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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
• • 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
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• • 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
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• Probe system for performing HLA DR typing, and typing method using said probes.
• the present invention relates to a method for determining the HLA class II genotype of an individual and relates more particularly to the detection of polymorphic HLA DR genes. This process is applicable in particular to HLA typing in transplantation, to medical diagnosis, to legal medicine, etc.
• the HLA (human lymphocyte antigen) system is encoded by the major histocompatibility complex in humans. It constitutes a very important constraint during organ transplants between individuals by making a distinction between the self and the non-self.
• HLA factors are involved in the predisposition to a large number of diseases.
• the antigens of the HLA system have therefore been used in typing methods to determine the characteristics between donors and recipients during organ transplants (FH BACH and JJ VAN ROOD, N. Engl. J. Med., 295, pages 806- 13 (1976)), as well as an individual's predisposition to certain diseases.
• the HLA system is well characterized and consists of a set of more or less polymorphic loci located in an interval of approximately 2 centimorgans (cM) on the short arm of chromosome 6.
• cM centimorgans
• HLA class II antigens represent a determining factor for the success of the transplant, that is to say to avoid graft rejection or the development of graft-host disease
• the polymorphism of the expression products of the HLA D region genes is usually defined by serological techniques based on the analysis by alloantisera of the HLA gene products expressed on the surface of the cells (JJ Van Rood and A. Van Leeuwen ( 1963) J. Clin Invest 42,1382 - JJ Van Rood, A. Van Leeuwen, JJ Koning, AB Van Oud Ablas (1975) Tissue Antigens 5, 73).
• the accuracy and reproducibility depend on the batches of will be available. However, even under the best conditions, a very large number of existing alleles cannot be detected by these serological techniques.
• the limits of the serological analysis result essentially from the absence of monospecific alloantisera, from incomplete discrimination with cross-reactivities between very similar specificities, for example DR3 and DRwl3 or else from an altered expression of the HLA class II molecules. on the surface of cells, for example leukemia cells.
• HLA class II polymorphism is distributed as follows: locus DRB1: 47 alleles, DRB3 locus: 4 alleles, DRB4 locus: 1 allele, DRB5 locus: 4 alleles, DQB1 locus: 17 alleles, DQA1 locus: 13 alleles, DPB1 locus: 21 alleles, DPA1 locus: 4.
• DR4 * 0401-0411 The limits of serological typing can be illustrated by the serological specificity DR4, now subdivided into 11 subtypes (DRB1 * 0401-0411) (see JG Bodmer, SGE Marsh, ED Albert, WF Bodmer, B. Dupont, HA Erlich, B. Mach, WR Mayr, P. Parham, T. Sasazuki, GMT Schreuder, JL Strominger, A. Svejgaard and PI Terasaki (1991) Tissue Antigens 37, 97), identifiable only at the DNA sequence level.
• DRwl3 and DRwl4 by some alloantisera actually contains 10 allelic sequences (DRB1 * 1301-1305 and DRB1 * 1401-1405 (see the publication of Bodmer JG cited above) which, here too, can only be discriminated by genotypic analysis at the DNA sequence level.
• Genotypic analysis is a new approach for analyzing the diversity of the HLA class II system directly at the gene level.
• the genotypic analysis is based on the principle of molecular hybridization and the first approach which was proposed is the so-called "RFLP" technique which consists in fragmenting DNA by using restriction enzymes and in analyzing the size of the fragments.
• RFLP restriction enzymes
• Specific DNA generated by these enzymes see CT Wake, EO Long and B. Mach (1982) Nature 300, 372 - J. Blhme, M; Andersson, G. Andersson, E. Miller, PA Peterson and L. Rask (1985 ) j. immunol 135, 2149 - JL Bidwell, EA Bidwell, DA Savage, D. Middleton, PT Klouda and BA Bradley (1988) Transplantation 45, 640).
• RFLP analysis only recognizes some of the allelic differences undetectable by serology, and this technique still has limitations. Indeed, an allele carrying a different sequence is identifiable only if the different nucleotide is in the recognition site of the restriction enzyme used in the analysis and therefore a large number of HLA class II alleles will not be recognized by this analysis. In addition, the RFLP analysis rarely highlights a modification in a coding sequence, and does not provide information on the exact nature of the modification. Finally, this technique is long and cumbersome because it involves using relatively large quantities of nucleic acids which must be digested with several restriction enzymes, electrophoresis and transfers on filters.
• oligonucleotide typing is the so-called "oligonucleotide typing" method. Thanks to the knowledge of the DNA sequences of HLA class II genes and in particular of the DR ⁇ genes, which are by far the most polymorphic, it is possible to use oligonucleotides which are specific for a given location of the gene sequence as tracers for the analysis of polymorphism by hybridization. These oligonucleotides are chosen so as to be as informative as possible and to allow the identification of the different alleles, on the basis of their sequence differences. In practice, any difference in sequence, even of a single nucleotide, can be detected.
• oligonucleotide typing technique can be applied to DNA as well, as described in the publication by Angelini et al., Proc. Natl. Acad. Sci. USA Vol. 83, pages 4489 - 4493 (1986), than to the RNA (see C. Ucla, J.J. Van Rood, J. Gorski and B. Mach (1987) J. Clin. Invest. 80, 1155).
• This new approach is based on the principle of molecular hybridization by using the characteristic properties of nucleic acids which are the possibilities of interacting with a complementary sequence via hydrogen bonds and thus of forming a stable hybrid, according to the laws.
• known pairings i.e. A-T, GC for DNA and A-U, GC for RNA.
• synthetic oligonucleotides corresponding to DNA or RNA sequences of known alleles can be used as probes to identify, in a sample, a nucleic sequence called target, containing a sequence complementary to that of the probe.
• the labeling of the hybrid formed between the target and the probe allows the detection and quantification of the target in the sample.
• This labeling is carried out by any known marker, such as an enzymatic, chemical or radioactive marker.
• any known marker such as an enzymatic, chemical or radioactive marker.
• the first application of typing by oligonucleotide for HLA class II was presented by Angelini et al. in the publication cited above, using the so-called "SOUTHERN" technique according to which the target DNA is deposited on a nylon membrane and the detection is carried out using a labeled oligonucleoditic probe. The technique was then applied to the detection of HLA class II alleles not identifiable by serology. routine (see JM Tiercy, J. Gorski, M. Jeannet and B. Mach (1988) Proc. Natl. Acad. Sci. USA 85, 198 - JM Tiercy, J.
• cell typing requires the detection of point mutations in the genome and involves the development of probes sensitive enough to detect and differentiate sequences homologous to a nucleotide closely, and it was found necessary to use short probes, generally of less than 30 nucleotides, which give the test a high specificity, while retaining a good sensitivity.
• short probes generally of less than 30 nucleotides, which give the test a high specificity, while retaining a good sensitivity.
• the use of short oligonucleotides provides a wide range of selectivity.
• the method of the invention can be implemented using a set of nucleotide probes chosen so as to allow typing with a minimum number of probes.
• This set of probes has the particular advantage of making it possible to operate at a single temperature, in particular at 37 ° C. (although it is possible to operate at another temperature, as will be seen in the experimental part below) after). Such a set of probes is also part of the invention.
• the set of probes of the invention which will be defined below can be used in the form of detection probes (labeled with a usual tracer agent) in techniques of the Southern type, or, preferably, in the form of capture probes (sandwich or reverse dot blot technique) immobilized on a solid support, either by passive fixation (adsorption) directly or via a ligand), such as a hydrophobic ligand (see for example the patent application European No. 0 405 913), either by the establishment of at least one covalent bond which can be made here again directly or by the intermediary of a ligand capable of fixing covalently on the support (see for example the PCT patent application No. WO 88/01302).
• the immobilization of the probes can be carried out either using known methods or using other methods which will be described below.
• probes of the invention will be described mainly in the form of nucleotide sequences. It is obvious to a person skilled in the art that, even for probes intended to detect point mutations, at a given temperature, it is possible to envisage the use of probes of variable length (number of nucleotides), in to a certain extent, thanks in particular to the use of solutions, buffers which more or less promote the stability of the hybridization complexes.
• the probes of the invention are therefore defined by a sequence which may generally be considered as maximum (in particular if it is desired to work at a relatively low temperature, for example at 37 ° C.), with in addition the indication of a minimum sequence which will still be usable at said temperature and which will be sensitive to a even a one-time mutation.
• nucleotide probes which can be used in oligonucleotide typing techniques to perform HLA DR typing, these probes being chosen from the following:
• the probe 101 makes it possible to identify the type DRB1 * 01.
• the probe 102 makes it possible to identify the type DRB1 * 02.
• the probe 103 makes it possible to identify the type DRB4 * 01 and the probe 104 is used in the identification of the type DRB1 * 1305.
• the invention also relates to a set of nucleotide probes, or to an HLA type kit, comprising at least one probe chosen from among probes 101 to 104.
• the subject of the invention is also such a set of probes further containing at least one probe chosen from the following:
• the probe 111 is used in its complete form, including the two T not underlined at the 3 'end. It has the same specificity as probe 45.
• Probe 115 is preferably used in the form of the underlined sequence increased by the two A's not underlined at the 5 'end. It has the same specificity as probe 28.
• the probes 105 to 110, 112 to 114, 116 and 117 are preferably used in the form of the probes having in the experimental part below the reference numbers 43, 9, 10, 14, 17, 44, 46, 48, 47, 24 and 27.
• the invention relates in particular to a set of probes as defined above, characterized in that it further comprises at least one of the following probes (the underlined part corresponding to the optimum sequence): - GAGGAGGACTTGCGCT - TACGGGGCTGTGGAG - GGAGCTGCGTAAGT
• the specific probes indicated above can be used in particular as capture or detection probes. They are preferably used in the form of capture probes immobilized or immobilizable on a solid support.
• the invention also relates to a method for determining the HLA DR ⁇ typing of an individual, according to the usual typing techniques by oligonucleotides, characterized in that one uses as capture or detection probes, either sequentially or simultaneously, at least part of the probes from the set of probes as defined above:
• the process of the invention therefore essentially comprises the stages consisting:
• the information collected is then used to determine the typing according to a pre-established typing plan, taking into account the probes used and knowledge of the HLA DR types and / or associated associated types.
• This work is simplified by the use of a typing plan, that is to say in practice a table giving directly the types and / or sub-types according to the positive responses (hybridization (s)) observed.
• a typing plan that is to say in practice a table giving directly the types and / or sub-types according to the positive responses (hybridization (s)) observed.
• s hybridization
• the invention relates in particular to a method as defined above, in which said probes are used as capture probes, this process being able to be characterized in that it comprises the steps consisting in: a) immobilizing each capture probe on a solid support, b) bringing each immobilized capture probe into contact with a liquid medium containing at least one target nucleic acid fragment, under predetermined conditions allowing hybridization if the sequence complementary to that of the probe is present in the target and c) detecting the presence of possibly formed hybrids.
• the probes of the invention make it possible to detect both target fragments of RNA and of DNA.
• all suitable probes in particular one of the probes described below in Example 5.
• the capture probe When the capture probe is very short, that is to say less than 20 nucleotides and in particular less than 17 nucleotides, it becomes necessary to use means making it possible to improve the fixation of the probe on a solid support .
• the attachment of the probe to the support is then carried out in the form of a derivative resulting from the covalent coupling of the probe with a ligand facilitating the attachment to the solid support.
• the ligand which may comprise a hydrophobic part, is in particular a ligand comprising at least one polar functional group, for exercises an amino group.
• the functional group can be used to fix the probe on the solid support by establishing a covalent bond. When the polar functional group does not react with the support, it improves the fixation by adsorption on the support, even if the support is hydrophobic.
• the ligand is for example chosen from proteins and compounds as represented respectively by formulas I and II below:
• Z represents an alkyl or alkenyl radical of 2 to 12 carbon atoms, linear or branched, unsubstituted or substituted by one or more groups chosen from hydroxy and / or amino groups, and m + represents in particular an alkali or ammonium ion.
• This ligand is preferably coupled to the 5 'end of the nucleotide sequence of the capture probe
• This ligand is preferably coupled to the 3 'end of the nucleotide sequence of the capture probe.
• the ligand is a protein, for example an albumin is chosen, preferably bovine serum albumin, which can be coupled to the 5 ′ or 3 * end of the nucleotide sequence of the capture probe.
• the support of the present invention can be any support making it possible to immobilize a nucleotide sequence or a derivative according to the invention, either by passive adsorption or by covalence.
• the supports can be made of any material usually used such as nitrocellulose, nylon, paper, or preferably, a hydrophobic material such as a styrene polymer or a styrene-based copolymer comprising at least 10% by weight of styrene units. .
• the solid support according to the invention can be without limitation in the form of a microtiter plate, a sheet, a tube, a cone, wells or the like.
• a sample containing a nucleic acid is obtained from an individual whose HLA DR genotype is to be determined.
• Any type of tissue containing HLA DR nucleic acid can be used in the context of the present invention. It is thus possible to use nucleic acid fragments (DNA or RNA) obtained after cutting by chemical, enzymatic or analogous means of the nucleic acid present in the sample of the individual.
• the incorporation of a prior step of amplification of the target DNA or RNA can facilitate the oligonucleotide typing method of the present invention.
• the principle of the analysis of the HLA polymorphism by hybridization of sequence-specific oligonucleotides remains the same, but a selective amplification step allows enrichment in target sequences, which simplifies the technique (RK Saiki, TL Bugawan, GT Horn, KB Mullis and HA Erlich (1986) Nature 324, 163 - JM Tiercy, M. Jeannet and B. Mach (1990) Eur. J. Immunol. 20, 237).
• Amplification can be obtained either from DNA or from RNA. It is obvious to a person skilled in the art that the amplification of the HLA DR target sequences in a sample can be accomplished by any known method which makes it possible to obtain sufficient amplification so that the target sequence can be detected by hybridization of nucleic acid to a probe.
• the nucleic acid in the sample will be DNA, most often genomic DNA.
• the present invention can also be implemented with other nucleic acids such as messenger RNA or cloned DNA, and the nucleic acid in the sample of the individual can be in the form of 'a single strand or a double strand.
• the acid nucleic acid is in the double-stranded form, it is necessary to practice a denaturation step to obtain a single-stranded nucleic acid.
• the probes used in the present invention are sequence specific oligonucleotides (OSS) which, under appropriate conditions, can specifically bind to their complementary sequences. If a particular probe can be used to identify only one allele, then the probe is called OSA, that is, an allele-specific oligonucleotide. A single probe may not be able to identify a specific DR ⁇ allele on its own due to the different nature between various DR ⁇ alleles.
• OSS sequence specific oligonucleotides
• the identity of an allele is deduced from a binding model of a set of probes, each individual probe of the set being specific for different parts of the HLA DR gene. Thanks to the choice of multiple probes corresponding to the DNA sequences of the known alleles, the specificity of the oligonucleotide typing method of the present invention makes it possible to identify all the alleles of the DRB1, DRB3 and DRB5 loci. Of course, the method of the present invention could be used to identify the alleles of other extremely polymorphic loci such as DQB1 and DPB1.
• the probes are chosen to be complementary to specific sequences localized in this region. In the event that new alleles are discovered, these are immediately listed in a HLA class II sequence register, which makes it possible to update the collection of informative tracers and therefore to adapt the methodology to the detection of any new allele.
• DR of patients on the waiting list for a kidney transplant or the typing of potential kidney donors the DR typing of leukemia patients for whom a bone marrow transplant is envisaged, as well as members of their family or unrelated potential donors , large-scale DR typing for the constitution of registers of voluntary marrow donors, to determine associations between diseases and the HLA system, for example in the case of insulin-dependent diabetes, for applications in predictive medicine or for research paternity and other judicial identifications.
• genotypic characteristics refers to the set of genotypic characteristics of an individual as opposed to the "phenotype” which are the characterizations of an individual as they emerge from the analysis of gene expression products and in particular proteins .
• oligonucleotide designates the primers, the probes, the fragments of nucleic acids to be detected, etc.
• the oligonucleotides can be prepared by any suitable known method.
• nucleotide probe represents a fragment of natural DNA or RNA, or a natural or synthetic oligonucleotide, or a fragment of synthetic DNA or RNA, unmodified or comprising one or more modified bases such as l 'inosine (symbolized by the letter I), methyl-5-deoxycytidine, dimethylamino-5- deoxyuridine, deoxyuridine, diamino-2, 6-purine, bromo-5-deoxyuridine or any other modified base allowing hybridization.
• modified bases such as l 'inosine (symbolized by the letter I), methyl-5-deoxycytidine, dimethylamino-5- deoxyuridine, deoxyuridine, diamino-2, 6-purine, bromo-5-deoxyuridine or any other modified base allowing hybridization.
• these optimal sequences can be extended at the 3 'and / or 5' end by at least one base.
• certain bases which can be optionally added have been indicated in parentheses as can be seen for example on reading the description above.
• ligands used in the present invention can be commercially available compounds such as in Table 1 below:
• An oligonucleotide is synthesized on an automatic 381 A device from the company APPLIED BIOSYSTEMS using phosphoramidite chemistry according to the manufacturer's protocol.
• the ligand carries a dimethoxytrityl protecting group, such as for compound d, it is necessary to carry out an additional step of deprotection of the trityl group with trichloroacetic acid at the end of the synthesis.
• the oligonucleotide is dried under vacuum and taken up in 1 ml of dH 2 O.
• an additional step of cleavage of the monomethoxytrityl group is carried out according to the manufacturer's protocol (CLONTECH and GLEN RESEARCH respectively) after deprotection.
• the automatic synthesis starts with the silica grafted with the ligand according to the standard protocol.
• the ligand and oligonucleotide coupling is carried out via the 3 'end of the latter.
• the oligonucleotides modified at their 5 ′ or 3 ′ ends are purified by high pressure liquid chromatography (HPLC) in reverse phase on a Brownlee RP18 column (10 mm - 25 cm).
• buffers A and B are as follows.
• Buffer B 50% Buffer A + 50% CH3CN.
• the conjugate is purified by ion exchange in HPLC on an AX300 column (BROWNLEE 4.6 x 100 mm) with an NaCl gradient (Table 1).
• the conjugate peak is dialyzed against water (2 x 1 liter) concentrated under empty, taken up in 1 ml H 2 O and stored at - 20 ° C.
• buffers A 'and B' are as follows:
• a ' 20 mM sodium phosphate, pH 7.00; 20% CH 3 CN.
• the alignments of amino acids of the various alleles of the DR Beta gene are shown in Table 2 in order to define the positions of the mutated amino acids with respect to the chosen consensus sequence (called "DR CONS").
• These mutations correspond to non-silent mutations at the DNA level, ie mutations which will induce an amino acid change.
• amino acids are encoded at the DNA level by base triplets.
• a mutation in the third position will generally not cause an amino acid change.
• the change of the second base will quite often induce a change of amino acid.
• a mutation on the first base will always lead to a modification of the amino acid.
• oligonucleotides were synthesized carrying either a ligand, as described in example 1 and which are summarized in table 4, or coupled to ASB, as described in example 2 and which are summarized in
• Tr represents the retention time in minutes (min) of the oligonucleotide in HPLC under the conditions described in Example 1 (BROWNLEE RP 18 column (4.6 mm-25 cm) flow rate 1 ml / min).
• the letter I in sequences 33, 34 and 34 bis represents inosine.
• Tr represents the retention time in minutes (min) of the oligonucleotide coupled to ASB in HPLC under the conditions described in Example 2.
• (1M) means that buffer B contains 1M NaCl.
• (2M) means that buffer B contains 2M NaCl.
• the oligonucleotide is quantified in picomoles by UV spectrometry by measuring the absorbance at 260 nm according to the APPLIED BIOSYSTEMS protocol.
• the ASB is assayed by the BRADFORD method (BRADFORD M.M., Anal.Biochem., 72.248 (1976)) in picomoles.
• the oligo / ASB ratio is the ratio of these 2 values.
• capture oligonucleotides have been defined which can be synthesized, without ligand, with a ligand or else can be coupled, for example, to ASB.
• the choice of the sequences of the oligonucleotides synthesized takes account of the alignment of the DNA sequences of the various alleles described in Table 3 of Example 3.
• the selected oligonucleotide probes, used for example as capture probes, make it possible to carry out a Typing plan as described in Table 6. It is obvious to those skilled in the art that other typing plans can be defined with other oligonucleotides.
• the sign + signifies that the subtype of the line considered in table 6 gives a hybridization with the probe of the corresponding column.
• Example 2 the activated oligonucleotide which is dried under vacuum is taken up in 1.25 10 -7 mole (5 mg) of horseradish peroxidase.
• the purification protocol is identical: the conjugate is stored at -20 ° C in a 50 mM tris HC1 buffer, pH 7.0, 40% glycerol.
• Table 7 summarizes the different conjugates used for HLA DR detection.
• Tr represents the retention time in minutes (min) of the oligonucleotide coupled to horseradish peroxidase (HRP) in HPLC under the conditions described in Example 2.
• (2M) means that buffer B contains 2M NaCl.
• the oligonucleotide is quantified in picomoles by UV spectrometry by measuring the absorbance at 260 nm according to the APPLIED BIOSYSTEMS protocol.
• Horseradish peroxidase (HRP) is dosed by UV at 402 nm in picomoles according to ATOR MA, J. Biol. Chem. , 31, 14954 (1987).
• the oligo / HRP ratio is the ratio of these 2 values.
• (GT) means that there is an equimolar mixture of the 2 bases G and T at this position.
• EXAMPLE 6 preparation of genetic material
• the extraction of nucleic acids from whole blood is carried out in an Applied Biosystems apparatus according to the following protocol: 2 to 6 ml of whole blood are taken up in TE buffer (10 mM Tris-HCl pH 8.00, 1 mM EDTA) (sufficient quantity for 6 ml) and are placed in a 30 ml extraction vial. A proteinase K solution (840 units in 20 mM Tris-HCl pH 8.5) is added. The whole is incubated for 1 hour at 55 ° C. with shaking. The excess protein present is eliminated by 2 simultaneous extractions (8.5 ml) with a mixture of phenol chloroform. The whole is stirred for 20 minutes at 60 ° C. After elimination of the organic phase, a new phenolic extraction is carried out.
• TE buffer 10 mM Tris-HCl pH 8.00, 1 mM EDTA
• a proteinase K solution 840 units in 20 mM Tris-HCl pH 8.5
• the whole is incubated for 1 hour at
• the excess phenol is removed by extraction with chloroform (9.5 ml), 10 minutes at 37 ° C.
• the DNA contained in the aqueous phase is precipitated by adding 0.5 ml of 3M sodium acetate pH 5.5 and 13.5 ml of isopropanol and then recovered on a filter. The DNA is then taken up in 1 ml of distilled water, then assayed spectrophotometrically at 260 nm.
• PCR polymerase chain reaction technique
• Thermocycler Perkin Elmer Ketus
• 35 temperature cycles will be carried out: - 0.5 minute denaturation at 95 ° C
• the primers used have the following sequence:
• primer 1 5'-CCGGATCCTTCGTGTCCCCACAGCACG-3 '
• a well of a polystyrene microtiter plate (Nunc 439454) are deposited 100 ⁇ l of a solution of an oligonucleotide for capturing a specific DR given at a concentration of 0.15 ⁇ M in PBS 3 x (0 , 45 M NaCl, 0.15 M sodium phosphate pH 7.0). There are as many wells filled as necessary for typing.
• the capture probe which is used as a positive control is present on all the alleles known to date and has the following sequence:
• the plate is washed 3 times with 300 ⁇ l of PBS Tween (0.15 M NaCl, 0.05 M sodium phosphate, pH 7.0; 0.5% Tween 20 (Merck
• the amplification product (100 ⁇ l) as described in Example 7 is denatured with 10 ⁇ l of 2N NaOH for 5 minutes with stirring at room temperature. 10 ⁇ l of 2N acetic acid then a volume of PEG buffer (0.1 M sodium phosphate, pH 7.0, 0.5 M NaCl, 0.65% Tween 20, salmon sperm DNA (Sigma D 9156)
• nx 50 ⁇ l 0.14 mg / ml, PEG 4,000 (Merck 8O7490) 2%) equivalent to nx 50 ⁇ l (n being the number of capture probes necessary for typing). are added successively to this solution. 50 ⁇ l of this solution are distributed per well followed by 50 ⁇ l of the detection probe (oligonucleotide peroxidase conjugate) at a concentration of 15 nM in the PEG buffer. The plate is incubated for 1 h at 37 ° C. and washed with 3 x 300 ⁇ l of PBS Tween.
• OPD substrate orthophenylenediamine Cambridge Medical Biotechnology ref / 456
• OPD buffer 0.05 M citric acid, 0.1 M Na 2 HPO 4 , pH 4.93
• H 2 O 2 is added immediately at 30 volumes in 1/1000, are added per well.
• the enzymatic activity is blocked by 100 ⁇ l of 1N H 2 SO 4 and the reading is carried out on Axia Microreader (bioMérieux) at 492 nm.
• Axia Microreader bioMérieux
• DNAs, prepared according to the method described in Example 6, are amplified according to the method described in Example 7.
• GATACTTCTATCACC 3' oligonucleotide of identical sequence but without ligand (referenced 545 nu)
• TGGACAACTACTG 3 ' oligonucleotide of identical sequence but without ligand (referenced 546 nu)
• the typing protocol conforms to the general protocol described in Example 8.
• the probes D1 and D2 (table 7) are used in a 50% - 50% mixture as detection probes.
• the typing protocol conforms to the general protocol described in Example 8.
• the probes D1 and D2 (table 7) are used in a 50% - 50% mixture as detection probes.
• the typing protocol includes the capture probes summarized in Table 9 below:
• the method described allows us to unambiguously type the 24 DNAs tested.
• the preferred hybridization temperature for the HLA DR typing described in the present invention is 37 ° C.
• Example 10 The following example is identical to Example 10 with the exception of the hybridization temperature which was changed from 37 ° C to 45 ° C. Typing is performed on 11 DNAs.
• the preferred hybridization temperature is 37 ° C. and the preferred hybridization buffer is the PEG buffer, but as the results of Examples 11 and 12 show, it can be seen that it is possible to vary both the hybridization temperature and hybridization buffer.
• the process of the present invention combines the following practical advantages: optimal specificity with possible discrimination of all the alleles,
• capture probes were prepared corresponding to the oligonucleotides designated by the reference numbers 101, 102, 103, 104, 115 and 111.
• capture probes of the invention can be used with the following detection probes:
• the preferred hybridization temperature is 37 ° C. and the preferred hybridization buffer is the PEG buffer, but as the results of Examples 11 and 12 show, it can be seen that it is possible to vary both the hybridization temperature and hybridization buffer.
• the process of the present invention combines the following practical advantages: optimal specificity with possible discrimination of all the alleles,
• capture probes were prepared corresponding to the oligonucleotides designated by the reference numbers 101, 102, 103, 104, 115 and 111.
• capture probes of the invention can be used with the following detection probes:

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• 1995
  • 1995-06-07 US US08/485,133 patent/US5976789A/en not_active Expired - Fee Related
• 1996
  • 1996-06-03 AU AU62289/96A patent/AU717296B2/en not_active Ceased
  • 1996-06-03 JP JP50018397A patent/JP3384806B2/ja not_active Expired - Fee Related
  • 1996-06-03 NZ NZ311058A patent/NZ311058A/xx unknown
  • 1996-06-03 IL IL12015896A patent/IL120158A/xx not_active IP Right Cessation
  • 1996-06-03 CA CA002196921A patent/CA2196921A1/fr not_active Abandoned
  • 1996-06-03 WO PCT/FR1996/000836 patent/WO1996040989A1/fr not_active Ceased
  • 1996-06-03 KR KR1019970700904A patent/KR970704891A/ko not_active Withdrawn
  • 1996-06-03 EP EP96920893A patent/EP0772694A1/de not_active Withdrawn
  • 1996-06-03 CN CN96190783A patent/CN1159211A/zh active Pending
  • 1996-06-03 PL PL96318536A patent/PL184298B1/pl unknown
• 1997
  • 1997-02-05 NO NO970518A patent/NO970518L/no not_active Application Discontinuation

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