EP1563090A2 - Procedes, compositions et logiciels informatiques pour interroger des variations de sequences dans les regions genomiques fonctionnelles - Google Patents

Procedes, compositions et logiciels informatiques pour interroger des variations de sequences dans les regions genomiques fonctionnelles

Info

Publication number
EP1563090A2
EP1563090A2 EP03768853A EP03768853A EP1563090A2 EP 1563090 A2 EP1563090 A2 EP 1563090A2 EP 03768853 A EP03768853 A EP 03768853A EP 03768853 A EP03768853 A EP 03768853A EP 1563090 A2 EP1563090 A2 EP 1563090A2
Authority
EP
European Patent Office
Prior art keywords
functional regions
functional
genome
determining
regions
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03768853A
Other languages
German (de)
English (en)
Other versions
EP1563090A4 (fr
Inventor
Thomas R. Gingeras
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Affymetrix Inc
Original Assignee
Affymetrix Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Affymetrix Inc filed Critical Affymetrix Inc
Publication of EP1563090A2 publication Critical patent/EP1563090A2/fr
Publication of EP1563090A4 publication Critical patent/EP1563090A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids

Definitions

  • the present invention relates to genetic analysis, genomics, biological assays and bioinformatics. Specifically, in one aspect of the invention, methods, compositions and computer software products are provided for analyzing genetic variations in functional regions.
  • RNA transcription sites, transcription factor binding sites, origins, methylation and chromatin modification sites, etc. are determined in biological samples. Typically, the samples may reflect various physiological, pathological, toxicological or pharmacological states.
  • the RNA transcription sites, transcription factor binding sites, origins, methylation and chromatin modification sites, etc. confer specific functions on these genomic regions and ascribe to them a priority status for analyzing the presence of sequence variations.
  • Regions such as these, associated with specific biological functions, are referred to as "functional regions" in this specification.
  • the functional regions can be determined using a variety of methods including the use of high density oligonucleotide probe arrays.
  • the sequence variations are determined on a large scale, for example, at least 500, 1000, 5000, 10000, or 100000 SNPs.
  • association or linkage studies highlight several regions of the genome as possible sites which may be involved in determining a trait in affected families or individuals, the presence of the functional regions in these regions can be empirically determined and can narrow down the possibilities for further analysis.
  • genomic and cDNA sequences in these regions can be empirically determined and can be analyzed by sequencing or SNP testing or Comparative Genomic Hybridization (CGH) testing in preference to other regions which will be important (but not exclusive) for sequence variations which are outside of coding regions.
  • CGH Comparative Genomic Hybridization
  • Transcription factor (TF) binding sites can be detected using a variety of methods including the use of high density oligonucleotide probe arrays.
  • DNA fragments protected by transcription binding factors are obtained using immunoprecipitation and interrogated using high density arrays to determine regions with DNA sequences that are bound with transcriptional binding factors.
  • WGS A is an assay that reduces the complexity of a genomic sample by obtaining representative restriction fragments.
  • the reduced-complexity genomic samples can be used for hybridization with high density oligonucleotide probe arrays to interrogate SNPs and perform resequencing (sequence variation detection).
  • sequences involved in various functional operations of the cell such as transcription factor bindings, origins, methylation and chromatin modification sites become defined, these sites should be examined for the presence of RE sites. This is so that for a specific functional class of sequences (e.g., TF binding sequences) the fewest number of REs could be identified which would allow for surveying the largest number of such sites across the genome for possible sequence variations present in these sequences.
  • TF binding sequences e.g., TF binding sequences
  • the invention relates to a method for interrogating genetic variations comprising obtaining a plurality of functional regions of the genome, wherein the functional regions comprise at least 10,000 bases and determining sequence variations of a plurality of individuals in the functional regions of the genome.
  • the functional regions comprise a plurality of transcription factor binding sites, a plurality of RNA:protein binding domains, a plurality of chromatin modification sites, a plurality of origins of replication, and/or a plurality of DNA methylation sites.
  • the obtaining comprises determining functional regions using microarrays, e.g., high density oligonucleotide arrays.
  • the microarrays comprise oligonucleotide probes tiling regions of the genome.
  • the determining comprises determining the sequences of the functional regions of a plurality of individuals.
  • the determining comprises determining the genotypes of the functional regions of a plurality of individuals, e.g., SNP genotypes.
  • the determining comprises performing a WGSA with at least one restriction enzyme that is suitable for interrogating at least one functional region, hi one embodiment, the determining comprises determining sequence copy number changes, one embodiment, the functional regions comprise at least 100000 bases or at least 500000 bases.
  • the invention also relates to a method for interrogating genetic variations comprising obtaining at least one interested genomic segment; obtaining a plurality of functional regions within the interested genomic segment, wherein the functional regions comprise at least 5,000 bases; and determining sequence variations of a plurality of individuals in the functional regions of the genome, hi one embodiment, the interested genomic region is determined by association or linkage analysis.
  • the functional regions comprise a plurality of transcription factor binding sites, a plurality of RNA:protein binding domains, a plurality of chromatin modification sites, a plurality of origins of replication and/or a plurality of DNA methylation sites.
  • the obtaining comprises determining functional regions using microarrays, e.g., high density oligonucleotide arrays.
  • the microarrays comprise oligonucleotide probes tiling regions of the genome.
  • the determining comprises determining the sequences of the functional regions of a plurality of individuals, h another embodiment, the determining comprises determining the genotypes of the functional regions of a plurality of individuals, hi one embodiment, the genotypes are SNP genotypes.
  • the determining comprises performing a WGSA with at least one restriction enzyme that is suitable for interrogating at least one functional region. In another embodiment, the determining comprises determining sequence copy number changes
  • Figure 1 is a schematic showing an exemplary process of genotyping functional regions in a genome.
  • Figure 2 is a schematic showing an exemplary process of determining sequence variations in functional regions of regions identified by association or linkage analysis.
  • an agent includes a plurality of agents, including mixtures thereof.
  • An individual is not limited to a human being but may also be other organisms including but not limited to mammals, plants, bacteria, or cells derived from any of the above.
  • the practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill in the art.
  • Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used.
  • Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual,
  • the present invention can employ solid substrates, including arrays in some preferred embodiments.
  • Patents that describe synthesis techniques in specific embodiments include United States Patent Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are described in many of the above patents, but the same techniques are applied to polypeptide arrays.
  • Nucleic acid arrays that are useful in the present invention include those that are commercially available from Affymetrix (Santa Clara, CA) under the brand name GeneChip®. Example arrays are shown on the website at affymetrix.com.
  • the present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Gene expression monitoring and profiling methods can be shown in United States Patents Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore are shown in USSN 60/319,253, 10/013,598 (U.S.
  • Patent Publication Number US-2003-0036069- Al United States Patent Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in United States Patents Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.
  • the present invention also contemplates sample preparation methods in certain preferred embodiments.
  • the genomic sample Prior to or concurrent with genotyping, the genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H.A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols:. A Guide to Methods and
  • LCR ligase chain reaction
  • LCR ligase chain reaction
  • Landegren et al. Science 241, 1077 (1988) and Barringer et al. Gene 89: 117 (1990)
  • transcription amplification Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO 88/10315
  • self-sustained sequence replication Guatelli et al, Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO 90/06995
  • selective amplification of target polynucleotide sequences United States Patent No.
  • Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al., Molecular Cloning: A Laboratory Manual (2 nd Ed. Cold Spring Harbor, N.Y, 1989); Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, CA, 1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. patent 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which is incorporated herein by reference
  • the present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See United States Patent Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601;
  • Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention.
  • Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc.
  • the computer executable instructions may be written in a suitable computer language or combination of several languages.
  • the present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, United States Patent Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.
  • the present invention may also make use of the several embodiments of the array or arrays and the processing described in United States Patent Nos. 5,545,531 and 5,874,219. These patents are incorporated herein by reference in their entireties for all purposes.
  • the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in United States Patent applications 10/063,559 (U.S. Patent Publication No. US- 2002-0183936-A1), 60/349,546, 60/376,003, 60/394,574, 60/403,381. II. Definitions
  • An "array” is an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically.
  • the molecules in the array can be identical or different from each other.
  • the array can assume a variety of formats, e.g., libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.
  • An “array plate” or a “plate” is a body having a plurality of arrays in which each array is separated from the other arrays by a physical barrier resistant to the passage of liquids and forming an area or space, referred to as a well.
  • a “nucleic acid library” or “array” is an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically and screened for biological activity in a variety of different formats (e.g., libraries of soluble molecules; and libraries of oligos tethered to resin beads, silica chips, or other solid supports).
  • nucleic acid refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs) as described in United States Patent No. 6, 156,501 that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein.
  • analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleoside sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution.
  • these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailored to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.
  • Biopolymer or biological polymer is intended to mean repeating units of biological or chemical moieties.
  • biopolymers include, but are not limited to, nucleic acids, oligonucleotides, amino acids, proteins, peptides, hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides, phospholipids, synthetic analogues of the foregoing, including, but not limited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, and combinations of the above.
  • Biopolymer synthesis is intended to encompass the synthetic production, both organic and inorganic, of a biopolymer.
  • bioploymer which is intended to mean a single unit of biopolymer, or a single unit which is not part of a biopolymer.
  • a nucleotide is a biomonomer within an oligonucleotide biopolymer
  • an amino acid is a biomonomer within a protein or peptide biopolymer
  • avidin, biotin, antibodies, antibody fragments, etc. are also biomonomers.
  • Initiation biomonomer or “initiator biomonomer” is meant to indicate the first biomonomer which is covalently attached via reactive nucleophiles to the surface of the polymer, or the first biomonomer which is attached to a linker or spacer arm attached to the polymer, the linker or spacer arm being attached to the polymer via reactive nucleophiles.
  • Complementary refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified.
  • Complementary nucleotides are, generally, A and T (or A and U), or C and G.
  • Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%.
  • substantial complementary exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement.
  • selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See, M. Kanehisa, Nucleic Acids Res.
  • a combinatorial synthesis strategy is an ordered strategy for parallel synthesis of diverse polymer sequences by sequential addition of reagents which may be represented by a reactant matrix and a switch matrix, the product of which is a product matrix.
  • a reactant matrix is a 1 column by m row matrix of the building blocks to be added.
  • the switch matrix is all or a subset of the binary numbers, preferably ordered, between 1 and m arranged in columns.
  • a "binary strategy" is one in which at least two successive steps illuminate a portion, often half, of a region of interest on the substrate. In a binary synthesis strategy, all possible compounds which can be formed from an ordered set of reactants are formed.
  • binary synthesis refers to a synthesis strategy which also factors a previous addition step. For example, a strategy in which a switch matrix for a masking strategy halves regions that were previously illuminated, illuminating about half of the previously illuminated region and protecting the remaining half (while also protecting about half of previously protected regions and illuminating about half of previously protected regions). It will be recognized that binary rounds may be interspersed with non-binary rounds and that only a portion of a substrate may be subjected to a binary scheme.
  • a combinatorial "masking" strategy is a synthesis which uses light or other spatially selective deprotecting or activating agents to remove protecting groups from materials for addition of other materials such as amino acids. "Effective amount” refers to an amount sufficient to induce a desired result.
  • Excitation energy refers to energy used to energize a detectable label for detection, for example illuminating a fluorescent label.
  • Devices for this use include coherent light or non-coherent light, such as lasers, UN light, light emitting diodes, an incandescent light source, or any other light or other electromagnetic source of energy having a wavelength in the excitation band of an excitable label, or capable of providing detectable transmitted, reflective, or diffused radiation.
  • Genome is all the genetic material in the chromosomes of an organism.
  • DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA.
  • a genomic library is a collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism.
  • Hybridization conditions will typically include salt concentrations of less than about IM, more usually less than about 500 mM and preferably less than about 200 mM.
  • Hybridization temperatures can be as low as 5°C, but are typically greater than
  • Hybridizations e.g., allele-specific probe hybridizations, are generally performed under stringent conditions. For example, conditions where the salt concentration is no more than about 1 Molar (M) and a temperature of at least 25 C, e.g., 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4 (5X SSPE) and a temperature of from about 25 C to about 30 C.
  • Hybridizations are usually performed under stringent conditions, for example, at
  • conditions of 5X SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30 C are suitable for allele-specific probe hybridizations.
  • stringent conditions see, for example, Sambrook, Fritsche and Maniatis. "Molecular Cloning: A laboratory Manual” 2 nd Ed. Cold Spring Harbor Press (1989) which is hereby incorporated by reference in its entirety for all purposes above.
  • hybridization refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide; triple-stranded hybridization is also theoretically possible. The resulting (usually) double-stranded polynucleotide is a "hybrid.” The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the "degree of hybridization.”
  • Hybridization probes are oligonucleotides capable of binding in a base-specific mamier to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al, Science 254, 1497-1500 (1991), and other nucleic acid analogs and nucleic acid mimetics. See U.S. Patent No. 6,156,501.
  • Hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • isolated nucleic acid is an object species invention that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).
  • Label includes” for example, a luminescent label, a light scattering label or a radioactive label.
  • Fluorescent labels include, inter alia, the commercially available fluorescein phosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (ABI). See United States Patent 6,287,778.
  • a ligand is a molecule that is recognized by a particular receptor.
  • the agent bound by or reacting with a receptor is called a "ligand,” a term which is definitionally meaningful only in terms of its counterpart receptor.
  • the term “ligand” does not imply any particular molecular size or other structural or compositional feature other than that the substance in question is capable of binding or otherwise interacting with the receptor.
  • a ligand may serve either as the natural ligand to which the receptor binds, or as a functional analogue that may act as an agonist or antagonist.
  • ligands that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, substrate analogs, transition state analogs, cofactors, drugs, proteins, and antibodies.
  • hormones e.g., opiates, steroids, etc.
  • hormone receptors e.g., opiates, steroids, etc.
  • hormone receptors e.g., opiates, steroids, etc.
  • peptides e.g., enzymes, enzyme substrates, substrate analogs, transition state analogs, cofactors, drugs, proteins, and antibodies.
  • Linkage disequilibrium or allelic association means the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. For example, if locus X has alleles a and b, which occur equally frequently, and linked locus Y has alleles c and d, which occur equally frequently, one would expect the combination ac to occur with a frequency of 0.25. If ac occurs more frequently, then alleles a and c are in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium with linked alleles.
  • Microtiter plates are arrays of discrete wells that come in standard formats (96, 384 and 1536 wells) which are used for examination of the physical, chemical or biological characteristics of a quantity of samples in parallel.
  • Mixed population or complex population refers to any sample containing both desired and undesired nucleic acids.
  • a complex population of nucleic acids maybe total genomic DNA, total genomic RNA or a combination thereof.
  • a complex population of nucleic acids may have been enriched for a given population but include other undesirable populations.
  • a complex population of nucleic acids maybe a sample which has been enriched for desired messenger RNA (mRNA) sequences but still includes some undesired ribosomal RNA sequences (rRNA).
  • mRNA messenger RNA
  • rRNA ribosomal RNA sequences
  • “Monomer” refers to any member of the set of molecules that can be joined together to form an oligomer or polymer.
  • the set of monomers useful in the present invention includes, but is not restricted to, for the example of (poly)peptide synthesis, the set of L-amino acids, D-amino acids, or synthetic amino acids.
  • “monomer” refers to any member of a basis set for synthesis of an oligomer. For example, dimers of L-amino acids form a basis set of 400 "monomers” for synthesis of polypeptides. Different basis sets of monomers maybe used at successive steps in the synthesis of a polymer.
  • mRNA or mRNA transcripts include, but not limited to pre- mRNA transcript(s), transcript processing intermediates, mature mRNA(s) ready for translation and transcripts of the gene or genes, or nucleic acids derived from the mRNA transcript(s). Transcript processing may include splicing, editing and degradation.
  • a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template.
  • a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc. are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample.
  • mRNA derived samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
  • Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally- occurring sources or may be artificially or synthetically produced, hi addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
  • oligonucleotide or “polynucleotide” is a nucleic acid ranging from at least 2, preferable at least 8, and more preferably at least 20 nucleotides in length or a compound that specifically hybridizes to a polynucleotide.
  • Polynucleotides of the present invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, recombinantly produced or artificially synthesized and mimetics thereof.
  • a further example of a polynucleotide of the present invention may be peptide nucleic acid (PNA).
  • the invention also encompasses situations in which there is a nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix.
  • Nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix.
  • Polynucleotide and oligonucleotide are used interchangeably in this application.
  • a probe is a surface-immobilized molecule that can be recognized by a particular target.
  • probes examples include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
  • hormones e.g., opioid peptides, steroids, etc.
  • hormone receptors e.g., opioid peptides, steroids, etc.
  • hormone receptors e.g., enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
  • Primer is a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions e.g., buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase.
  • the length of the primer in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 20, 25, 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template.
  • the primer site is the area of the template to which a primer hybridizes.
  • the primer pair is a set of primers including a 5' upstream primer that hybridizes with the 5' end of the sequence to be amplified and a 3' downstream primer that hybridizes with the complement of the 3' end of the sequence
  • Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • a polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion.
  • a polymorphic locus may be as small as one base pair.
  • Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, mmisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • the first identified allelic form is arbitrarily designated as the reference form, and other allelic forms are designated as alternative or variant alleles.
  • the allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms.
  • a diallelic polymorphism has two forms.
  • a triallelic polymorphism has three forms. Single nucleotide polymorphisms (SNPs) are included in polymorphisms.
  • Reader or “plate reader” is a device which is used to identify hybridization events on an array, such as the hybridization between a nucleic acid probe on the array and a fluorescently labeled target. Readers are known in the art and are commercially available through Affymetrix, Santa Clara CA and other companies. Generally, they involve the use of an excitation energy (such as a laser) to illuminate a fluorescently labeled target nucleic acid that has hybridized to the probe. Then, the reemitted radiation (at a different wavelength than the excitation energy) is detected using devices such as a CCD, PMT, photodiode, or similar devices to register the collected emissions. See United States Patent No. 6,225,625.
  • Receptor is a molecule that has an affinity for a given ligand. Receptors may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance.
  • receptors which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles.
  • Receptors are sometimes referred to in the art as anti-ligands. As the term receptors is used herein, no difference in meaning is intended.
  • a "Ligand Receptor Pair" is formed when two macromolecules have combined through molecular recognition to form a complex.
  • Solid support refers to a material or group of materials having a rigid or semi-rigid surface or surfaces, hi many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like.
  • the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See U.S. Patent No. 5,744,305 for exemplary substrates.
  • Target is a molecule that has an affinity for a given probe.
  • Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance.
  • targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes refe ⁇ ed to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended.
  • a "Probe Target Pair" is formed when two macromolecules have combined through molecular recognition to form a complex.
  • WGSA Whole Genome Sampling Assay
  • genomic DNA for example, is digested with a restriction enzyme of interest and adaptors are ligated to the digested fragments.
  • a single primer corresponding to the adaptor sequence is used to amplify fragments of a desired size, for example, 500-2000 bp.
  • the processed target is then hybridized to nucleic acid arrays comprising SNP-containing fragments/probes.
  • WGSA is disclosed in, for example, US Provisional Application Serial Nos.
  • methods are provided for facilitating searches for sequence variations (SNPs, amplifications, deletions etc.) in functional regions of the genome without recourse to annotations.
  • Mapping RNA and transcription factor binding sites etc. in genomic regions of affected and unaffected individuals confers specific functions on these genomic regions and ascribes to them a priority status for analyzing the presence of sequence variations.
  • the methods are particularly useful for analyzing large regions of the genome, for example, for analyzing at least 10,000 bases, 100,000 bases, 1 Mbases or 5 Mbases of the genome.
  • Functional regions of a genome maybe determined using a variety of methods.
  • Preferred methods include mapping with high density oligonucleotide probe arrays. Methods for mapping functional regions of a genome are described, for example, in the following US Patent Applications and Provisional Patent Applications: 60/339,655,
  • Functional regions may be dynamic in nature. For example, transcription sites, transcription factor binding sites may change in different physiological, pathological, toxicological and pharmacological state of a sample (see, e.g., U.S. Provisional Application Serial Number 60/486,376, "Differential Regulation of Novel Transcripts", incorporated herein by reference).
  • the functional regions may also be tissue specific.
  • the functional regions may be profiled in a variety of samples of interested states to determine functional region profiles for sequence variation analysis.
  • the information about functional regions (such as functional region profiles) may be stored in a computerized data base. Sequence variation detection assays, such as WGSA assays, may be designed using such a data base.
  • the genomic and cDNA sequences in the functional regions can be analyzed by sequencing or SNP testing or Comparative Genomic Hybridization (CGH) testing in preference to other regions which will be important (but not exclusive) for sequence variations which are outside of coding regions.
  • the functional regions to be analyzed for sequence variations are at least 1000 bases, 10000 bases, 100000 bases, 1 Mbases, or 5 Mbases of the genome. Sequencing can be performed by traditional Sanger sequencing, Sequencing by
  • Hybridization or microarray based resequencing can be used to detect sequence variations in genomic regions.
  • resequencing microarrays Affymetrix, Santa Clara, CA
  • SNP genotyping can be performed by a variety of methods (for a review of
  • SNP genotyping methods see, e.g., Pui-Yan Kwok, 2001, Methods For Genotyping Single Nucleotide Polymorphisms, Annual Review of Genomics and Human Genetics 2:235-258, and Tsuchihashi and Dracopoli, 2002, Progress in high throughput SNP genotyping methods, Pharmacogenomics J. 2002;2(2):103-10, all incorporated here by reference).
  • One particularly preferred method is the Whole Genome Sampling Assay (WGSA) and high density oligonucleotide probe arrays. Patent specifications disclosing WGSA have been previously incorporated by reference. The method is also described in, e.g., Kennedy et al, Large-scale genotyping of complex DNA, Nat Biotechnol.
  • WGSA assays As the lists of sequences involved in various functional operations of the cell such as transcription factor bindings, origins, methylation and chromatin modification sites become defined, these sites should be examined, computationally, for the presence of restriction endonuclease (RE) sites ( Figure 1) to design a WGSA assay.
  • RE restriction endonuclease
  • the computer software products typically contain a computer readable medium having computer codes that perform the method of retrieving information about functional regions, analyzing RE sites suitable for interrogating the functional regions and optionally selecting probes for interrogating SNPs within the regions.
  • Oligonucleotides for interrogating SNPs within the functional regions are also provided.
  • the probes are typically designed using a computer software to identify the SNPs to be interrogated.
  • the probes are selected according to previously disclosed tiling strategies (See, e.g., Kennedy et al., Large-scale genotyping of complex DNA, Nat Biotechnol. 2003 Oct; 21(10):1233-7, incorporated herein by reference) or other suitable detection strategies.
  • the probes are typically immobilized on a substrate, beads or optical fibers, hi preferred embodiments, the probes are immobilized on a substrate at high densities, such as more than 1000, 100,000, 1000000 different probes per cm 2 . Methods for manufacturing high density oligonucleotide probe arrays are described in patent specifications previously incorporated by reference.
  • Comparative genomic hybridization is a molecular cytogenetic technique that allows detection of DNA sequence copy number changes throughout the genome or in specific regions of the genome in a single hybridization.
  • CGH Comparative genomic hybridization
  • sequence variations in functional genomic regions has practical utility in, for example, drug target identification, and diagnostics.
  • Genetic association and linkage analysis are frequently used to identify genomic regions related to a trait of interest (such as a site related to a disease).
  • Association analysis is a method of genetic analysis that compares the frequency of alleles between affected and unaffected individuals (as used herein, an individual can be a human, an animal, a plant, etc).
  • a given allele is considered to be associated with the disease of interest if that allele occurs at a significantly higher frequency among affected individuals.
  • Linkage analysis is commonly used to identify the presence of a disease allele at a locus that is co-inherited with a closely linked marker, such as an SNP.
  • association and linkage analysis can be performed using, e.g., WGSA, to identify a genomic region of interest.
  • association or linkage studies highlight several regions of the genome as possible sites which may be involved in determining a trait in affected families or individuals, the presence of the functional regions in these regions can be empirically determined and can narrow down the possibilities for further analysis ( Figure 2).
  • the genomic and cDNA sequences in these regions can be empirically determined and can be analyzed by sequencing or SNP testing or Comparative Genomic Hybridization (CGH testing in preference to other regions which will be important (but not exclusive) for sequence variations which are outside of coding regions.

Abstract

Dans un mode de réalisation de l'invention, des procédés, des compositions et des logiciels informatiques permettent de détecter des variations génétiques dans des régions fonctionnelles d'un génome. Dans d'autres modes de réalisation, des réseaux de sonde de juxtaposition d'oligonucléotides sont utilisés pour détecter des régions transcrites, des régions de fixation du facteur de transcription, etc. Les variations génétiques dans lesdites régions peuvent être déterminées au moyen, par exemple, d'un essai biologique de prélèvement du génome entier.
EP03768853A 2002-11-12 2003-11-12 Procedes, compositions et logiciels informatiques pour interroger des variations de sequences dans les regions genomiques fonctionnelles Withdrawn EP1563090A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US42587902P 2002-11-12 2002-11-12
US42588002P 2002-11-12 2002-11-12
US425880P 2002-11-12
US425879P 2002-11-12
PCT/US2003/035819 WO2004044700A2 (fr) 2002-11-12 2003-11-12 Procedes, compositions et logiciels informatiques pour interroger des variations de sequences dans les regions genomiques fonctionnelles

Publications (2)

Publication Number Publication Date
EP1563090A2 true EP1563090A2 (fr) 2005-08-17
EP1563090A4 EP1563090A4 (fr) 2007-07-11

Family

ID=32314612

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03768853A Withdrawn EP1563090A4 (fr) 2002-11-12 2003-11-12 Procedes, compositions et logiciels informatiques pour interroger des variations de sequences dans les regions genomiques fonctionnelles

Country Status (4)

Country Link
EP (1) EP1563090A4 (fr)
CN (1) CN1711358A (fr)
AU (1) AU2003291453A1 (fr)
WO (1) WO2004044700A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015035212A (ja) * 2013-07-29 2015-02-19 アジレント・テクノロジーズ・インクＡｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｉｎｃ． ターゲットシークエンシングパネルから変異を見つける方法
CN107808073B (zh) * 2017-10-31 2018-11-13 广东美格基因科技有限公司 高通量微生物功能基因微阵列处理方法及电子设备

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030186280A1 (en) * 2002-03-28 2003-10-02 Affymetrix, Inc. Methods for detecting genomic regions of biological significance

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6468742B2 (en) * 1993-11-01 2002-10-22 Nanogen, Inc. Methods for determination of single nucleic acid polymorphisms using bioelectronic microchip
US5972608A (en) * 1997-08-27 1999-10-26 University Of Massachusetts Assays and reagents for chromatin remodeling enzymes and their modulators
US6306643B1 (en) * 1998-08-24 2001-10-23 Affymetrix, Inc. Methods of using an array of pooled probes in genetic analysis
US6322985B1 (en) * 1999-12-27 2001-11-27 Technion Research And Development Foundation Ltd. Abundant, well distributed and hyperpolymorphic simple sequence repeats in prokaryote genomes and use of same for prokaryote classification and typing

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030186280A1 (en) * 2002-03-28 2003-10-02 Affymetrix, Inc. Methods for detecting genomic regions of biological significance

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CUTLER DAVID J ET AL: "High-throughput variation detection and genotyping using microarrays" GENOME RESEARCH, COLD SPRING HARBOR LABORATORY PRESS, WOODBURY, NY, US, vol. 11, no. 11, November 2001 (2001-11), pages 1913-1925, XP002311211 ISSN: 1088-9051 *
FAN J-B ET AL: "Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays" GENOME RESEARCH, COLD SPRING HARBOR LABORATORY PRESS, WOODBURY, NY, US, vol. 10, no. 6, 2000, pages 853-860, XP002236784 ISSN: 1088-9051 *
KENNEDY G C ET AL.: "Large-scale genotyping of complex DNA" NATURE BIOTECHNOLOGY, vol. 21, no. 10, October 2003 (2003-10), pages 1233-1237, XP002434032 *
LINDBLAD-TOH K ET AL: "LOSS-OF-HETEROZYGOSITY ANALYSIS OF SMALL-CELL LUNG CARCINOMAS USING SINGLE-NUCLEOTIDE POLYMORPHISM ARRAYS" NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 18, no. 9, September 2000 (2000-09), pages 1001-1005, XP001063318 ISSN: 1087-0156 *
LIU N-P ET AL.: "Physical and genetic mapping of the macular corneal dystrophy locus on chromosome 16q and exclusion of TAT and LCAT as candidate genes" MOLECULAR VISION, vol. 6, 2000, pages 95-100, XP002434030 *
MCDONALD O G KRYNETSKI E Y EVANS W E: "Molecular haplotyping of genomic DNA for multiple single nucleotide polymorphisms located kilobases apart using long range polymerase chain reaction and intramolecular ligation" PHARMACOGENETICS, CHAPMAN & HALL, LONDON, GB, vol. 12, no. 2, February 2002 (2002-02), pages 93-99, XP002956448 ISSN: 0960-314X *
SCHUBERT E L ET AL.: "Single nucleotide polymorphism array analysis of flow-sorted epithelial cells from frozen versus fixed tissues for whole genome analysis of allelic loss in breast cancer" AMERICAN JOURNAL OF PATHOLOGY, vol. 160, no. 1, January 2002 (2002-01), pages 73-79, XP008078927 *
See also references of WO2004044700A2 *
WANG D G ET AL: "Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 280, 1998, pages 1077-1082, XP002089398 ISSN: 0036-8075 *
WANG M ET AL.: "Construction and characterization of a human chromosome 2-specific BAC library" GENOMICS, vol. 24, 1994, pages 527-534, XP002434029 *
XIANG F I ET AL.: "A Huntington disease-like neurodegenrative disorder maps to chromosome 20p" AMERICAN JOURNAL OF HUMAN GENETICS, vol. 63, 1998, pages 1431-1438, XP008078926 *

Also Published As

Publication number Publication date
EP1563090A4 (fr) 2007-07-11
WO2004044700A3 (fr) 2005-05-06
AU2003291453A8 (en) 2004-06-03
WO2004044700A2 (fr) 2004-05-27
AU2003291453A1 (en) 2004-06-03
CN1711358A (zh) 2005-12-21

Similar Documents

Publication Publication Date Title
US7374927B2 (en) Methods of analysis of degraded nucleic acid samples
US7361468B2 (en) Methods for genotyping polymorphisms in humans
US7341835B2 (en) Methods of analysis of alternative splicing in mouse
US8133667B2 (en) Methods for genotyping with selective adaptor ligation
US20060194243A1 (en) Methods for Identifying DNA Copy Number Changes
US20050244851A1 (en) Methods of analysis of alternative splicing in human
US20040191810A1 (en) Immersed microarrays in conical wells
EP1645640A2 (fr) Procédé pour l'amplification et l'analyse d'acides nucléiques
US20050106591A1 (en) Methods and kits for preparing nucleic acid samples
US8392355B2 (en) Computer software for visualizing genotyping data
US20050208555A1 (en) Methods of genotyping
US20040161779A1 (en) Methods, compositions and computer software products for interrogating sequence variations in functional genomic regions
US7312035B2 (en) Methods of genetic analysis of yeast
US7629164B2 (en) Methods for genotyping polymorphisms in humans
US20110160092A1 (en) Methods for Selecting a Collection of Single Nucleotide Polymorphisms
US20040115644A1 (en) Methods of direct amplification and complexity reduction for genomic DNA
US20040171167A1 (en) Chip-in-a-well scanning
US20040259124A1 (en) Methods for oligonucleotide probe design
US20050074799A1 (en) Use of guanine analogs in high-complexity genotyping
EP1563090A2 (fr) Procedes, compositions et logiciels informatiques pour interroger des variations de sequences dans les regions genomiques fonctionnelles
US20060147940A1 (en) Combinatorial affinity selection
US20050136452A1 (en) Methods for monitoring expression of polymorphic alleles
US20040110132A1 (en) Method for concentrate nucleic acids
US20070184454A1 (en) Methods and compositons for enhancing discrimination between perfect match and mismatch hybridization
US8815510B2 (en) Combinatorial affinity selection

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050602

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20070612

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20070602