Classifications

• • 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
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • 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
• • 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
  • C12Q2600/16—Primer sets for multiplex assays

Definitions

• the present invention relates to an in vitro assay and method characterized by the simultaneous genotyping of Calcitonin Receptor and Collagen I Al genes. More specifically, in preferred embodiments the assay further allows genotyping of Vitamin D Receptor and Estrogen Receptor and, in still more preferred embodiments, the genotyping also of Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes in ciinicai samples.
• the present invention relates to genotyping of polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al, and of Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T.
• the present invention relates to an assay and method comprising independent genotyping of the group of genes Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al genes; and of the group of genes Osteoprotegerin, LRP5, CYP 17, CYP19 and MTHFR.
• the invention also relates to diagnostic kits comprising above-mentioned methods and their use for diagnosing predisposition to osteoporosis and other bone pathological conditions, as well as to treatment responsiveness.
• Osteoporosis is a disorder in which loss of bone strength leads to fragility fractures (Raisz, 2005, J. Clin. Invest 115(12):3318-25).
• the fundamental pathogenetic mechanisms underlying this disorder include: (a) failure to achieve a skeleton of optimal strength during growth and development; (b) excessive bone resorption resulting in loss of bone mass and disruption of architecture; and (c) failure to replace lost bone due to defects in bone formation.
• Estrogen deficiency is known to play a critical role in the development of osteoporosis, while calcium and vitamin D deficiencies and secondary hyperparathyroidism also contribute.
• Certain clinical trials have assessed that there is a relationship between the presence of certain genotypes of Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor, Collagen I Al, Osteoprotegerin (OPG), LRP5, Cytochrome P450cl7alpha (CYP17), Aromatase (CYP19) and Methylenetetrahydrofolate reductase (MTHFR) genes, and the predisposition to bone diseases such as osteoporosis, as well as to the effectiveness of treatment.
• OPG Osteoprotegerin
• LRP5 Cytochrome P450cl7alpha
• CYP19 Aromatase
• MTHFR Methylenetetrahydrofolate reductase
• VDR vitamin D receptor
• VDR genotypes a prospective baseline-controlled clinical trial was carried out wherein sixty-eight Italian osteoporotic women were enrolled and treated with alendronate at a dose of 10 mg/day for 12 months. (Palomba et al., 2003, Clin
• polymorphisms in genes involved in synthesis of sex steroids such as polymorphism CYP19 C(1558)-T of the CYP19 gene encoding aromatase, are associated with the magnitude of bone gain in response to hormone replacement therapy (Tofteng et al., 2004, Calcif Tissue Int. 74(i):25-34).
• MTHFR methylenetetrahydrofolate reductase
• Braga et al., 2002, Calcif Tissue Int. 70(6):457-62 displays a method for co-detection of Calcitonin Receptor, COLlAl and Vitamin D Receptor which consists of the individual gene amplification reactions, followed by restriction enzyme analysis.
• Bandres et al., 2005, J Endocrinol Invest. 28(4):3I2-21 displays detection of COLlAl, Vitamin D Receptor, Estrogen Receptor and Calcitonin Receptor genes, also by means of DNA extraction, followed by individual PCR amplification and restriction enzyme analysis.
• CoIlAl shows correlation with the prevalence of osteoporotic fractures in a postmenopausal Spanish women cohort, that Calcitonin Receptor and Vitamin D Receptor show correlation with BMD, while Estrogen Receptor does not shown any such correlation in the same study.
• (1) a method in which a gene fragment containing the polymorphism site is isolated and the base sequence of the site is determined or the polymorphism site is directly detected by use of a specific probe or primer, (2) a method in which a difference in higher level structure of a gene fragment containing the polymorphism site is used to distinguish polymorphisms based on electrophoretic mobility, and (3) a method in which the possibility of cleavage at the polymorphism site with a restriction enzyme is used to distinguish polymorphisms based on electrophoretic mobility.
• a sequencing method a sequence specific oligonucleotide probe (SSOP) method, a mutant allele-specific amplification (MASA) method, etc.
• SSOP sequence specific oligonucleotide probe
• MASA mutant allele-specific amplification
• examples are provided wherein a VDR gene fragment, an ER gene fragment, and an ApoE gene fragment are separately PCR amplified using the genome DNA as a template and specific primer pairs for each gene fragment.
• Each amplification reaction mixture is then treated with restriction enzymes with specific cleavage sites at the polymorphism site, followed by agarose gel electrophoresis. Polymorphisms of the VDR, ER, and ApoE genes are discriminated based on the band patterns.
• polymorphism identification is carried out by incubation of the amplified products with polyT-added probes coated at separate sites on a single sheet of nylon membrane.
• Tempfer et al. 2004, Fertil Steril, 82(1): 132-7, displays an analysis of some SNPs associated with risks and benefits of estrogen therapy and hormone therapy, wherein genomic regions containing the specific SNPs are amplified through multiplex PCR. These regions include CYP17, CYP19, estrogen receptor, MTHFR and Vitamin D Receptor, among others.
• the PCR products After amplification through multiplex PCR, the PCR products have then to be purified, and subjected to primer extension combined with fluorophore incorporation.
• the fluorophore- labeled single strands are hybridized to an ailele-specific oiigo array bound to a glass support.
• aspects of the invention allow the provision of a reliable and high through-put assay that allows the simultaneous detection of Calcitonin Receptor and Collagen I Al in a sample.
• the solution is based on the multiplex amplification reaction of the Calcitonin Receptor and Collagen I Al genes followed by hybridisation of the amplified fragments with ailele-specific probes.
• a first aspect of the present invention relates to an assay comprising genotyping Calcitonin Receptor and Collagen I Al in a sample, characterized in that a multiplex amplification reaction of the Calcitonin Receptor and Collagen I Al genes is performed on the sample.
• the assay further comprises multiplex amplification of the Vitamin D Receptor and/or the Estrogen Receptor genes; preferably in the same multiplex amplification as that of Calcitonin Receptor and Collagen I Al genes.
• the assay further to the multiplex amplification reaction of the group of genes i), comprising Calcitonin Receptor and Collagen I Al, and optionally, Vitamin D Receptor and/or Estrogen Receptor, comprises an additional multiplex amplification reaction of one or more genes being selected from the group of genes ii) comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR.
• genotypes that are determined correspond to polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al, and of Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T.
• Genbank accession numbers of the various genes are as follows.
• Calcitonin Receptor NM_001742; Vitamin D Receptor: AY342401; Estrogen Receptor: AY425004; Collagen I Al: AF017178; Osteoprotegerin: AB008822; LRP5: NM_002335; CYP17: NM_000102; CYP19: NMJB1226; MTHFR: NM_005957.
• the polymorphisms are known to those of skill in the art; selected probes useful for detecting the polymorphisms are set out in the tables disclosed herein.
• Second and third aspects of the present invention relate to a solid support comprising a plurality of specific probes for polymorphisms of Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al, Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR, as well as to a reaction vessel comprising the solid support.
• a fourth aspect of the present invention relates to a diagnostic kit comprising above-mentioned assay and yet a fifth aspect relates to the use of the kit for diagnosing predisposition to pathological bone conditions, as well as for anticipating response to treatment. Additional aspects of the present invention relate to specific probes and amplification primers.
• the present invention further provides a method for simultaneous genotyping of Calcitonin Receptor, Collagen I Al, as well as Vitamin D Receptor and/or Estrogen Receptor, also allowing the possibility to further genotype Osteoprotegerin, LRP5, CYPl 7, CYP 19 and MTHFR genes.
• the assay of the present invention makes possible above-mentioned genotyping without the drawbacks of the methods of the state of the art, wherein individual amplification followed by genotyping of each gene of interest is required.
• the use of such the assay of the present invention allows the diagnosis of individual predisposition to osteoporosis and to other bone pathologies, as well as the anticipation of responsiveness to treatment.
• the genotyping assay comprises: performing a nucleic acid amplification reaction on a sample, obtaining single stranded oligonucleotides from the amplification products, allowing single stranded oligonucleotides to hybridise with alleie-specific probes corresponding to the genes of interest, which are immobilised on a solid support, and detecting hybridised oligonucleotides.
• the amplification reaction is preferably PCR
• Single stranded oligonucleotides may be obtained by denaturing any double stranded oligonucleotides present, for example by heating, Allele-specific probes are preferably selected to specifically bind to the single-stranded oligonucleotides from amplification products under the same hybridisation conditions for ail probes.
• the selected probe must hybridize with the region of the amplified fragment in which the polymorphism is located, being able to distinguish between the two genotypes of a polymorphism.
• the probes are conveniently 15 to 30 nt in length, more preferably 15 to 23 nt. In a more preferred embodiment, the probe length is 15 to 20 nt. And in the particular case of polymorphism COLlAl SpI of gene Collagen I Al, the most suitable probe length is 19 nt. All probes need not be the same length.
• Preferred probes are selected from the group comprising SEQ ID NO 11 to SEQ ID NO 29, SEQ ID NO 44 to SEQ ID NO 211 and SEQ ID NO 212-222. The probes may be duplicated on the solid support, to provide for multiple detection locations for redundancy.
• One or more control sequences may also be immobilised to the solid support; for example, a probe which does not hybridise to the target sequence from any allele of the selected polymorphisms.
• the sample and the solid support are contained within a reaction vessel; for example, that described in US2005064469.
• M probes for location reference (Marker- 1 [GCA GTA TAA GAT TAT TGA TGC CGG AAC]; Marker-2 [GTC AAA ACC TGG GAT AGT AGT TTT ACC]).
• Figure 2 Photograph of an array tube used in the present invention. The arrangement of probes corresponds to the one displayed in Figure 1.
• Figure 3 Details corresponding to Example 5.
• Panel A Multiplex PCR conditions.
• Panel B Multiplex PCR cycling profile.
• Panel C Visualization of the amplification products by means of agarose gei electrophoresis (2,5%),
• Figure 4 Details corresponding to Example 6.
• Panel A Probes designed for hybridisation with genotypes S or s of gene Collagen I Al.
• Panel B Results obtained in an array tube assay with the probes of Panel A and the seven samples: op42 ss, op90 ss, op61 ss, op87 SS, op55 Ss, op72 Ss, op58 Ss (Values in Arbitrary Units).
• FIG. 5 Details corresponding to Example 7.
• Panel A Probes designed for hybridisation with the amplified fragment of gene Collagen I Al, in a region common to genotypes S and s.
• Panel B Results obtained in an array tube assay with the probes of Panel A and the seven samples: op42 ss, op90 ss, op61 ss, op87 SS, op55 Ss, op72 Ss, op58 Ss (Values in Arbitrary Units).
• Figure 6 Comparison of SEQ ID No 217 and SEQ ID No 15 in detecting Collagen I Al gene.
• Figure 7 Comparison of SEQ ID No 14, 218, and 219 in detecting Estrogen Receptor gene.
• - Amplification Primers Nucleic acids or nucleic acid analogs that bind to and allow amplification of one or more target sequences.
• Multiplex amplification reaction reaction that allows amplification of two or more nucleic acid sequences if present.
• Nucleic acid with ability to specifically bind a target nucleic acid sequence This includes DNA, RNA, PNA, and so forth.
• a reaction vessel which has a shape and size typical of a laboratory reaction vessel (for example, a 1.5 ml Eppendorf tube) with a microarray arranged therein in which microarray based tests can be carried out.
• a set of reaction vessels usually 8, or multiples of 8, each with a microarray arranged therein, in which microarray based tests can be carried OUt.
• the present invention relates to a method and a kit for the simultaneous genotyping of Calcitonin Receptor and Collagen I Al genes. More specifically, the assay further allows genotyping of Vitamin D Receptor and Estrogen Receptor and, in a more preferred embodiment, additionally the genotyping of Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes in clinical samples.
• the gene regions to be amplified comprise the polymorphisms: i) AIuI of the Calcitonin Receptor; Fokl and/or Bsml of the Vitamin D Receptor; Xbal and/or PvuII of the Estrogen Receptor; COLlAl SpI of Collagen I Al; ii) Osteoprotegerin OPG 209 G>A, OPG 245 T>G and/or OPG163 A>G; LRP5 C171346A, C135242T and/or G138351A; CYP17 T(27)-C; CYP19 C(1558)- T; MTHFR C677T.
• these two groups are amplified in two separate multiplex reactions.
• multiplexes involving genes from both groups simultaneously are less effective. There may be two sequential multiplexes carried out on the same sample; or the sample may be split and separate multiplexes carried out on each portion of the sample.
• Amplification of sample DNA DNA obtained from clinical samples is amplified, preferably by PCR. Although PCR is the preferred amplification method, amplification of target sequences in a sample may be accomplished by any other method known in the art (ligase chain reaction, transcription-based amplification system, strand displacement amplification, etc).
• the group of genes i) comprising Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al is amplified in a first reaction tube, while the group of genes ii) comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR is amplified in a second reaction tube.
• Amplification primers may preferably be selected from those displayed in Tables 1 and 3, respectively.
• Nucleotides of the sequences of Tables 1 to 5 are designated as follows: G for Guanine, A for Adenine, T for Thymine, C for Cytosine, M for nucleotides A or C, and Y for nucleotides T or C.
• the nucleotides as used in the present invention may be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridization characteristics.
• a label is introduced in the amplified DNA during its amplification to allow further detection, preferably a label that provides a signal that may be detected by colorimetric methods.
• At least one of the primers used is labelled at the 5' end with biotin.
• any other kind of label known in the art may be used (e. g. digoxigenin).
• labelling of amplified DNA may be alternatively achieved by adding modified nucleotides bearing a label (e. g. biotinylated or digoxigenin dUTP derivatives) in the amplification mixture. Radioactive labels may be used, or fluorophores, in certain embodiments.
• Hybridization amplified DNA from step (i) is denatured (e.g. by heat). Other ways to prepare single stranded DNA after amplification may be used as well; for example, chemical means.
• the single stranded DNA is then incubated with a plurality of target-specific probes provided on a solid support. At least one, but preferably more than one probe with ability to hybridise with each target sequence, are provided on the solid support.
• the solid support is not necessary, and the single stranded DNA may be incubated with target-specific probes provided in solution; however, it is preferred that a solid support be used.
• the solid support is located within a reaction vessel suitable for containing the sample.
• the probes of the present invention are provided on a solid support located within an array tube. A single array tube or, alternatively, an independent array tube for each set of genes may be used.
• the probes of the present invention are provided on a solid support located within an array strip. In a preferred embodiment, the array strip of the present invention has 8 wells.
• the solid support is a coated glass slide.
• the probes of the present invention contain an amino group and the support comprises epoxy groups; this promotes binding of the probes to the support.
• one or more probes contained in the array tube are selected from the probes shown in Tables 2, 4 and 5.
• DNA hybrids may be detected by recognition of the labei by specific binding to a l ⁇ gand or by immunodetection.
• biotin label is detected by specific binding to streptavidin conjugated with horse-radish-peroxidase (HRP) and the subsequent conversion of tetramethyibenzidine (TMB) to a blue pigment that precipitates in the location where the corresponding specific probe was bound.
• HRP horse-radish-peroxidase
• TMB tetramethyibenzidine
• Other kind of conjugates well known in the art may also be suitable for purposes of the present invention (e. g. streptavidin-Au conjugate).
• Fluorescently labelled detection systems may instead be used, either indirectly or directiy labelled. Alternatively, other enzyme-based systems may be used.
• array tubes so processed can be read using simple optical devices, such as an optical microscope or ATROl and ATS readers manufactured by CLONDIAG chip technologies GmbH (Jena, Germany).
• a preferred device for processing array strips is ASP, manufactured by CLONDIAG chip technologies GmbH (Jena, Germany).
• Table 1 Amplification Primers for Calcitonin Receptor, Vitamin D Receptor, Estrogen Receptor and Collagen I Al genes.
• Table 2 Detection probes for polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al.
• Table 3 Amplification primers for Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR.
• Table 4 Detection probes for polymorphisms Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T.
• Table 5 Additional detection probes for polymorphisms of Collagen I Al, Estrogen Receptor and Vitamin D Receptor.
• the solid support used in the present invention may comprise one or more allele-specific probes selected from nucleotide sequences from the sequence lists of Tables 2, 4 and 5.
• the probes may be selected only from table 2; only from table 4; only from table 5; or from any combination of tables 2, 4 and 5.
• Probe sequences are represented as single stranded DNA oligonucleotides from the 5' to the 3' end.
• any of these probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U), as long as this change of sequence does not affect dramatically its functionality, Such probes are all within the scope of the present invention.
• any probe of tables 2, 4 or 5, which has been prepared by adding or changing one or more nucleotides of its sequence without dramatically affecting to its functionality is also within the scope of the present invention. Preferably, no more than 5, 4, 3, 2, 1 nucleotides are modified.
• Specific probes for SNPs associated with osteoporosis were selected as follows: The design of the probes was made based on the sequence of each gene deposited in GenBank, for each amplified region. The design was performed using a conventional nucleic acid program, such as Oligo (6 version). Potential sequences of oligonucleotides to be used as specific probes were selected from these sequence regions, preferably having following features: G+C balanced in both sides of the SNP, size of the probe, preferably an approximate length between 15-30 nt, more preferably 15-23 nt; preferably with no secondary structures or strings of consecutive same nucleotide longer than 4; preferably with a G+C ratio between 45 and 60 % and a Tm as much similar among all selected probes as possible.
• the present invention provides probes for specific detection of polymorphisms AIuI of the Calcitonin Receptor, Fokl and Bsml of the Vitamin D Receptor, Xbal and PvuII of the Estrogen Receptor and COLlAl SpI of Collagen I Al (Table 2; SEQ ID MO 11-29; Table 5; SEQ ID NO 212-222), and of Osteoprotegerin OPG 209 G>A, OPG 245 T>G and OPG163 A>G, LRP5 C171346A, C135242T and G138351A, CYP17 T(27)-C; CYP19 C(1558)-T and MTHFR C677T (Table 4, SEQ ID NO 44-211).
• the solid support may comprise one or more probes for specific detection of controls such as PCR reaction control or adequacy of the DNA from the sample control.
• it may also comprise one or more labelled oligonucleotides (e.g. biotin modified oligonucleotides) for positive control of the detection reaction and for positioning reference so that all remaining probes can be located.
• Oligonucleotide sequences Marker-1 [GCA GTA TAA GAT TAT TGA TGC CGG AAC] and Marker-2 [GTC AAA ACC TGG GAT AGT AGT TTT ACC], have no significant homology to any of the amplified products of the present invention,
• biotin modified oligonucleotides Marker-1 and Marker-2 serve as positive control of the PCR products detection reaction and as positioning reference so that all remaining probes can be located.
• the probes of the present invention can be obtained by different methods, such as chemical synthesis (e. g. by the conventional phosphotriester method) or genetic engineering techniques, for example by molecular cloning of recombinant plasmids in which corresponding nucleotide sequences have been inserted and can be later obtained by digestion with nucleases.
• chemical synthesis e. g. by the conventional phosphotriester method
• genetic engineering techniques for example by molecular cloning of recombinant plasmids in which corresponding nucleotide sequences have been inserted and can be later obtained by digestion with nucleases.
• 5' amine-linked oligonucleotide probes are bound to the surface of a solid support in known distinct locations.
• Said probes may be immobilized individually or as mixtures to delineated locations on the solid support.
• Said probes or mixtures of probes may be immobilized in a single location of the solid support, preferably in two distinct locations of the solid support and more preferably in three distinct locations of the solid support.
• Figure 1 exemplifies a schematic representation of an arrangement of probes on the surface of the microarray. The numbers refer to the SEQ ID Nos as given in the above tables, while M represents markers, and O represents no probe.
• the probes are provided on a solid support located within an array tube or within an array strip.
• the array tube and array strip of the present invention may comprise one or more probes selected from nucleotide sequences SEQ ID N 0 11 to SEQ ID N 0 29, SEQ ID N°44 to SEQ ID N 0 211, and SEQ ID N°212 to SEQ ID N 0 222.
• they may comprise one or more probes for specific detection of controls such as PCR reaction control or adequacy of the DNA from the sample control.
• they may also comprise one or more labelled oligonucleotides (e.g. biotin modified oligonucleotides) for positive control of the detection reaction and for positioning reference so that all remaining probes can be located.
• DNA hybrids After incubation of single stranded DNA with a plurality of target-specific probes, DNA hybrids may be detected by recognition of the label by specific binding to a ligand or by immunodetection.
• biotin label is detected by specific binding to streptavidin conjugated with horse-radish-peroxidase (HRP) and the subsequent conversion of tetramethylbenzidine (TMB) to a blue pigment that precipitates in the concrete location where corresponding specific probe was bound.
• HRP horse-radish-peroxidase
• TMB tetramethylbenzidine
• Other kind of conjugates well known in the art may also be suitable for purposes of the present invention (e. g. streptavidin-Au conjugate).
• Fluorescently labelled detection systems may instead be used, either indirectly or directly labelled. Alternatively, other enzyme-based systems may be used.
• visualization of array tubes and array strips consists of the following steps; first, the image of the array is captured using an optical device, then, the image is analysed, and finally, a report containing an interpretation of the result is provided.
• the image is analysed by means of appropriate software. Any device suitable for this processing can be used. Preferred devices for processing array tubes are ATROl and ATS readers manufactured by CLONDIAG chip technologies GmbH (Jena, Germany). A preferred device for processing array strips is ASP, manufactured by CLONDIAG chip technologies GmbH (Jena, Germany). In a preferred embodiment, processing of the array strips can be automated in a specific workstation. In a more preferred embodiment such processing comprises all the washing and incubation steps of the method, starting from the PCR and ending with visualization of the result.
• the present invention relates to an in vitro diagnostic kit for genotyping Calcitonin Receptor and Collagen I Al genes.
• a kit might also allow genotyping of Vitamin D Receptor and/or Estrogen Receptor genes.
• the present invention also relates to an in vitro diagnostic kit for genotyping Calcitonin Receptor and Collagen I Al, and optionally, Vitamin D Receptor and/or Estrogen Receptor, further allowing genotyping of one or more genes selected from the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR.
• the present invention also relates to an in vitro diagnostic kit for genotyping Calcitonin Receptor, Collagen I Al, Vitamin D Receptor and Estrogen Receptor genes and of Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR genes, in clinical samples.
• the mentioned kit comprises any or all of the following components: one or more amplification mix(es), including amplification buffer, dNTPs, primers, and control plasmid; wash buffer; detection reagents; array tube(s) including a solid support including allele-specific probes; reagents for obtaining and preparing a sample.
• amplification mix(es) including amplification buffer, dNTPs, primers, and control plasmid
• wash buffer detection reagents
• array tube(s) including a solid support including allele-specific probes reagents for obtaining and preparing a sample.
• suitable kit components and buffer compositions will depend on the exact conditions under which the kit is intended to be used, although the skilled person will be able to determine suitable kit components and buffer compositions.
• the kit comprises: i) a nucleic acid amplification mix comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al, ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor and at least another probe is specific for Collagen I Al, and iii) reagents for use in visualising hybridisation of nucleic acids to the probes of the solid support.
• nucleic acid amplification mix comprising four or more pairs of amplification primers, wherein at least one pair allows amplification of
• Calcitonin Receptor at least another pair allows amplification of Collagen I Al, at least another pair allows amplification of Vitamin D Receptor and at least another pair allows amplification of Estrogen Receptor,
• reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor; at least another probe is specific for Collagen I
• At least another probe is specific for Vitamin D Receptor; and at least another probe is specific for Estrogen Receptor, and
• the kit comprises: i) two nucleic acid amplification mixes, one of them comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al; the other amplification mix comprising two or more pairs of amplification primers that allow amplification of two or more of the genes of the group of genes comprising Osteoprotegerih, LRP5, CYP17, CYP19 and MTHFR, ii) a reaction vessel or set of reaction vessels containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor, at least another probe is specific for Collagen I Al, and at least two or more probes are specific for the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR amplifyabie by the amplification mixture of i), ii
• nucleic acid amplification mixes one of them comprising two or more pairs of amplification primers, wherein at least one pair allows amplification of Calcitonin Receptor and at least another pair allows amplification of Collagen I Al; the other amplification mix comprising two or more pairs of amplification primers that allow amplification of two or more of the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR, ii) two reaction vessels or sets of reaction vessels, one containing a solid support that comprises target-specific probes, wherein at least one probe is specific for Calcitonin Receptor, at least another probe is specific for Collagen I Al, the other containing a support comprising at least two or more probes specific for the genes of the group of genes comprising Osteoprotegerin, LRP5, CYP17, CYP19 and MTHFR amplifyabie by the amplification mixture of i), iii)
• Array tubes of the present invention were manufactured at CLONDIAG chip Technologies GmbH (Jena, Germany) as follows.
• a standard reaction test tube from Eppendorf made of polypropylene and having a nominal receiving volume of 1,5 ml was modified by re-melting, so that, an opened recess for the microarray support with an adhesive edge was modelled into the tube.
• Microarrays to be inserted into these tubes were produced by using a MicroGrid II Arrayer (BioRobotics, Cambridge, Great Britain). Probes consisting of 5' end amino-modified oligonucleotides having a sequence from the sequence list were deposited at defined sites on an epoxidized glass surface of a slide (slide size: 75 mm x 25 mm) and covalently immobilised.
• locations have a spacing of 0.2 mm, so that the DNA library included in each microarray covered an area of 2.4 mm x 2.4 mm and, in total, more than 100 identical DNA libraries could be produced in this way per slide.
• either one single probe or a mixture of them could be deposited at each one of these locations.
• single probes were deposited at each location when specificity and sensitivity experiments for probes selection were carried out.
• mixtures of probes capable of hybridizing in separate regions of the amplified product of a specific polymorphism genotype associated with osteoporosis could be deposited in the same location when identification of polymorphism genotypes assays were performed.
• Figure 2 shows the localization of the probes within microarrays used for this invention.
• the schematic representation of the arrangement of probes corresponding to Figure 2 is displayed in Figure 1.
• Four replicates for each probe or mixture of probes were included in each microarray.
• microarrays included reference markers at several locations consisting of 5' end biotin modified oligonucleotides with no significant homology for any of the amplified sequences from this invention (Marker-1 [GCA GTA TAA GAT TAT TGA TGC CGG AAC] and Marker-2 [GTC AAA ACC TGG GAT AGT AGT TTT ACC]). These reference markers served both for verifying proper performance of the detection reaction and for optical orientation of the image by the reader so all remaining probes can be located and the data analyzed.
• oligonucleotides were deposited on the slide from a Ix QMT Spotting Solution I (Quantifoil Micro Tools GmbH, Jena, Germany). Total concentration of oligonucleotides in each spotting solution ranged from 2,5 mM for reference markers to 20 mM for specific probes. Oligonucleotides were then covalently linked to the epoxide groups on the glass surface by baking at 6O 0 C for 30 minutes followed by a multi-step washing process. Dried slides were cut into 3.15 mm x 3.15 mm glass pieces which, strictly speaking, are what we name microarrays. In the final step for array tubes manufacturing, these microarrays were then inserted into the aforementioned modified Eppendorf tubes and glued to the adhesive edge.
• a commercial kit (NucleoSpin® Tissue kit Catalogue No. 635966 from BD Biosciences Clontech, Palo Alto, CA, USA) designed for DNA isolation from samples from a variety of sources was used to process blood samples.
• protocol was performed following manufacturer specifications for isolation of genomic DNA from 200 ⁇ l of total blood: 25 ⁇ l of Proteinase K was added to
• the column was transferred to a 1.5 ml Eppendorf tube and 60 ⁇ l of Buffer BE (prewarmed at 7O 0 C) was added and incubated for 3 minutes at room temperature. The column was centrifuged at IiOOOg for 1 minute and supernatant was transferred to a clean and sterile tube. An Aliquot of 5 ⁇ l was subsequently used in the PCR reaction.
• Buffer BE prewarmed at 7O 0 C
• PCR amplification using primers described as SEQ ID N 0 I, 2, 3, 4, 5, 6, 7, 8, 9, and 10 was performed. Briefly, PCR amplification was carried out in a 50 ⁇ l final volume reaction containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1 mM MgCI2, 0.2 ⁇ M each primer of SEQ ID NO I 1 2, 3, 4, 5, 6, 7, 8, 9, and 10, 500 ⁇ M of dNTPs (dATP, dTTP, dGTP, dCTP), 5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), and 5 ⁇ l of each clinical sample DNA from Example 2.1.
• dNTPs dATP, dTTP, dGTP, dCTP
• AmpliTaq Gold DNA polymerase Applied Biosystems, Foster City, CA, USA
• PCR amplification of sample DNAs was carried out in a 50 ⁇ l final volume reaction using 0.13-0.15 ⁇ M of each primer of SEQ ID NO 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42 and 43 in the presence of 500 ⁇ M of dNTPs
• Negative controls constituted of 5 ⁇ l of blank samples from Example 2.1. or 5 ⁇ l of deionised water were processed in parallel with the sample DNA. The use of these kinds of negative controls serves to check that contamination does not occur at any point in sample handling or in PCR reaction setting up and all positive results represent true presence of DNA in the sample.
• PCR reactions were run in a Mastercycler thermocycler (Eppendorf, Hamburg, Germany) programmed with the following cycling profile: one initial denaturing cycle at 95 0 C for 12 minutes, 40 cycles of 30 seconds at 95 0 C, 40 seconds at 55-56 0 C and 60 seconds at 72 0 C, and one final extension cycle at 6O 0 C for 30 minutes. After amplification, 5 ⁇ l of each reaction were used for subsequent detection with specific probes.
• Array tubes were pre-washed just before its use by addition of 300 ⁇ l of 0.5X PBS-Tween 20 buffer to each tube and inverting them several times. All liquid from inside each tube was removed using a Pasteur pipette connected with a vacuum system, Five microliters of amplification reactions from Example 3 were added to 95 ⁇ l of hybridization solution (250 mM sodium phosphate buffer, pH 7.2; SSC IX; SDS 4.5%; 10 mM EDTA, pH 8.0), This mixture was denatured by heating them to 95 0 C for 10 minutes and, immediately after, was applied to the array tube prepared in Example 1 prewarmed at 5O 0 C in a Thermomixer comfort (Eppendorf, Hamburg, Germany) for i or 2 minutes.
• hybridization solution 250 mM sodium phosphate buffer, pH 7.2; SSC IX; SDS 4.5%; 10 mM EDTA, pH 8.0
• Hybridization reaction was carried out in a Thermomixer comfort (Eppendorf, Hamburg, Germany) by incubating the array tubes at 5O 0 C for one hour and 30 minutes with shaking at 550 rpm. After incubation period, hybridization reaction was removed using a Pasteur pipette connected with a vacuum system and a washing step with 300 ⁇ l of 0,5X PBS-Tween 20 buffer was carried out. Hybridized DNA was detected by incubation in 100 ⁇ l of a 0.075 ⁇ g/ml PoIy- HRP Streptavidin (Pierce Biotechnology Inc., Rockford, IL, USA) solution at 3O 0 C for 15 minutes with shaking at 550 rpm.
• ATS reader may have specific software installed for automatic processing of the sample analysis result obtained with the array tube developed in the present invention.
• EXAMPLE 5 Importance of the design of amplification primers on the result of the Multiplex PCR.
• the five pairs of primers corresponding to SEQ ID N 0 1-10 were designed as amplification primers for fragments containing the polymorphisms AIuI of the Calcitonin Receptor (SEQ ID N 0 7 & 8), Fokl and Bsml of the Vitamin D Receptor (SEQ ID N 0 9 & 10 and 1 & 2, respetively), Xbal and PvuII of the Estrogen Receptor (SEQ ID N 0 3 & 4, which serve the purpose of amplification of a fragment containing both polymorphisms) and COLlAl SpI of Collagen I Al (SEQ ID N 0 5 & 6).
• the lower amplification levels corresponding to Sample 2 may be due to the fact that the concentration of the DNA extracted from this sample was lower than the optimal value of 40 ng/ ⁇ l. Differently, the concentration of the DNA extracted from the samples 1 and 3 was 40 ng/ ⁇ l.
• Amplification fragments corresponding to Calcitonin Receptor as well as to polymorphism Fokl of the Vitamin D Receptor are 157 and 188 bp long, respectively. Their similar size is the reason why they appear as a single band.
• EXAMPLE 6 Analysis of probes designed for the detection of genotypes S or s corresponding to the polymorphism COLlAl SpI of gene Collagen I Al.
• allelic variants of the polymorphism COLlAl SpI of gene Collagen I Al are:
• Genotype of samples op42 ss, op90 ss, op ⁇ l ss is ss; Genotype of sample op87 SS is SS; Genotype of samples op55 Ss, op72 Ss, op58 Ss is Ss, Samples were subjected to DNA amplification in a Multiplex-PCR according to conditions described in example 5, the hybridization temperature being of 55 0 C, Amplification products were denatured and incubated with an array tube comprising the probes of Figure 4, Panel A, under the conditions already described in Example 4. The results obtained after visualization are displayed in Figure 4, Panel B (Values in Arbitrary Units).
• EXAMPLE 7 Analysis of probes designed for the detection of the amplified fragment corresponding to polymorphism COLiAl SpI of gene Collagen I Al.
• Probes were designed that might hybridise with the amplification fragment of the Multiplex-PCR corresponding to polymorphism COLlAl SpI of gene Collagen I Al, independently of the genotype that might be present. Thus, a requisite of such probes was that they had the ability to hybridise with a region of the amplification fragment wherein the polymorphism would not be contained.
• Probe of SEQ ID N 0 217 was again compared with another probe, probe of SEQ ID N 0 15, both of which were designed to hybridise with the amplification fragment of the Muitiplex-PCR corresponding to polymorphism COLlAl SpI of gene Collagen I Al independently of the genotype that might be present.
• a requisite of such probes is that they have the ability to hybridise with a region of the amplification fragment wherein the polymorphism is not contained.
• probe of SEQ ID N 0 217 provides a better hybridization than probe of SEQ ID N 0 15, in 14 out of the 21 samples.
• Probe of SEQ ID N 0 217 provides better hybridization levels than probe of SEQ ID N 0 15 in the detection of gene CoIlAl.
• Genotype distribution was as follows: SS: 8 samples; Ss: 7 samples; ss: 6 samples.
• probes of SEQ ID N 0 212, 213 and 214 do not provide a good alternative for the detection of allele H S" of COLlAl SpI, they constitute an alternative for the discrimination of allele "s".
• both polymorphisms Xbal and PvuII are contained.
• Probes of SEQ ID N 0 14, 218 and 219 were designed to hybridise with the amplification fragment of the Multipiex-PCR corresponding to the Estrogen Receptor gene. A requisite of such probes is that they have the ability to hybridise with a region of the amplification fragment wherein no polymorphism is contained.
• Genotype distribution was as follows: PP: 5 samples; Pp: 5 samples; pp: 5 samples.
• the probe of SEQ ID N 0 220 improves the detection of genotype ⁇ pp" with respect to SEQ ID N 0 19, in 2 out of 5 samples. Further, the probe of SEQ ID N 0 220 improves the detection of genotype "Pp" in 5 out of 5 samples, when compared with the probe of SEQ ID N 0 19. Finally, detection of genotype "PP" of the final 5 samples remained the same both with probes of SEQ ID N 0 220 and 19.
• probe of SEQ ID N 0 220 provides a better detection of allele "p" of polymorphism PvuII of the Estrogen Receptor gene, than probe of SEQ ID N 0 19.
• Genotype distribution was as follows: FF: 7 samples; Ff: 7 samples; ff: 2 samples.
• both probes of SEQ ID N 0 16 and SEQ ID N 0 222 are able to discriminate the presence or absence of allele "f". Nevertheless, the probe of SEQ ID N 0 222 provides higher hybridization levels in 8 out of 9 samples.
• probe of SEQ ID N 0 16 and of SEQ ID N 0 222 are able to discriminate the presence or absence of allele "f" of polymorphism Fokl of gene Vitamin D Receptor, probe of SEQ ID N 0 222 provides higher hybridization levels than probe of SEQ ID N 0 16 in the detection of allele "f".
• Genotype distribution was as follows: BB: 4 samples; Bb: 6 samples; bb: 6 samples.

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