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WO2000055363A2 - Analyse de l'expression genique differentielle - Google Patents

Analyse de l'expression genique differentielle Download PDF

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Publication number
WO2000055363A2
WO2000055363A2 PCT/GB2000/000807 GB0000807W WO0055363A2 WO 2000055363 A2 WO2000055363 A2 WO 2000055363A2 GB 0000807 W GB0000807 W GB 0000807W WO 0055363 A2 WO0055363 A2 WO 0055363A2
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WO
WIPO (PCT)
Prior art keywords
beads
reagent
target
nucleic acids
bead
Prior art date
Application number
PCT/GB2000/000807
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English (en)
Other versions
WO2000055363A3 (fr
Inventor
Nicholas Thomas
Alan Waggoner
Original Assignee
Amersham Pharmacia Biotech Uk Ltd
Amersham Pharmacia Biotech Inc
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Filing date
Publication date
Application filed by Amersham Pharmacia Biotech Uk Ltd, Amersham Pharmacia Biotech Inc filed Critical Amersham Pharmacia Biotech Uk Ltd
Priority to EP00907817A priority Critical patent/EP1163367A2/fr
Priority to JP2000605779A priority patent/JP2002538836A/ja
Priority to AU29284/00A priority patent/AU2928400A/en
Publication of WO2000055363A2 publication Critical patent/WO2000055363A2/fr
Publication of WO2000055363A3 publication Critical patent/WO2000055363A3/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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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
    • 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
    • B01J2219/005—Beads
    • 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
    • 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
    • B01J2219/00664—Three-dimensional arrays

Definitions

  • This invention relates to methods for detecting the differential expression or presence of two analytes. and more specifically to procedures which provide for rapid and efficient analysis of gene expression in biological systems.
  • Such approaches have largely been displaced by more powerful, reproducible and informative methods based on the use of arrays of nucleic acids in which large numbers of specific sequences are laid down in an ordered pattern on a solid surface and form the target for hybridisation and capture of labelled mRNA or cDNA from the cells under study.
  • arrays have been constructed on a variety of supports, ranging from nylon membranes to glass and silicon wafers. Whatever the support, the essential method of use is the same: firstly know n sequences, complementary to cellular mRNAs, either in the form of synthetic oligonucleotides or as PCR products, arc laid down on the solid support in spots at defined locations.
  • sequences are then exposed to sequences (probes) extracted from the cells or tissue under study, where the probes are tagged with some form of label which can be detected in subsequent analysis.
  • probes In early techniques, radioactive labels were used to probe targets on nylon membranes using techniques developed from Southern and Northern blotting methods. However these methods required the hybridisation process to be performed twice, once for the control sample and once for the test sample. They were subsequently overtaken by more elegant procedures based on the use of different coloured fluorophors to label control and test probes and using either PCR products coupled to glass (Schena M. et al, (1996), Proc. Natl. Acad.
  • the mRNA sequences extracted from control and test cells or tissues are either labelled directly or are first converted or amplified to yield equivalent cDNA sequences which are subsequently labelled.
  • the fluorescent labels attached to the probes are detected, either by scanning or by imaging, and quantified to yield data on the amounts of different mRNAs present in the test and control samples. Since sequences from test and control cells are labelled with different fluorophors, both samples can be applied and hybridised simultaneously and the resulting pattern and intensity of hybridised probes determined using detection instrumentation tuned to distinguish between the emission wavelengths of the fluorophors used.
  • the hybridisation process is also subject to a number of problems arising from the geometry of the system and the temperature required for the hybridisation process.
  • This invention provides an alternative to micro-array systems for analysis of gene expression. Means are provided for performing analyses using a particle based technique so as to replace ordered 2D arrays with randomly oriented 3D arrays which can be quickly and easily modified to include new target sequences.
  • the technique provides favourable geometry and kinetics for promoting efficient hybridisation, that can be performed in a standard reaction tube, and that allows measurement of hybridised probe to several thousand target sequences to be accomplished in a few seconds.
  • the invention provides a method of detecting and analysing differences between nucleic acids from two sources, which method comprises: a. providing nucleic acids from two sources as labelled probes; b.
  • each reagent is a population of beads carrying a polynucleotide target, the target of one reagent being different from the target of another reagent, the beads of one reagent being distinguishable from the beads of another reagent; c. incubating the mixture under conditions to promote specific hybridisation between probes and targets; and, d. analysing beads in the mixture by flow cytometry.
  • a polynucleotide target is partly or wholly single-stranded and is capable of specific hybridisation. Oligonucleotides of at least 8 residues are preferred.
  • cDNA sequences derived, e.g. by RT-PCR amplification, from cellular mRNA.
  • the pooled reagents may comprise one bead, or preferably a plurality of beads, of each reagent.
  • features of the invention include: a) gene expression assays are performed on carrier beads; b) individually identifiable beads or populations of beads each carrying a different target sequence are prepared; c) selected beads, or populations of beads, are pooled together in suspension to provide a randomly oriented 3D array of particles carrying all sequences of interest for an individual investigation; d) mRNAs or cDNAs prepared from control and test cells or tissues are labelled with fluorescent tags to identify their source; e) labelled probe species are mixed with the pooled suspension of target carrying beads under conditions which promote specific hybridisation between probes and targets; f) the bead mixture is analysed by flow cytometry to simultaneously determine the identity of each bead analysed (and hence the identity of the target sequence carried by the bead) and to quantify the amounts of both control and test probes bound to each bead; and g) data is analysed to yield information on the relative and absolute abundance of each mRNA in the control and test samples.
  • the beads of one reagent can be distinguished from the beads of another reagent by a number of different means. Suitable distinguishing means include differences in size, colour or fluorescence or the nature or concentration of markers attached to the beads. Beads of one reagent can be distinguished from beads of other reagents using one or more of such means.
  • Figure 1 Schematic illustration of 2D ordered array and 3D random array.
  • Figure 2 Flowchart illustrating the principle of the bead based flow cytometry gene expression process.
  • Figure 3 Schematic representation of the bead based flow cytometry gene expression process.
  • FIG. 4 Schematic representation of method for analysis of differential expression of TNF and GAPDH genes in LPS stimulated THP-1 cells.
  • each target spot immobilised on a planar surface are defined by x,y coordinates and hence target sequences are identified by the same co-ordinates.
  • a 3D array formed from particles dispersed in a space defined by dimensions x,y,z if each particle is individually identifiable by some inherent characteristic, it is not necessary to use x,y,z locations to specify the identity of each bead and the particles can be randomly distributed throughout the volume as in a suspension of beads in liquid. It follows that if each bead is individually identifiable, then any target sequence previously coupled to that bead is also identifiable. Therefore if a number of differing beads, or discrete populations of beads, are individually prepared where each bead carries a different target sequence and then selectively pooled, the pooled beads can form a 3D array which can be used for gene expression analysis.
  • Beads suitable for use in the method of the invention are those which can be readily identified during analysis by flow cytometry; such beads have been previously developed and used for diagnostic assays to measure a wide range of analytes in blood and other biological fluids by immunoassay.
  • a desire to have a higher throughput in these applications has led to the development of multiplex methods which allow more than one analyte to be measured simultaneously by means of flow cytometry analysis. Multiplexing is achieved by carrying out solid phase linked assays using plastic or latex beads as assay substrates.
  • each bead type carries reagents for one assay
  • standard flow cytometer instrumentation may be used both to identify the bead type and to measure the assay signal associated with each bead. Discrimination between bead populations can be achieved by size (Frengen I. et al (1995), Journal of Immunological Methods, Volume 178, pi 41 ), by colour or fluorescence (Fulwyier M.J. UK Patent 1 ,561 ,042) or by electronic means (Mandecki W. US Patent 5,641,634).
  • Selected target cDNA sequences are prepared by standard PCR methods incorporating a means to allow coupling of target sequences to beads.
  • One suitable method would utilise a 5'-biotin on one of the PCR primers, yielding a 5'- biotinylated DNA suitable for coupling to streptavidin-coated beads.
  • alternative chemical coupling strategies are available. Such alternative strategies may include, for example, synthesising oligonucleotides having a chemical group such as an amino group at the 5' end thus rendering them suitable for crosslinking to beads which have been modified to have, for example, carboxyl groups on their surface. It will also be appreciated that oligonucleotides synthesised with a terminal biotin or other coupling group could readily be used in place of PCR generated DNA sequences.
  • each target sequence is separately coupled to a corresponding discrete population of beads (Bead 1 to Bead n respectively). Aliquots are then removed from each population and pooled to form a mixed suspension of beads constituting a randomly orientated 3D array of target sequences.
  • the 3D array is then hybridised with fluorescently labelled probes (RNA or cDNA) prepared from the control and target cells or tissues which have been labelled with two different fluorophors (Fluor A and Fluor B respectively).
  • the mixed population of beads is analysed by flow cytometry; as each bead is analysed info ⁇ nation from the flow cytometer detectors is used to identify the bead and to measure the amounts of Fluor A (control mRNA) and Fluor B (sample mRNA) bound to the complementary target sequence carried by the bead. These measurements are then used to determine the relative expression of each mRNA in the samples.
  • Control (1) and test (2) probes are prepared and labelled using standard methods and aliquots mixed in a tube (3) containing a mixture of beads (4) carrying the desired target sequences and the tube sealed. Hybridisation of probe and target sequences are promoted by incubating the mixture under conditions of heat, pH and salt concentration which are known to allow the formation of specific nucleic acid hybrids. Following hybridisation, the bead mixture is analysed by flow cytometry using multiple channel fluorescence detection. In the embodiment illustrated, two fluorescence channels are used to identify beads and two further channels are used to measure control and test probe fluorescence.
  • Bead identity is determined by measuring the amounts of two different fluorophors (bead Fluor 1 and bead Fluor 2) incorporated within the bead during manufacture. Plotting the intensities of the two fluorophors on x,y axes (8 & 9) separates the different bead populations used.
  • the number of possible target sequences that can be measured in a single assay will necessarily be limited by the number of bead populations which it is possible to discriminate in a mixture. With current flow cytometry instrumentation this does not pose a limitation on the utility of the procedure.
  • Typical modern flow cytometry instruments are capable of simultaneously measuring fluoresce.nce at four wavelengths together with other parameters, for example light scattering which is a measure of the size of particles under analysis.
  • the dynamic range of fluorescence detection is high and fluorescence may be accurately measured over several orders of magnitude.
  • Plotting the intensity of probe fluorophors on the z axis of two different plots (10 & 1 1 ) shows the amounts of the control (10) and test (1 1 ) probes bound to each bead population. This allows the production of a table of the amounts bound to each target sequence in the analysis (14).
  • one mRNA species (12) is expressed at a lower level in the test sample than in the control (13). Other differences in expression can be readily identified as differences in the heights of equivalent peaks in the two plots.
  • DNA or oligonucleotide target sequences are typically applied to a solid surface as discrete areas of dimensions in the range 10-100 ⁇ m, with dimensions of 50-100 ⁇ m being typical of DNA spots applied as liquid droplets, and smaller areas being used in techniques utilising photo-lithographic oligonucleotide synthesis.
  • Beads used for flow cytometry typically have diameters in the range from 1 -10 ⁇ m and therefore individually do not have sufficient surface area to substitute for a typical micro-array.
  • Beads used for flow cytometry typically have diameters in the range from 1 -10 ⁇ m and therefore individually do not have sufficient surface area to substitute for a typical micro-array.
  • Beads to carry each target sequence it is possible to achieve equivalence in target presentation as shown in the following example:
  • spot area ⁇ r
  • the total volume required for the assay is 1 ⁇ l. If desired, larger volumes may be used for convenience in hybridisation or analysis; for example using beads at a concentration of 1 % v/v would give a 10 ⁇ l total volume.
  • the method of the invention would allow larger numbers of beads to be used with a consequent increase in capacity for binding a greater mass of target sequence spread over the total bead population. This would allow the user if desired to increase the amount of probe bound to the bead populations to increase the sensitivity of the process for detecting rare species. Alternatively, it enables an increase in the number of samples which may be analysed simultaneously, for example, to measure expression of a panel of genes simultaneously in a control and more than one test sample, where as described previously each control or test sample is labelled with a different fluorophor. Such increases in assay complexity are not achievable with conventional arrays on solid surfaces without reducing sensitivity due to the finite capacity of array spots for binding complementary sequences.
  • the method of the present invention provides a number of significant advantages over previously described procedures for gene expression analysis which are based on 2D arrays: a) the basic components for the bead based assay are readily prepared by coupling solutions of cDNA or oligonucleotides to commercially available beads using standard coupling methods, b) no specialised equipment is required for preparation or analysis, in contrast to the dedicated array production and scanning equipment required for micro- arrays, c) the design of investigations can be easily modified with target sequences being added or deleted at will without the requirement to scrap existing materials, d) hybridisation is performed in suspension in standard reaction vessels, thereby avoiding problems with evaporation associated with thin films of liquid covering micro-arrays and promoting hybridisation through efficient mixing of probe and target sequences, and e) analysis speed is significantly improved: flow cytometers typically analyse beads at rates of 1,000-10,000 beads/second allowing processing of a 100 sequence gene expression analysis in a few seconds.
  • FIG. 4 shows a scheme of a method for the analysis of differential Tumour Necrosis Factor (TNF) expression in THP-1 cells either treated in the presence (test) or absence (control) of bacterial lipopolysaccharide (LPS) (Su S. etal BioTechniques 1997, 22: 1 107-1 1 13).
  • LPS bacterial lipopolysaccharide
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • RNA isolation RNA isolation will be recognised by someone skilled in the art and include the use of commercially available reagents or kits (e.g. RNeasy, Qiagen).
  • RNA molecules are converted to cDNA by means of the enzyme, reverse transcriptase, using the standard method set out in Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press 1989, pp 8.1 1 -8.13.
  • cDNA molecules obtained from each sample are then used in separate multiplex PCR reactions using standard conditions (Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory Press 1989 pp 14.5-14.20) and primer pairs which amplify GAPDH and TNF (Su S. etal BioTechniques 1997,
  • One primer of each primer pair is modified at the 5 ' end with a biotin molecule to aid in strand separation in subsequent steps in the process. This is readily achieved using standard techniques during oligonucleotide synthesis with modified phosphoramidites, for example : 5'-Biotin Phosphoramidite (Glen Research). Fluorescently labelled nucleotides are incorporated into the PCR reactions in order to label the amplified PCR products such that their origin from the test or control RNA populations may be established.
  • the PCR reaction mix containing cDNAs prepared from the control cells includes Cy3TM-dCTP and the reaction mix containing cDNAs prepared from test cells includes Cy5TM-dCTP (Cy3TM-dCTP and Cy5TM-dCTP are obtained from Amersham Pharmacia Biotech).
  • Cy3TM-dCTP and Cy5TM-dCTP are obtained from Amersham Pharmacia Biotech.
  • Double stranded cDNA products of the PCR reactions are converted to single stranded cDNA molecules prior to further analysis by binding the PCR products via the biotin at the 5' end of each PCR product to streptavidin-coated magnetic beads (MagneSphere, Promega).
  • the double stranded PCR products are denatured by addition of 0.2 volume of 2M NaOH, incubating for 10 minutes at room temperature in order to release the non-biotinylatcd strands from the beads.
  • the beads are separated from the solution by attraction to a magnet and the clarified solution, which contains the single stranded labelled PCR products, is removed and made neutral by addition of 1 volume of 0.4M HC1.
  • Other methods for separating double-stranded DNA molecules are known to those skilled in the art.
  • populations of beads or particles suitable for performing analysis of differential gene expression are prepared using beads containing fluorescent dyes (SPHEROTM, Spherotech Inc.). Yellow fluorescent streptavidin-coated beads are coated with the 5 ' -biotin primer for GAPDH and blue fluorescent streptavidin-coated beads are coated with the 5 '-biotin primer for TNF (using the same 5' -biotin primers as were used earlier).
  • these populations of beads have two distinguishing characteristics i.e. each bead population is distinguishable from all other bead populations by its fluorescence characteristics and each bead population is capable of recognising a single cDNA species (either TNF or GAPDH).
  • the separately prepared beads are mixed together, further mixed with the Cy 3 and Cy 5-labelled single stranded cDNA products prepared previously and incubated in a hybridisation buffer (0.1M Tris.HCl pH7.4, 750mM NaCl) at 45° for 2 hours prior to analysis of the bead populations by flow cytometry.
  • a hybridisation buffer 0.1M Tris.HCl pH7.4, 750mM NaCl
  • cDNA products from the mixed population will bind specifically to their complementary capture sequences carried on beads such that each bead will ultimately be decorated with a mixture of labelled cDNAs of a single species arising from the test and control populations, and where the relative abundance of the labels reflects the relative abundance of single species in the original test and control populations.
  • the relative abundance of GAPDH transcripts in the population of molecules derived from control and test samples is compared by detecting the fluorescence of yellow beads at 460nm/ 480nm (excitation/emission); and, within the population of yellow beads, bound Cy3 labelled transcripts are detected at 550nm/570nm (excitation emission) and bound Cy5 labelled transcripts are detected at 650nm/670nm (excitation/emission).
  • the relative abundance of TNF transcripts in the molecules derived from test and control samples is determined by detecting the fluorescence of blue beads at 650nm/710nm (excitation/emission); and further detecting bound Cy3 labelled transcripts at 550nm/570nm (excitation/emission) and bound Cy5 labelled transcripts at 650nm/670nm (excitation/emission).
  • bead fluorescence is used to assign the identity of the gene associated with each bead
  • label fluorescence is used to determine the relative abundance of cD As arising from the test and control samples which are attached to the bead, the latter yielding information on the differential expression of that gene under the conditions used in treatment of the original cell or tissue sample.

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Abstract

La présente invention concerne des procédés de détection de l'expression différentielle ou de la présence de deux analytes et, plus spécifiquement, des procédures qui permettent une analyse rapide et efficace de l'expression génique dans des systèmes biologiques. Elle concerne en particulier un procédé de détection et d'analyse de différences entre des acides nucléiques provenant de deux sources, ce procédé consistant a) à mettre les acides nucléiques des deux sources sous forme de sondes marquées, b) à former un mélange des sondes marquées avec des réactifs regroupés, dans lequel chaque réactif est sous la forme d'une population de billes portant une cible polynucléotide, la cible d'un réactif étant différente de la cible d'un autre réactif, les billes d'un réactif pouvant être distinguées des billes d'un autre réactif, c) à incuber le mélange dans des conditions permettant de favoriser l'hybridation entre sondes et cibles, et d) à analyser les billes dans le mélange à l'aide de la cytométrie de flux.
PCT/GB2000/000807 1999-03-12 2000-03-09 Analyse de l'expression genique differentielle WO2000055363A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP00907817A EP1163367A2 (fr) 1999-03-12 2000-03-09 Analyse de l'expression genique differentielle
JP2000605779A JP2002538836A (ja) 1999-03-12 2000-03-09 遺伝子発現の変化の分析
AU29284/00A AU2928400A (en) 1999-03-12 2000-03-09 Analysis of differential gene expression

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9905807.5 1999-03-12
GBGB9905807.5A GB9905807D0 (en) 1999-03-12 1999-03-12 Analysis of differential gene expression

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WO2000055363A2 true WO2000055363A2 (fr) 2000-09-21
WO2000055363A3 WO2000055363A3 (fr) 2000-12-21

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WO (1) WO2000055363A2 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027328A1 (fr) * 1999-10-08 2001-04-19 University Of Utah Research Foundation Dosage d'analyse de particules pour la quantification de biomoleculaire
WO2002064829A3 (fr) * 2001-02-13 2002-12-12 Smartbead Technologies Ltd Methode biochimique et appareil associe permettant de detecter des caracteristiques genetiques
WO2002065123A3 (fr) * 2001-02-13 2002-12-12 Smartbead Technologies Ltd Procede et appareil biochimiques de detection de caracteristiques de proteines
WO2004106931A1 (fr) * 2003-05-26 2004-12-09 Institut Virion\Serion Gmbh Procede et systeme d'essai pour l'analyse et/ou la detection de biomolecules et/ou de substances actives dans des echantillons liquides
WO2005071412A2 (fr) 2004-01-09 2005-08-04 Applera Corporation Billes codees avec des particules de phosphore
US6994971B1 (en) 1999-10-08 2006-02-07 University Of Utah Research Foundation Particle analysis assay for biomolecular quantification
EP2196544A1 (fr) 2001-11-21 2010-06-16 Applied Biosystems, LLC Réactions de détection de ligature utilisant des marqueurs codés
US8129115B2 (en) 2006-06-06 2012-03-06 Panasonic Corporation Method of modifying nucleotide chain
EP2975123A4 (fr) * 2013-03-12 2016-11-30 Hitachi Ltd Dispositif à réseau cellulaire bidimensionnel et appareil pour la quantification de gènes et l'analyse de séquences

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5604097A (en) * 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
AU7398996A (en) * 1995-10-11 1997-04-30 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
US6060240A (en) * 1996-12-13 2000-05-09 Arcaris, Inc. Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom
AU1080999A (en) * 1997-10-14 1999-05-03 Luminex Corporation Precision fluorescently dyed particles and methods of making and using same
WO1999064867A1 (fr) * 1997-12-04 1999-12-16 Amersham Pharmacia Biotech Uk Limited Procede pour dosages multiples
US6265163B1 (en) * 1998-01-09 2001-07-24 Lynx Therapeutics, Inc. Solid phase selection of differentially expressed genes

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001027328A1 (fr) * 1999-10-08 2001-04-19 University Of Utah Research Foundation Dosage d'analyse de particules pour la quantification de biomoleculaire
US6994971B1 (en) 1999-10-08 2006-02-07 University Of Utah Research Foundation Particle analysis assay for biomolecular quantification
US8034555B2 (en) 1999-10-08 2011-10-11 University Of Utah Research Foundation Particle analysis assay for biomolecular quantification
WO2002064829A3 (fr) * 2001-02-13 2002-12-12 Smartbead Technologies Ltd Methode biochimique et appareil associe permettant de detecter des caracteristiques genetiques
WO2002065123A3 (fr) * 2001-02-13 2002-12-12 Smartbead Technologies Ltd Procede et appareil biochimiques de detection de caracteristiques de proteines
EP2196544A1 (fr) 2001-11-21 2010-06-16 Applied Biosystems, LLC Réactions de détection de ligature utilisant des marqueurs codés
WO2004106931A1 (fr) * 2003-05-26 2004-12-09 Institut Virion\Serion Gmbh Procede et systeme d'essai pour l'analyse et/ou la detection de biomolecules et/ou de substances actives dans des echantillons liquides
WO2005071412A2 (fr) 2004-01-09 2005-08-04 Applera Corporation Billes codees avec des particules de phosphore
US8129115B2 (en) 2006-06-06 2012-03-06 Panasonic Corporation Method of modifying nucleotide chain
EP2975123A4 (fr) * 2013-03-12 2016-11-30 Hitachi Ltd Dispositif à réseau cellulaire bidimensionnel et appareil pour la quantification de gènes et l'analyse de séquences
US10030240B2 (en) 2013-03-12 2018-07-24 Hitachi, Ltd. Two-dimensional cell array device and apparatus for gene quantification and sequence analysis

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AU2928400A (en) 2000-10-04
JP2002538836A (ja) 2002-11-19
EP1163367A2 (fr) 2001-12-19
WO2000055363A3 (fr) 2000-12-21
GB9905807D0 (en) 1999-05-05

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