EP1784500A1 - Procede d'analyse de mutations ponctuelles - Google Patents

Procede d'analyse de mutations ponctuelles

Info

Publication number
EP1784500A1
EP1784500A1 EP04764692A EP04764692A EP1784500A1 EP 1784500 A1 EP1784500 A1 EP 1784500A1 EP 04764692 A EP04764692 A EP 04764692A EP 04764692 A EP04764692 A EP 04764692A EP 1784500 A1 EP1784500 A1 EP 1784500A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
dna
probe
chip
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04764692A
Other languages
German (de)
English (en)
Inventor
Holger Dr. Klapproth
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP1784500A1 publication Critical patent/EP1784500A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides

Definitions

  • SNPs single nucleotide polymorphisms
  • many genetic diseases such as e.g.
  • SNPs can be detected by means of PCR or sequencing of the corresponding ⁇ s.
  • sequencing is not that easy or at least much more expensive.
  • areas of the gene in the DNA sequencer are no longer evaluable. If several possible SNPs are present on a gene, the analysis by means of PCR becomes very difficult and again very expensive.
  • oligonucleotide microarrays are on these microarrays diverse short DNA fragments (oligonucleotides) bound, in which the center of the strand is the base that is complementary to the sought SNP. Due to the resulting differences in the melting points with the DNA of the analyte, it can now be determined whether the 100% complementary DNA has bound or not.
  • the usual detection lies in the comparison of the signal strengths between the perfect matches (100% complementary) and the SNP mismatches (about 5% false binding). These analyzes were mostly performed by hybridizing the DNA to the chips at a fixed temperature, and then the binding intensity was measured and compared. If a large number of SNPs are analyzed simultaneously on one chip, it is almost impossible to adapt all probes to the same analysis temperature, since, in addition, the methods for calculating the melting point for DNA hybrids do not correspond to the measured values.
  • Such methods can be especially determined by determining the Generate the melting point of the hybrids on the chip.
  • suitable for this purpose are devices with which these analyzes can be carried out real-time, such as, for example, the ATR reader, which has been developed by the Fraunhofer ⁇ esellschat (FH ⁇ -IPM) (see FIG. 1).
  • the ATR reader analysis method proves to be relatively expensive, since the melting curves of the nucleic acids are analyzed in order to determine the so-called melting points (Tm) of the nucleic acid hybrids.
  • Tm melting points
  • the melting points of DNA probes must be calculated and subsequently determined experimentally. In particular, this technique makes the design of the chips relatively difficult for many probes per chip.
  • the invention describes the surprising finding that for the analysis of SNPs on ATR chips no probe design in the true sense is necessary, but sufficient, these probes in a preselected length, which preferably does not differ too much from probe to probe, and then the Include melting point curve respectively. Subsequently, the curves of the wild-type probes, which are also referred to below as wt probes, and with the probes of the possibly occurring mutants are compared. This comparison is achieved by the subtraction of the melting point curve of the Wildi ⁇ p probe from the melting point curve of one or more mutations. The result is shown as a graph (see FIG. 2). The location of this curve now indicates which genotype is present. A reliable allocation is achieved, above all, by comparison with reference data.
  • the base to be examined is located in the middle of the DNA probe sequence.
  • the chip is heated in stages of 1 0 C, for example, 30 0 C to 80 0 C.
  • the signal is recorded and stored on each probe. To As a result, reaching the upper selected temperature range, a complete melting point curve is recorded.
  • the data of the variations to be examined are then compared with the data of the so-called wild-type sequence. For this purpose, the data of the variations are subtracted from the data of the wild-type sequence. If the DNA analyzed is homozygous for the wild type, the resulting curve will have positive values in the region of the melting point of the DNA, but at least significantly more positive values than in the case of a heterozygous sample. If the sample is heterozygous, the resulting curve will ideally approach a straight line along the zero point. If the sample for the mutated DNA is homozygous, the melting point at the probe of the mutated DNA (where there is 100% binding) is higher than the melting point at the wild-type probe (there is no 100% binding). Consequently, the curve which is calculated by subtracting the melting curves is characterized by a deflection in the direction of the negative y-axis. The location of these curves explicitly describes the composition of the sample DNA with respect to the mutation to be studied.
  • the sensors are preferably waveguide chips, which may be object carrier-shaped chips into which the excitation light for a TIRF excitation is coupled via an edge. But it can also be chips that are created in the form of thin-film waveguides.
  • Such thin film waveguide chips are e.g. Tantalum pentoxide-coated glass substrate carriers into which the light is coupled via optical gratings.
  • DNA probe molecules are preferably formed on such chips via chemical adhesion promoters, such as e.g. Bonded silane or polymer coatings. Such compounds are described, for example, in EP 1 132 739 B1.
  • DNA probes suitable for binding to such surfaces are preferably provided with a polythymidine spacer consisting of at least 10 thymidine molecules.
  • the probe molecules are preferably covalently coupled. Suitable methods for this purpose are described in WO 00/43539 A2.
  • the preparation of suitable samples and the marking is described, for example, in WO 01/53822 and Lehr (Lehr 2002).
  • the surface of the support is coated with a UV-reactive polymer.
  • This polymer is then passed through Irradiation of the coated support covalently coupled to UV light on the surface.
  • polymer supports such polymer thin layers can be applied by dipcoating in a dilute solution (analogous to EP 176422 A1)
  • DNA molecules can be applied to these surfaces by means of polythymidine spatters and UV crosslinkers, particular preference being given to copolymers having a limited hydrophilicity
  • a polymer of methacrylic acid, styrene, and benzophenone methacrylate is provided with these properties, and of course, other copolymers, and in particular, block copolymers, are also suitable
  • it is expedient to silanize these substrates beforehand in order to improve the adhesion of the polymer layers Such silanization is described, for example, in EP 1 1 32 739 B1 and in US Pat
  • Suitable supports for analysis may also be integrated systems (DE 10 245 435 A2, such as CMOS chips with additionally applied waveguide structures). These waveguides are preferably located above the opto-sensors and are connected to the optochip by means of a thin layer of a substrate of lower optical density than the waveguide.
  • the biomolecules are applied to polymer polymer thin-film waveguides.
  • Such waveguides consist of a carrier polymer with a low optical density and a waveguide terpolymer with a high optical density.
  • a carrier polymer with a low optical density for example, a polymer used in the manufacture of plastic eyeglass lenses. Suitable polymers of correspondingly low optical density are sufficiently known to the person skilled in the art.
  • the chip is rinsed after a measurement with buffer and heated so that the bound nucleic acids are largely removed. After that, the chip can be used again. In addition to heating, the chip can also be regenerated by denaturing agents, such as 0.1% NaOH.
  • This method also makes it possible to hybridize the chip with reference nucleic acids so as to calibrate each chip before, during or after the measurement. In this way it is possible to compare the measured values of different experiments with one another and to calculate out individual differences, which may be due, for example, to the production of the chips. Furthermore, this method allows the quality control of the chips during use and allows the determination of whether a chip is still suitable for further experiment or not.
  • chips are used for such experiments in which the initial hybridization can not be completely resolved, such chips can be saturated by blocking with an unlabeled sample that binds to all nucleic acid probes, so that hybridized after the actual experiment and the detachment Samples no residual fluorescence signal remains.
  • Detection of mutation H63D of Haemochromatose gene mutation H63D The base exchange of a cytosine for a ⁇ uanine (C ⁇ G, transversion) at position 187 in exon 2 of the HFE gene leads to an amino acid exchange of a histidine to aspartic acid at position 63 of the HFE protein. This mutation is referred to in the literature as H63D (Feder et al 1996).
  • the mutation H63D involves the replacement of a cytosine by a guanine by a transversion.
  • a pyrimidine is replaced by a purine base and there is a steric hindrance at the site of base mismatch. Since no binding can form in the base mismatches present in transversions, these are easier to detect.
  • the probes for detecting the mutation H63D carry at a central point of the specific sequence a C (wt probe) or a ⁇ (mut probe).
  • the sample DNA carries a ⁇ (wt sample, H63D (+ / +)), a C (mut sample, H63D (- / -)), 50% ⁇ and 50% C, respectively (het sample; H63D (+/-)).
  • oligonucleotides On PMMA carriers of dimension 7 ⁇ x25xl mm with a lateral edge of 70 ° for coupling the excitation light are printed with a top-spot printer or other suitable device (Lehr 2002) oligonucleotides.
  • the pressure buffer used is a 5% DMSO solution in water.
  • the oligonucleotides are present in a concentration of 10 ⁇ M.
  • the oligonucleotides consist of a recognition sequence of the target DNA of 17 nucleotides in length and the desired mutation in the middle of the sequence. At the 5 'end of the oligonucleotide is a polythymidine strand of at least 10 nucleotides in length.
  • the freshly printed chips are irradiated for 10 minutes in a UV crosslink oven (Stratalinker, Stratagene) for 10 minutes with UV light having a wavelength of 260 nm. Subsequently, with water to which a detergent is added (0.1% SDS) thoroughly rinsed rinsed with distilled water and blown dry in a stream of nitrogen.
  • a detergent 0.1% SDS
  • chips were made with a 17mer probe pair (63_17wt and ⁇ 3_17mut).
  • the chips were hybridized in the TIRF measuring instrument with DNA samples of the different types a) Wf sample, b) mut sample, c) het sample), stained and the dissociation behavior of the probe pair was measured.
  • the fluorescence intensities at the Wt and mut probe were observed over a temperature range of 20 ° C-80 ° C in 2 ° C increments and shown in the following diagrams.
  • the DNA purification was carried out from 10 ml of human blood by means of the nucleon extraction and purification kit from Amersham Biotech.
  • the absorption of UV radiation of wavelength 260 and 280 nm was measured by a nucleic acid solution, against the respective solvent, in a spectrophotometer.
  • a A 2 ⁇ o unit corresponds to a double-stranded DNA concentration of 50 ⁇ g / ml and a single-stranded DNA concentration of 33 ⁇ g / ml.
  • the ratio of absorbances at 260 and 280 nm is an indicator of the purity of the DNA. It assumes a value of 1.8 with optimum purity.
  • the primers store at a temperature which is close to the melting temperature Tm of the primer is applied to the single-stranded DNA.
  • modified nucleotides can also be incorporated into the PCR.
  • the nucleotides may e.g. Fluorescent dyes or biotin, biotinylated nucleotides being much easier and more efficient to incorporate than e.g. Cy5-labeled nucleotides.
  • biotin! ⁇ -dUTP incorporated into the DNA.
  • the biotin-dUTP was incorporated instead of dTTP.
  • direct Cy-5 dUTP can also be installed.
  • PCR products were used which were prepared with a thioate-modified primer and an unmodified primer. These PCR products were mixed in a volume of 25 .mu.l with 1 ul 17 gene exonuclease from Amersham and incubated at 37 ° C for 30 min. To complete the reaction, the batch was heated at 85 ° C for 10 min.
  • the hybridization was carried out by adding the amplified sample in hybridization buffer to the measuring field of the sensor chip.
  • the hybridization solution was added directly to a hybridization cuvette in the TIRF meter. Subsequently, the carriers in the hybridization cuvette of the TIRF apparatus were rinsed for several seconds with commercially available washing buffer.
  • the staining of the bound biotinylated PCR fragments was carried out by incubation with a streptavidin-Cy5 conjugate.
  • the stock solution was taken up according to manufacturer's instructions in dyeing buffer. This was added directly into the hybridization cuvette of the TIRF meter.
  • Fig. 1 describes the TIRF measuring optics.
  • Laser light from a semiconductor laser diode (1) is focused by a lens (2) (3) and blasted onto the coupling edge of a TIRF measuring chip (4).
  • biomolecules (5, 6) labeled with a luminescent dye.
  • the luminescent dyes are excited and emit luminescent light (5,7), which can be collected by means of suitable detectors.
  • Luminescent dyes which are outside the evanescent field are not excited by luminescent dyes ( ⁇ ) and accordingly emit no light.
  • the fluorescent light (7) can be recorded by means of commercially available CCD or CMOS cameras.
  • Fig. 2 shows the analysis according to the invention of three experiments in comparison to each other.
  • the temperature in ° C and on the ordinate the signal difference on a 17mer probe pair (mutation H ⁇ 3D in hybridization with different enenolypenes) are plotted.
  • the analyte used was a DNA containing only wild-type alleles.
  • the chip When the temperature is reached, the chip is recorded with the camera and the image of the fluorescence is stored. Subsequently, the signal of the fluorescence images of different temperature levels is evaluated. For this purpose, the signal sity at the points where the probes are immobilized quantified. At the same time, reference points on the chip are evaluated on which fluoreszenz ⁇ labeled DNA is immobilized. This signal decreases when the chip is heated (because the fluorescence signal is reduced by heat). This decrease is determined on a percentage basis and the recorded signal curves are corrected by this decrease. After correction, the signal of the probe of the desired mutation is now subtracted from the probe of wild-type DNA. The result is plotted as a graph.
  • the middle curve represents the case when both wild-type DNA and mutated DNA are present in the analyte.
  • the curve then ideally forms a straight line along the zero point.
  • the lower curve now represents the case when only mutated DNA is present in the analyte.
  • the difference in the binding curve of wild-type DNA to the binding curve of the mutated DNA is negant, since the mutated DNA binds to the wild-type probe at a much lower binding energy than it binds to the probe of the mutated DNA sequence.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de déterminer des mutations ponctuelles au moyen de "puces à ADN" et par excitation de fluorescence TIRF. Ce procédé consiste à mesurer la liaison d'acides nucléiques à des sondes ADN courtes sur une puce à ADN à différentes températures. Des courbes de points de fusion sont générées à partir de ces valeurs de mesure et la différence des courbes de points de fusion entre la sonde pour l'ADN de type sauvage et la sonde pour l'ADN muté correspondant est générée. L'état de ces courbes permet de déterminer sans équivoque s'il s'agit d'un ADN homozygote ou d'un ADN hétérozygote pour cette mutation ponctuelle, ainsi que le type d'homozygotie.
EP04764692A 2004-09-01 2004-09-01 Procede d'analyse de mutations ponctuelles Withdrawn EP1784500A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2004/009730 WO2006024314A1 (fr) 2004-09-01 2004-09-01 Procede d'analyse de mutations ponctuelles

Publications (1)

Publication Number Publication Date
EP1784500A1 true EP1784500A1 (fr) 2007-05-16

Family

ID=34958460

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04764692A Withdrawn EP1784500A1 (fr) 2004-09-01 2004-09-01 Procede d'analyse de mutations ponctuelles

Country Status (3)

Country Link
US (1) US20080085839A1 (fr)
EP (1) EP1784500A1 (fr)
WO (1) WO2006024314A1 (fr)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11001881B2 (en) 2006-08-24 2021-05-11 California Institute Of Technology Methods for detecting analytes
US8637436B2 (en) 2006-08-24 2014-01-28 California Institute Of Technology Integrated semiconductor bioarray
US11525156B2 (en) 2006-07-28 2022-12-13 California Institute Of Technology Multiplex Q-PCR arrays
WO2008014485A2 (fr) 2006-07-28 2008-01-31 California Institute Of Technology Ensembles de pcr-q multiplex
US11560588B2 (en) 2006-08-24 2023-01-24 California Institute Of Technology Multiplex Q-PCR arrays
DE102007031710B3 (de) * 2007-07-06 2008-11-06 Klapproth, Holger, Dr. Verfahren und Vorrichtung zum Nachweisen und/oder Bestimmen der Konzentration von Nukleinsäure-Liganden in einer Probe
US8538733B2 (en) * 2008-01-25 2013-09-17 Life Technologies Corporation Methods for the analysis of dissociation melt curve data
US9657338B2 (en) 2008-10-01 2017-05-23 Koninklijke Philips N.V. Method for immobilizing nucleic acids on a support
CN102054644B (zh) * 2010-12-13 2012-05-30 电子科技大学 一种起伏状波导慢波结构
CN102409088B (zh) * 2011-09-22 2014-11-12 郭奇伟 一种基因拷贝数变异的检测方法
SG11201508345TA (en) * 2013-04-11 2015-11-27 Agency Science Tech & Res Nanoprobe-based genetic testing
WO2015041603A1 (fr) 2013-09-17 2015-03-26 Agency For Science, Technology And Research Procédé de détection d'un variant génétique
US9708647B2 (en) * 2015-03-23 2017-07-18 Insilixa, Inc. Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays
US9499861B1 (en) 2015-09-10 2016-11-22 Insilixa, Inc. Methods and systems for multiplex quantitative nucleic acid amplification
WO2017155858A1 (fr) 2016-03-07 2017-09-14 Insilixa, Inc. Identification de séquence d'acide nucléique à l'aide d'une extension de base unique cyclique en phase solide
AU2018321760A1 (en) * 2017-08-25 2020-02-27 Zoetis Services Llc A nucleic acid probe, a method of immobilizing the nucleic acid to a solid support using UV light, a solid support comprising an immobilized nucleic acid probes, and a test device comprising a solid support
EP3937780A4 (fr) 2019-03-14 2022-12-07 InSilixa, Inc. Procédés et systèmes pour une détection à base de fluorescence résolue en temps
US20240069317A1 (en) * 2020-12-29 2024-02-29 Interherence GmbH Optoelectronic chip

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6221586B1 (en) * 1997-04-09 2001-04-24 California Institute Of Technology Electrochemical sensor using intercalative, redox-active moieties
US6850846B2 (en) * 2000-01-11 2005-02-01 Affymetrix, Inc. Computer software for genotyping analysis using pattern recognition
DE10002566A1 (de) * 2000-01-21 2001-08-02 Fraunhofer Ges Forschung Verfahren und Einrichtung zur Bestimmung des Schmelzpunktes und/oder der Bindungskonstante von Substanzen, wie z. B. DNA-Sequenzen, in einer Probe
EP1132484A3 (fr) * 2000-03-08 2003-05-28 Fuji Photo Film Co., Ltd. Procédé pour tester la complémentation des acides nucléiques
EP1176422B1 (fr) * 2000-07-27 2004-10-06 Micronas Holding GmbH Puces détectrices à multicouches composées de polysiloxanes
DE10245435B4 (de) * 2002-09-27 2006-03-16 Micronas Gmbh Vorrichtung zur Detektion mindestens eines in einer zu untersuchenden Probe enthaltenen Liganden

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2006024314A1 *

Also Published As

Publication number Publication date
US20080085839A1 (en) 2008-04-10
WO2006024314A1 (fr) 2006-03-09

Similar Documents

Publication Publication Date Title
EP1784500A1 (fr) Procede d'analyse de mutations ponctuelles
DE19844931C1 (de) Verfahren zur DNS- oder RNS-Sequenzierung
EP2266699B1 (fr) Appareil pour l'amplification et la détection d'acide nucléique
DE69928683T2 (de) Verfahren zur amplifikation und zur sequenzierung von nukleinsäuren
DE69031665T2 (de) Nachweis von Nukleinsäuresequenzen unter Verwendung von Fluoreszenz-Polarisation
EP1975246A1 (fr) Séquençage sans marquage sur une surface solide en utilisant un transitor à effet de champ
EP2182077B1 (fr) Procédé de détection de polymorphisme et de mutation d'un nucléotide unique à l'aide de microréseau de réaction en chaîne de polymérase en temps réel
DE19935772C2 (de) Verfahren zur relativen Quantifizierung der Methylierung von Cytosin Basen in DNA-Proben
WO2002038806A2 (fr) Identification de polymorphismes d'acide nucleique
WO2017205827A1 (fr) Réseaux pour la détection de molécule unique et leur utilisation
US20120040853A1 (en) Real time multiplex pcr detection on solid surfaces using double stranded nucleic acid specific dyes
DE602004012273T2 (de) Bestimmung der sequenz eines polynukleotids
DE69814848T2 (de) Untersuchung von nukleotiden in loesung mit hilfe von pna-sonden
EP2167685B1 (fr) Procédé et système de sondes et d'amorces pour la mise en évidence en temps réel d'un d'acide nucléique cible
JP2005525787A (ja) プローブとの相互作用による遺伝子ハプロタイプの検出方法
DE102004025694A1 (de) Verfahren und Oberfläche zu hochparallelen Analysen von Nukleinsäureketten
DE102004025695A1 (de) Verfahren und Oberfläche zur parallelen Sequenzierung von Nukleinsäureketten
DE102004025745A1 (de) Oberfläche für die Analysen an einzelnen Molekülen
WO2008110622A1 (fr) Procédé et kit de test permettant la détection rapide de séquences d'acides nucléiques spécifiques, notamment la détection de mutations ou de polymorphismes d'un seul nucléotide (snp)
EP2852458B1 (fr) Procédé pour détecter en temps réel des amas de molécules et/ou pour surveiller le processus de production d'un microréseau de molécules
DE10036174B4 (de) Verfahren und Vorrichtung zum Analysieren von chemischen oder biologischen Proben
DE10111420A1 (de) Bestimmung von Analyten durch Fluoreszenz-Korrelationsspektroskopie
US20060118754A1 (en) Stabilizing a polyelectrolyte multilayer
DE10309526A1 (de) Verfahren zum Analysieren von Punktmutationen
DE102005029811B4 (de) Oligonukleotidanordnungen, Verfahren zu deren Einsatz und deren Verwendung

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20070327

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20071019

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20090909