Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • B01J2219/00277—Apparatus
  • B01J2219/00497—Features relating to the solid phase supports
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  • B01J2219/00277—Apparatus
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  • B01J2219/005—Beads
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  • 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
  • B01J2219/00497—Features relating to the solid phase supports
  • B01J2219/00527—Sheets
  • B01J2219/00529—DNA chips
• • 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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  • B01J2219/00583—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
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  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00596—Solid-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
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  • 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
  • B01J2219/00608—DNA chips
• • 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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  • B01J2219/00583—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
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  • 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
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  • B01J2219/00583—Features relative to the processes being carried out
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  • 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
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  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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  • 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
  • B01J2219/00623—Immobilisation or binding
  • B01J2219/00626—Covalent
• • 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
  • B01J2219/00632—Introduction of reactive groups to the surface
  • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
• • 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
  • B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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  • 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
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  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
  • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
  • C07B2200/11—Compounds covalently bound to a solid support
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

• the present invention relates to a process for the preparation of an activated biochip for the covalent attachment of oligonucleotide probes to a solid support by means of a spacer compound of the NHS-PEG-VS type, as well as the biochips capable of be obtained by such a process.
• the invention also includes methods for detecting nucleic acids in a sample or methods for screening for compounds capable of specifically binding to oligonucleotide probes in which the biochips according to the invention are used.
• the present invention also relates to kits for the detection, quantitative or qualitative analysis of nucleic acids in a sample, comprising such biochips as well as the use of the latter as affinity matrix for the purification of nucleic acid. , for nucleic acid sequencing, for the qualitative or quantitative analysis of gene expression or even for the study and detection of genetic polymorphism.
• nucleic acids such as biochips, or DNA chips (also called “micro- or macroarrays”, or even “DNA chip”)
• biochips can in particular be produced from a support, generally solid, functionalized on which have been fixed by covalent bond and localized given nucleic acids (nucleic probes) and on which nucleic probes will be specifically fixed respectively by pairing ( or specific hybridization) or by recognition of an affinity site the nucleic acids which it is desired to detect or identify in the biological sample.
• a support generally solid, functionalized on which have been fixed by covalent bond and localized given nucleic acids (nucleic probes) and on which nucleic probes will be specifically fixed respectively by pairing ( or specific hybridization) or by recognition of an affinity site the nucleic acids which it is desired to detect or identify in the biological sample.
• polymer brushes which makes it possible to increase the grafting density .
• These polymers can be obtained from hydroxyethyl methacrylate, acrylamide, or vinyl pyrrolidone;
• the international patent application published under the number WO 00/36145 describes, for its part, a method of manufacturing DNA chips, comprising the polymerization on a metal layer type substrate, of a copolymer of pyrrole and of functionalized pyrrole, fixing a crosslinking agent on the functionalized pyrrole, then fixing a biological probe (such as an oligonucleotide).
• the crosslinking agent can be bifunctional, and for example have an ester function of N-hydroxysuccinimide and a maleimide function;
• PCR ranging from 200 to a few thousand base pairs, are the subject of great interest.
• burrs syndromes of fluorescence around the spots after hybridization
• biochip whose protocol allowing the immobilization of the oligonucleotide probes on a support, such as glass, for the manufacture of these biochips is a simple protocol (minimum of stages, if possible of "light” chemistry), rapid (this point is all the more important as the volumes used on each "spot” are very small and evaporate quickly, it is therefore essential that the covalent grafting be rapid and that excess probes can be easily removed from the surface (without leaving streaks)) and reproducible.
• the subject of the present invention is a method for preparing an activated biochip for the covalent attachment of oligonucleotide probes, said biochip comprising a solid support previously functionalized with a thiol or amine function, characterized in that it comprises a step of covalent fixing under the appropriate conditions on said functionalized support of an NHS-PEG-VS spacer compound of formula (I):
• n is an integer chosen such that the molecular mass of the NHS-PEG-VS compound of formula (I) is between 500 and 5000, preferably between 2000 and 4000, more preferably close to 3400.
• activated biochip in the present description, a solid support as defined below, on which will have been fixed by covalent bonding the spacer compounds of formula (I) capable of interacting with the nucleic probes , but not yet coated with said probes.
• nucleic acid nucleic probe, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, allowing to define a fragment or a region of a nucleic acid, with or without unnatural nucleotides, and which can correspond as well to double stranded DNA, single stranded DNA, a PNA (for “Peptid Nucleic Acid”) or LNA (for "Locked Nucleic Acid”) as transcripts of said DNAs such as TARN.
• PNA for “Peptid Nucleic Acid”
• LNA for “Locked Nucleic Acid
• oligonucleotide probe or nucleic probe is intended to denote here the functionalized oligonucleotide which will be deposited (or “spotted”) and fixed by covalent bond to said spacer compound on the functionalized solid support, this as opposed to the target nucleic acid derived from the biological sample that one seeks to detect or identify.
• the method for preparing an activated biochip according to the invention is characterized in that said solid support is selected from glass solid supports, plastic, Nylon ®, Kevlar ®, silicone , in silicon, or in polyoses or poly (hetero-oses), such as cellulose, preferably in glass.
• This support can be of any shape (flat blade, microbeads, ).
• the process for preparing an activated biochip according to the invention is characterized in that said solid glass support is functionalized by silanization.
• the method for preparing an activated biochip according to the invention is characterized in that said solid support is functionalized with an amino function when said oligonucleotide probes intended to be fixed are functionalized with a terminal thiol function .
• the method for preparing an activated biochip according to the invention is characterized in that said solid support is functionalized with a thiol function when said oligonucleotide probes intended to be fixed are functionalized with a terminal amino function .
• said solid support in particular made of glass, is functionalized with a thiol function
• the present invention also relates to a process for the preparation of a deactivated biochip for the covalent attachment of oligonucleotide probes functionalized with a terminal amine function, characterized in that it comprises the following stages: a) activation of the biochip by a process for the preparation of an activated biochip for the covalent attachment of oligonucleotide probes functionalized with a terminal amino function; and b) hydrolysis in the presence of an aqueous solution, preferably of pure water, of the free NHS functions of the NHS-PEG-VS spacer compounds of formula (I) fixed on the solid support.
• the present invention also relates to a process for the preparation of a regenerated biochip for the covalent attachment of oligonucleotide probes functionalized with a terminal amine function, characterized in that it comprises the following steps:
• EDC 1-ethyl-3 [3 (dimethylamino) propyl] carbodiimide hydrochloride
• NHS carbodiimide hydrochloride
• the process for preparing an activated, deactivated or regenerated biochip according to the invention is characterized in that it comprises a step in which the biochip obtained is frozen, dried or lyophilized, preferably dried under an inert atmosphere, such as under nitrogen, and away from humidity.
• the present invention relates to a process for the preparation of a biochip coated with oligonucleotide probes, characterized in that it comprises the following steps: ⁇ ) the preparation of a biochip activated or regenerated by a process according to the invention; ⁇ ) deposition and fixation by covalent bond under the appropriate conditions: - either of said oligonucleotide probes previously functionalized with a thiol function, if said solid support has been functionalized with an amino function, or
• oligonucleotide probes previously functionalized with an amino function, if said solid support has been functionalized with a thiol function; and optionally, ⁇ ) elimination of the oligonucleotide probes not fixed on the support by at least one step of rinsing the support under appropriate conditions, preferably in deionized water.
• the present invention further relates to a process for preparing a biochip comprising a solid support previously functionalized with a thiol function, then activated by deposition of NHS-PEG-VS of formula (I) and coated with oligonucleotide probes according to invention, characterized in that it further comprises the following step: ⁇ ) deactivation in the presence of amino compounds under the appropriate conditions of the NHS functions of the spacer compound which has not interacted with the amino functions of the oligonucleotide probes.
• said compound allowing the deactivation of the NHS functions of the spacer compound in step ⁇ ) is chosen from amino compounds having a primary amine, preferably ethanolamine or methoxy-PEG-NH 2 , in particular such as available for the latter from Shearwater
• the present invention also relates to a process for preparing a biochip comprising a solid support previously functionalized with an amine function then activated by deposition of NHS-PEG-VS of formula (I) and coated with oligonucleotide probes, characterized in that that it further comprises the following step: ⁇ ) the reduction, under appropriate conditions, of surface charges in the presence of anionic compounds or which are capable of establishing covalent bonds with the amino groups and lead to a neutral or negative species under the appropriate conditions, preferably in the presence of methyl N-succinimidyl adipate (MSA).
• MSA methyl N-succinimidyl adipate
• the present invention also relates to a process for the preparation of a biochip coated with oligonucleotide probes according to the invention, characterized in that it comprises a step in which the biochip obtained is stored away from humidity, light and / or in an inert atmosphere.
• said oligonucleotide probes are single-stranded DNAs or RNAs, preferably DNAs or RNAs, the size of which is between 15 and 7000 bp, preferably between 20 and 1000 bp, between 20 and 500 bp, between 20 and 250 bp, between 20 and 100 bp, between 20 and 80 bp or between 35 and 80 bp.
• probe DNAs or RNAs can be obtained by chemical synthesis, or from genomic DNA, RNA, or mRNA, or their fragments, extracted from cells, in particular for cDNAs after reverse transcription of these RNAs, or also in the form of a PCR fragment obtained by RT-PCR from these RNAs, or by PCR from these genomic DNAs (“RT-PCR” for a method called reverse transcription followed by a polymerization chain reaction).
• the methods for preparing a biochip coated with oligonucleotide probes according to the invention are characterized in that said oligonucleotide probes are deposited in the form of spots whose average diameter is between 20 ⁇ m and 500 ⁇ m, preferably between 50 ⁇ m and 200 ⁇ m, and, where appropriate, in that the average distance between the center of each of the spots of oligonucleotide probes is between 80 ⁇ m and 400 ⁇ m.
• the methods for preparing a biochip coated with oligonucleotide probes according to the invention are characterized in that the number of said spots of oligonucleotide probes is between 2 and 10 5 , preferably between 2 and 10 4 , 2 and 10 3 , 2 and 4 10 2 , 2 and 10 2 , even more preferably between 50 and 10 3 and between 50 and 4 10 2 per cm 2 .
• the subject of the present invention is a biochip comprising a solid support previously functionalized with a thiol or amino function, characterized in that it comprises an NHS-PEG-VS spacer compound of formula (I):
• PEG denotes poly (ethylene glycol) of formula HO- (CH 2 CH 2 O) n CH 2 CH 2 -OH, where n is an integer chosen such that the molecular mass of the NHS-PEG-VS compound of formula (I) is between 500 and 5000, preferably between 200 and 4000 and close to 3400, said spacer compound being fixed on said solid support by a covalent bond resulting either from the interaction between the thiol function of said functionalized support and the vinyl sulfone function of the spacer compound of formula (I) or either from the interaction between the amino function of said functionalized support and the NHS function of the spacer compound of formula (I).
• the biochip according to the invention is characterized in that it further comprises at least one oligonucleotide probe, previously functionalized with a thiol or amine function, attached to said solid support by a covalent bond resulting either from the interaction between the free NHS function of said spacer compound of formula (I) and an amine function of said oligonucleotide probe, or either of the interaction between the free vinyl sulfone function of the spacer compound of formula (I) and a thiol function of said oligonucleotide probe.
• the biochip according to the invention is characterized in that said fixed oligonucleotide probes are DNAs or single-stranded RNAs, preferably DNAs or RNAs, the size of which is between 15 and 7000 bp, preferably between 20 and 1000 bp, between 20 and 500 bp, between 20 and 250 bp, between 20 and 100 bp, between 20 and 80 bp or between 35 and 80 bp.
• said fixed oligonucleotide probes are DNAs or single-stranded RNAs, preferably DNAs or RNAs, the size of which is between 15 and 7000 bp, preferably between 20 and 1000 bp, between 20 and 500 bp, between 20 and 250 bp, between 20 and 100 bp, between 20 and 80 bp or between 35 and 80 bp.
• the biochip according to the invention is characterized in that said oligonucleotide probes are deposited in the form of spots whose diameter is between 20 ⁇ m and 500 ⁇ m, preferably between 50 ⁇ m and 200 ⁇ m and, if necessary , in that the average distance between the center of each of the spots of oligonucleotide probes is between 80 ⁇ m and 400 ⁇ m.
• the biochip according to the invention is characterized in that the number of said spots of oligonucleotide probes is between 2 and 10 5 , preferably between 2 and 10 4 , 2 and 10 3 , 2 and 4 10 2 , 2 and 10 2 , even more preferably between 50 and 10 3 and between 50 and 4 10 2 per cm2.
• the biochip according to the invention is characterized in that said solid support is chosen from solid supports made of glass, plastic, Nylon, Kevlar, silicone, silicon, polyoses or polyhetero-oses, preferably glass, preferably silanized.
• the present invention also relates to an activated, deactivated, regenerated biochip, or else coated with oligonucleotide probes, capable of being obtained by a method according to the invention.
• the present invention includes the use of a biochip according to the invention for the detection of nucleic acids in a sample.
• the present invention relates to a kit or necessary for the detection, qualitative or quantitative analysis of nucleic acids in a sample, characterized in that it comprises a biochip according to the invention.
• the present invention also relates to a method for the detection of nucleic acids in a sample, characterized in that it comprises the following steps: a) the deposition of the sample containing the target nucleic acids whose presence on a biochip coated with oligonucleotide probes according to the invention, under conditions allowing specific hybridization of these target nucleic acids with said oligonucleotide probes; b) if necessary, rinsing the biochip obtained in step a) under the appropriate conditions in order to remove the nucleic acids from the sample not captured by hybridization; and c) detecting the nucleic acids captured on the biochip by hybridization.
• these are preferably conditions of high stringency in particular as defined below or as cited, without being limited to, in the examples below. -after.
• Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two complementary DNA or RNA / DNA fragments.
• conditions of high stringency of the step Hybridization for the purpose of defining the hybridization conditions described above are advantageously the following.
• DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS.
• / last wash is carried out in 0.1 x SSC + 0.1% SDS for 30 minutes at 60 ° C for a probe of size> 100 nucleotides.
• the high stringency hybridization conditions described above for a polynucleotide of defined size can be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al. (1989, Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor).
• the invention also includes a method for the detection of nucleic acids in a sample according to the invention, characterized in that the nucleic acids whose presence is sought to be detected are marked beforehand at one of their ends with a marker capable of generating directly or indirectly a detectable signal, preferably detectable by fluorescence.
• the invention also includes a method for the detection of nucleic acids according to the present invention, characterized in that the nucleic acids whose presence is sought to be detected are marked beforehand for at least two of them by a different marker.
• said markers are chosen from cyanine derivatives, preferably chosen from sulfonated cyanine derivatives, in particular compounds Cy5 or Cy3, nanocrystals (Migyong Han et al, Nature Biotechnology, 19, 631-635, 2001 ) or nanoparticles (Genicon Science company).
• the present invention also relates to the use of a biochip according to the invention as an affinity matrix or for the purification of nucleic acid.
• the present invention also relates to the use of a biochip according to the invention for the sequencing of nucleic acid, for the qualitative or quantitative analysis of gene expression or also for the study and detection of polymorphisms genetics (also called SNP's for “Single Nucleotide Polymorphism” or SNIPS).
• DNA probes corresponding to known genes are deposited on the biochips according to the invention.
• Each deposit or spot may contain several thousand oligonucleotide probes corresponding to the same gene and from one spot to another the oligonucleotide probes will correspond to another gene, or to a gene having a different polymorphism.
• Genomic DNAs, or messenger RNAs, or their fragments, from the tissue or cell which it is desired to study may be extracted and then labeled with fluorochromes (the DNAs or mRNAs may in particular be transformed into complementary DNAs (cDNAs) by reverse transcription. , and, where appropriate, multiplied by PCR or RT-PCR techniques).
• fluorochromes the DNAs or mRNAs may in particular be transformed into complementary DNAs (cDNAs) by reverse transcription. , and, where appropriate, multiplied by PCR or RT-PCR techniques).
• DNAs, cDNAs, or RNAs will then be deposited on the biochip coated with oligonucleotide probes and, if necessary, bind by specific hybridization with the previously deposited oligonucleotide probes which correspond to them.
• the applications of these DNA biochips are therefore numerous, such as transcription studies, diagnosis (search for mutation), search for therapeutic targets, genetic mapping of individuals.
• the invention relates to a method for screening for compounds or cells capable of binding specifically to a given oligonucleotide, characterized in that it comprises the following steps: a) bringing said compound into contact to be tested on a biochip according to the invention, under the conditions allowing the possible specific fixation of said compound or of said cell to be tested with said given oligonucleotide, said biochip comprising at least one spot of oligonucleotide probes containing said given oligonucleotides fixed on its solid support and, where appropriate, said compound or said cell being labeled with a label capable of directly or indirectly generating a detectable signal; b) elimination by at least one washing step under the appropriate conditions of the compounds or cells to be tested not specifically bound to said given given oligonucleotide
• the present invention relates to a diagnostic or research instrument or device comprising a biochip according to the invention.
• Fifiures 1 A to ID Grafting of oligonucleotides functionalized by an amino group (NH 2 -terminated)
• Figure 1 A glass surface (Si-OH silanols).
• Figure 1B glass surface functionalized with mercaptosilanes (SH- terminated).
• Figure 1C grafting of heterobifunctional PEG (NHS-PEG-VS).
• the NHS ends remain free and reactive;
• Figure ID fixation of oligonucleotides functionalized by an amino group (NH -terminated).
• the surface is firstly silanized (functionalization) so as to obtain SH (thiol at the surface) functions.
• SH thiol at the surface
• the VS function of the heterobifunctional PEG which is then deposited (activation) will react with the thiols on the surface to form a covalent bond.
• the oligonucleotides carrying an amino function at one of their ends are finally deposited. This amino function will react with the NHS function of the heterobifunctional PEG to form a covalent bond.
• GOLD SEAL glass slides are used. These slides are cleaned using a mixture of sulfuric acid-hydrogen peroxide (70/30, v / v) with a volume of 300 ml for 15 minutes (Piranha mixture).
• the slides are rinsed with pure water and then with methanol.
• the silanization bath is composed of:
• the slides are immersed for 2 hours in this bath.
• the slides are then rinsed with pure methanol and then dried with argon, finally the slides are placed for 15 minutes in an oven at 94 ° C.
• Example 2 Support for oligonucleotide probes functionalized by a thiol group: Method 2
• the slides are, as for the experimental protocol of Example 1, washed with the Piranha mixture.
• the composition of the silanization bath changes:
• composition of the solution (for one slide): - 2 mg of NHS -PEG -VS MW 3400 Shearwater Polymers;
• Carbonate-Bicarbonate (CB) buffer pH 8.4.
• the pH of 8.4 is optimal for the chemical reaction between the NHS function of PEG and the NH2 functions of the silanized surface.
• the solution is deposited on the surface of the slide to be treated for 45 minutes.
• Example 3 Support deactivated then regenerated for oligonucleotide or peptide probes functionalized with an amino group
• the NHS function is sensitive to humidity and deactivates within a few days. To remedy this, a method has been developed which consists in deactivating the function in a stable form and then regenerating it when the oligonucleotide probes are deposited.
• the slides are first prepared as for method 1. Then the NHS function is hydrolyzed by immersing the slides in a bath of pure water until the moment of deactivation. The NHS function is transformed into carboxylate (COO " ). B) Regeneration
• Example 4 Passivation (or “capping”) of the regions of the support having active NHS groups of the spacer compound which have not interacted with the probes for method 1 1.
• Passivation or “capping” of the regions of the support having active NHS groups of the spacer compound which have not interacted with the probes for method 1 1.
• the regions around these deposits always have active groups of NHS or VS type depending on the method used. This is particularly troublesome for method 1 as described in example 1 for which the active NHS group is reactive with the amino groups.
• these amino groups are found in certain nucleotide bases. These amino groups are, in particular for the bases, much less reactive than the primary amino groups which serve as a hooking function for the oligonucleotides.
• the base amines do not compete (or very little) with the terminal amines of the functionalized oligonucleotide probes.
• the target nucleic acids such as the cDNAs, which are deposited on the support only have the amines constituting their bases. There is therefore a risk of a reaction between the base amines of the target nucleic acids and the NHS remaining around the deposits of the oligonucleotide probes, which has the effect of increasing the background noise. It is therefore preferable to deactivate these NHS sites that are still active.
• the slides are placed for 15 min in the blocking solution at 50 ° C: - rinsing with milliQ water for 4 min;
• Protocol 2 using a monofunctional PEG as an agent for deactivating NHS groups which have not interacted with the probes.
• a monofunctional PEG having an amino group at one of its ends is used. This amino group reacts with NHS by forming a covalent bond. The surface around the spots on which the probes have been placed is then covered with PEG.
• a solution is prepared containing: - 2 ml of phosphate buffer (HPO 4 / H2PO 4 ) 150 mM at pH 8.2; and
• the slide is immersed in this solution for 45 minutes, then it is rinsed with pure water and dried with argon.
• thiolated compounds for example N-ethylmaleimide, iodoacetate derivatives (sodium tetrathionate, Ellman reagent), aziridines, acryloyl derivatives, can be used.
• Oligonucleotide probes of 50 bases corresponding to fragments of mouse genes were deposited on functionalized glass slides on which the spacer compound NHS-PEG-VS was previously fixed. Three nucleic acid sequences were used:
• the oligonucleotides are deposited using a "spotter” from the company GeneMachines (OmniGrid). This spotter is a spotter with tip (Majer Precision) which allows deposits of a few ni. Between each deposit, the tip is washed to avoid contamination from one deposit to another.
• spotter from the company GeneMachines (OmniGrid). This spotter is a spotter with tip (Majer Precision) which allows deposits of a few ni. Between each deposit, the tip is washed to avoid contamination from one deposit to another.
• the blades according to the present invention are prepared according to method 1 and method 2. The results obtained with these blades were compared with those obtained under the same conditions of use with blades marketed by the company SurModics (SurModics, Inc., Eden Prairie, MN) under the reference “3D-LINK TM activated slides” (Lot DN01B058 package N ° 19), activated slides capable of fixing NH functionalized nucleotide probes having a free amine function (“amine-binding slides”).
• the blades of the SurModics Company are known to be the most efficient of the commercial blades currently available.
• the oligonucleotides are resuspended in pure water (milliQ) at a concentration of 0.5 mM.
• concentration of the oligonucleotide solutions is adjusted to 5 ⁇ M for the values of pH 6.8; 7.7; and 8.3 in 150 mM phosphate buffer, in a total volume of 12 ⁇ l.
• the biochips thus coated with oligonucleotide probes are hybridized with a solution containing the target oligonucleotides of sequences complementary to the deposited oligonucleotide probes.
• Three target oligonucleotides of sequences complementary to the oligonucleotide probes thus spotted were mixed:
• target oligonucleotides carry a 5 'fluorochrome (Cy5). These complementary target oligonucleotides are first resuspended in pure water at a concentration of 50 ⁇ M.
• each complementary target oligonucleotide ie a total of 3 ⁇ l.
• a mixture of the three target oligonucleotides carrying a fluorochrome is obtained
• the slides are rinsed as below.
• the slides are first placed in a 4X SSC solution in order to drop the slides, then:
• the scanner used has two settings for reading: - the setting of the excitation intensity (LASER); and
• the intensity of these two adjustments is expressed in an arbitrary unit. At the most the two values are at 100. For certain slides these maximum values saturate the fluorescence signal, it is then necessary to decrease the intensities of the LASER and / or of the PMT.
• Hybridization experiment on a biochip prepared according to method 1 pH test.
• NH 2 functionalized olignucleotide probes (“oligo NH2”) and non-functionalized were deposited at different pH values.
• the chip was hybridized with a mixture of target oligonucleotides complementary to those deposited, fluorescently labeled. 2 matrices of 54 spots were deposited.
• the fluorescence intensity reports obtained for the functionalized and non-functionalized oligonucleotide probes show the very good selectivity of the support for the functionalized oligonucleotide probes which are grafted by covalent bond.
• the signal to noise ratio is excellent (5,000), the background noise being very low.
• the spots are of very good size with a very small dispersion of the diameters (low wettability) especially for the oligonucleotide probes functionalized with amino groups.
• the scanner was set to 100/100.
• the results of the experiment are presented in Table 2 below.
• Oligonucleotide probes functionalized with an SH group (“Oligo SH”) and non-functionalized (“Oligo non-functionalized”) were deposited in solution at different pH values.
• the chip was hybridized with a mixture of target oligonucleotides complementary to the deposited probes, fluorescently labeled.
• the oligonucleotide probes functionalized with an SH group are better fixed than the non-functionalized oligonucleotide probes but the selectivity of the support is less strong than for the slides obtained with method 1.
• the pH has little influence on grafting.
• the scanner setting is 100/100.
• Oligonucleotide probes functionalized with an amino group (“Oligo NH2”) and nonfunctionalized (“Oligo nonfunctionalized”) were deposited in solution at different pH values.
• the biochip has been hybridized with a mixture target oligonucleotides complementary to the deposited probes, fluorescently labeled.
• the intensity of the fluorescence is low compared to the intensities measured on the slides according to the present invention, especially since the scanner settings are very different for the two experiments.
• the grafting seems more effective in basic medium (pH 8.3).
• the signal to noise ratio appears acceptable but much less important than for the blades according to the invention obtained by method 1 (7 times less). It is the advantage of having a good fluorescence signal which makes it possible to reduce the intensity of the excitation and detection LASER and thus to reduce the background noise.
• spot diameters are quite wide (greater wettability of the “SURMODICS” blades than that of the blades according to the present invention).
• the slides are prepared according to method 1 and compared to Surmodics slides.
• Three oligonucleotide sequences (the same as in Example 6) and for each sequence two types of modified oligonucleotides (functionalized NH2 and not functionalized) are deposited.
• Each oligonucleotide is deposited for 5 concentration values (2; 5; 10; 20; and 40 ⁇ M).
• Each sample is spotted twice.
• 2 matrices of 2 x 3 x 5 x 2 45 spots of oligonucleotides will have been deposited.
• a double deposit of buffer is carried out in order to eliminate any risk of contamination.
• the scanner setting is 90/95.
• the results of the experiments are given in Table 4 below.
• NH 2 functionalized olignucleotide probes (“oligo NH2”) and non-functionalized were deposited at different concentrations.
• the chip was hybridized with a mixture of target oligonucleotides complementary to those deposited, marked 0 in fluorescence. 2 matrices of 45 spots were deposited.
• the scanner setting is 90/95.
• the experimental results are given in Table 5 below.
• the intensities are lower than for the slides of method 1.
• the Surmodics blades have a fluorescence signal 4 times higher (31,000 against 7200) and a signal-to-noise ratio 5 times higher for a concentration of oligonucleotides 8 times lower (5 ⁇ M).
• the slides according to the invention make it possible to use fewer oligonucleotides than the Surmodics slides for a higher signal intensity.
• the size of the spots on the Surmodics blades is larger than for the blades according to the invention with a fairly narrow distribution.
• biochips obtained by the preparation methods according to the invention prove to be particularly effective compared to other commercial supports, in particular compared to supports of the “Surmodics” type which refer to the subject.

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