EP1629117A2 - Procede pour analyser des polymorphismes de nucleotide simple sur des biopuces presentant des zones oligonucleotides - Google Patents

Procede pour analyser des polymorphismes de nucleotide simple sur des biopuces presentant des zones oligonucleotides

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Publication number
EP1629117A2
EP1629117A2 EP04739568A EP04739568A EP1629117A2 EP 1629117 A2 EP1629117 A2 EP 1629117A2 EP 04739568 A EP04739568 A EP 04739568A EP 04739568 A EP04739568 A EP 04739568A EP 1629117 A2 EP1629117 A2 EP 1629117A2
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EP
European Patent Office
Prior art keywords
hybridization
probe
molecules
sample
molecule
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.)
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EP04739568A
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German (de)
English (en)
Inventor
Dirk Fischer
Jörg GEISTLINGER
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Array-on GmbH
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Array-on GmbH
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Publication date
Application filed by Array-on GmbH filed Critical Array-on GmbH
Publication of EP1629117A2 publication Critical patent/EP1629117A2/fr
Withdrawn legal-status Critical Current

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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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a method for the multiparallel detection of nucleotide polymorphisms on a polydimensional array. Furthermore, the invention relates to a method for the detection of many individual nucleotide polymorphisms, the nucleotide polymorphisms of multiple individuals on the array being able to be detected multiparallel.
  • the hybridization of one probe molecule each with one sample molecule in each case takes place in a hybridization field on the array, which is separated from surrounding hybridization fields.
  • SNPs single nucleotide polymorphisms
  • An SNP is a single DNA base exchange or an insertion / deletion of individual bases in a specific position within a genomic section, such as a gene. For example, some individuals in a population may have base adenine, while other individuals may have base cytosine in the same location within a gene. It is believed that more than 5 million of such SNPs exist in the human genome, whose allele with the rarer nucleotide occurs in more than 10% of the individuals examined. Around 90,000 of these are said to be located in protein-coding regions, which makes these SNPs particularly interesting with regard to medical and pharmacological issues. If you look at several SNPs in an individual, this offers the possibility of creating a genetic fingerprint for the individual concerned.
  • SNPs DNA microarrays specially designed for SNP analysis.
  • the Analysis of SNPs is generally useful for answering molecular genetic questions. For example, the result of an SNP analysis can provide valuable information about an individual's predisposition to a particular disease. In this way it is possible to estimate how the patient will react to a certain class of active ingredient before medication to a patient.
  • SNP analysis is not only of particular interest for medical genetics and pharmacogenetics, the determination of whether a certain SNP and thus a certain property is present or absent in an individual is general for them Characterization of individuals, be they human, animal, vegetable or microbial, useful. For example, SNP genotyping has been an integral part of modern plant breeding for several years.
  • the SNP analysis is now routinely carried out, particularly in the field of biomedicine, and is offered as a service by appropriately oriented companies. Suitable reaction kits from various providers for SNP analysis are already available on the market, but not for arrays.
  • oligonucleotides which are arranged on the chip in the form of a so-called array, i.e. a predetermined arrangement, in order to detect individual base polymorphisms in a DNA sample to be examined either by hybridization or by hybridization followed by a DNA polymerase Detect dependent primer extension of the arranged oligonucleotides.
  • the probe is the respective oligonucleotide arranged and fixed on the chip in a specific position, while the nucleic acid molecules to be examined in the sample are in the form of a hybridization solution.
  • This solution is brought into contact with the chip and incubated, as a result of which the DNA molecules of the sample contained in the solution find their suitable hybridization partners, namely the oligonucleotide probe suitable for the respective molecule, on the surface of the biochip and hybridize with them.
  • DNA chip biochip and (micro) array are often used interchangeably, as is also the case in the present application.
  • microarray simply means that molecules are arranged in a high density at certain positions within an array or raster.
  • microarrays can have up to several hundred thousand positions (often referred to as spots) on a support or matrix.
  • the chip is the actual array substrate, i.e. the carrier of one or usually many microarrays.
  • Slides or other substrates made of glass and wafers are used.
  • sample molecules or loci to be examined must be from the same individual originate, since when two or more individuals or their samples are used, cross-hybridization of the homologous sample molecules would automatically take place with the same probe and the signals would thus no longer be distinguishable and assignable.
  • a typical example of such methods is the use of microarrays for the detection of microorganisms in samples in biomedical diagnostics.
  • rRNA ribosomal RNA
  • These species-characteristic sequences are applied to a microarray in the form of single-stranded DNA oligonucleotides.
  • the target DNA molecules to be examined are first isolated from the sample to be examined and provided with fluorescent markers.
  • the labeled target DNA molecules are then incubated in a solution with the probes applied to the microarray, interactions which occur unspecifically are removed by appropriate washing steps and specific interactions are detected by fluorescence-optical evaluation.
  • the object of the invention is therefore to make the parallel analysis of several nucleic acids possible simultaneously and comparably on an array by hybridization, the sources of error (e.g. cross-hybridization) being eliminated as far as possible and no high demands being placed on the analysis parameters such as e.g. B. Sample concentration can be provided.
  • sources of error e.g. cross-hybridization
  • sample concentration can be provided.
  • the invention is based on the fact that in separate hybridization fields on the biochip only one sample molecule (in the sense of a certain nucleic acid sequence) hybridizes with a complementary probe molecule immobilized on the chip (in the sense of a certain nucleic acid sequence).
  • a locally targeted hybridization takes place, in which undesired hybridization reactions are excluded by the spatial distance of the individual hybridization fields and areas from one another and by optional drying of the hybrids formed.
  • the methods of the prior art also require that the sample molecules of a single individual are pooled in the hybridization solution and the hybridization solution is brought into contact with the chip on which the oligonucleotide probes are arranged, which leads to different sample molecules of an individual have to find their hybridization partner under numerous probes, which is particularly problematic when the number of loci analyzed increases.
  • the invention is based on the fact that no sample pool is brought to hybridization with the respective probe, but rather each individual sample molecule (in the sense of a specific nucleic acid sequence) is hybridized exclusively with the probe molecule complementary to it in a separate hybridization field.
  • hybridization In this way, hybridizations with other sample molecules (in the sense of a specific nucleic acid sequence) (cross hybridization) are prevented.
  • the hybridization therefore takes place in a targeted manner between a sample molecule and the complementary probe molecule without the different sample molecules being able to mix.
  • the hybrid of the probe molecule and the sample molecule is formed individually for each locus to be examined and each individual to be examined.
  • the hybridization takes place in hybridization fields on the chip that are spatially separated from one another. The fact that the position of the oligonucleotide probe was determined beforehand enables the hybrid formation of the probe and sample to be reliably established.
  • the hybrids formed can be stabilized by adding, for example, spermidine or polyethylene glycol or preserved under alcohol.
  • the stabilization of the hybrid structure i.e. the cohesion of the single strands in the double strand, is also supported by drying, that is to say by reducing the volume.
  • the hybrids of oligonucleotide probe and sample molecule are dried in order to obtain further protection against the mixing of the different sample molecules. These dried hybrids are rehydrated as part of the detection reaction.
  • the hybridization areas are additionally separated from one another by applying a separating matrix made of plastic, which can be removed again before the detection reaction.
  • the method according to the invention for SNP detection can in principle also be extended to other microarray-based detection methods.
  • the target molecules amplified by PCR can also be hybridized on the chip with a microorganism-specific rDNA probe without the target molecules mixing.
  • competition reactions from different probes for targets can be eliminated in a simple manner and cross-hybridizations can be avoided.
  • hybridization can also be carried out more efficiently, which leads to better signal-to-noise ratios in the evaluation.
  • problems that arise due to the simultaneous diffusion of many targets to many probes in evaluations according to the prior art are eliminated.
  • mRNA ie the expressed genetic information
  • mRNA is first quantitatively converted into corresponding cDNAs by reverse transcription.
  • an amplified cDNA with a probe in each case can then spatially in a method according to the invention by hybridization defined hybridization field and evaluation of the hybridization can be concluded on the gene activity of the respective cell etc.
  • single-stranded mRNA e.g. of highly expressed or strongly induced genes
  • probe molecules used directly as a sample molecule and hybridized with the probe molecules in order to examine the expression of these genes.
  • the use of the method according to the invention for analyzes at the RNA level is therefore expressly provided.
  • the invention thus relates to a method for the detection of nucleic acids, comprising the following steps in the order given:
  • the hybrids forming in a hybridization field are dried during or before the probe and sample molecule are brought into contact with one another in the adjacent hybridization field.
  • the hybrids which form in a hybridization field are dried during or before the probe and sample molecule are brought into contact with one another in the adjacent hybridization field, a separation matrix being applied between the individual hybridization areas and separating the hybridization areas from one another.
  • the method of the invention is suitable for the parallel detection of e.g. several sample molecules in a mixture, for the parallel detection of several organisms, preferably microorganisms in one sample, for analyzing the
  • Gene activity of several genes of an organism or e.g. a cell or to detect the expression of a gene is also possible.
  • the invention relates to a method for detecting a nucleotide polymorphism (SNP), comprising the following steps in the order given:
  • the hybrids forming in a hybridization field are dried during or before the probe and sample molecule are brought into contact with one another in the adjacent hybridization field.
  • the hybrids forming in a hybridization field are dried during or before the probe and sample molecule are brought into contact with one another in the adjacent hybridization field, a separation matrix being applied between the individual hybridization areas and separating the hybridization areas from one another.
  • the method of the invention is thus suitable for the detection or genotyping of the presence, absence or identity of an individual nucleotide polymorphism in a specific position within the genome of an individual.
  • several to many individuals are examined simultaneously on a common support for a specific nucleotide variation.
  • a very large number of individuals are particularly preferably examined simultaneously with regard to a few nucleotide variations.
  • the method of the invention is also suitable for the detection or genotyping of the presence, absence or identity of insertions or deletions of one or more bases in a specific position within the genome of an individual.
  • the invention further relates to a method for multiparallel SNP analysis of several individuals and several loci in the genome of an individual, comprising the following steps in the order given:
  • Nucleic acid molecules the sequence of which represents a specific locus in the genome of an individual, in hybridization areas with a minimum size of 0.5 mm on a support, b) mixing and hybridizing in each case a specific probe molecule in a spatially defined hybridization field within the
  • Hybridization areas each with a second single-stranded nucleic acid molecule (sample molecule) which has the SNP to be detected and whose sequence is at least partially complementary to that of the probe molecule, so that the two nucleic acid molecules can hybridize with one another at least via the complementary sequence, sample molecules of the same locus being located within a hybridization area , but of different origins in different hybridization fields can hybridize with the same probe molecule, c) enzymatic template-dependent extension of the probe molecule, which acts as a primer within the hybrid, with the incorporation of a nucleotide or analog leading to chain termination, whereby probe molecules, each having different loci represent, and sample molecules from several individuals are used for parallel analysis.
  • the hybrids forming in a hybridization field are dried during or before the probe and sample molecule are brought into contact with one another in the adjacent hybridization field.
  • the hybrids which form in a hybridization field are dried during or before the probe and sample molecule are brought into contact with one another in the adjacent hybridization field, a separation matrix being applied between the individual hybridization areas and separating the hybridization areas from one another.
  • each hybridization area only one sample molecule is hybridized in each hybridization area with the probe molecule present in the area, it being possible for each area to be spatially delimited from the other areas by a separation matrix.
  • probe molecules which each represent different loci of different individuals, can also be used for parallel analysis.
  • locus is understood to mean a genomic section in which the genetic polymorphism to be examined, such as e.g. the SNP, the insertion or deletion, lies.
  • immobilization refers to the process by which a molecule is carried on or on a solid support stabilized layer or surface is applied by covalent or non-covalent interaction such that the molecule can no longer move freely on the support or diffuse away from the support in solution.
  • the immobilization of the probe molecules in areas creates a flat coating with the oligonucleotide probe in the context of the present invention.
  • carrier refers to devices on which molecules can be applied by covalent or non-covalent interactions.
  • carrier or substrate for nucleic acid molecules come e.g. Slides or others
  • Materials made of glass, plastic, ceramic or metal which can be coated planar, two-dimensional, three-dimensional or spherical, as well as wafers. Three or four dimensional coatings for substrates are commercially available e.g. under the names CodeLink Bioarray (from Amersham Pharmacia) or MGX TM 4D-Array (from Metrigenix).
  • the different hybridization areas are separated from one another by a separation matrix made of chemically inert material, in contrast to supports with wells (wells) or other depressions, as are known in the prior art.
  • hybridization area refers to the area of the chip in which an oligonucleotide probe of a certain sequence has been arranged and immobilized.
  • a chip preferably contains a maximum of 192 areas, particularly preferably a maximum of 96 areas, particularly preferably a maximum of 48 areas and most preferably a maximum of twelve areas
  • the area of the areas is at least 0.5 mm, preferably it is at least 4 mm, particularly preferably it is at least 16 mm and most preferably it is at least 256 mm within these areas, several hybridizations can be spaced apart from one another Hybridization fields take place.
  • hybridization field is the area within the hybridization area in which a sample molecule with a defined nucleic acid sequence from a particular individual is hybridized with a probe molecule whose nucleic acid sequence is at least partially complementary to that of the sample molecule.
  • the localization on the carrier, the matrix or the substrate takes place in a defined spatial arrangement, this arrangement is often also referred to as an array.
  • a specific position of the array, in this case the hybridization field, is usually also referred to as a spot.
  • chip usually denotes the arrangement of a molecule in array format on a carrier.
  • a “separation matrix” is a lattice which separates separate hybridization areas from one another on the chip.
  • the material of the separation matrix can be any chemically inert material which effectively prevents the passage of liquid and reliably seals off the separate areas from that In addition, the material should adhere reversibly to the support, be tear-resistant and, after hybridization, should preferably be able to be removed in its entirety without residue. Chemically inert polymers and plastics are preferred.
  • the material of the separation matrix is particularly preferably silicone rubber.
  • the width the separation matrix is preferably 0.2 to 5 mm, particularly preferably 0.5 to 3 mm and most preferably 0.8 to 1 mm.
  • the height of the separation matrix is preferably 0.2 to 2 mm, particularly preferably 0.8 to 1.8 mm and most preferably 1.2 to 1.6 mm.
  • the detection of an SNP in a sample molecule hybridized with the probe and immobilized on a support can be carried out by various detection methods familiar to the person skilled in the art.
  • the most established and most frequently used method for the detection of SNPs in sample-probe hybrids is the so-called primer extension.
  • the primer extension i.e. the extension of the probe molecule serving as a primer in a template-dependent manner, the sample molecule being the template, can be carried out in a conventional manner, as described, for example, in EP 0648 280 B1, EP 0 705 349 B1 and WO 98 / 59066 AI. Reference is hereby expressly made to the disclosure of primer extension contained in these documents.
  • primer molecules which have a polynucleotide sequence complementary to one or more nucleotide sequences of a genomic DNA segment of an individual, the genomic segment being immediately 3 ′ distal to an SNP , Y, is localized by template-dependent extension of the nucleic acid primer molecule by a single nucleotide or nucleotide analog R which is complementary to the nucleotide Y of the SNP allele.
  • the template-dependent extension of the primer which is often referred to in the art as an intergration primer, in the presence of one or more dideoxynucleoside triphosphate derivatives or analogs selected from the group consisting of ddATP, ddTTP, ddCTP and ddGTP, or other base analogues leading to chain termination, but in the absence of dATP, dTTP, dCTP and dGTP instead, the non-renewable dideoxynucleotide triphosphate derivative can be detected in the position of the SNP and thus the SNP itself in a conventional manner.
  • the step of primer extension which enables SNP detection directly, has been described many times in the prior art and can also be carried out by a person skilled in the art using reagents and reaction kits available for this purpose on the market.
  • the oligonucleotide which is used as a probe molecule for the formation of the hybrid of probe molecule and sample molecule, usually also represents the primer, which, depending on the polymerase, extends the nucleotide in question by the nucleotide in question, which corresponds to the SNP allele becomes.
  • detection methods known to the person skilled in the art for detecting the SNPs in the hybrid are e.g. the allele-specific primer extension (see below), the allele-specific hybridization (see below) and the mass spectrometry.
  • hybrids of probe molecule and sample molecule formed on the chip according to the present invention are never denatured before the extension, but rather directly represent the substrate for the polymerase, which attaches a detectable, usually labeled nucleotide to the probe molecule in question , Since the sample molecules to be examined hybridize individually and separately from one another with the respective probe molecules and are not pooled in a hybridization solution, as in the prior art, any cross-hybridization is excluded. This is the first time that
  • each locus of each individual to be examined is individually amplified by PCR and hybridized with a probe characteristic of a locus separately from the other probes and samples in different hybridization fields within the areas on the chip becomes.
  • this analysis can be carried out by the user himself, by prefabricated chips, which carry the probe molecules immobilized in hybridization areas and possibly a removable separation matrix, are hybridized by the user himself with his previously amplified sample molecules and the polymorphism is detected.
  • This is made possible by spatially separated hybridization fields, which allow the sample molecules to be applied to the chip using a handheld pipette or a pipetting robot, without these mixing up and resulting in cross-hybridizations. This makes it possible for the user to be inexpensive, time-saving and reproducible perform the analysis of a large number of individuals yourself.
  • the method according to the invention also enables a certain degree of automation, since the probe molecules can be applied to the matrix with a microarray or pipetting robot in hybridization areas and the sample molecules not necessarily with a handheld pipette, but also with a pipetting or a microarray robot can be placed in any hybridization field. Up to now, such automation was only possible if the poly-dimensionality was lost.
  • kits which contain a prefabricated chip coated with one or more probe molecules in hybridization areas, optionally with a removable separation matrix.
  • this kit can also contain primers for the PCR amplification of the sample molecules and the reagents for generating single strands from the PCR products.
  • the kit can also contain the reagents for the template-dependent extension of the probe molecule.
  • a kit contains a prefabricated chip coated with one or more probe molecules in hybridization areas with a removable separation matrix, primers for the PCR amplification of the sample molecules and the reagents for generating single strands from the PCR products and reagents for the template -dependent extension of the probe molecule.
  • the sample nucleic acid here also called the target nucleic acid or target nucleic acid, which is suspected to contain the variable nucleotide residue, i.e. the SNP, and which should therefore be analyzed for this, can be a human, animal, vegetable act fungal or microbial nucleic acid (DNA or RNA).
  • the sample nucleic acid can be isolated from biological samples using conventional nucleic acid purification methods, or can also be present in an unpurified form within a biological sample.
  • RNA can first be transcribed into a cDNA by reverse transcription, which is then again used as a template in e.g. can serve a PCR.
  • PCR-independent enrichment steps can also be carried out, which are based, for example, on affinity chromatography, NAT (Nucleic Acid Amplification Testing), Ampliphi or Genomiphi amplification techniques (see product information from Amersham Pharmacia) or magnetic microparticles (eg Dynabeads TM).
  • Samples that are not purified, enriched or amplified can also be used under certain circumstances, but later require an amplification step for the fluorescence signals (signal amplification). For more effective hybridization of the sample and probe molecule, it is advantageous if the sample molecule is single-stranded.
  • primer modifications are e.g. B. 5'-PTO (5'-phosphoro-thioate nucleotides), in which the phosphate groups 5 '-terminal nucleotides of the primer are replaced by phosphoro-thioate groups.
  • modified primers are not degraded by the T7 Gen 6 5'-exonuclease or the 5'-exonuclease of the lambda phage.
  • other 5 'protecting groups or PNA (peptide nucleic acid) primers can also be used to protect the hybridizing strand.
  • asymmetric PCR Another method for producing largely single-stranded sample molecules that is not based on modification followed by enzymatic degradation is the so-called "asymmetric PCR".
  • the primer of the required strand is added in multiple excess to the primer of the strand not required. This leads to the preferred synthesis of the strand required for the hybridization.
  • the hybridization is not hindered by the essentially non-amplified counter strand.
  • Single-stranded sample molecules can also be obtained by enrichment methods with magnetic particles or affinity chromatography.
  • These substrates contain nucleotides complementary to the target sequence on their surface and fish the desired sequences from a denatured, fragmented genomic DNA and enrich it (eg the Kingfischer system from Hybaid).
  • sample material does not necessarily have to be a separate single strand, but can also be obtained by denaturation and be available together with its complement. The same applies to asymmetric DNA.
  • the probe molecules are oligonucleotides or polynucleotides, which due to their nucleotide sequence can hybridize with sample molecules present in the sample.
  • the molecules can be DNA or RNA.
  • the probe molecules can be made using techniques well known in the art. They can also be obtained from suppliers who commercially produce oligonucleotides and polynucleotides.
  • the probe molecules are usually artificially synthesized single-stranded oligonucleotides which, if the primer extension is used as the detection method, end with their last 3'-nucleotide directly in front of the polymorphic position in the sequence of the individual to be examined. They are complementary to at least part of the protected amplified PCR strand in order to guarantee the matching of probe and sample. Furthermore, the extension primers have a modification at their 5 'end, with which they are bound to the chip surface, via which the binding takes place.
  • the modification is a so-called spacer (placeholder; mostly 6 to 24 C atoms; but also polyA or polyT spacers are known), at the end of which is remote from the primer there is an amino modification ,
  • This amino group reacts under UV Irradiation with the epoxy coating of the chip and thus chemically covalently binds the probe molecule to the coating.
  • the support is provided with a three-dimensional layer, such as the CodeLink bioarray from Amersham Pharmacia, a spacer may also be dispensed with and the modification may be applied directly to the probe.
  • the probe molecules represent the so-called interrogation primers, also called detection step primers in the prior art, which are extended by primer extension through the activity of a polymerase in a template-dependent manner.
  • the primer ie the probe molecule, is complementary to the nucleotide sequence 3 'of the variable nucleotide (SNP) in the associated sample molecule.
  • the primer which thus acts as the starting point for the template-dependent elongation by a DNA polymerase, is selected in such a way that it hybridizes with a nucleotide sequence immediately next to or in the vicinity of the variable nucleotide which is to be detected in the context of the SNP analysis .
  • the primer ie the probe molecule, can be selected so that it is complementary to either the coding or the non-coding strand of a double-stranded target molecule, depending on which strand is to be present in the hybrid of probe molecule and sample molecule ,
  • the selection of the probe molecule is determined by the nature of the nucleotide variation to be analyzed.
  • the interrogation primer is selected and produced in such a way that it lies directly next to the variable nucleotide to be detected when the hybrid has formed from the primer and the sample molecule.
  • the interrogation primer is selected such that it hybridizes n nucleotide residues to a probe molecule away from the SNP to be detected.
  • the number n of nucleotide residues between the 3 'end of the primer and the variable nucleotide it should only be noted that there is no nucleotide residue within the n nucleotide residues that is identical to the nucleotide to be detected.
  • the single-stranded extension primers used can themselves have hybrid nature in the simia that they are complementary only in their 3 'area of the sample, but in their 5' area they have additional sequences (again approx. 20 bp) that only go to the PCR primers used are complementary (artificially complementary sequences).
  • a so-called address system can be set up which supports hybridization and strengthens the nucleotide pairings (due to the increased amount of hydrogen bonds).
  • oligonucleotides synthetic probe molecules
  • the immobilization of oligonucleotides (synthetic probe molecules) on biochips is largely standardized.
  • the cheapest alternative is amino-modified probes that react with the epoxysilane (3-glycidoxypropyltrimethoxysilane) coated surface of fluorescence-free microscope slides.
  • Other coatings and associated modifications of the probes are commercially available. These coatings include e.g. Aldehyde, aminosilane (3-aminopropyltrimethoxysilane), polylysine or isothiocyanate coatings.
  • the substrates mentioned preferably react with amino groups at the end of the spacers of probe molecules or the probe molecules themselves. Another alternative would be a hybrid bond via the biotin-streptavidin system. It is clear to the person skilled in the art that the functional groups of the coatings and the linker could also be interchanged accordingly.
  • the chemical bonding of amino groups of the probe molecules and epoxy groups on the chip is done by simple UV crosslinking.
  • the chips can also be incubated in a moist chamber or baked hot.
  • not only microscope slides made of glass can be surface-coated, but also other materials, primarily plastic or ceramic, can be used for coating.
  • the materials are typically optically transparent or mirrored.
  • the probe molecules are immobilized in surface-coated hybridization areas with a minimum size of 0.5 mm 2 , which in a preferred embodiment are separated from one another by a separation matrix.
  • this is applied to the chip before or after coating the chip with the probe molecules, so that areas separated from one another are formed on the chip, in each of which a probe molecule with a defined nucleic acid sequence is immobilized over the surface.
  • a separation matrix it can either be pressed onto the carrier in solid form or applied in liquid form in order to then allow it to harden.
  • the separation matrix can be removed again, e.g. is pulled off the chip in its entirety with tweezers. This enables a common detection reaction for all hybrids.
  • hybridization means that two strands of nucleic acid molecules form hydrogen bonds in a sequence-dependent manner.
  • complementary nucleotide sequences can hybridize with one another to form double-stranded DNA or RNA, or a double-stranded hybrid of RNA and DNA. See also Sambrook et al., Vide supra; Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, New York (1989); or Higgins and Hames, Nucleic Acid Hybridization, A Practical Approach, EAL Press Oxford, Washington DC (1985), which are expressly attached here for reference.
  • hybridization means the formation of double-stranded nucleic acid molecules from complementary single-stranded nucleic acid molecules, the complementarity resulting from the base sequences of the single-stranded molecules.
  • the double-stranded nucleic acid molecules can e.g. are DNA-DNA, DNA-RNA or RNA-RNA duplex molecules. Hybridization experiments are usually used to demonstrate complementarity between different single-stranded nucleic acid molecules.
  • annealing is the process in which two single-stranded nucleic acid molecules are attached to one another due to their base complementarity, interact with one another and form double-stranded structures or a double-stranded helix or a duplex.
  • the interaction between the single-stranded nucleic acid molecules is based on the hydrogen bond between complementary base pairs in the individual strands. In this way, double-stranded DNA-DNA helices, DNA-RNA helices or RNA-RNA helices can be formed.
  • a hybrid is referred to as a double-stranded nucleic acid molecule, the single strands of which come from different nucleic acid molecules and which is formed by forming a hydrogen bond between these complementary single strands.
  • the individual sample molecules that are single-stranded by the 5'-exonuclease treatment are preferably synthesized with the appropriate single-stranded with the help of a microarray robot Probe molecules in spatially defined hybridization fields within the hybridization areas brought into contact on the chip.
  • the hybrids that form are dried by allowing the reaction liquid to evaporate slowly. This can be done under normal conditions, by gentle heating (25 to 45 ° C) or by vacuum application.
  • the aforementioned primer extension that is to say the enzymatic extension of the primer, which preferably represents the probe molecule, takes place in a template-dependent manner.
  • the primer extension is generally carried out as described in the prior art, it being possible to use commercially available enzymes, reagents and kits.
  • a dried hybrid is rehydrated by adding the extension mixture.
  • the probe-sample hybrid is contacted with one or more nucleoside triphosphates, including at least one labeled or modified nucleoside triphosphate, together with a polymerizing agent under conditions that favor extension of the primer.
  • a polymerizing agent Either labeled deoxyribonucleoside triphosphates (dNTPs) or labeled chain-terminating dideoxyribonucleoside triphosphates (ddNTPs) can be used.
  • the polymerizing agent will extend the primer with the nucleoside triphosphate complementary to the variable nucleotide next to the primer.
  • labeled nucleoside triphosphate includes any nucleoside triphosphate, deoxy or dideoxynucleoside triphosphate which is provided with a detectable label or which is modified such that it comprises a group or a residue which is capable of a detectable label to tie. It does not matter in the context of the invention which marker is used for the detection. However, different markers naturally differ in terms of their manageability, their cost and their sensitivity. It is important, however, that the detectable marker does not hinder or make incorrect the incorporation of the labeled nucleoside triphosphate during the polymerization reaction, which leads to the extension of the primer.
  • cyanine e.g. Cy3 or Cy5
  • Renaissance e.g. ROX or Rl
  • fluorescein dyes e.g. FAM or FITC
  • Radioactive labels such as P 32 , the biotin-streptavidin system or antigen-antibody systems which are coupled to enzymes such as horseradish peroxidase can be used as alternative labels.
  • the detection can be carried out with different numbers of labeled nucleotides or nucleotide analogs.
  • Fluorescence markers are usually used as markers (see above), usually either 2 or 4 labeled nucleotides or analogs being used. Accordingly, one speaks of the 2-color and 4-color approach.
  • probe extension protocols are also used.
  • a very important method is the allele-specific primer extension. The most important difference from that already shown The method can be seen in the probes used.
  • the 5'-amino-modified probe molecules are designed in such a way that they do not end a nucleotide 3 'before the polymorphic position, but with their last 3' nucleotide they lie exactly on the polymorphic position.
  • 4 probes of identical sequence are required for each locus to be analyzed, which differ only in the last 3 'nucleotide (the SNP nucleotide).
  • extension primers After application to four different areas and hybridization with the sample molecule in the four different hybridization areas, only one of these 4 extension primers is extended during the extension reaction, namely the one with the complementary SNP nucleotide at its 3 'end. The other 3 extension primers are not extended either because the last 3 'nucleotide cannot pair with the sample molecule (extension does not occur).
  • dNTPs nucleotides A, C, G and T
  • the identity of the SNP nucleotide is determined by which of the 4 extension primers emits a fluorescence signal, ie which carries the correct nucleotide in the last 3 'position.
  • the specificity of the hybridization is regulated via so-called swj ⁇ tc / z oligonucleotides (WO89 / 10977; Southern et al. (1992), Genomics, 13, 1008-1017). Up to 20 oligonucleotides are required per locus to be analyzed (due to permutation of the mismatch positions).
  • This detection method is also suitable for the method according to the invention.
  • Insertion or deletions can also be detected by primer extension analysis.
  • the deletion it should be noted that the first base in the deletion is not identical to the first base after the deletion.
  • the first base of the insertion is not identical to the first base after the insertion.
  • polymerizing agent stands for any enzyme that is capable of extending nucleic acids in a template-dependent manner. Suitable enzymes include e.g. Sequenase, T7 DNA polymerase, T4 DNA polymerase, the Klenow fragment of DNA polymerase from Escherichia coli and other suitable DNA polymerases, reverse transcriptase and polymerases from thermophilic microorganisms such as Thermos aquaticus and Thermos thermophilus.
  • the polymerizing agent is Sequenase TM.
  • the hybrids for the template-dependent primer extension are firmly bound in array format on the chip surface and can also be stored in the dried state.
  • the implementation of the multiparallel primer extension in which thousands of analyzes in e.g. 50 ⁇ l volume can be carried out on the chip represents an effective rationalization and cost reduction process. If the extension were carried out in the MTP, much larger amounts of expensive reagents such as Sequenase TM and fluorescence-labeled ddNTPs would have to be used.
  • the extension on the biochip begins by bringing the array and extension solution into contact in an evaporation-proof chamber on the chip.
  • the reaction mixture which contains, for example, 0.2 units of Sequenase TM per ⁇ l and 6-8 ⁇ M fluorescence-labeled ddNTPs in a reaction buffer supplied with the Sequenase TM, and the array are kept for approx. 1 h at the optimum temperature for the polymerizing agent used under these circumstances, which, however, should not be higher than 5 ° C below the melting temperature of the area complementary between the probe molecule and the sample molecule, and the primer extension is carried out.
  • the extension solution can contain all four ddNTPs or analogues labeled with four different dyes, or only two ddNTPs labeled with different dyes, since SNPs are often binary markers and in many applications the aim is only to determine which of the two possible nucleotide states is present. Since the labeled ddNTPs are firmly attached to the extension primer (probe molecule) by the polymerizing agent and the probe is firmly attached to the support (the chip), the attached ddNTP is also irreversibly bound to the chip.
  • Excitation / detection system that analyzes the fluorochromes, typically a resolution of 2 ⁇ m and a sensitivity of 0.5 fluorochromes per ⁇ m.
  • two (or four) fluorochrome types are chosen whose excitation or emission wavelengths are as far apart as possible.
  • Cy5 is excited at 650 nm and emitted at 667 nm (filter 675 nm ⁇ 10)
  • Rl 10 is excited with a 488 nm laser and emitted at 525 nm (filter 512nm ⁇ 15). This ensures that the signals of the two dyes do not overlap, since they can be clearly separated from one another by the corresponding filter systems.
  • the analysis program compatible with the scanner recognizes the wavelength (color) and intensity of each sample point and stores the results directly in a database. As already stated, several genomic loci of several individuals can be detected simultaneously in the method according to the invention.
  • probes which can each be used for the detection of different genomic loci and which have been immobilized in different hybridization areas, are hybridized with the complementary sample molecule in each case on the carrier of the chip in hybridization fields within the hybridization areas, which are at a distance from one another.
  • a maximum of 192 different probes preferably a maximum of 96 different probes, particularly preferably a maximum of 48 different probes, particularly preferably a maximum of different probes, most preferably a maximum of twelve different probes are immobilized on the carrier.
  • the maximum number of probes that can be applied to an array is determined by the synthesis method of the array and the minimum size of the hybridization areas.
  • the probe molecules have a length of at least 10 to 100 nucleotides, preferably of at least 15 to 75 nucleotides, particularly preferably of at least 18 to 50 nucleotides, particularly preferably of at least 20 to 40 nucleotides, likewise particularly preferably of 20 to 35 nucleotides and most preferably from at least 20 to 30 nucleotides.
  • the sample molecules have a length of at least 40 to 500 nucleotides, preferably of at least 50 to 250 nucleotides, particularly preferably of at least 50 to 150 nucleotides, particularly preferably of at least 50 to 100 nucleotides and most preferably of at least 50 to 90 nucleotides.
  • at least 48 different individuals preferably at least 150 different individuals, particularly preferably at least 625 different individuals, likewise particularly preferably at least 1250 different individuals, particularly preferably at least 2500 different individuals and most preferably at least 5000 different individuals, are examined on the carrier ,
  • At least 48 different individuals preferably at least 150 different individuals, particularly preferably at least 625 different individuals, likewise particularly preferably at least 1250 different individuals, particularly preferably at least 2500 different individuals, most preferably at least 5000 different individuals with respect to a maximum of 192 different loci, are used in the method , preferably a maximum of 96 different loci, particularly preferably a maximum of 48 different loci, particularly preferably a maximum of 24 different loci and most preferably a maximum of twelve different loci examined in parallel.
  • SNPs genetic polymorphisms
  • SNPs genetic polymorphisms
  • These include e.g. Microorganisms, preferably E. coli and / or human pathogens
  • Microorganisms fungi, preferably Saccharomyces cerevisiae, Schizosaccharomyces pombe and / or phytopathogenic fungi, rats, cattle, mice, humans or plants.
  • Useful, ornamental, food or feed plants characterized by the method according to the invention.
  • Examples of monocotyledonous plants are plants belonging to the genera Avena (oat), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, sorghum (millet), Zea (corn ) and the like.
  • Dicotyledons include cotton, Legumes such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, potato, goat plants and trees.
  • Other useful plants can include fruit (in particular apples, pears, cherries, grapes, citrus, pineapples and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal and, in the case of medicinal plants, rauwolfia and digitalis.
  • the cereals wheat, rye, oats, barley, rice, corn and millet, sugar beet, rapeseed, soybeans, tomato, potatoes and tobacco are particularly preferred.
  • Further useful plants can be found in US Pat. No. 6,137,030.
  • Arabidopsis are most preferably investigated according to the invention as a model system for dicotyledonous plants and rice as a model system for monocotyledonous plants.
  • One or more human individuals can be examined, for example, with regard to one or more polymorphisms, inter alia in the genes for 5-lipoxygenase or cytochrome P450, which result in a modified metabolism of active substances. This would allow the drug therapy to be individually adapted to the genotype of the individual concerned.
  • the spatially defined hybridization of one sample molecule each with one probe molecule in each case in a hybridization field can of course also be carried out for nucleic acid analogs such as PNAs.
  • the sample and probe can also be of a completely different nature, e.g. Proteins or peptides in antibody screening or carbohydrates such as sugar chains on glycoproteins for screening low molecular weight ligands (antigens) of all classes of substances.
  • the method according to the invention can thus be applied to all detection methods in which the specific interaction between two molecules (probe and sample), which have mutually complementary binding properties, is detected by hybrid formation.
  • the method according to the invention is not limited to the detection of SNPs, but can also be used to detect several species in parallel in a sample, for example by rDNA hybridization.
  • the gene activity of a cell and the expression of certain genes can also be detected by the method according to the invention.
  • the detection of the hybrids on the chip can be carried out by a number of additional detection methods which have not previously been mentioned.
  • the sample molecules can e.g. by using appropriately labeled primers already during amplification e.g. be labeled with the fluorophores already mentioned.
  • the detection method consists of determining whether labeled hybrids can be detected on the microarray. The same detection method can be used in the analysis of gene activity if appropriately labeled primers are used during the PCR amplification of the cDNA (see above). Other detection methods are known to the person skilled in the art.
  • a method according to the invention can usually comprise the following steps:
  • SNPs are determined using bioinformatics and public sequence databases. Different genome projects mean that millions of sequences of expressed genes (cDNAs) as well as entire genomes are available. In addition, there are databases (e.g. at NCBI, http://www.ncbi.nlm.nih.gov) that explicitly state the position of SNPs in the genome of different organisms. Based on these SNP databases or by comparing your own sequence data with the corresponding reference sequences in publicly accessible databases, SNPs can be identified.
  • primers for a PCR are then derived, which amplify the genomic fragment which bears the polymorphism to be detected.
  • a partial area of one of the two primers must be sequence-identical to a partial area of the future extension primer (the probe) in order to guarantee the hybridization of the probe with the target with 100% certainty.
  • the primer used to amplify the strand required for hybridization can carry a modification that prevents it from being degraded by a 5'-exonuclease (e.g. a phosphoro-thioate modification).
  • Primers can e.g. with the software Primer3 (Whitehead Institute, MIT, Steve Rozen, Helen J. Skaletsky, 1996 and 1997; available at http://www.genome.wi.mit.edu/genome_software /other/primer3.html).
  • a comparative sequencing of the amplified fragments can be carried out in different genetic backgrounds for verification, quality control and selection of suitable markers.
  • 5'-amino-modified probe molecules are also designed and synthesized using the Primer3 software.
  • the order is placed with well-known manufacturers such as Metabion, Martinsried, Germany.
  • the probes are selected so that they end at their 3 'end exactly one nucleotide in front of the polymorphic position of the polymorphism to be examined.
  • the single-stranded probe molecules are used to coat biochips in areas. This coating is carried out by companies that specialize in the coating of surfaces with DNA molecules, such as the RoboScreen, für, Germany.
  • epoxy-modified biochip blanks e.g. epoxy slides, from Quantifoil, Jena
  • a separation matrix e.g. B. made of silicone rubber, which additionally separates the hybridization areas from each other.
  • the different genomic loci of the various individuals to be examined are then amplified by PCR.
  • the annealing temperature of the primers is between 50 and 68 ° C, preferably between 55 and 60 ° C.
  • the size of the amplified fragments is between 40 to 500 base pairs, preferably between 50 and 250 base pairs, particularly preferably between 50 and 150 base pairs, in particular between 50 and 100 base pairs and most preferably between 50 and 90 base pairs.
  • Typical reaction conditions include:
  • the polymerase and the reaction buffer are usually obtained from Qiagen, Germany.
  • the PCR temperature protocol usually looks like this:
  • the unprotected amplified strand which carries the same sequence as the probe molecule, is then digested by an exonuclease which is selective with regard to the PTO modification, e.g. T7 Gen 6 5'-exonuclease, according to the manufacturer's instructions (Amersham-Pharmacia Biotech).
  • an exonuclease which is selective with regard to the PTO modification, e.g. T7 Gen 6 5'-exonuclease, according to the manufacturer's instructions (Amersham-Pharmacia Biotech).
  • the single-strand sample with the complementary probe oligonucleotide is then in a hybridization field of the hybridization area with the aid of a microarray (eg Microgrid® II 600, from Biorobotics) or pipetting robot (eg Hamilton Microlab® Star) or brought into contact by a hand pipette, thereby triggering the hybridization.
  • a microarray eg Microgrid® II 600, from Biorobotics
  • pipetting robot eg Hamilton Microlab® Star
  • the resulting hybrids are dried by allowing the liquid to evaporate.
  • the separation matrix After the hybridization, the separation matrix, if present, can be removed by pulling it off the carrier with tweezers without leaving any residue. This creates a single, large reaction field in which the detection reaction is carried out. If different extension mixtures are to be used in the individual areas, the separation matrix can remain on the slide. If the same extension mixture is to be used in more than one but not all areas, more than one separation matrix can be applied, with the extension being removed for some separation matrices and some remaining on the support. This enables the most cost-effective analysis possible.
  • the extension reaction on the chip is carried out with Sequenase TM and differently fluorescence-labeled ddNTPs in Sequenase TM buffer at 50-70 ° C, in any case below the melting temperature of the hybrid.
  • the chip with the reaction mixture is sealed against evaporation. This is done either by the remaining separation matrix or, if no separation matrix was used or the separation matrix was removed after hybridization, by a silicone rubber seal surrounding the slide (e.g. Casil 40 IT), which seals the slide against a lid (second slide or cover glass ) seals.
  • the SNP detection is carried out by extending the hybrid molecule on the chip surface by a fluorescence-labeled nucleotide (so-called “single base extension”), the probe oligonucleotide acting as a primer and the hybridized single-strand sample as a template. Because the nucleotides have different fluorescence labels are and no longer within the scope of
  • Extension reaction can be extended, only the nucleotide that is complementary to the respective SNP is inserted.
  • the chip is washed once with hot distilled water, which can contain up to 0.1-2% SDS and once with 1x SSC plus 0.1% SDS, with distilled water. Rinsed water and dried in a stream of nitrogen.
  • the chip is then placed in a laser scanner (e.g. GSI LS IV, GSI Lumonics or Typhoon, Amersham Pharmacia) or another scanner (e.g. StormReader, Molecular Dynamics or ImageScanner, Amersham Pharmacia ), which in a few seconds emitted the color ( Wavelength) of the extended probe molecule and determines the identity of the SNP at the respective sample point on the chip based on the measured wavelength.
  • the physical properties of the dyes are taken into account when selecting the laser / filter systems according to the manufacturer's recommendation (e.g. GSI Lumonics). The results are called the intensity values of the
  • Fluorescence (e.g. 65536 grayscale at the wavelengths of the fluorescence-labeled ddNTPs) is stored directly in a database.
  • the detection of 48 loci in 4 barley cultivars is described below.
  • the position of the primers used for PCR with respect to the sequence of one of the examined loci is shown as an example in FIGS. 1 to 3.
  • an SNP is examined for thymidine (T) and cytosine (C).
  • sequence information of 48 singular nucleotide polymorphisms (SNPs) from Hordeum vulgar L was determined using bioinformatics in EST and genomic sequence databases at the NCBI (http://www.ncbi.nlm.nih.gov).
  • the example locus has the accession code: gi9410596.
  • genomic PCR primers for the amplification of the sample molecule were designed. Compiling the source code under SuSE Linux, Kernel 2.4. was done according to the programmer in the documentation for the source code and from the website (http://www.genome.wi.mit.edu/genome_software/other/primer3.html).
  • the genomic primers were designed in such a way that they amplify genomic fragments that carry the SNPs to be examined ( Figure (Fig.) 1, gray background Y). One of the two primers bears phosphorothioate binding modifications at the 5 'end and the other, unmodified primer corresponds in sequence to the later extension primer (probe, Fig. 3 and 4
  • oligonucleotide in
  • PBox in order to guarantee error-free hybridization of the probe from the 3 'end with the then single-stranded PCR fragment after the amplification.
  • the reverse primer (PTO primer) which forms the hybridizing half-strand, requires a modification in order to protect it from degradation by a 5 'exonuclease.
  • a comparative sequencing (ABI Sequencer 3700, Rhodamine Sequencing Kit, Applied Biosystems, USA) was then carried out on the amplified PCR fragments, each with one of the two PCR primers in different genetic backgrounds for verification, quality control and selection of suitable markers.
  • probe molecules were designed that end 3 'exactly one nucleotide before the polymorphic position (software Primer3, see above; Figs. 3 and 4,
  • the synthesis of the probes was carried out at the
  • the probe molecules carried an NH 2 group which was coupled to the probe via a C 1 spacer. These probe molecules were used to produce 48 oligonucleotide areas with an area of 16 mm 2 and a distance of 1 mm. For this purpose, a solution of at least 500 fmol / ⁇ l of the Cn-amino-modified oligonucleotide was used in order to ensure that the binding frequency of one of 1000 oligonucleotide molecules is sufficient to achieve the
  • Detection limit of the fluorescence reader (GSI scanner IV, Packard Bioscience). This solution was applied to the slide (s) using a microarray robot using 50% QMT TM buffer (from Quantifoil, Jena) in points of approximately 150 ⁇ m in diameter at a distance of 100 to 150 ⁇ m, so that the drops settled could combine and a continuous coating in square areas (a) was achieved (Fig. 5/1). As a result, a density of the probe molecules of 0.5 to 5 molecules per ⁇ m 2 was achieved.
  • the epoxy group of the chip reacts covalently with the 5'-amino groups on the Ci 2 spacer of the probe molecules and thereby fixes them on the chip surface.
  • the lattice-shaped trerm matrix (g) was applied (from the transparent and thermostable elastomer C ASIL 401 T; from the
  • Hybridization areas (a) were additionally separated from each other (Fig. 5 / II).
  • the 48 PCR fragments were then amplified from genomic DNA from the 4 cultivars.
  • An example of genomic primers at the locus is given in Fig. 2.
  • the annealing temperature was 51 ° C.
  • the amplified fragment size was 174 base pairs.
  • the PCR was carried out according to the instructions of the Polymerase manufacturer (Qiagen GmbH, Germany) according to the following protocol:
  • the amplified PCR fragments were then precipitated by adding 2 volumes (vol) of pure ethanol and 1/10 vol 3 M sodium acetate pH 4.5 at 4 ° C, and centrifuged at 14000 ⁇ m and 4 ° C for 45 min. The precipitate was then washed twice by adding 70% ethanol and again in between
  • the 5'-ex nuclease was inactivated by heating for 20 min at 85 ° C.
  • the solution with the single-stranded sample molecule in the exonuclease buffer was mixed with polyethylene glycol (with a degree of polymerization of 3000-5000 subunits) to a final concentration of 0.2%.
  • the extension reaction took place at 50 ° C for 1 h in the evaporation-proof chamber construction of the thermal cycler in situ PCR System 1,000 (Perkin Elmer in situ PCR System 1,000 plus accessories).
  • the evaporation-proof chamber construction results from the fact that the remaining separation matrix (g) seals the slide (s) against a second slide, which serves as a lid (d) (Fig. 5 / IV).
  • the lid was placed on one side of the separation matrix and the extension mixture without air bubbles and fixed with a clamp (AmpliCover Clip, PerkinElmer).
  • Fig. 4 shows the extension reaction for the described SNP Y.
  • the chip was then chilled on ice for 5 seconds and immediately after opening the
  • the chip was analyzed using the GSI LS IV laser scanner (from GSI Lumonics). For this purpose, the sample points were excited with the now fluorescence-labeled extended extension primers with monochromatic light at 488 nm and 650 nm and the emitted light at 525 nm and 667 nm measured and registered. The physical properties of the dyes were selected when selecting the laser / filter systems and the laser energies based on the recommendation of the device manufacturer (Manual from Genomic Solutions, USA) considered. The color (emitted wavelength of the fluorescence-labeled nucleotide) of the extended probe molecule and thus the identity of the SNP at the respective sample point on the chip was determined.
  • results were stored directly as intensity values of the fluorescence in a database.
  • the software used was GT Scan software, GSI Analyzer (and associated documentation).
  • Fig. 1 Hordeum vulgäre L. (barley) -EST locus from the NCBI gene bank database (http://www.ncbi.nlm.nih.gov/ database entry: gi9410596, bold, underlined: Primersite. Bold, italic , underlined: complementary PTO primer site, Y: SNP)
  • Fig. 2 Genomic PCR of the fragment containing SNP (size: 174 bp, forward primer: gray, black background, PTO reverse primer: gray background; nucleotides linked by phosphorothioate bonds: white, bold, black background)
  • Fig. 3 Attachment (PTO-protected fragment to the extension primer fixed on the area) in the hybridization field of the area and before the extension: formation of a hybrid molecule between extension primer (
  • Fig. 5 Process steps that are carried out with the slide (note: the drawing is true to scale with the exception of the vertical extent of the area (a) in Fig. 5 / IV and V, which is only shown for clarification) I. Surface coating of the Slide (s) with oligonucleotide probes in

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Abstract

L'invention concerne un procédé de détection multiparallèle de polymorphismes de nucléotide simple sur un jeu ordonné d'échantillons polydimensionnel. L'invention concerne en outre un procédé de détection de nombreux polymorphismes de nucléotide simple individuels, selon lequel les polymorphismes de nucléotide de multiples individus peuvent être détectés de manière multiparallèle sur le jeu ordonné d'échantillons. Dans le cadre du procédé selon l'invention, l'hybridation, dans chaque cas, d'une molécule-sonde avec, dans chaque cas, une molécule-échantillon, s'effectue dans un champ d'hybridation sur le jeu ordonné d'échantillons, qui est séparé des champs d'hybridation environnants.
EP04739568A 2003-06-03 2004-06-03 Procede pour analyser des polymorphismes de nucleotide simple sur des biopuces presentant des zones oligonucleotides Withdrawn EP1629117A2 (fr)

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DE10325098A DE10325098B3 (de) 2003-06-03 2003-06-03 Verfahren zur SNP-Analyse auf Biochips mit Oligonukleotid-Arealen
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AUPO427996A0 (en) * 1996-12-20 1997-01-23 Co-Operative Research Centre For Diagnostic Technologies Method for detecting a nucleotide at a specific location within a polynucleotide sequence and apparatus therefor
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US6238869B1 (en) * 1997-12-19 2001-05-29 High Throughput Genomics, Inc. High throughput assay system
US6235483B1 (en) * 2000-01-31 2001-05-22 Agilent Technologies, Inc. Methods and kits for indirect labeling of nucleic acids
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