetric determination of the signal oligonucleotides labeled with ferrocene redox labels.
  • the value obtained is compared with a previously determined reference value at which the SNP-ID ligand was associated under the same hybridization conditions (amount, concentration, duration of hybridization) to an identical modified surface, but in the absence of nucleic acid oligomer ligands.
  • the corresponding nucleic acid oligomer ligand which was complementary to the ligate nucleic acid oligomer, was available and was a perfect match for the 20 nucleotides that the ligate can cover.
  • Example 1 Representation of the N-hydroxysuccinimide active esters of the redox (or fluorophore) labels
  • Example 2 Representation of the amino-modified oligonucleotides for coupling the active ester labels from example 1 or of thiol-modified oligonucleotides for anchoring to gold as ligate nucleic acid oligomers
  • oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 ⁇ mol synthesis.
  • the synthesis of the (signal) nucleic acid oligomer ligands is carried out as standard on A-CPG as the carrier material. Modifications to the 5 ' position of the oligonucleotides are carried out with a coupling step which is extended to 5 minutes.
  • the amino modifier C2 dT (Glen Research 10-1037) is built into the sequences with the respective standard protocol.
  • 3'-thiol-modified ligate oligonucleotides (or HO- (CH 2 ) 2 -SS- (CH 2 ) 2 OPO 3 -oligonucleotides) are prepared on a 1-O-dimethoxytrityl-propyl-disulfide-CPG support (Glen Research 20-2933) analogous to standard protocols, the oxidation steps being carried out with a 0.02 M iodine solution in order to avoid oxidative cleavage of the disulfide bridge.
  • the coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically.
  • the oligonucleotides are deprotected with concentrated ammonia (30%) at 37 ° C for 16 h.
  • the oligonucleotides are purified using RP-HPL chromatography according to standard protocols (eluent: 0.1 M triethylammonium acetate buffer, acetonitrile), and the characterization is carried out using MALDI-TOF MS.
  • Example 3 Implementation of the amino-modified oligonucleotides (Example 2) with the N-hydroxy active esters (Example 1)
  • the amino-modified oligonucleotides are dissolved in 0.1 M borate buffer (pH 8.5) and reacted with the N-hydroxysuccinimide active esters dissolved in DMSO in accordance with the Molecular Probes protocol (Labeling Amine-Modified Oligonucleotides).
  • the oligonucleotides are purified using RP-HPL chromatography according to standard protocols (eluent: 0.1 M triethylammonium acetate buffer, acetonitrile), and the characterization is carried out using MALDI-TOF MS.
  • Example 4 Production of the oligonucleotide electrode Au-SfCH ⁇ ⁇ z-ss-oligo
  • the production of Au-S (CH 2 ) 2 -ss-oligo is divided into two sections, namely the representation of the conductive surface and the derivatization of the surface with the ligate oligonucleotide in the presence of a suitable monofunctional linker (incubation step).
  • the carrier material for the covalent attachment of the double-stranded oligonucleotides is formed by an approximately 100 nm thin gold film on mica (muscovite platelet).
  • mica muscovite platelet
  • gold (99.99%) is applied in a layer thickness of approx. 100 nm by electrical discharge.
  • the gold film is then freed of surface impurities with 30% H 2 O 2 /70% H 2 SO 4 (oxidation of organic deposits) and immersed in ethanol for about 20 minutes in order to displace oxygen adsorbed on the surface.
  • a modified 20 bp single-strand oligonucleotide of the sequence 5'-TAG CGG ATA ACA CAG TCA CC-3 ' is used, which at the phosphate group of the 3' end with (HO- (CH 2 ) 2 -S) 2 to the PO - (CH 2 ) 2 -SS- (CH 2 ) r OH (see Ex. 2) is esterified.
  • a 5x10 "5 molar solution of this oligonucleotide in HEPES buffer (0.1 molar in water, pH 7.5 with 0.7 molar addition of TEATFB) is an approximately 10 ⁇ 5 to 10 " 1 molar propanethiol solution (or another thiol or disulfide suitable chain length) added, the gold Test site surface completely wetted and incubated for 2 - 24 h.
  • the disulfide spacer PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH of the oligonucleotide is cleaved homolytically.
  • the spacer forms a covalent Au-S bond with the Au atoms of the surface, which leads to a 1: 1 coadsorption of the SS oligonucleotide and the split off 2-hydroxy-mercaptoethanol.
  • the free propanethiol which is simultaneously present in the incubation solution is also adsorbed by forming an Au-S bond (incubation step).
  • this single strand can also be hybridized with its unmodified complementary strand.
  • Example 5 Alternative preparation of the oligonucleotide electrode Au-S (CH_) _- ss-oligo
  • the carrier material for the covalent attachment of the ligate oligonucleotides is formed by an approximately 100 nm thin gold film on mica (muscovite plate), cf. Ex. 4.
  • a modified 20 bp single-strand oligonucleotide of the sequence 5'-TAG CGG ATA ACA CAG TCA CC-3 ' is used, which at the phosphate group of the 3' end with (HO- (CH 2 ) 2 -S) 2 to the PO - (CH 2 ) 2 -SS- (CH 2 ) OH is esterified.
  • the gold surface of a test site is wetted with an approximately 5x10 "5 molar solution of this oligonucleotide in HEPES buffer (0.1 molar in water, pH 7.5) and incubated for 2-24 h.
  • the disulfide spacer PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH of the oligonucleotide is homolytically cleaved, whereby the spacer forms a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss oligonucleotide and the cleaved 2- Hydroxy-mercaptoethanol comes (incubation step).
  • the gold electrode modified in this way is completely wetted with an approximately 10 "5 to 10 " 1 molar propanethiol solution (in water or buffer, pH 7-7.5) or with another thiol or disulfide (suitable chain length) and 2 - Incubated 24h. After the incubation step, the free propanethiol covers the free gold surface by forming an Au-S bond.
  • Example 6 Chronocoulometric measurement on the Au-ss-oligo / ferrocene-modified nucleic acid tetramer system in the absence and presence of nucleic acid oligomer ligands (complementary to ss-oligo in Au-ss-oligo)
  • a probe electrode is produced.
  • the HO- (CH 2 ) 2 -SS- (CH 2 ) 2 -modified oligonucleotide (sequence TAG CGG ATA ACA CAG TCA CC) described above is immobilized on gold (50 ⁇ mol oligonucleotide in phosphate buffer (500 mM K 2 HPO / KH 2 PO 4 pH 7), subsequent coating with 1 mM propanethiol in water).
  • the diameter of the gold electrode used is 6 mm, ie an area of 0.28 cm 2 is available for the immobilization of the ligate nucleic acid oligomers.
  • the integrals of curves 1 and 2 result in a difference of 70 x 10 "8 C (0.7 ⁇ C).
  • This value corresponds to 2.5 ⁇ C / cm 2 or 1.6 x 10 13 electrons / cm 2 Assuming a maximum occupancy with ligate oligonucleotides, ie an occupancy with 7 x 10 12 ligate oligonucleotides per cm 2 , at least about 2.2 electrons per ligate oligonucleotide were converted on average, ie 2.2 ferrocene labels by hybridization of the ligate nucleic acid oligomer with the nucleic acid oligomer ligand, thus displacing an average of 1.1 tetramers from the ligate oligonucleotide.

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Abstract

La présente invention concerne un procédé destiné à la détection d'hybridations d'oligomères d'acide nucléique, ledit procédé comprenant les étapes suivantes: mise à disposition d'une surface modifiée, la modification correspondant à la liaison d'au moins un type d'oligomères d'acide nucléique de ligature; mise à disposition de ligands d'oligomères d'acide nucléique signal; mise à disposition d'un échantillon comprenant des ligands d'oligomères d'acide nucléique; mise en contact d'une quantité définie des ligands d'oligomères d'acide nucléique signal avec la surface modifiée, et mise en contact de l'échantillon et des ligands d'oligomères d'acide nucléique qu'il contient, avec la surface modifiée; détection des ligands d'oligomères d'acide nucléique signal; et comparaison des valeurs obtenues pour la détection des ligands d'oligomères d'acide nucléique signal avec des valeurs de référence.
PCT/DE2002/003122 2001-08-25 2002-08-26 Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique WO2003018834A2 (fr)

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US10/487,816 US20060228703A1 (en) 2001-08-25 2002-08-26 Displacement assay for the detection of nucleic acid oligomer hybridization events
EP02762252A EP1554396B1 (fr) 2001-08-25 2002-08-26 Essai de deplacement destine a la detection d'hybridations d'oligomeres d'acide nucleique
AT02762252T ATE513058T1 (de) 2001-08-25 2002-08-26 Verdrängungsassay zur detektion von nukleinsäureoligomer-hybridisierungsereignissen

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DE10141691A DE10141691A1 (de) 2001-08-25 2001-08-25 Verdrängungsassay zur Detektion von Ligat-Ligand-Assoziationsereignissen
DE10141691.1 2001-08-25
PCT/DE2002/001269 WO2003019194A2 (fr) 2001-08-25 2002-04-06 Essai de deplacement pour la detection d'evenements d'association ligat-ligand
DEPCT/DE02/01269 2002-04-06

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JP2005031066A (ja) * 2003-04-28 2005-02-03 Canon Inc 固定化核酸プローブの処理方法、核酸の検出方法、核酸濃度分析方法、および核酸検出用キット
EP2039781A1 (fr) 2007-09-18 2009-03-25 Fidicula GmbH Essai de suppression destiné à la détection d'événements d'hybridation d'oligomères d'acide nucléique
EP2130930A2 (fr) 2008-06-06 2009-12-09 FRIZ Biochem Gesellschaft für Bioanalytik mbH Substrat électrique destiné à l'utilisation comme support de biomolécules
WO2011069501A1 (fr) 2009-12-07 2011-06-16 Friz Biochem Gesellschaft Für Bioanalytik Mbh Essai de compétition pour détecter des événements d'hybridation d'oligomères d'acide nucléique
WO2013091614A1 (fr) 2011-12-19 2013-06-27 Friz Biochem Gesellschaft Für Bioanalytik Mbh Procédé de détection électrochimique d'événements d'hybridation d'oligomères d'acide nucléique

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005031066A (ja) * 2003-04-28 2005-02-03 Canon Inc 固定化核酸プローブの処理方法、核酸の検出方法、核酸濃度分析方法、および核酸検出用キット
EP2039781A1 (fr) 2007-09-18 2009-03-25 Fidicula GmbH Essai de suppression destiné à la détection d'événements d'hybridation d'oligomères d'acide nucléique
DE102007044664A1 (de) 2007-09-18 2009-04-02 Fidicula Gmbh Verdrängungsassay zur Detektion von Nukleinsäureoligomer-Hybridisierungsereignissen
EP2130930A2 (fr) 2008-06-06 2009-12-09 FRIZ Biochem Gesellschaft für Bioanalytik mbH Substrat électrique destiné à l'utilisation comme support de biomolécules
WO2011069501A1 (fr) 2009-12-07 2011-06-16 Friz Biochem Gesellschaft Für Bioanalytik Mbh Essai de compétition pour détecter des événements d'hybridation d'oligomères d'acide nucléique
WO2013091614A1 (fr) 2011-12-19 2013-06-27 Friz Biochem Gesellschaft Für Bioanalytik Mbh Procédé de détection électrochimique d'événements d'hybridation d'oligomères d'acide nucléique

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