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WO2005098031A1 - Dosage d'hybridation en phase liquide - Google Patents

Dosage d'hybridation en phase liquide Download PDF

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Publication number
WO2005098031A1
WO2005098031A1 PCT/EP2005/003534 EP2005003534W WO2005098031A1 WO 2005098031 A1 WO2005098031 A1 WO 2005098031A1 EP 2005003534 W EP2005003534 W EP 2005003534W WO 2005098031 A1 WO2005098031 A1 WO 2005098031A1
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WIPO (PCT)
Prior art keywords
sample
probe
assay
nucleic acid
foregoing
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PCT/EP2005/003534
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English (en)
Inventor
Lajos Nyarsik
Anna Guerasimova
Michal Janitz
Regine Schwartz
Hans Lehrach
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2005098031A1 publication Critical patent/WO2005098031A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00378—Piezoelectric or ink jet dispensers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00495—Means for heating or cooling the reaction vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00277—Apparatus
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00572—Chemical means
    • B01J2219/00576—Chemical means fluorophore
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00599—Solution-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G01N2035/1046—Levitated, suspended drops

Definitions

  • the present invention relates to a method of fluorescence- based detecting of hybridization events in an assay containing an intercalating dye, a sample RNA sequence and a probe nucleic acid sequence labelled with a marker dye. Further- more, the present invention relates to a device for fluorescence-based detecting of hybridization events according to the above method.
  • PNAs fluorescently labelled peptide nucleic acids
  • FRET fluorescence resonance energy transfer
  • the iFRET method has the advantage of providing a good signal to background ratio on immobilized probe-target duplexes using a double-strand DNA-specific intercalating dye (e.g., SYBR Green I) as the FRET donor and a conventional FRET ac- ceptor attached to the DNA probe molecules.
  • a double-strand DNA-specific intercalating dye e.g., SYBR Green I
  • the iFRET configuration combines the advantages of intercalating dyes, such as relative high signal strengths and low cost, with maintaining the specificity and multiplex potential provided by traditional FRET-based detection systems.
  • the conventional fluorescence-based detec- ting methods and in particular the iFRET method have the following disadvantages.
  • the conventional microarrays are not suitable for "screening" experiments (multiple probes against multiple targets) , since a single slide can be hybridized only with one probe at the same time. Finally, the molecules can be immobilized on a solid phase surface as a single layer only. Therefore, for a readout a very high sensitive detector has to be used rendering almost impossible conventional fluorescent detection or time-resolved detection.
  • a further object of the present invention is to provide a new device for fluorescence-based detecting of hybridization events.
  • the present invention provides a method of detecting hybridi- zation events in an assay containing an intercalating dye, a sample nucleic acid sequence and a probe nucleic acid sequence labelled with a marker dye, comprising a step of illuminating the assay and detecting a fluorescence signal of at least one of the marker dye and the intercalating dye, said fluorescence signal depending on a number of hybridization events in the assay, characterized in that the sample nucleic acid sequence is an RNA sequence which is contained in the assay in a liquid phase condition.
  • the present method uses a liquid-based hybridization assay with the sample nucleic acid sequence free in solution.
  • the present method avoids the disadvantages of the conventional prior art methods as outlined above.
  • RNA sequence instead of a DNA sequence offers several additional advantages, in particular in a short probes hybridization assay which is a preferred embodiment of the present invention as detailed below.
  • Short probes hybridization is a potent DNA characterization technique due to its high specificity and possibility of thermodynamical discrimination of mismatches.
  • any probing of a long DNA sample e.g. DNA clones
  • a short oligo probe requires the DNA to be in a single-stranded condition.
  • this is achieved by alkali denaturation and physical fixation to the membrane of the analyte.
  • Other approaches to immobilization of DNA involve strand separation e.g. via biotin-labelling of one of the strands.
  • RNA duplexes are more stable than DNA: DNA homoduplexes is beneficial for hybridization with very short probes, such as 7- or 6-mers, which produce less stable hybridization complexes than longer ones.
  • the sample RNA sequence may be an original RNA sample, such as native mRNA or genomic RNA from a retrovirus, but usually it will be generated by enzymatic in vi tro transcription of an original DNA sample.
  • a large number of suitable enzymes i.e. DNA-dependent RNA polymerases such as E. coli RNA poly- merase and T7 RNA polymerase, are commercially available and suitable reaction conditions for in vi tro transcriptions are well known in the art.
  • the original sample DNA such as a DNA clone
  • the original sample DNA may have been subjected to a PCR amplification step prior to the transcription.
  • An additional advantage of said in vi tro transcription resides in the concomitant amplification of the nucleic acid template for the subsequent hybridization reaction, since several RNA copies from each DNA molecule are generated during the transcription. Thus, the sensitivity of the hybrid!- zation assay of the present invention is increased.
  • the intercalating dye to be used in the present invention is a double-strand specific dye, which means that its fluorescence in the presence of double-stranded nucleic acid is much greater, preferably at least 10 times greater, than its fluorescence in free solution or associated with single-stranded nucleic acid.
  • Suitable dyes are known in the art and commercially available. Specific, but not limiting examples are SYBR Green I (Molecular Probes) and Vistra Green (Amersham) .
  • the intercalating dye may be added before or after the hybridization reaction of sample and probe nucleic acid sequences .
  • the marker dye attached to the probe nucleic acid may be any suitable fluorescent dye, in particular a dye which is known as a FRET acceptor dye.
  • a green emitting intercalating dye in conjunction with a red emitting fluorescent dye is used.
  • Suitable oligo probes for use in the present invention preferably comprise short sequences of 6 to 10 nucleotides, more preferably 6 to 8 nucleotides. Oligo probes even with shorter sequences of e. g. 5 nucleotides can be used as well.
  • the reliable hybridization of such short oligos in the method of the present invention enables the use thereof for rese- quencing by hybridization or SNP-detection.
  • the oligo probes are oligo DNAs or modified nucleic acids, such as locked nucleic acids (LNAs) or peptide nucleic acids (PNAs) .
  • LNAs locked nucleic acids
  • PNAs peptide nucleic acids
  • a detergent is added to the assay to enhance the specific iFRET signal.
  • the detergent is a non-ionic detergent, more preferably a polysorbate detergent, in particular Tween 20.
  • the detergent may be added to the solution in one of the droplets or to the mixed droplet.
  • the assay of the present invention permits the simultaneous or sequential analysis of a plurality of samples. In particular, its permits the simultaneous use of a plurality of samples and/or probes with different labeling (e.g. different marker dyes with different colors) in order to generate and evaluate a multiplex signal.
  • the present method is suitable for rapid screening of multiple probes against multiple targets.
  • the present liquid-based hybridization assay permits real time detection of hybridization events in miniaturized high density array platforms. This method enhances the throughput by large scale applications, reduces the costs for probe con- sumption, and improves the detection signal quality. By using nanodispensing techniques the reaction volume can be reduced down to 1000 to 1 nl or below 1 nl .
  • the method according to the pre- sent invention combines miniaturized reaction well and droplet based hybridization in solution with real time iFRET fluorescent detection.
  • sample and probe solutions are mixed directly together in the droplet form without the need of sample nucleic acid immobilization.
  • the droplets may added to each other by simultaneous or sequential depositing of sample and probe droplets on a common substrate.
  • Said substrate may be a flat surface, such as a glass surface, a nanowell, a mi- crochannel plate or a cuvette.
  • the glass surface enables direct monitoring of the hybridization reaction.
  • the droplets are distributed on a flat surface or are placed into arrayed nanowells by non-contact nanodispens- ing (piezodispensing or valve dispensing) .
  • the probe application can be performed in two directions thus allowing mixing inside the well (or on the surface) for reaction. Therefore the first set of probes can be placed in a column and the second in a row. Any type of droplet distribution is possible.
  • Such screening in droplets is well suited for high throughput screening, such as, e.g., oligo finger printing (100 oligoprobes x 100000 DNA or RNA samples) .
  • the droplet size is dependent on the dispensing technique (appr. piezodispensing 1 nl, valve dispensing 50 nl) , the probe concentration and the detection sensitivity.
  • a pitch below 500 ⁇ m can be achieved with nanodispensing technique (pitch 500 ⁇ m: 4 droplets/mm 2 , 400 droplets/cm 2 ). Using the pitch of 500 ⁇ m, 10000 screening droplets can be placed on the chip of size 50x50 mm.
  • a square format is more advantageous for screening in droplets because of symmetrical arrangement.
  • reaction droplets are closed in a small chamber or under oil to avoid evaporation of liquid during the measurement (from 1-60 in up to several hours) .
  • the assay sensitivity is increased by varying the temperature of the hybridization reaction and recording the corresponding fluorescence signal.
  • the annealing of oligos to form DNA duplexes will be impaired and the iFret signal induced by an intercalating dye is decreased. After complete melting, only the background signal is left. Performing multiple heating-cooling cycles provides several data points for the same hybridization reaction and improves the signal quality and reliability.
  • a thin chip is used in this embodiment which allows for a rapid thermal regulation. Hence the detection time is shorter, which is an important factor for high throughput applications .
  • the step of detecting a fluorescence signal comprises a polarisation-sensitive measurement of fluorescence emission.
  • at least one of the sample and probe is labelled with a dye for detection of polarisation as known in the art.
  • the step of detecting a fluorescence signal comprises a time- resolved measurement of fluorescence emission.
  • This embodiment involves application of different fluorescence dyes with short and long term emission signals as known in the art.
  • the present invention also provides a device for detecting hybridization events in an liquid-based assay according to the present invention.
  • a device for detecting hybridization events in an assay containing an intercalating dye and sample and probe nucleic acid sequences comprising: a carrier substrate for accommodating the assay containing the sample and probe nucleic acid sequences in a liquid phase condition, an illumination device for illuminating the assay, a detection device for detecting a fluorescence signal from the assay, and an evaluation device for evaluating the fluo- rescence signal.
  • the carrier substrate in said device may comprise a structured surface containing nanowells for arrayed positioning of mixed droplets.
  • the carrier substrate may comprise a plane surface for arrayed positioning of mixed droplets.
  • the carrier substrate may comprise micro-channels or a separate reaction chamber for spectral measurements.
  • the reaction chamber may comprise a cuvette or capillary.
  • the carrier substrate comprises a cover in order to reduce evaporation of liquid from the assay.
  • the device of the present invention preferably comprises a preparation device for combining the sample and probe nucleic acid sequences in the assay.
  • the preparation device comprises a non-contact droplet dispensing device.
  • the non-contact droplet dispensing device comprises a piezoelectric dispenser or a liquid droplet dispenser being operated like an inkjet dispenser.
  • the non-contact droplet dispensing device is adapted for dispensing droplets with a volume in the range of 1 nl to 1000 nl.
  • the carrier substrate is connected with a temperature setting device.
  • the detection device preferably is adapted for detecting at least one spec- tral line or a spectral emission range.
  • the different parts of the above device can be integrated into a closed workstation. This gives the possibility to built a benchtop sys- tem.
  • the probes are placed in microplates within the system (probe A in microplate A, probe B in microplate B) .
  • the closed system After defining the .protocol, the closed system carries out the screening operation and generates the data.
  • a high density droplet array can be detected by a CCD matrix camera.
  • Fig. 1 shows different arrangements for mixing of two droplets in a cuvette (A), nanowells (B) or. in a microchannel plate (C) .
  • Fig. 2 shows the mixing and illuminating of droplets on a flat surface.
  • Fig. 3 shows the mixing and illuminating of droplets on a flat surface, wherein the mixed droplet is covered by a transparent plate ("sandwich"-format) .
  • Fig. 4 schematically shows an arrangement of probe and sample dispenser devices.
  • Fig. 5 is a schematic diagram of the principal steps in the method of the present invention.
  • Fig. 6 is a schematic diagram of an embodiment of a device according to the invention.
  • Fig. 7 is a iFRET-fluorescence spectrum in solution, showing the simultaneous detection of two oligos labelled with different marker dyes (Cy5 and ROX) hybridized to the same sample nucleic acid (multiplexing) .
  • Fig. 8 is a schematic diagram of melting curves obtained according to the invention.
  • two droplets with the sample and the probe, resp., are mixed in a common cuvette.
  • the droplets are mixed in common nanowells (B) or in a micro- channel plate (C) .
  • illumination and detection can be performed with an illumination and detection device positioned above the surface.
  • the reproducibility of the measurements can be improved if the droplet is covered with a plate as shown in Figure 3. Between the surface and the plate, a gap is formed accommodating the mixed droplets.
  • probe and sample dispenser devices 20, 30 An exemplary arrangement of probe and sample dispenser devices 20, 30 is shown in Figure 4.
  • the probe and sample dis- an devices for depositing the probe and sample droplets, resp., are movable in directions orthogonal to each other and parallel to the surface of a carrier substrate 10.
  • the probe and sample dispenser devices are known in the art, e.g. pie- zoelectric dispensers are used.
  • the principal steps in the method of the present invention comprise the probe and sample preparation and mixing, followed by the hybridization in liquid phase ( Figure 5) . Subse- quently, the fluorescence of the assay is measured and evaluated.
  • Reference numeral 70 refers to a temperature control device.
  • the sample nucleic acid was dispensed into nanowells ( Figure 1 B) with a nanodispersing system (1-10 nl) .
  • the probes were deposited- in parallell lines (column or row) .
  • sample nucleic acids were dispensed and subsequently the oligo probes were deposited orthogonally to the DNA probes.
  • the nanowells were covered with an oil layer to avoid evapora- tion. A thin oil layer can be applied just after sample dispensing.
  • the probes were mixed immediately (instantly) or after centrifugation by applying an interlayer.
  • the nanowell plate was placed on a thermal block. For 7mer oligonucleotide probes the signal was monitored at temperatures between 10- 40 °C to create a melting curve.
  • a 96 channel dispensing head can accelerate the sample processing.
  • the templates used in the above examples were synthetic DNAs of 25-40nt length designed to carry the matched sites for the respective oligo-probe sequences or transcribed DNA clones of l-2kb length with known sequence.
  • DNA clones were PCR amplified, purified with the Qiagen Kit and converted into RNA using the Message Amp tm Kit from Ambion with subsequent fragmentation as described by the Affimetrix protocol (supplier" s manual) .
  • the oligo probes used were 7mer-10mers labelled with the fluorescent dyes ROXl, Cy5 or Bodipy. Said oligo probes were hybridized separately or pooled with RNA templates, complexed with the intercalating dye Sybr Green I (Molecular Probes) . An iFRET signal was monitored in 4 wavelengths regions: 550-600 nm, 600-650 nm (ROX), 650-700 nm (Cy5) , 700-750 nm.
  • Figure 8 shows temperature cycling curves obtained according to the invention with various RNA samples. With increasing hybridization temperature, the iFRET signal induced by an intercalating dye is decreased. After complete melting, only the background signal is left. Performing multiple heating- cooling cycles improves the signal quality and reliability.
  • the method of the invention can be applied with differential hybridisation of oligonucleotides for distinguishing between variants through distinct pattern of hybridisation to a known set of oligonucleotides.
  • Differential hybridisation has been described by R. Drmanac et al. in “Meth. Mol. Biol.” (vol. 170, 2001, p. 173-179) and by J. Shendure et al . in “Nature Reviews Genetics” (vol. 5, 2004, p. 335 -344).
  • Successful sequence determination with a set of 5-mer probes has already been reported for 100 bp fragments (I. Pe'er, et al. in "Nu- cleic Acids Res.” (vol. 31(19), 2003, p.
  • the present method is closely relevant to the above Sequenc- ing-by-Hybridisation concept of R. Drmanac et al . with the difference that a more actual task of so called "re- sequencing" avoids complete sequence determining de novo but proves information by comparison with the known reference.
  • This technology is suitable to query genotype of the focused regions (sets of genomic positions) of practical length (100- 1000 nt) . Provided these regions are known one can design a comprehensive set of oligonucleotide probes of fixed length- short enough to offer specificity and accuracy but long e- nough to provide necessary stability in hybridisation. E.g. 7 to lOmer probes would be preferred for such applications. Those applications may include diagnostic tests for highly polymorphic genes (e.g. Cystic fibrosis gene), typing of pathogens, identification of somatic mutations, identifica- tion of splice variants and polymorphisms in non-coding part of genome.
  • highly polymorphic genes e.g. Cystic fibrosis gene
  • Fluorescence-based method of signal detection is sufficient and compatible with the throughput needed for re-sequencing and possibility to perform analysis in a liquid phase should considerably speed up the analysis.

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Abstract

La présente invention a trait à un procédé de détection d'événements d'hybridation dans un dosage contenant un colorant intercalant, une séquence d'acides nucléotidiques d'échantillon et une séquence d'acides nucléotidiques de sonde marquée avec un colorant de marquage, comprenant une étape d'éclairage du dosage et la détection d'un signal de fluorescence d'au moins parmi le colorant de marquage et le colorant intercalant, ledit signal de fluorescence dépendant du nombre d'événements d'hybridation dans le dosage, la séquence d'acides nucléotidiques d'échantillon étant contenue dans le dosage dans un état de phase liquide.
PCT/EP2005/003534 2004-04-06 2005-04-04 Dosage d'hybridation en phase liquide WO2005098031A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007087336A3 (fr) * 2006-01-23 2008-04-10 Quest Diagnostics Invest Inc Procedes de detection d’acides nucleiques au moyen de signaux multiples
US7785786B2 (en) 2006-01-23 2010-08-31 Quest Diagnostics Investments Incorporated Methods for detecting nucleic acids using multiple signals
US8039216B2 (en) 2006-01-23 2011-10-18 Quest Diagnostics Investments Incorporated Methods for detecting nucleic acids using multiple signals

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