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WO2001051670A2 - Acides nucleiques renfermant des polymorphismes de nucleotide simple et procede d'utilisation associe - Google Patents

Acides nucleiques renfermant des polymorphismes de nucleotide simple et procede d'utilisation associe Download PDF

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Publication number
WO2001051670A2
WO2001051670A2 PCT/US2001/000322 US0100322W WO0151670A2 WO 2001051670 A2 WO2001051670 A2 WO 2001051670A2 US 0100322 W US0100322 W US 0100322W WO 0151670 A2 WO0151670 A2 WO 0151670A2
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Prior art keywords
sequence
polymoφhic
nucleotide
complement
nucleic acid
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PCT/US2001/000322
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English (en)
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WO2001051670A3 (fr
Inventor
Richard A. Shimkets
Martin D. Leach
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Curagen Corporation
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Priority to CA002395607A priority Critical patent/CA2395607A1/fr
Priority to AU26308/01A priority patent/AU2630801A/en
Priority to EP01900897A priority patent/EP1250456A2/fr
Publication of WO2001051670A2 publication Critical patent/WO2001051670A2/fr
Publication of WO2001051670A3 publication Critical patent/WO2001051670A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—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

Definitions

  • Sequence polymorphism-based analysis of nucleic acid sequences can augment or replace previously known methods for determining the identity and relatedness of individuals.
  • the approach is generally based on alterations in nucleic acid sequences between related individuals.
  • This analysis has been widely used in a variety of genetic, diagnostic, and forensic applications. For example, polymorphism analyses are used in identity and paternity analysis, and in genetic mapping studies.
  • RFLPS restriction fragment length polymorphism
  • RFLP restriction fragment length polymorphism
  • STR sequences typically include tandem repeats of 2, 3, or 4 nucleotide sequences that are present in a nucleic acid from one individual but absent from a second, related individual at the corresponding genomic location.
  • SNPs single nucleotide polymorphisms
  • cSNP single nucleotide polymorphisms
  • SNPs can arise in several ways.
  • a single nucleotide polymorphism may arise due to a substitution of one nucleotide for another at the polymorphic site.
  • Substitutions can be transitions or transversions.
  • a transition is the replacement of one purine nucleotide by another purine nucleotide, or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine, or the converse.
  • Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • the polymorphic site is a site at which one allele bears a gap with respect to a single nucleotide in another allele.
  • Some SNPs occur within, or near genes.
  • One such class includes SNPs falling within regions of genes encoding for a polypeptide product. These SNPs may result in an alteration of the amino acid sequence of the polypeptide product and give rise to the expression of a defective or other variant protein.
  • Such variant products can, in some cases result in a pathological condition, e.g. , genetic disease.
  • genes in which a polymorphism within a coding sequence gives rise to genetic disease include sickle cell anemia and cystic fibrosis.
  • Other SNPs do not result in alteration of the polypeptide product.
  • SNPs can also occur in noncoding regions of genes.
  • SNPs tend to occur with great frequency and are spaced uniformly throughout the genome.
  • the frequency and uniformity of SNPs means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest.
  • the invention is based in part on the discovery of novel single nucleotide polymorphisms (SNPs) in regions of human DNA.
  • SNPs single nucleotide polymorphisms
  • the invention provides an isolated polynucleotide which includes one or more of the SNPs described herein.
  • the polynucleotide can be, e.g., a nucleotide sequence which includes one or more of the polymorphic sequences shown in Table 1 and the Sequence Listing (SEQ ID NOS: 1 - 650) and which includes a polymorphic sequence, or a fragment of the polymorphic sequence, as long as it includes the polymorphic site.
  • the polynucleotide may alternatively contain a nucleotide sequence which includes a sequence complementary to one or more of the sequences (SEQ ID NOS: 1-650), or a fragment of the complementary nucleotide sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence.
  • the polynucleotide can be, e.g. , DNA or RNA, and can be between about 10 and about
  • nucleotides e.g 10-90, 10-75, 10-51, 10-40, or 10-30, nucleotides in length.
  • the polymorphic site in the polymorphic sequence includes a nucleotide other than the nucleotide listed in Table 1, column 5 for the polymorphic sequence, e.g., the polymorphic site includes the nucleotide listed in Table 1, column 6 for the polymorphic sequence.
  • the complement of the polymorphic site includes a nucleotide other than the complement of the nucleotide listed in Table 1, column 5 for the complement of the polymorphic sequence, e.g., the complement of the nucleotide listed in Table 1, column 6 for the polymorphic sequence.
  • the polymorphic sequence is associated with a polypeptide related to one of the protein families disclosed herein.
  • the nucleic acid may be associated with a polypeptide related to an ATPase associated protein, a cadherin, or any of the other proteins identified in Table 1, column 10.
  • the invention provides an isolated allele-specific oligonucleotide that hybridizes to a first polynucleotide containing a polymorphic site.
  • the first polynucleotide can be, e.g. , a nucleotide sequence comprising one or more polymorphic sequences (SEQ ID NOS: 1 - 650), provided that the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1 , column 5 for the polymorphic sequence.
  • the first polynucleotide can be a nucleotide sequence that is a fragment of the polymorphic sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence, or a complementary nucleotide sequence which includes a sequence complementary to one or more polymorphic sequences (SEQ ID NOST - 650), provided that the complementary nucleotide sequence includes a nucleotide other than the complement of the nucleotide recited in Table 1 , column 5.
  • the first polynucleotide may in addition include a nucleotide sequence that is a fragment of the complementary sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence.
  • the oligonucleotide does not hybridize under stringent conditions to a second polynucleotide.
  • the second polynucleotide can be, e.g., (a) a nucleotide sequence comprising one or more polymorphic sequences (SEQ ID NOST - 650), wherein the polymorphic sequence includes the nucleotide listed in Table 1, column 5 for the polymorphic sequence; (b) a nucleotide sequence that is a fragment of any of the polymorpliic sequences; (c) a complementary nucleotide sequence including a sequence complementary to one or more polymorphic sequences (SEQ ID NOST - 650), wherein the polymorphic sequence includes the complement of the nucleotide listed in Table 1, column 5; and (d) a nucleotide sequence that is a fragment of the complementary sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence.
  • the oligonucleotide can be, e.g., between about 10 and about 100 bases in length. In some embodiments, the oligonucleotide is between about 10 and 75 bases, 10 and 51 bases, 10 and about 40 bases, or about 1-5 and 30 bases in length.
  • the invention also provides a method of detecting a polymorphic site in a nucleic acid.
  • the method includes contacting the nucleic acid with an oligonucleotide that hybridizes to a polymorphic sequence selected from the group consisting of SEQ ID NOS: 1-650, or its complement, provided that the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1, column 5 for the polymorphic sequence, or the complement includes a nucleotide other than the complement of the nucleotide recited in Table 1, column 5.
  • the method also includes determining whether the nucleic acid and the oligonucleotide hybridize. Hybridization of the oligonucleotide to the nucleic acid sequence indicates the presence of the polymorphic site in the nucleic acid.
  • the oligonucleotide does not hybridize to the polymorphic sequence when the polymorphic sequence includes the nucleotide recited in Table 1, column 5 for the polymorphic sequence, or when the complement of the polymorphic sequence includes the complement of the nucleotide recited in Table I, column 5 for the polymorphic sequence.
  • the oligonucleotide can be, e.g., between about 10 and about 100 bases in lengtli. In some embodiments, the oligonucleotide is between about 10 and 75 bases, 10 and 51 bases, 10 and about 40 bases, or about 15 and 30 bases in length.
  • the polymorphic sequence identified by the oligonucleotide is associated with a polypeptide related to one of the protein families disclosed herein.
  • the nucleic acid may be associated polypeptide related to an ATPase associated protein, cadherin, or any of the other protein families identified in Table 1, column 10.
  • the method includes determining if a sequence polymorphism is the present in a subject, such as a human.
  • the method includes providing a nucleic acid from the subject and contacting the nucleic acid with an oligonucleotide that hybridizes to a polymorphic sequence selected from the group consisting of SEQ ID NOS: 1-650, or its complement, provided that the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1, column 5 for said polymorphic sequence, or the complement includes a nucleotide other than the complement of the nucleotide recited in Table 1, column 5. Hybridization between the nucleic acid and the oligonucleotide is then determined.
  • Hybridization of the oligonucleotide to the nucleic acid sequence indicates the presence of the polymorphism in said subject.
  • the invention provides a method of determining the relatedness of a first and second nucleic acid. The method includes providing a first nucleic acid and a second nucleic acid and contacting the first nucleic acid and the second nucleic acid with an oligonucleotide that hybridizes to a polymorphic sequence selected from the group consisting of SEQ ID NOS: 1-650, or its complement, provided that the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1, column 5 for the polymorphic sequence, or the complement includes a nucleotide other than the complement of the nucleotide recited in Table 1, column 5.
  • the method also includes determining whether the first nucleic acid and the second nucleic acid hybridize to the oligonucleotide, and comparing hybridization of the first and second nucleic acids to the oligonucleotide. Hybridization of first and second nucleic acids to the nucleic acid indicates the first and second subjects are related.
  • the oligonucleotide does not hybridize to the polymorphic sequence when the polymorphic sequence includes the nucleotide recited in Table 1 , column 5 for the polymorphic sequence, or when the complement of the polymorphic sequence includes the complement of the nucleotide recited in Table 1, column 5 for the polymorphic sequence.
  • the oligonucleotide can be, e.g., between about 10 and about 100 bases in length. In some embodiments, the oligonucleotide is between about 10 and 75 bases, 10 and 51 bases, 10 and about 40 bases, or about 15 and 30 bases in length.
  • the method can be used in a variety of applications.
  • the first nucleic acid may be isolated from physical evidence gathered at a crime scene
  • the second nucleic acid may be obtained from a person suspected of having committed the crime. Matching the two nucleic acids using the method can establish whether the physical evidence originated from the person.
  • the first sample may be from a human male suspected of being the father of a child and the second sample may be from the child. Establishing a match using the described method can establish whether the male is the father of the child.
  • the invention provides an isolated polypeptide comprising a polymorphic site at one or more amino acid residues, and wherein the protein is encoded by a polynucleotide including one of the polymorphic sequences SEQ ID NOS: 1-650, or their complement, provided that the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1, column 5 for the polymorphic sequence, or the complement includes a nucleotide other than the complement of the nucleotide recited in Table 1, column 5.
  • the polypeptide can be, e.g., related to one of the protein families disclosed herein. For example, polypeptide can be related to an ATPase associated protein, cadherin, or any of the other proteins provided in Table 1, column 10.
  • the polypeptide is translated in the same open reading frame as is a wild type protein whose amino acid sequence is identical to the amino acid sequence of the polymorphic protein except at the site of the polymorphism.
  • the polypeptide encoded by the polymorphic sequence, or its complement includes the nucleotide listed in Table 1, column 6 for the polymorphic sequence, or the complement includes the complement of the nucleotide listed in Table 1, column 6.
  • the invention also provides an antibody that binds specifically to a polypeptide encoded by a polynucleotide comprising a nucleotide sequence encoded by a polynucleotide selected from the group consisting of polymorphic sequences SEQ ID NOS: 1-650, or its complement.
  • the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1 , column 5 for the polymorphic sequence, or the complement includes a nucleotide other than the complement of the nucleotide recited in Table 1, column 5.
  • the antibody binds specifically to a polypeptide encoded by a polymorphic sequence which includes the nucleotide listed in Table 1, column 6 for the polymorphic sequence.
  • the antibody does not bind specifically to a polypeptide encoded by a polymorphic sequence which includes the nucleotide listed in Table 1, column 5 for the polymorphic sequence.
  • the invention further provides a method of detecting the presence of a polypeptide having one or more amino acid residue polymorphisms in a subject.
  • the method includes providing a protein sample from the subject and contacting the sample with the above-described antibody under conditions that allow for the formation of antibody-antigen complexes. The antibody-antigen complexes are then detected. The presence of the complexes indicates the presence of the polypeptide.
  • the invention also provides a method of treating a subject suffering from, at risk for, or suspected of, suffering from a pathology ascribed to the presence of a sequence polymorphism in a subject, e.g., a human, non-human primate, cat, dog, rat, mouse, cow, pig, goat, or rabbit.
  • the method includes providing a subject suffering from a pathology associated with aberrant expression of a first nucleic acid comprising a polymorphic sequence selected from the group consisting of SEQ ID NOST - 650, or its complement, and treating the subject by administering to the subject an effective dose of a therapeutic agent.
  • Aberrant expression can include qualitative alterations in expression of a gene, e.g., expression of a gene encoding a polypeptide having an altered amino acid sequence with respect to its wild-type counterpart.
  • Qualitatively different polypeptides can include, shorter, longer, or altered polypeptides relative to the amino acid sequence of the wild-type polypeptide.
  • Aberrant expression can also include quantitative alterations in expression of a gene. Examples of quantitative alterations in gene expression include lower or higher levels of expression of the gene relative to its wild-type counterpart, or alterations in the temporal or tissue-specific expression pattern of a gene.
  • aberrant expression may also include a combination of qualitative and quantitative alterations in gene expression.
  • the therapeutic agent can include, e.g., second nucleic acid comprising the polymorphic sequence, provided that the second nucleic acid comprises the nucleotide present in the wild type allele.
  • the second nucleic acid sequence comprises a polymorphic sequence which includes nucleotide listed in Table 1 , column 5 for the polymorphic sequence.
  • the therapeutic agent can be a polypeptide encoded by a polynucleotide comprising polymorphic sequence selected from the group consisting of SEQ ID NOST - 650, or by a polynucleotide comprising a nucleotide sequence that is complementary to any one of polymorphic sequences SEQ ID NOS.T - 650, provided that the polymorphic sequence includes the nucleotide listed in Table 1, column 6 for the polymorphic sequence.
  • the therapeutic agent may further include an antibody as herein described, or an oligonucleotide comprising a polymorphic sequence selected from the group consisting of SEQ ID NOST - 650, or by a polynucleotide comprising a nucleotide sequence that is complementary to any one of polymorphic sequences SEQ ID NOST - 650, provided that the polymorphic sequence includes the nucleotide listed in Table 1, column 5 or Table 1, column 6 for the polymorphic sequence.
  • the invention provides an oligonucleotide array comprising one or more oligonucleotides hybridizing to a first polynucleotide at a polymorphic site encompassed therein.
  • the first polynucleotide can be, e.g., a nucleotide sequence comprising one or more polymorphic sequences (SEQ ID NOST - 650); a nucleotide sequence that is a fragment of any of the nucleotide sequences, provided that the fragment includes a polymorphic site in the polymorphic sequence; a complementary nucleotide sequence comprising a sequence complementary to one or more polymorphic sequences (SEQ ID NOST - 650); or a nucleotide sequence that is a fragment of the complementary sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence.
  • the array comprises 10; 100; 1,000; 10,000; 100,000 or more oligonucleotides.
  • the invention also provides a kit comprising one or more of the herein-described nucleic acids.
  • the kit can include, e.g., a polynucleotide which includes one or more of the SNPs described herein.
  • the polynucleotide can be, e.g., a nucleotide sequence which includes one or more of the polymorphic sequences shown in Table 1 and the Sequence Listing (SEQ ID NOS: 1 - 650) and which includes a polymorphic sequence, or a fragment of the polymorphic sequence, as long as it includes the polymorphic site.
  • the polynucleotide may alternatively contain a nucleotide sequence which includes a sequence complementary to one or more of the sequences (SEQ ID NOS: 1-650), or a fragment of the complementary nucleotide sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence.
  • the invention provides an isolated allele-specific oligonucleotide that hybridizes to a first polynucleotide containing a polymorphic site.
  • the first polynucleotide can be, e.g., a nucleotide sequence comprising one or more polymorphic sequences (SEQ ID NOST - 650), provided that the polymorphic sequence includes a nucleotide other than the nucleotide recited in Table 1, column 5 for the polymorphic sequence.
  • the first polynucleotide can be a nucleotide sequence that is a fragment of the polymorphic sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence, or a complementary nucleotide sequence which includes a sequence complementary to one or more polymorphic sequences (SEQ ID NOST - 650), provided that the complementary nucleotide sequence includes a nucleotide other than the complement of the nucleotide recited in Table 1, column 5.
  • the first polynucleotide may in addition include a nucleotide sequence that is a fragment of the complementary sequence, provided that the fragment includes a polymorphic site in the polymorphic sequence.
  • the invention provides human SNPs in sequences which are transcribed, i.e., are cSNPs. Many SNPs have been identified in genes related to polypeptides of known function. If desired, SNPs associated with various polypeptides can be used together. For example, SNPs can be grouped according to whether they are derived from a nucleic acid encoding a polypeptide
  • SNPs can be grouped according to the functions played by their gene products. Such functions include, structural proteins, proteins from which associated with metabolic pathways fatty acid metabolism, glycolysis, intermediary metabolism, calcium metabolism, proteases, and amino acid metabolism, etc.
  • the present invention provides a large number of human 0 cSNP's based on at least one gene product that has not been previously identified.
  • the cSNP's involve nucleic acid sequences that are assembled from at least one known sequence.
  • the present invention provides a large number of human cSNP's based on at least one gene product that has not been previously identified.
  • the cSNP's involve nucleic acid sequences that are assembled from at least one known sequence.
  • 650 distinct polymorphic sites were identified by the present inventors, using the following procedure.
  • Raw traces underlying sequence data were drawn from public databases and from the proprietary database of the Assignee of the present invention. The sequences were 0 obtained by calling the bases from these traces, and included assigning "Phred" quality scores for each called base.
  • allelic set at the polynucleotide level, four or more nucleotide sequences were identified having at least partial overlap with one another.
  • these four or more sequences could be clustered and assembled to make a consensus contig that included an ORF.
  • the inventors found that the 5 assembled contigs defined associated sets of two, or possibly more than two, alleles defined by an SNP at a particular polymorphic site.
  • the nucleotide change from the consensus sequence had to occur in at least two individual sequences, and had to have a "Phred" score of 23 or higher at the site of the presumed SNP.
  • no more than 50% mismatching with the consensus sequence was allowed.
  • the SNP alleles occur in polynucleotides found in public databases. Furthermore, it was found that the assembled contigs defined associated sets of two, or possibly more than two, alleles defined by an SNP at a particular polymorphic site. These associations were not previously known. The SNPs are presented in Table 1.
  • allelic sets in which one allele defines a known polypeptide sequence that includes the polymorphic site and another polypeptide allele is not previously known. Then, various associations of alleles are possible. For example, it is possible that an allelic pair is defined in a noncoding region of the contig containing an ORF. In such cases the inventors believe that the invention resides in the recognition of the allelic pair; this association has not heretofore been made. Alternatively, sets of allelic contigs may exist in which the polymorphic site is within an ORF, but does not result in an amino acid change among the allelic polypeptides.
  • the invention resides in the recognition of the allelic pair; and that this association has not heretofore been made.
  • the polymorphic site resides within an ORF and results in an amino acid change, or a frameshift, among the alleles of the allelic set.
  • at least one of the alleles at the polypeptide level is a known protein.
  • At least one of the remaining allele or alleles in the set, carrying a variant amino acid at the polymorphic site is a novel polypeptide not heretofore known.
  • the invention resides at least in the recognition of the polymorphic allele as being a variant of the known reference polypeptide.
  • Table 1 provides information concerning the allelic sequences.
  • One of the sequences may be termed a reference polymorphic sequence, and the corresponding second sequence includes the variant SNP at the polymorphic site.
  • the Sequence Listing accompanying this application provides the sequences.
  • the SEQ ID Nos are also cross-referenced in Table 1.
  • a reference to the SEQ ID NOs that correspond to the translated amino acid sequences are also given.
  • the Table includes thirteen columns that provide descriptive information for each cSNP, each of which occupies one row in the Table. The column headings, and a description of each, are given below.
  • SNPs disclosed in Table 1 were detected by aligning large numbers of sequences from genetically diverse sources of publicly available mRNA libraries (Clontech). Software designed specifically to look for multiple examples of variant bases differing from a consensus sequence was created and deployed. A criteria of a minimum of 2 occurrences of a sequence differing from the consensus in high quality sequence reads was used to identify an SNP.
  • the SNPs described herein may be useful in diagnostic kits, for DNA arrays on chips and for other uses that involve hybridization of the SNP.
  • Specific SNPs may have utility where a disease has already been associated with that gene. Examples of possible disease correlations between the claimed SNPs with members of the genes of each classification are listed below:
  • Amylase is responsible for endohydrolysis of 1,4-alpha-glucosidic linkages in oligosaccharides and polysaccharides. Variations in amylase gene may be indicative of delayed maturation and ofvarious amylase producing neoplasms and carcinomas.
  • the serum amyloid A (SAA) proteins comprise a family of vertebrate proteins that associate predominantly with high density lipoproteins (HDL). The synthesis of certain members of the family is greatly increased in inflammation. Prolonged elevation of plasma SAA levels, as in chronic inflammation, 15 results in a pathological condition, called amyloidosis, which affectsthe liver, kidney and spleen and which is characterized by the highly insoluble accumulation of SAA in these tissues. Amyloid selectively inhibits insulin-stimulated glucose utilization and glycogen deposition in muscle, while not affecting adipocyte glucose metabolism.
  • SAA serum amyloid A
  • Deposition of fibrillar amyloid proteins intraneuronally, as neurofibrillary tangles, extracellularly, as plaques and in blood vessels, is characteristic of both Alzheimer's disease and aged Down's syndrome. Amyloid deposition is also associated with type II diabetes mellitus.
  • angiogenesis is also an essential step in tumor growth in order for the tumor to get the blood supply it needs to expand. Variation in these genes may be predictive of any form of heart disease, numerous blood clotting disorders, stroke, hypertension and predisposition to tumor formation and metastasis. In particular, these variants may be predictive of the response to various antihypertensive drugs and chemotherapeutic and anti-tumor agents.
  • apoptosis Active cell suicide
  • apoptosis is induced by events such as growth factor withdrawal and toxins. It is controlled by regulators, which have either an inhibitory effect on programmed cell death (anti-apoptotic) or block the protective effect of inhibitors (pro-apoptotic).
  • regulators which have either an inhibitory effect on programmed cell death (anti-apoptotic) or block the protective effect of inhibitors (pro-apoptotic).
  • anti-apoptotic an inhibitory effect on programmed cell death
  • pro-apoptotic block the protective effect of inhibitors
  • Many viruses have found a way of countering defensive apoptosis by encoding their own anti-apoptosis genes preventing their target-cells from dying too soon. Variants of apoptosis related genes may be useful in formulation of antiaging drugs.
  • cyclins Members of the cell division/cell cycle pathways such as cyclins, many transcription factors and kinases, DNA polymerases, histones, helicases and other oncogenes play a critical role in carcinogenesis where the uncontrolled proliferation of cells leads to tumor formation and eventually metastasis.
  • Variation in these genes may be predictive of predisposition to any form of cancer, from increased risk of tumor formation to increased rate of metastasis. In particular, these variants may be predictive of the response to various chemotherapeutic and anti-tumor agents.
  • Granulocyte/macrophage colony-stimulating factors are cytokines that act in hematopoiesis by controlling the production, differentiation, and function of 2 related white cell populations of the blood, the granulocytes and the monocytes-macrophages.
  • Complement proteins are immune associated cytotoxic agents, acting in a chain reaction to exterminate target cells to that were opsonized (primed) with antibodies, by forming a membrane attack complex (MAC). The mechanism of killing is by opening pores in the target cell membrane.
  • Variations in 20 complement genes or their inhibitors are associated with many autoimmune disorders. Modified serum levels of complement products cause edemas ofvarious tissues, lupus (SLE), vasculitis, glomerulonephritis, renal failure, hemolytic anemia, thrombocytopenia, and arthritis. They interfere with mechanisms of ADCC (antibody dependent cell cytotoxicity), severely impair immune competence and reduce phagocytic ability.
  • Variants of complement genes may also be indicative of type I diabetes mellitus, meningitis neurological disorders such as Nemaline myopathy, Neonatal hypotonia, muscular disorders such as congenital myopathy and other diseases.
  • the respiratory chain is a key biochemical pathway which is essential to all aerobic cells.
  • cytochromes involved in the chain. These are heme bound proteins which serve as electron carriers. Modifications in these genes may be predictive of ataxia areflexia, dementia and myopathic and neuropathic changes in muscles. Also, association with various types of solid tumors.
  • Kinesins are tubulin molecular motors that function to transport organelles within cells and to move chromosomes along microtubules during cell division. Modifications of these genes may be indicative of neurological disorders such as Pick disease of the brain, tuberous sclerosis.
  • Cytokines such as erythropoietin are cell-specific in their growth stimulation; erythropoietin is useful for the stimulation of the proliferation of erythroblasts.
  • Variants in cytokines may be predictive for a wide variety of diseases, including cancer predisposition.
  • G-protein coupled receptors also called R7G are an extensive group of hormones, neurotransmitters, odorants and light receptors which transduce extracellular signals by interaction with guanine nucleotide-binding (G) proteins. Alterations in genes coding for G-coupled proteins may be involved in and indicative of a vast number of physiological conditions. These include blood pressure regulation, renal dysfunctions, male infertility, dopamine associated cognitive, emotional, and endocrine functions, hypercalcemia, chondrodysplasia and osteoporosis, pseudohypoparathyroidism, growth retardation and dwarfism. Thioesterases
  • Eukaryotic thiol proteases are a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. Variants of thioester associated genes may be predictive of neuronal disorders and mental illnesses such as Ceroid Lipoffiscinosis, Neuronal 1, Infantile, Santavuori disease and more.
  • SNPs are shown in Table 1 and the Sequence Listing. Both provide a summary of the polymorphic sequences disclosed herein.
  • a "SNP" is a polymorphic site embedded in a polymorphic sequence.
  • the polymorphic site is occupied by a single nucleotide, which is the position of nucleotide variation between the wild type and polymorphic allelic sequences.
  • the site is usually preceded by and followed by relatively highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
  • a polymorphic sequence can include one or more of the following sequences: (1) a sequence having the nucleotide denoted in Table 1, column 5 at the polymorphic site in the polymorphic sequence; or (2) a sequence having a nucleotide other than the nucleotide denoted in Table 1, column 5 at the polymo ⁇ hic site in the polymorphic sequence.
  • An example of the latter sequence is a polymorphic sequence having the nucleotide denoted in Table 1, column 6 at the polymo ⁇ hic site in the polymo ⁇ hic sequence.
  • Nucleotide sequences for a referenced-polymo ⁇ hic pair are presented in Table 1. Each cSNP entry provides information concerning the wild type nucleotide sequence as well as the corresponding sequence that includes the SNP at the polymo ⁇ hic site.
  • the Sequence Listing accompanying this application provides the sequences.
  • the SEQ ID NOs: are also cross- referenced in Table 1. A reference to the SEQ ID NOs: giving the translated amino acid sequences are also given if appropriate.
  • the Table includes thirteen columns that provide descriptive information for each cSNP, each of which occupies one row in the Table. The column headings, and an explanation for each, are given below.
  • SEQ ID provides the cross-references to the two nucleotide SEQ ID NOs: for the cognate pair, which are numbered consecutively, and, as explained below, amino acid SEQ ID NOs: as well, in the Sequence Listing of the application. Conversely, each sequence entry in the Sequence Listing also includes a cross-reference to the CuraGen sequence ID, under the label "CuraGen sequence ID”.
  • the first pair of SEQ ID NOs: given in the first column of each row of the Table are the SEQ ID NOs: identifying the nucleic acid sequences for the polymo ⁇ hisms.
  • a polymo ⁇ hism carries an entry for the amino acid portion of the row
  • a third SEQ ID NO: appears in parentheses in the column "Amino acid before” (see below) for the reference amino acid sequence
  • a fourth SEQ ID NO: appears in parentheses in the column “Amino acid after” (see below) for the polymo ⁇ hic amino acid sequence .
  • the latter SEQ ID NOs: refer to amino acid sequences giving the cognate reference and polymo ⁇ hic amino acid sequences that are the translation of the nucleotide polymo ⁇ hism. If a polymo ⁇ hism carries no entry for the protein portion of the row, only one pair SEQ ID NOs: is provided, in the first column.
  • Base pos. of SNP gives the numerical position of the nucleotide in the nucleic acid at which the cSNP is found, as identified in this invention.
  • Polymo ⁇ hic sequence provides a 51 -base sequence with the polymo ⁇ hic site at the 26 th base in the sequence, as well as 25 bases from the reference sequence on the 5' side and the 3' side of the polymo ⁇ hic site.
  • the designation at the polymo ⁇ hic site is enclosed in square brackets, and provides first, the reference nucleotide; second, a "slash (/)"; and third, the polymo ⁇ hic nucleotide.
  • the polymo ⁇ hism is an insertion or a deletion. In that case, the position which is "unfilled" (i.e., the reference or the polymo ⁇ hic position) is indicated by the word "gap".
  • Base before provides the nucleotide present in the reference sequence at the position at which the polymo ⁇ hism is found.
  • Base after provides the altered nucleotide at the position of the polymo ⁇ hism.
  • amino acid before provides the amino acid in the reference protein, if the polymo ⁇ hism occurs in a coding region.
  • This column also includes the SEQ ID NO: in parentheses for the translated reference amino acid sequence if the polymo ⁇ hism occurs in a coding region.
  • amino acid after provides the amino acid in the polymo ⁇ hic protein, if the polymo ⁇ hism occurs in a coding region.
  • This column also includes the SEQ ID NO: in parentheses for the translated polymo ⁇ hic amino acid sequence if the polymo ⁇ hism occurs in a coding region.
  • Type of change provides information on the nature of the polymo ⁇ hism.
  • SILENT-NONCODING is used if the polymo ⁇ hism occurs in a noncoding region of a nucleic acid.
  • SILENT-CODING is used if the polymo ⁇ hism occurs in a coding region of a nucleic acid of a nucleic acid and results in no change of amino acid in the translated polymo ⁇ hic protein.
  • CONSERVATIVE is used if the polymo ⁇ hism occurs in a coding region of a nucleic acid and provides a change in which the altered amino acid falls in the same class as the reference amino acid.
  • the classes are:
  • Acidic Asp, Glu, Asn, Gin;
  • End defines a termination codon
  • NONCONSERVATIVE is used if the polymo ⁇ hism occurs in a coding region of a nucleic acid and provides a change in which the altered amino acid falls in a different class than the reference amino acid.
  • FRAMESHIFT relates to an insertion or a deletion. If the frameshift occurs in a coding region, the Table provides the translation of the frameshifted codons 3' to the polymo ⁇ hic site.
  • Protein classification of CuraGen gene provides a generic class into which the protein is classified. During the course of the work leading to the filing of the four applications identified above, approximately 100 classes of proteins were identified. They are described further below.
  • Similarity (pvalue) following a BLASTX analysis provides the pvalue, a statistical measure from the BLASTX analysis that the polymo ⁇ hic sequence is similar to, and therefore an allele of, the reference, or wild-type, sequence.
  • a cutoff of pvalue > 1 x 10 "50 is used to establish that the reference- polymo ⁇ hic cognate pairs are novel.
  • Map location provides any information available at the time of filing related to localization of a gene on a chromosome.
  • the polymo ⁇ hisms are arranged in the Table in the following order.
  • SEQ ID Nos: 1-98 are nucleotide sequences for SNPs that lead to conservative amino acid changes.
  • SEQ ID Nos: 99-348 are nucleotide sequences for SNPs that lead to nonconservative amino acid changes.
  • SEQ ID NOs: 349-650 are nucleotide sequences for SNPs that involve a gap.
  • the allelic cSNP introduces an additional nucleotide (an insertion) or deletes a nucleotide (a deletion).
  • An SNP that involves a gap generates a frame shift.
  • SEQ ID NOs: 651-748 are the amino acid sequences centered at the polymo ⁇ hic amino acid residue for the protein products provided by SNPs that lead to conservative amino acid changes. 7 or 8 amino acids on either side of the polymo ⁇ hic site are shown. The order in which these sequences appear mirrors the order of presentation of the cognate nucleotide sequences, and is set forth in the Table.
  • SEQ ID NOs: 749-998 are the amino acid sequences centered at the polymo ⁇ hic amino acid residue for the protein products provided by SNPs that lead to nonconservative amino acid changes. 7 or 8 amino acids on either side of the polymo ⁇ hic site are shown. The order in which these sequences appear mirrors the order of presentation of the cognate nucleotide sequences, and is set forth in the Table.
  • SEQ ID NOs: 999-1300 are the amino acid sequences centered at the polymo ⁇ hic amino acid residue for the protein products provided by SNPs that lead to frameshift-induced amino acid changes. 7 or 8 amino acids on either side of the polymo ⁇ hic site are shown. The order in which these sequences appear mirrors the order of presentation of the cognate nucleotide sequences, and is set forth in the Table.
  • compositions which include, or are capable of detecting, nucleic acid sequences having these polymo ⁇ hisms, as well as methods of using nucleic acids.
  • polymo ⁇ hic alleles of the invention may be detected at either the DNA, the RNA, or the protein level using a variety of teclmiques that are well known in the art. Strategies for identification and detection are described in e.g., EP 730,663, EP 717,113, and PCT US97/02102.
  • the present methods usually employ pre-characterized polymo ⁇ hisms. That is, the genotyping location and nature of polymo ⁇ hic forms present at a site have already been determined. The availability of this information allows sets of probes to be designed for specific identification of the known polymo ⁇ hic forms.
  • PCR DNA Amplification
  • PCR Protocols A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR
  • recombinant protein refers to a peptide or protein produced using non-native cells that do not have an endogenous copy of DNA able to express the protein.
  • a recombinantly produced protein relates to the gene product of a polymo ⁇ hic allele, i.e., a "polymo ⁇ hic protein” containing an altered amino acid at the site of translation of the nucleotide polymo ⁇ hism.
  • the cells produce the protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequence.
  • the recombinant protein will not be found in association with proteins and other subcellular components normally associated with the cells producing the protein.
  • nucleic acid and “polypeptide” are used interchangeably herein.
  • substantially purified or “isolated” when referring to a nucleic acid, peptide or protein, means that the chemical composition is in a milieu containing fewer, or preferably, essentially none, of other cellular components with which it is naturally associated.
  • isolated or “substantially pure” refers to nucleic acid preparations that lack at least one protein or nucleic acid normally associated with the nucleic acid in a host cell. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution.
  • nucleic acid or protein Purity and homogeneity are typically determined using analytical chemistry techniques such as gel electrophoresis or high performance liquid chromatography.
  • a substantially purified or isolated nucleic acid or protein will comprise more than 80% of all macromolecular species present in the preparation.
  • the nucleic acid or protein is purified to represent greater than 90% of all macromolecular species present. More preferably the nucleic acid or protein is purified to greater than 95%), and most preferably the nucleic acid or protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional analytical procedures.
  • the genomic DNA used for the diagnosis may be obtained from any nucleated cells of the body, such as those present in peripheral blood, urine, saliva, buccal samples, surgical specimen, and autopsy specimens.
  • the DNA may be used directly or may be amplified enzymatically in vitro through use of PCR (Saiki et al. Science 239:487-491 (1988)) or other in vitro amplification methods such as the ligase chain reaction (LCR) (Wu and Wallace Genomics 4:560-569 (1989)), strand displacement amplification (SDA) (Walker et al. Proc. Natl. Acad. Sci. U.S.A. 89:392-396 (1992)), self-sustained sequence replication (3SR) (Fahy et al. PCR Methods P&J& 1:25-33 (1992)), prior to mutation analysis.
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • 3SR self-sustained sequence replication
  • nucleic acid is a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, including known analogs of natural nucleotides unless otherwise indicated.
  • nucleic acids refers to either DNA or RNA.
  • Nucleic acid sequence or “polynucleotide sequence” refers to a single-stranded sequence of deoxyribonucleotide or ribonucleotide bases read from the 5' end to the 3' end.
  • RNA transcripts The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA.and which are beyond the 5' end of the RNA transcript in the 5' direction are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are beyond the 3' end of the RNA transcript in the 3' direction are referred to as "downstream sequences".
  • upstream sequences sequence regions on the DNA strand having the same sequence as the RNA and which are beyond the 3' end of the RNA transcript in the 3' direction are referred to as "downstream sequences”.
  • upstream sequences sequence regions on the DNA strand having the same sequence as the RNA and which are beyond the 3' end of the RNA transcript in the 3' direction are referred to as "downstream sequences”.
  • upstream sequences sequence regions on the DNA strand having the same sequence as the RNA and which are beyond the 3' end of the
  • polymo ⁇ hisms in specific DNA sequences can be accomplished by a variety of methods including, but not limited to, restriction-fragment-length-polymo ⁇ hism detection based on allele-specific restriction-endonuclease cleavage (Kan and Dozy Lancet ii:910-912 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al. Nucl. Acids Res. 6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al. Proc. Natl. Acad. SCI. USA.
  • Specific hybridization refers to the binding, or duplexing, of a nucleic acid molecule only to a second particular nucleotide sequence to which the nucleic acid is complementary, under suitably stringent conditions when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA).
  • Stringent conditions are conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter ones.
  • stringent conditions are selected such that the temperature is about 5°C lower than the thermal melting point (Tm) for the specific sequence to which hybridization is intended to occur at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50%) of the target sequence hybridizes to the complementary probe at equilibrium.
  • stringent conditions include a salt concentration of at least about 0.01 to about 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3.
  • the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) .
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-specific probe hybridization.
  • “Complementary” or “target” nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., or Current Protocols in Molecular Biology, F. Ausubel et al., ed., Greene Publishing and Wiley- Interscience, New York (1987).
  • a perfectly matched probe has a sequence perfectly complementary to a particular target sequence.
  • the test probe is typically perfectly complementary to a portion of the target sequence.
  • a "polymo ⁇ hic" marker or site is the locus at which a sequence difference occurs with respect to a reference sequence.
  • Polymo ⁇ hic markers include restriction fragment length polymo ⁇ hisms, variable number of tandem repeats (VNTR'-s), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • the reference allelic form may be, for example, the most abundant form in a population, or the first allelic form to be identified, and other allelic forms are designated as alternative, variant or polymo ⁇ hic alleles.
  • the allelic form occurring most frequently in a selected population is sometimes referred to as the "wild type" form, and herein may also be referred to as the "reference" form.
  • Diploid organisms may be homozygous or heterozygous for allelic forms.
  • a diallelic polymo ⁇ hism has two distinguishable forms (i.e., base sequences), and a triallelic polymo ⁇ hism has three such forms.
  • an "oligonucleotide” is a single-stranded nucleic acid ranging in length from 2 to about 60 bases. Oligonucleotides are often synthetic but can also be produced from naturally occurring polynucleotides.
  • a probe is an oligonucleotide capable of binding to a target nucleic acid of a complementary sequence through one or more types of chemical bonds, usually through complementary base pairing via hydrogen bond formation. Oligonucleotides probes are often between 5 and 60 bases, and, in specific embodiments, may be between 10-40, or 15-30 bases long.
  • An oligonucleotide probe may include natural (i.e.
  • A, G, C, or T or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in an oligonucleotide probe may be joined by a linkage other than a phosphodiester bond, such as a phosphoramidite linkage or a phosphorothioate linkage, or they may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than by phosphodiester bonds, so long as it does not interfere with hybridization.
  • primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • a polymerization agent such as DNA polymerase, RNA polymerase or reverse transcriptase
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not be perfectly complementary to the exact sequence of the template, but should be sufficiently complementary to hybridize with it.
  • primer site refers to the sequence of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of
  • Oligonucleotides for use as primers or probes are chemically synthesized by methods known in the field of the chemical synthesis of polynucleotides, including by way of non- limiting example the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett 22:1859-1 862 (1981) and the triester method provided by Matteucci, et al., J. Am. Chem. Soc, 103:3185 (1981) both inco ⁇ orated herein by reference. These syntheses may employ an automated synthesizer, as described in Needham-VanDevanter, D.R., et al., Nucleic Acids Res. 12:61596168 (1984).
  • oligonucleotides Purification of oligonucleotides may be carried out by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J.D. and Regnier, F.E., ,J. Chrom,, 255:137-149 (1983).
  • a double stranded fragment may then be obtained, if desired, by annealing appropriate complementary single strands together under suitable conditions or by synthesizing the complementary strand using a DNA polymerase with an appropriate primer sequence.
  • a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid.
  • sequence of the synthetic oligonucleotide or of any nucleic acid fragment can be can be obtained using either the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al. Molecular Cloning - a Laboratory Manual ( " 2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989), which is inco ⁇ orated herein by reference. This manual is hereinafter referred to as "Sambrook et al.” ; Zyskind et al.,
  • Oligonucleotides useful in diagnostic assays are typically at least 8 consecutive nucleotides in length, and may range upwards of 18 nucleotides in length to greater than 100 or more consecutive nucleotides.
  • antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the SNP- containing nucleotide sequences of the invention, or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, about 25, about 50, or about 60 nucleotides or an entire SNP coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a polymo ⁇ hic nucleotide sequence of the invention.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence of the invention.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can generally be complementary to the entire coding region of an mRNA, but more preferably as embodied herein, it is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of the mRNA.
  • An antisense oligonucleotide can range in length between about 5 and about 60 nucleotides, preferably between about 10 and about 45 nucleotides, more preferably between about 15 and 40 nucleotides, and still more preferably between about 15 and 30 in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantbine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycar
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following section).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polymo ⁇ hic protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementary to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual -uni ts, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) FEBS Lett 115: 311-330).
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence.
  • Optimal alignment of sequences for aligning a comparison window may, for example, be conducted by the local homology algorithm of Smith and Waterman Adv. Appl.
  • nucleic acid sequence encoding refers to a nucleic acid which directs the expression of a specific protein, peptide or amino acid sequence.
  • the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein, peptide or amino acid sequence.
  • the nucleic acid sequences include both the full length nucleic acid sequences disclosed herein as well as non-full length sequences derived from the full length protein. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Consequently, the principles of probe selection and array design can readily be extended to analyze more complex polymo ⁇ hisms (see EP 730,663). For example, to characterize a triallelic SNP polymo ⁇ hism, three groups of probes can be designed tiled on the three polymo ⁇ hic forms as described above.
  • Genomic DNA is typically amplified before analysis. Amplification is usually effected by PCR using primers flanking a suitable fragment e.g., of 50-500 nucleotides containing the locus of the polymo ⁇ hism to be analyzed. Target is usually labeled in the course of amplification.
  • the amplification product can be RNA or DNA, single stranded or double Stranded. If double stranded, the amplification product is typically denatured before application to an array. If genomic DNA is analyzed without amplification, it may be desirable to remove RNA from the sample before applying it to the array. Such can be accomplished by digestion with DNase-free RNase.
  • the SNPs disclosed herein can be used to determine which forms of a characterized polymo ⁇ hism are present in individuals under analysis.
  • Allele-specific probes for analyzing polymo ⁇ hisms is described by e.g., Saiki et al., Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymo ⁇ hic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymo ⁇ hic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 7, 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms.
  • Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymo ⁇ hisms within the same target sequence.
  • the polymo ⁇ hisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in published PCT application WO 95/11995.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymo ⁇ hism.
  • Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence.
  • the second group of probes is designed by the same principles, except that the probes exhibit complementarity to the second reference sequence.
  • a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e.g., two or more mutations within 9 to 21 bases).
  • An allele-specific primer hybridizes to a site on a target DNA overlapping a polymo ⁇ hism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two-primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymo ⁇ hic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymo ⁇ hism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co New York, 1992, Chapter 7).
  • Alleles of target sequences can be differentiated using single-strand conformation polymo ⁇ hism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).
  • Amplified PCR products can be generated and heated or otherwise denatured, to form single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • the genotype of an individual with respect to a pathology suspected of being caused by a genetic polymo ⁇ hism may be assessed by association analysis.
  • Phenotypic traits suitable for association analysis include diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulinemia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent po ⁇ hyria).
  • diseases that have known but hitherto unmapped genetic components e.g., agammaglobulinemia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy,
  • Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms.
  • autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non- independent), systemic lupus erythematosus and Graves disease.
  • cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, oral cavity, ovary, pancreas, prostate, skin, stomach, leukemia, liver, lung, and uterus.
  • Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments.
  • polymo ⁇ hisms of the invention are often used in conjunction with polymo ⁇ hisms in distal genes.
  • Preferred polymo ⁇ hisms for use in forensics are diallelic because the population frequencies of two polymo ⁇ hic forms can usually be determined with greater accuracy than those of multiple polymo ⁇ hic forms at multi-allelic loci.
  • the capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymo ⁇ hic forms occupying selected polymo ⁇ hic sites is the same in the suspect and the sample. If the set of polymo ⁇ hic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source of the sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene. If frequencies of the polymo ⁇ hic forms at the loci tested have been determined (e.g., by analysis of a suitable population of individuals), one can perform a statistical analysis to determine the probability that a match of suspect and crime scene sample would occur by chance.
  • p(ID) is the probability that two random individuals have the same polymo ⁇ hic or allelic form at a given polymo ⁇ hic site. In diallelic loci, four genotypes are possible: AA, AB, BA, and BB. If alleles A and B occur in a haploid genome of the organism with frequencies x and y, the probability of each genotype in a diploid organism are (see WO 95/12607):
  • the probability of identity p(ID) for a 3-allele system where the alleles have the frequencies in the population of x, y and z, respectively, is equal to the sum of the squares of the genotype frequencies:
  • p(ID) x 4+ (lxy) 2+ (lyz) + (lxz) + Z 4+ y 4
  • the appropriate binomial expansion is used to calculate p(ID) and p(exc).
  • the cumulative probability of identity (cum p(ID)) for each of multiple unlinked loci is determined by multiplying the probabilities provided by each locus:
  • the object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the putative father. Paternity testing can be performed by analyzing sets of polymo ⁇ hisms in the putative father and the child.
  • x and y are the population frequencies of alleles A and B of a diallelic polymo ⁇ hic site.
  • p(exc) xy(l-xy)+ yz(l-yz)+ xz(l-xz)+ 3xyz(l-xyz))
  • x, y and z and the respective population frequencies of alleles A, B and C are:
  • the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymo ⁇ hic marker set matches the child's polymo ⁇ hic marker set attributable to his/her father.
  • the polymo ⁇ hisms of the invention may contribute to the phenotype of an organism in different ways. Some polymo ⁇ hisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymo ⁇ hisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymo ⁇ hism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymo ⁇ hisms in different genes. Further, some polymo ⁇ hisms predispose an individual to a distinct mutation that is causally related to a certain phenotype.
  • Phenotypic traits include diseases that have known but hitherto unmapped genetic components. Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms. Some examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non-independent), systemic lupus erythematosus and Graves disease.
  • cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, sldn, stomach and uterus.
  • Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments. Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymo ⁇ hic marker sets. To perform such analysis, the presence or absence of a set of polymo ⁇ hisms (i.e.
  • a polymo ⁇ hic set is determined for a set of the individuals, some of whom exhibit a particular trait, and some of whom exhibit lack of the trait.
  • the alleles of each polymo ⁇ hism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest. Correlation can be performed by standard statistical methods and statistically significant correlations between polymo ⁇ hic form(s) and phenotypic characteristics are noted. For example, it might be found that the presence of allele Al at polymo ⁇ hism A correlates with heart disease. As a further example, it might be found that the combined presence of allele Al at polymo ⁇ hism A and allele Bl at polymo ⁇ hism B correlates with increased milk production of a farm animal.
  • Such correlations can be exploited in several ways.
  • detection of the polymo ⁇ hic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient.
  • Detection of a polymo ⁇ hic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions.
  • the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymo ⁇ hism from her husband to her offspring.
  • the present section describes identification of a physical linkage between a genetic locus associated with a trait of interest and polymo ⁇ hic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it.
  • Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al., Proc. Natl. Acad. Sci. (USA) 83, 7353-7357 (1986); Lander et al, Proc. Natl Acad. Sci.
  • Linkage studies are typically performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for a set of polymo ⁇ hic markers. The distribution of polymo ⁇ hic markers in an informative meiosis is then analyzed to determine which polymo ⁇ hic markers co-segregate with a phenotypic trait. See, e.g., Kerem et al., Science 245, 1073-1080 (1989); Monaco et al., Nature 316, 842 (1985); Yamoka et al., Neurology 40, 222-226 (1990); Rossiter et al., FASEB Journal 5, 21-27 (1991).
  • Linkage is analyzed by calculation of LOD (log of the odds) values.
  • a lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction RF, versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome” in The Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4).
  • the likelihood at a given value of RF is: probability of data if loci linked at i? to probability of data if loci unlinked.
  • the computed likelihood is usually expressed as the logjo of this ratio (i.e., a lod score). For example, a lod score of 3 indicates
  • lod scores for differing values of RF (e.g., LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)).
  • RF e.g., LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)
  • a recombination fraction may be determined from mathematical tables. See Smith et al., Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann. Hum. Genet. 32, 127-150 (1968). The value of RF at which the lod score is the highest is considered to be the best estimate of the recombination fraction.
  • Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search focuses on the remaining non- excluded chromosomal locations.
  • the invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated.
  • Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinj ecting the construct into a zygote. See Hogan et al., "Manipulating the Mouse Embryo, A Laboratory Manual," Cold Spring Harbor Laboratory. (1989).
  • Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker.
  • transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems.
  • the invention further provides methods for assessing the pharmacogenomic susceptibility of a subject harboring a single nucleotide polymo ⁇ hism to a particular pharmaceutical compound, or to a class of such compounds.
  • Genetic polymo ⁇ hism in drug-metabolizing enzymes, drug transporters, receptors for pharmaceutical agents, and other drug targets have been correlated with individual differences based on distinction in the efficacy and toxicity of the pharmaceutical agent administered to a subject.
  • Pharmocogenomic characterization of a subjects susceptibility to a drug enhances the ability to tailor a dosing regimen to the particular genetic constitution of the subject, thereby enhancing and optimizing the therapeutic effectiveness of the therapy.
  • method of treating such a condition includes administering to a subject experiencing the pathology the wild type cognate of the polymo ⁇ hic protein. Once administered in an effective dosing regimen, the wild type cognate provides complementation or remediation of the defect due to the polymo ⁇ hic protein. The subject's condition is ameliorated by this protein therapy.
  • a subject suspected of suffering from a pathology ascribable to a polymo ⁇ hic protein that arises from a cSNP is to be diagnosed using any of a variety of diagnostic methods capable of identifying the presence of the cSNP in the nucleic acid, or of the cognate polymo ⁇ hic protein, in a suitable clinical sample taken from the subject.
  • the subject is treated with a pharmaceutical composition that includes a nucleic acid that harbors the correcting wild-type gene, or a fragment containing a correcting sequence of the wild-type gene.
  • Non-limiting examples of ways in which such a nucleic acid may be administered include inco ⁇ orating the wild-type gene in a viral vector, such as an adenovirus or adeno associated virus, and administration of a naked DNA in a pharmaceutical composition that promotes intracellular uptake of the administered nucleic acid.
  • a viral vector such as an adenovirus or adeno associated virus
  • administration of a naked DNA in a pharmaceutical composition that promotes intracellular uptake of the administered nucleic acid Once the nucleic acid that includes the gene coding for the wild-type allele of the polymo ⁇ hism is inco ⁇ orated within a cell of the subject, it will initiate de novo biosynthesis of the wild-type gene product. If the nucleic acid is further inco ⁇ orated into the genome of the subject, the treatment will have long-term effects, providing de novo synthesis of the wild-type protein for a prolonged duration. The synthesis of the wild-type protein in the cells of the subject will contribute to a therapeutic enhancement of the
  • a subject suffering from a pathology ascribed to a SNP may be treated so as to correct the genetic defect.
  • Such a subject is identified by any method that can detect the polymo ⁇ hism in a sample drawn from the subject.
  • Such a genetic defect may be permanently corrected by administering to such a subject a nucleic acid fragment inco ⁇ orating a repair sequence that supplies the wild-type nucleotide at the position of the SNP.
  • This site-specific repair sequence encompasses an RNA/DNA oligonucleotide which operates to promote endogenous repair of a subject's genomic DNA.
  • a genetic defect leading to an inborn pathology may be overcome, as the chimeric oligonucleotides induces inco ⁇ oration of the wild-type sequence into the subject's genome.
  • the wild-type gene product is expressed, and the replacement is propagated, thereby engendering a permanent repair.
  • kits comprising at least one allele-specific oligonucleotide as described above.
  • the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymo ⁇ hism.
  • the allele-specific oligonucleotides are provided immobilized to a substrate.
  • the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100, 1000 or all of the polymo ⁇ hisms shown in the Table.
  • kits include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions.
  • the kit also contains instructions for carrying out the hybridizing methods.
  • nucleic acids comprising a SNP of the inventions.
  • DNA is isolated from a genomic or cDNA library using labeled oligonucleotide probes having sequences complementary to the sequences disclosed herein.
  • probes can be used directly in hybridization assays.
  • probes can be designed for use in amplification techniques such as PCR.
  • mRNA is isolated from tissue such as heart or pancreas, preferably a tissue wherein expression of the gene or gene family is likely to occur.
  • cDNA is prepared from the mRNA and ligated into a recombinant vector. The vector is transfected into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known, See Gubler, U. and Hoffman, B.J. Gene 25:263-269 (1983) and Sambrook et al.
  • the DNA is extracted from tissue and either mechanically sheared or enzymatically digested to yield fragments of about 12-20 kb. The fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors. These vectors and phage are packaged in vitro, as described in Sambrook, et al. Recombinant phage are analyzed by plaque hybridization as described in Benton and Davis, Science 196:180-1 82 (1977). Colony hybridization is carried out as generally described in M. Grunstein et al. Proc. Natl. Acad. Sci. USA. 72:3961-3965 (1975).
  • DNA of interest is identified in either cDNA or genomic libraries by its ability to hybridize with nucleic acid probes, for example on Southern blots, and these DNA regions are isolated by standard methods familiar to those of skill in the art. See Sambrook, et al. In PCR techniques, oligonucleotide primers complementary to the two 3' borders of the
  • DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See PCR Protocols: a Guide to Methods and Applications (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990). Primers can be selected to amplify the entire regions encoding a full-length sequence of interest or to amplify smaller DNA segments as desired. PCR can be used in a variety of protocols to isolate cDNAs encoding a sequence of interest. In these protocols, appropriate primers and probes for amplifying DNA encoding a sequence of interest are generated from analysis of the DNA sequences listed herein. Once such regions are PCR-amplified, they can be sequenced and oligonucleotide probes can be prepared from the sequence.
  • DNA encoding a sequence comprising a cSNP is isolated and cloned, one can express the encoded polymo ⁇ hic proteins in a variety of recombinantly engineered cells. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of DNA encoding a sequence of interest. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes is made here.
  • the expression of natural or synthetic nucleic acids encoding a sequence of interest will typically be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by inco ⁇ oration into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
  • Typical expression vectors contain initiation sequences, transcription and translation terminators, and promoters useful for regulation of the expression of a polynucleotide sequence of interest.
  • expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription translation terminator.
  • the expression vectors may also comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems. See Sambrook et al.
  • prokaryotic expression systems may be used to express the polymo ⁇ hic proteins of the invention. Examples include E. coli, Bacillus, Streptomyces, and the like.
  • expression plasmids which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription translation terminator.
  • regulatory regions suitable for this pu ⁇ ose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky, C, J. Bacterial. 158:1018-1024 (1984) and the leftward promoter of phage lambda as described by A, I. and Hagen. P.. Ann. Rev. Genet. 14:399-445 (1980).
  • selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. See Sambrook et al. for details concerning selection markers for use in E. coli.
  • the expressed protein may first be denatured and then renatured. This can be accomplished by solubilizing the bacterially produced proteins in a chaotropic agent such as guanidine HCI and reducing all the cysteine residues with a reducing agent such as beta- mercaptoethanol. The protein is then renatured, either by slow dialysis or by gel filtration. See U.S. Patent No. 4,511,503. Detection of the expressed antigen is achieved by methods known in the art as radioimmunoassay, or Western blotting techniques or immunoprecipitation. Purification from E. coli can be achieved following procedures such as those described in U.S. Patent No. 4,511,503.
  • Any of a variety of eukaryotic expression systems such as yeast, insect cell lines, bird, fish, and mammalian cells, may also be used to express a polymo ⁇ hic protein of the invention.
  • a nucleotide sequence harboring a cSNP may be expressed in these eukaryotic systems. Synthesis of heterologous proteins in yeast is well known. Methods in Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods available to produce the protein in yeast.
  • Suitable vectors usually have expression control sequences, such as promoters, including 3 -phosphogty cerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • promoters including 3 -phosphogty cerate kinase or other glycolytic enzymes
  • origin of replication termination sequences and the like as desired.
  • suitable vectors are described in the literature (Botstein, et al., Gene 8:17-24 (1979); Broach, et al, Gene 8:121-133 (1979)).
  • yeast cells are first converted into protoplasts using zymolyase, lyticase or glusulase, followed by addition of DNA and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG-treated protoplasts are then regenerated in a 3% agar medium under selective conditions. Details of this procedure are given in the papers by J.D. Beggs, Nature (London) 275:104-109 (1978); and Hinnen, A., et al., Proc. Natl. Acad. Sci. USA, 75:1929-1933 (1978).
  • the second procedure does not involve removal of the cell wall.
  • the purification process can be monitored by using Western blot techniques or radioimmunoassay or other standard techniques.
  • the sequences encoding the proteins of the invention can also be ligated to various immunoassay expression vectors for use in transforming cell cultures of, for instance, mammalian, insect, bird or fish origin.
  • Illustrative of cell cultures useful for the production of the polypeptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines, and various human cells such as COS cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen et al. Immunol. Rev. 89:49 (1986)) and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
  • a promoter e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kina
  • vectors for expressing the proteins of the invention in insect cells are usually derived from baculo virus.
  • Insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider J. Embryol. Exp. Mo ⁇ hol., 27:353-365 (1987).
  • the vector e.g., a plasmid, which is used to transform the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the protein. These sequences are referred to as expression control sequences.
  • polyadenylation or transcription terminator sequences from known mammalian genes need to be inco ⁇ orated into the vector.
  • An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV4O (Sprague, J. et a/., J. Virol. 45: 773-781 (1983)).
  • gene sequences to control replication in the host cell may be Saveria-Campo, M., 1985, "Bovine Papilloma virus DNA a Eukaryotic Cloning Vector" in DNA Cloning Vol.
  • the host cells are competent or rendered competent for transformation by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation and micro-injection of the DNA directly into the cells.
  • the transformed cells are cultured by means well known in the art (Biochemical Methods in Cell Culture and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., (1977)).
  • the expressed polypeptides are isolated from cells grown as suspensions or as monolayers. The latter are recovered by well known mechanical, chemical or enzymatic means.
  • operably linked refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence.
  • operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the gene encoding the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression sequence.
  • vector refers to viral expression systems, autonomous self-replicating circular DNA (plasmids), and includes both expression and nonexpression plasmids.
  • gene as used herein is intended to refer to a nucleic acid sequence which encodes a polypeptide. This definition includes various sequence polymo ⁇ hisms, mutations, and/or sequence variants wherein such alterations do not affect the function of the gene product.
  • gene is intended to include not only coding sequences but also regulatory regions such as promoters, enliancers, termination regions and similar untranslated nucleotide sequences. The term further includes all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A43 1 cells, human Col 0205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL- 60, U937, HaK or Jurkat cells.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida or any yeast strain capable of expressing heterologous proteins.
  • Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein.
  • the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available * in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac ⁇ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), inco ⁇ orated herein by reference.
  • an insect cell capable of expressing_a polynucleotide of the present invention is "transformed.”
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the polymo ⁇ hic protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
  • the protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art.
  • the polymo ⁇ hic proteins produced by recombinant DNA technology may be purified by techniques commonly employed to isolate or purify recombinant proteins.
  • Recombinantly produced proteins can be directly expressed or expressed as a fusion protein.
  • the protein is then purified by a combination of cell lysis (e.g., sonication) and affinity chromatography.
  • cell lysis e.g., sonication
  • affinity chromatography e.g., affinity chromatography
  • subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired polypeptide.
  • the polypeptides of this invention may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R.
  • antibodies may be raised to the proteins of the invention as described herein.
  • Cell membranes are isolated from a cell line expressing the recombinant protein, the protein is extracted from the membranes and immunoprecipitated. The proteins may then be further purified by standard protein chemistry techniques as described above.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-Toyopearl@ or Cibacrom blue 3GA
  • Sepharose B one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will facilitate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from Kodak (New Haven, CT).
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
  • the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen, such as polymo ⁇ hic.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, F ab and F (ab')2 fragments, and an F a b expression library.
  • antibodies to human polymo ⁇ hic proteins are disclosed.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassay s are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, a Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • Antibodies that immunospecifically bind to polymo ⁇ hic gene products but not to the corresponding prototypical or "wild-type" gene products are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide.
  • Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product.
  • An isolated polymo ⁇ hic protein, or a portion or fragment thereof, can be used as an immunogen to generate the antibody that binds the polymo ⁇ hic protein using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length polymo ⁇ hic protein can be used or, alternatively, the invention provides antigenic peptide fragments of polymo ⁇ hic for use as immunogens.
  • the antigenic peptide of a polymo ⁇ hic protein of the invention comprises at least 8 amino acid residues of the amino acid sequence encompassing the polymo ⁇ hic amino acid and encompasses an epitope of the polymo ⁇ hic protein such that an antibody raised against the peptide forms a specific immune complex with the polymo ⁇ hic protein.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of polymo ⁇ hic that are located on the surface of the protein, e.g., hydrophilic regions.
  • polymo ⁇ hic protein For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the polymo ⁇ hic protein.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed polymo ⁇ hic protein or a chemically synthesized polymo ⁇ hic polypeptide. The preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyois, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • the antibody molecules directed against polymo ⁇ hic proteins can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography, to obtain the IgG fraction.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that originates from the clone of a singly hybridoma cell, and that contains only one type of antigen binding site capable of immunoreacting with a particular epitope of a polymo ⁇ hic protein.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polymo ⁇ hic protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized.
  • Such techniques include, but are not limited to, the hybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al, 1983.
  • techniques can be adapted for the production of single-chain antibodies specific to a polymo ⁇ hic protein (see e.g., U.S. Patent No. 4,946,778).
  • methodologies can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a polymo ⁇ hic protein or derivatives, fragments, analogs or homologs thereof.
  • Non-human antibodies can be "humanized" by techniques well known in the art. See e.g., U.S. Patent No. 5,225,539.
  • Antibody fragments that contain the idiotypes to a polymo ⁇ hic protein may be produced by techniques known in the art including, but not limited to: (i) an F (ab' ) 2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F (ab' ) 2 fragment; (Hi) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • anti-polymo ⁇ hic protein antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International
  • methodologies for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme-linked immunosorbent assay
  • Anti-polymo ⁇ hic protein antibodies may be used in methods known within the art relating to the detection, quantitation and/or cellular or tissue localization of a polymo ⁇ hic protein (e.g., for use in measuring levels of the polymo ⁇ hic protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies for polymo ⁇ hic proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody-derived CDR are utilized as pharmacologically-active compounds in therapeutic applications intended to treat a pathology in a subject that arises from the presence of the cSNP allele in the subject.
  • An anti-polymo ⁇ hic protein antibody (e.g., monoclonal antibody) can be used to isolate polymo ⁇ hic proteins by a variety of immunochemical techniques, such as immunoaffinity chromatography or immunoprecipitation.
  • An anti-polymo ⁇ hic protein antibody can facilitate the purification of natural polymo ⁇ hic protein from cells and of recombinantly produced polymo ⁇ hic proteins expressed in host cells.
  • an anti-polymo ⁇ hic protein antibody can be used to detect polymo ⁇ hic protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polymo ⁇ hic protein.
  • Anti-polymo ⁇ hic antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H.

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Abstract

L'invention concerne des acides nucléiques renfermant des polymorphismes de nucléotide simple identifiés pour des séquences humaines transcrites, ainsi que des procédés d'utilisation de ces acides nucléiques.
PCT/US2001/000322 2000-01-07 2001-01-05 Acides nucleiques renfermant des polymorphismes de nucleotide simple et procede d'utilisation associe WO2001051670A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002395607A CA2395607A1 (fr) 2000-01-07 2001-01-05 Acides nucleiques renfermant des polymorphismes de nucleotide simple et procede d'utilisation associe
AU26308/01A AU2630801A (en) 2000-01-07 2001-01-05 Nucleic acids containing single nucleotide polymorphisms and methods of use thereof
EP01900897A EP1250456A2 (fr) 2000-01-07 2001-01-05 Acides nucleiques renfermant des polymorphismes de nucleotide simple et procede d'utilisation associe

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17496200P 2000-01-07 2000-01-07
US60/174,962 2000-01-07

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WO2001051670A2 true WO2001051670A2 (fr) 2001-07-19
WO2001051670A3 WO2001051670A3 (fr) 2002-02-28

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003027321A3 (fr) * 2001-09-24 2003-11-06 Univ Aarhus Methodes de diagnostic et de traitement de maladies associees a une expression alteree de neurogranine
WO2003040369A3 (fr) * 2001-09-17 2004-07-29 Molecular Engines Lab Sequences impliquees dans les phenomenes de suppression tumorale, reversion tumorale, apoptose et/ou resistance aux virus et leur utilisation comme medicaments
WO2019048555A1 (fr) * 2017-09-06 2019-03-14 Jaerver Peter Oligonucléotides simple brin inhibant l'endocytose

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5858659A (en) * 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US5856104A (en) * 1996-10-28 1999-01-05 Affymetrix, Inc. Polymorphisms in the glucose-6 phosphate dehydrogenase locus
WO1998020165A2 (fr) * 1996-11-06 1998-05-14 Whitehead Institute For Biomedical Research Marqueurs bialleliques
US6054632A (en) * 1996-11-15 2000-04-25 New York Blood Center, Inc. Method of making monoclonal antibodies using polymorphic transgenic animals
CA2274317A1 (fr) * 1996-12-02 1998-07-16 Biocem S.A. Sequences vegetales comprenant un site polymorphe et utilisation de celles-ci
WO1998038846A2 (fr) * 1997-03-07 1998-09-11 Affymetrix, Inc. Compositions genetiques et procedes

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040369A3 (fr) * 2001-09-17 2004-07-29 Molecular Engines Lab Sequences impliquees dans les phenomenes de suppression tumorale, reversion tumorale, apoptose et/ou resistance aux virus et leur utilisation comme medicaments
WO2003027321A3 (fr) * 2001-09-24 2003-11-06 Univ Aarhus Methodes de diagnostic et de traitement de maladies associees a une expression alteree de neurogranine
WO2019048555A1 (fr) * 2017-09-06 2019-03-14 Jaerver Peter Oligonucléotides simple brin inhibant l'endocytose
US11345916B2 (en) 2017-09-06 2022-05-31 Tirmed Pharma Ab Single stranded oligonucleotides inhibiting endocytosis

Also Published As

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WO2001051670A3 (fr) 2002-02-28
EP1250456A2 (fr) 2002-10-23
AU2630801A (en) 2001-07-24
CA2395607A1 (fr) 2001-07-19

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