EP1068354A2 - Marqueurs bialleliques - Google Patents

Marqueurs bialleliques

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Publication number
EP1068354A2
EP1068354A2 EP99915117A EP99915117A EP1068354A2 EP 1068354 A2 EP1068354 A2 EP 1068354A2 EP 99915117 A EP99915117 A EP 99915117A EP 99915117 A EP99915117 A EP 99915117A EP 1068354 A2 EP1068354 A2 EP 1068354A2
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European Patent Office
Prior art keywords
wiaf
polymorphic
segment
allele
column
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German (de)
English (en)
Inventor
Eric S. Lander
David Wang
Thomas Hudson
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Whitehead Institute for Biomedical Research
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Whitehead Institute for Biomedical Research
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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
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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

  • the variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral. In some instances, a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism. In other instances, a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form. In many instances, both progenitor and variant form(s) survive and co-exist in a species population. The coexistence of multiple forms of a sequence gives rise to polymorphisms.
  • RFLP RFLP
  • the restriction fragment length polymorphism may create or delete a restriction site, thus changing the length of the restriction fragment.
  • RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; W090/11369; Donis-Keller , Cell 51, 319-337 (1987); Lander et al . , Genetics 121, 85-99 (1989)).
  • a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.
  • VNTR variable number tandem repeat
  • polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs . Some single nucleotide polymorphisms occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Examples of genes, in which polymorphisms within coding sequences give rise to genetic disease include ⁇ -globin (sickle cell anemia) and CFTR (cystic fibrosis) . Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing) . Other single nucleotide polymorphisms have no phenotypic effects .
  • Single nucleotide polymorphisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages .
  • Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism.
  • the greater frequency and uniformity of single nucleotide polymorphisms means that there is a greater probability that such a polymorphism will be found in close proximity to a genetic locus of interest than would be the case for other polymorphisms .
  • the different forms of characterized single nucleotide polymorphisms are often easier to distinguish than other types of polymorphism (e.g., by use of assays employing allele-specific hybridization probes or primers) .
  • the invention provides nucleic acid sequences comprising nucleic acid segments of from about 10 to about 200 bases as shown in the Table, column 7, including a polymorphic site. Complements of these segments are also included.
  • the segments can be DNA or RNA, and can be double- or single-stranded. Segments can be, for example, 10-20, 10-50 or 10-100 bases long. Preferred segments include a biallelic polymorphic site.
  • the base occupying the polymorphic site in the segments can be the reference (Table, column 3) or an alternative base (Table, column 4) .
  • the invention further provides allele-specific oligonucleotides that hybridize to a segment of a fragment shown in the Table, column 7, or its complement.
  • oligonucleotides can be probes or primers.
  • isolated nucleic acids comprising a sequence shown in the Table, column 7, or the complement thereto, in which the polymorphic site within the sequence is occupied by a base other than the reference base shown in the Table, column 3.
  • the invention further provides a method of analyzing a nucleic acid from an individual.
  • the method determines which base is present at any one of the polymorphic sites shown in the Table.
  • a set of bases occupying a set of the polymorphic sites shown in the Table is determined. This type of analysis can be performed on a number of individuals, who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymorphic sites in the individuals tested.
  • An oligonucleotide can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means.
  • the oligonucleotides of the present invention can comprise all of an oligonucleotide sequence presented in column 7 of the Table or a segment of such an oligonucleotide which includes a polymorphic site.
  • Oligonucleotides can be all of a nucleic acid segment as represented in column 7 of the Table; a nucleic acid sequence which comprises a nucleic acid segment represented in column 7 of the Table and additional nucleic acids (present at either or both ends of a nucleic acid segment of column 7); or a portion (fragment) of a nucleic acid segment represented in column 7 of the Table which includes a polymorphic site.
  • Preferred oligonucleotides of the invention include segments of DNA, or their complements, which include any one of the polymorphic sites shown in the Table. The segments can be between 5 and 250 bases, and, in specific embodiments, are between 5-10, 5-20, 10-20, 10- 50, 20-50 or 10-100 bases.
  • segments of the invention can be 5 , 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 nucleotides in length.
  • the polymorphic site can occur within any position of the segment.
  • the segments can be from any of the allelic forms of DNA shown in the Table.
  • the nucleotide at the polymorphic site is different from the nucleotide in a corresponding reference or wild-type allele.
  • Hybridization probes are oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al . , Science 254, 1497-1500 (1991) .
  • 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 an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • appropriate conditions e . g. , in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or 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 reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template.
  • primer site refers to the area 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.
  • linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. It can be measured by percent recombination between the two genes, alleles, loci or genetic markers.
  • 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 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, minisatellites, 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 or biallelic polymorphism has two forms.
  • a triallelic polymorphism has three forms.
  • a single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences.
  • the site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations) .
  • a single nucleotide polymorphism usually arises due to substitution of one nucleotide for another at the polymorphic site.
  • a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine or vice versa.
  • Single nucleotide polymorphisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Typically the polymorphic site is occupied by a base other than the reference base.
  • the altered allele can contain a "C", "G” or "A” at the polymorphic site.
  • Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • conditions of 5X SSPE 750 mM NaCl, 50 mM NaPhosphate", 5 mM EDTA, pH 7.4 and a temperature of 25- 30°C, or equivalent conditions, are suitable for allele- specific probe hybridizations.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.
  • an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances) , buffer system or reagent mix.
  • the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC .
  • an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.
  • the novel polymorphisms of the invention are listed in the Table.
  • the first column of the Table lists the names assigned to the fragments in which the polymorphisms occur.
  • the fragments are all human genomic fragments .
  • the sequence of one allelic form of each of the fragments (arbitrarily referred to as the prototypical or reference form) has been previously published. These sequences are listed at http://www-genome.wi.mit.edu/ (all STS ' s (sequence tag sites)); http://shgc.stanford.edu (Stanford STS's); and http://ww.tigr.org/ (TIGR STS's).
  • the Web sites also list primers for amplification of the fragments, and the genomic location of fragments. Some fragments are expressed sequence tags, and some are random genomic fragments. All information in the websites concerning the fragments listed in the Table is incorporated by reference in its entirety for all purposes.
  • the second column lists the position in the fragment in which a polymorphic site has been found. Positions are numbered consecutively with the first base of the fragment sequence as listed in one of the above databases being assigned the number one.
  • the third column lists the base occupying the polymorphic site in the sequence in the data base. This base is arbitrarily designated the reference or prototypical form, but it is not necessarily the most frequently occurring form.
  • the fourth column in the Table lists the alternative base(s) at the polymorphic site.
  • the fifth column of the Table lists a 5 ' (upstream or forward) primer that hybridizes with the 5' end of the DNA sequence to be amplified.
  • the sixth column of the Table lists a 3' (downstream or reverse) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • the seventh column of the Table lists a number of bases of sequence on either side of the polymorphic site in each fragment.
  • the indicated sequences can be either DNA or RNA. In the latter, the T's shown in the Table are replaced by U's.
  • the base occupying the polymorphic site is indicated in EUPAC-IUB ambiguity code.
  • Polymorphisms are detected in a target nucleic acid from an individual being analyzed.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
  • the target nucleic acid is a cytochrome P450
  • the liver is a suitable source .
  • Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally 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
  • NASBA nucleic acid based sequence amplification
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • B. Detection of Polymorphisms in Target DNA There are two distinct types of analysis of target DNA for detecting polymorphisms.
  • the first type of analysis sometimes referred to as de novo characterization, is carried out to identify polymorphic sites not previously characterized (i.e., to identify new polymorphisms) . This analysis compares target sequences in different individuals to identify points of variation, i.e., polymorphic sites.
  • allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender.
  • the de novo identification of polymorphisms of the invention is described in the Examples section.
  • the second type of analysis determines which form(s) of a characterized (known) polymorphism are present in individuals under test. There are a variety of suitable procedures, which are discussed in turn.
  • Allele-specific probes for analyzing polymorphisms 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 polymorphic 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 polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 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 polymorphisms within the same target sequence.
  • the polymorphisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995.
  • One form of such arrays is described in the Examples section in connection with de novo identification of polymorphisms.
  • the same array or a different array can be used for analysis of characterized polymorphisms.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymorphism.
  • 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 as described in the Examples, except that the probes exhibit complementarity to the second reference sequence.
  • a second group 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 target DNA overlapping a polymorphism 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 polymorphic 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 polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • the direct analysis of the sequence of polymorphisms of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al . , Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al . , Recombinant DNA Laboratory Manual , (Acad. Press, 1988) ) .
  • Denaturing Gradient Gel Electrophoresis 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.
  • this information can be used in a number of methods .
  • polymorphisms of the invention are often used in conjunction with polymorphisms in distal genes.
  • Preferred polymorphisms for use in forensics are biallelic because the population frequencies of two polymorphic forms can usually be determined with greater accuracy than those of multiple polymorphic 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 polymorphic forms occupying selected polymorphic sites is the same in the suspect and the sample. If the set of polymorphic 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.
  • p(ID) is the probability that two random individuals have the same polymorphic or allelic form at a given polymorphic site. In biallelic 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 is (see WO 95/12607) :
  • the appropriate binomial expansion is used to calculate p(ID) and p(exc) .
  • 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 polymorphisms in the putative father and the child.
  • the set of polymorphisms in the child attributable to the father does not match the set of polymorphisms of the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymorphisms in the child attributable to the father does match the set of polymorphisms of the putative father, a statistical calculation can be performed to determine the probability of coincidental match.
  • the polymorphisms of the invention may contribute to the phenotype of an organism in different ways. Some polymorphisms 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 polymorphisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymorphism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymorphisms in different genes. Further, some polymorphisms 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 (e.g., agammaglobulimenia, 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 porphyria) .
  • agammaglobulimenia e.g., diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease
  • 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.
  • Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, 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 polymorphic markers sets.
  • a set of polymorphisms i.e. a polymorphic set
  • the alleles of each polymorphism 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 such as a ⁇ -squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted.
  • allele Al at polymorphism A correlates with heart disease.
  • allele Bl at polymorphism B correlates with increased milk production of a farm animal .
  • Such correlations can be exploited in several ways. In the case of a strong correlation between a set of one or more polymorphic forms and a disease for which treatment is available, detection of the polymorphic 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 polymorphic 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 polymorphism from her husband to her offspring.
  • immediate therapeutic intervention or monitoring may not be justified.
  • the patient can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles.
  • Identification of a polymorphic set in a patient correlated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed.
  • Y_ kpcountry ⁇ + S X + P : + X k + ⁇ x + ... ⁇ 17 + PE n + a n +e p
  • Y llknp is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record
  • ⁇ is an overall mean
  • YS 1 is the effect common to all cows calving in year-season
  • X k is the effect common to cows in either the high or average selection line
  • ⁇ x to ⁇ 17 are the binomial regressions of production record on mtDNA D-loop sequence polymorphisms
  • PE n is permanent environmental effect common to all records of cow n
  • a n is effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect
  • e p is a random residual. It was found that eleven of seventeen polymorphisms tested influenced at least one production trait. Bovines having the
  • D. Genetic Mapping of Phenotypic Traits The previous section concerns identifying correlations between phenotypic traits and polymorphisms that directly or indirectly contribute to those traits.
  • the present section describes identification of a physical linkage between a genetic locus associated with a trait of interest and polymorphic 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 .
  • 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 ⁇ , 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
  • the likelihood at a given value of ⁇ is: probability of data if loci linked at ⁇ to probability of data if loci unlinked.
  • the computed likelihoods are usually expressed as the log 10 of this ratio (i.e., a lod score) . For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence.
  • the use of logarithms allows data collected from different families to be combined by simple addition.
  • lod scores for differing values of ⁇ (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 ⁇ at which the lod score is the highest is considered to be the best estimate of the recombination fraction.
  • the invention further provides variant forms of nucleic acids and corresponding proteins.
  • the nucleic acids comprise one of the sequences described in the Table, column 8, in which the polymorphic position is occupied by one of the alternative bases for that position.
  • Some nucleic acids encode full-length variant forms of proteins.
  • variant proteins have the prototypical amino acid sequences encoded by nucleic acid sequences shown in the Table, column 8, (read so as to be in-frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one of the polymorphic positions shown in the Table. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in the Table.
  • Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter.
  • the promoter is a eukaryotic promoter for expression in a mammalian cell.
  • the transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host.
  • the selection of an appropriate promoter for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected.
  • Commercially available expression vectors can be used. Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
  • the means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra .
  • a wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli , yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e . g. , mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post- translational modification, and the like.
  • the protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984) ; Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer-Verlag, New York (1987); and Deutscher (ed) , Guide to Protein
  • the protein If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.
  • 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 microinjecting the construct into a zygote.
  • 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. See Capecchi, Science 244, 1288-1292 (1989) .
  • the 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 present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides.
  • Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding.
  • Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
  • Antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof .
  • 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. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.
  • 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 polymorphism.
  • 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 or all of the polymorphisms shown in the Table.
  • kits include, for example, restriction enzymes, reverse-transcriptase or poly erase, 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 methods.
  • the polymorphisms shown in the Table were identified by resequencing of target sequences from three to ten unrelated individuals of diverse ethnic and geographic backgrounds by hybridization to probes immobilized to microfabricated arrays or conventional sequencing.
  • the strategy and principles for design and use of such arrays are generally described in WO 95/11995.
  • the strategy provides arrays of probes for analysis of target sequences showing a high degree of sequence identity to the reference sequences of the fragments shown in the Table, column 1.
  • the reference sequences were sequence-tagged sites (STSs) developed in the course of the Human Genome Project ( see, e . g. , Science 270, 1945-1954 (1995); Nature 380, 152-154 (1996)). Most STS's ranged from 100 bp to 300 bp in size.
  • a typical probe array used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence.
  • a first probe set comprises a plurality of probes exhibiting perfect complementarily with one of the reference sequences.
  • Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two.
  • For each probe in the first set there are three corresponding probes from three additional probe sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence.
  • probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets .
  • probes were 25 nucleotides long. Arrays tiled for multiple different references sequences were included on the same substrate.
  • target sequences from an individual were amplified from human genomic DNA using primers for the fragments indicated in the listed Web sites.
  • the amplified target sequences were fluorescently labelled during or after PCR.
  • the labelled target sequences were hybridized with a substrate bearing immobilized arrays of probes. The amount of lable bound to probes was measured. Analysis of the pattern of label revealed the nature and position of differences between the target and reference sequence. For example, comparison of the intensities of four corresponding probes reveals the identity of a corresponding nucleotide in the target sequences aligned with the interrogation position of the probes.
  • the corresponding nucleotide is the complement of the nucleotide occupying the interrogation position of the probe showing the highest intensity (see WO 95/11995) .
  • the existence of a polymorphism is also manifested by differences in normalized hybridization intensities of probes flanking the polymorphism when the probes hybridized to corresponding targets from different individuals. For example, relative loss of hybridization intensity in a "footprint" of probes flanking a polymorphism signals a difference between the target and reference (i.e., a polymorphism) (see EP 717,113).
  • hybridization intensities for corresponding targets from different individuals can be classified into groups or clusters suggested by the data, not defined a priori , such that isolates in a give cluster tend to be similar and isolates in different clusters tend to be dissimilar. Hybridizations to samples from different individuals were performed separately. The Table summarizes the data obtained for target sequences in comparison with a reference sequence for the individuals tested.
  • the invention includes a number of general uses that can be expressed concisely as follows.
  • the invention provides for the use of any of the nucleic acid segments described above in the diagnosis or monitoring of diseases, such as cancer, inflammation, heart disease, diseases of the CNS, and susceptibility to infection by microorganisms.
  • the invention further provides for the use of any of the nucleic acid segments in the manufacture of a medicament for the treatment or prophylaxis of such diseases.
  • the invention further provides for the use of any of the DNA segments as a pharmaceutical.
  • KST291092 49 TATTGGGCAACAGGCTGCAGGTGAGGGGGCTGACAGGAGGAGGGT
  • WIAF-3648 AAAGGGTCATTAAAAACAAAACAAAATTGTGAAAAAA [A/G] AGAAATAAGAATGTGTCT stSG28751 97 CTGTTGCACAACTGCATTCTATCCTT
  • WIAF-2688 CCTTGGTCCAACACCCTATTGCTCCCCACCTTAAAATTGCTATGTCTATGTACAATCTTT WTR-866f 369 A AAGGAGTT [A/G] CTTATTGGGGCAGTTTCAAACTCAGAAAT
  • WIAF-3142 CAGATGAAATGAATACTTGAGAATTCTTACATAAAGGTGTAAAAATATAGTTATGGTTTT WIR-1275b 56 TCGCTTAGGGATAATTCCTGTTTCTGGCACTTTTATTTACATCCC
  • WIAF-3233 AGCAACATAAGCCAAAT [C/G] TTTGTGTTTATCTTACACGAAAGAGACCAGTATCTTTC WTR-1635a 77 TTTTGGCTTGGGTGGCTCACAACTTTTGTTCTTTAAATGAGTGGATATCCAAGGAAAAA
  • WTAF-3239 AGCAACATAAGCCAAATCTTTGTGTTTATCTTACACGAAAGAGACCAGTATCTTTCTTTT WTR-1635d 127 GGCTTGG [G/T] TGGCTCACAACTTTTGTTCTTTAAATGAGTGGATATCCAAGGAAAAA
  • WIAF-3053 TTAAAAGGCAGAGTGTCAAGCTGGATAAAGAAGCAAGAGCCAAAGCTATGCTGTCTTTAA WTR-1563a 96 GAGATTCATCTCACATGAAATGACACTCAGGCTCAATAAATAAATAAAAGGGGA
  • WIAF-3750 CTTTCTGTCACTGAATAACTGACCCATAAAACAATAGAGCATATTGACCTAGTGTGCAGG stSG29781a GTAAGCAATGAG
  • AAATTGGAGTCCTTCAAC CATGACAATGGAATGTCTCTTTAGTTACT ⁇ AGGTCTC WIR-2087a TACTTTCTCTCTGCAGTGTTTTGTTGCTTGTTTG
  • WIAF-3381 AAATTGGAGTCCTTCAACGCATGACA [A/G] TGGAATGTCTCTTTAGTTACTTAGGTCTC WlR-2087b TACTTTCTCTCTGCAGTGTTTTGTTGCTTGTTTG
  • WTAF-3383 AAATTGGAGTCCTTCAACGCATGACAATGGAATGTCT [C/G] TTTAGTTACTTAGGTCTC WIR-2087c_ TACTTTCTCTCTGCAGTGTTTTGTTGCTTGTTTG
  • WlAF-1682 ACATAGAAAAGGTACAGTAAAAAATACGGTATTATGGGGCCACCATCATATGTTTGGTCT stSGl 0082a 48 GTCGTGGACTAAAATGTCATTAT
  • WIAF-1683, /C TAGAAAAGGTACAGTAAAAAATACGGTATTATGGGGCCACCATCATAT GTTTGGTCT SLSG10082b GTCGTGGACTAAAATGTCATTAT _ _ _ _ _
  • WIAF-3801 TCCAGACTTCTGAGATAGCCTGGGATGAGCAATCCTGTTA [C/T] AGTACATCTGGACCT stSG26818 100 TCCCTACCTGGGCTCTG
  • WIAF-63 AAGGNCTAAAAGGAAATGTAATTCAAAATTCCTCTTCTTATGTG [C/T] ATACATTGATT MR14813b_ 164_ T TTTTTTTCTTTAACTTAGTGTTTCCAACAGTCAAATGACTC WIAF-3822, GCCTCTT [C/T] CCCTGGAAGCTGCACATCACGCAGAAGGACAACTACAGGGTCTACAAC stSG26825 67__ T ACGATGCC
  • WIAF-3224 AGAAAGGTTGAGTAAGTTTGACACAGTTTGACTTTAACATGTCAGTGAAAGTTGAAGGTA WIR-1441b 223 ACAAAGTTTCATTTGCAGTTAGAGGTGTCTCAATAGAGCGGAAGTATCTGCATTGA
  • WIAF-3369 TTATGAAAACACATTGGAAAAATTAAATAACCCAAGAGTTTCTCAGCACAGGAAAAACAT stSG15609a 46 GGCCTGGNCCTCTCTGCATGTGCACTGTGCTGCCGTGACAA
  • WIAF-3371 TTATGAAAACACATTGGAAAAATTAAATAACCCAAGAGTTTCTCAGCACAGGAAAAACAT stSG15609b 64 GGCCTGGNCCTCTCTGCATGTGCACTGTGCTGCCGTGACAA
  • WIAF-3373 TTATGAAAACACATTGGAAAAATTAAATAACCCAAGAGTTTCTCAGCACAGGAAAAACAT stSG15609c 104 GGCCTGGNCCTCTCTGCATGTGCACTGTGCTGCCGTGACAA
  • WIAF-1580 ATTCTGTGTGGGGTTTGGGACAGGAACACTGACCCCTGAAGTCGAGCCTGGGGGGTCTAA stSG3277a 43 CCATAGTGGGTCATTTGTCCNAGCCTGTTTTATGGGAAGGAACT
  • WIAF-158 TTTTTTCCAAAAAAGATGCAAATCTTTTCCTTATATGGATCAATACAGTAGTCAATCTTT MR5689a 90 T GTATTGGTTGATTAGAACTCCTGGAATGTA
  • WTAF-3200 TAATTGGAACTGTAGAATGAGAATGCTGATTCTTATTCTGGTTCTGTCAA [T/A] ATTTT WTR-1869 350 GCAAACCTTTACCTAATTGTGCTACCTTATTGGTCCCTGAAAACAGGA WIAF-31 , CTCCCAGTCTCTCTCTCTCTGCA [T/G] AATCTGGTCAAGCAGACGAGAAAAATCAGTTTGT MRI 1901 21 CTCTTTT NCTTTCCAAMTTAAGATTAATGGGAGAAAAGCATTT
  • WIAF-2990 GAGAAACAGACAAACAGGTCTGGGTTCTCTGACTAGGAGTTCCGCTTCTGAGATACAGTC WTR-258a 134 TTCTCAGTTTATGCTTGCTCTGTTCTGATACCAGTCGGGGTTGT
  • WIAF-3580 AAAATTTTTAAAGCTTCTTTTCAATCTACTTTTTGCTCAGNAAGGAATGTCTTCCCAACA stSG16412a 39 TCTTTCAATGCA
  • WIAF-3583 AAAATTTTTAAAGCTTCTTTTCAATCTACTTTTTGCTCAGNAAGGAATGTCTTCCCAACA stSG16412b 98 TCTTTCAATGCA
  • GTTACTGCATGCTTTTTTTCTT [G/A] CTGTTTTTTGAATTCTCTCTTTGTCTTTGATGT stSG21940 182 TAGATAGTTTGACTATAATGTACCATGGAGAATGCCTTTTTGCATTTCATCTGCCTGGGG
  • HSC15C072b TGCCAGGGGACCCTTATAGGCCTCTGTCTTTAAACCTGTAATGGTATATTAATCCTTGG ⁇
  • WIAF-1135 CACTATGAACGCTTCTTTCCCAGGACAGAAAATGTGTAGTCTACCTTTATTTTTTATTAA 7314c, WI- CAAAACTTGTTTTTTAAAAAGATGATTGCTGGTCTTAACTTTAGGTAACTCTGCTGTGCT 7314c GGAGATCATCTTTAAGGGCAAAGGAGTTGGATT _ ____ _
  • WIAI 2512 TTAGTGGCGATTTGTGTGATTTTGGTGCACCCATTACCCAAGGAGTATACACTGCACCAT STS R53533b, ACTCGGTCTTTTATCCCTCGCCCCTCTCCCACTTTTCCCCTCAAGTCCCAAAAGRCCATT WT 21961b GTATCATTCTTATGCCTTG _
  • WIAF-1227 TTATAGTTTACATTGATATCTAGACATATATCTTAAACAGTCTCCAAATTTNCTTTAATT
  • HSC0ZA102b AATCA [A/+] AGTATGTTAATGTCACTTGGAATTCTACATGGAAAAGCCAACAAAATAAC
  • WI-9497b TAAAACTTGACTAATGAAGATCAGCGTCACTAATAAAAG
  • STS-R53533c GGTCTTTTATCCCTCGCCCC [T/G] CTCCCACTTTTCCCCTCAAGTCCCAAAAG CCATT
  • WIAF-1212 AAATTCAAAGACTATCTGCAGCTAGTGTGTTTCTTCTTTACACACATATACACACAGACA 7338m2b
  • WIAF-1213 TCAAAGACTATCTGCAGCTAGTGTGTTTCTTCTTTACACAC [A/G] TATACACACAGACA 7338m2c
  • WIAF-4038 TCCTGATCTATTGGGAACTTCCTCCTAATAGATCAGGAAAATCCACCTCATTTAATCATG MH598, WI- GACAACNNAAAAGGAATA [T/C] GATCCCGCATGCAACATTTATTCAGTGAAAACATGAT 884 198 GAAAATGAACATAATGGTACTACTGAAAATGNGAGCACACCAGAAAAATTATAAATTAA
  • WIAF-1110 TA [C/T] AGTGGACAACAGCAATCAAATTATGGACACATGAAAGGGGGCAGTTTTGGTGG 7301m5b, WI- AAGAAGCTCGGGCAGTCCCTATGGTGGTGGTTATGGATCTGGTGGTGGAAGTGGTGGATA 7301b 182 TGG AGCAGAAGGTTCTAAAAACAGCAGGAAAAGGGCTACAG
  • WIAF-1111 TACAGTGGACAACAGCAATCAAATTATGGAC [A/C] CATGAAAGGGGGCAGTTTTGGTGG 7301 5c, WI- AAGAAGCTCGGGCAGTCCCTATGGTGGTGGTTATGGATCTGGTGGTGGAAGTGGTGGATA 7301c 1211 TGGTAGCAGAAGGTTCTAAAAACAGCAGGAAAAGGGCTACAG
  • WIAF -2267 CATAGAAAGGAGTCTTTGAGTATTGTACAGTTTTGAAAATTCTCTTTGAGATAATTGATT
  • A005I.24a AAT [A/G] AAAACCCAATTTCTCTTTCACCATTT GTTTGATTATCATCTGGATTTTCAC
  • A005D24a 123 AATTAAGGAAACAATACCAGTGTTGATAAAGACAATACCAGTGTTGATAAAGATAATACCAGTGTTGATAAAGATA
  • UTR-03180a AACCAGCGACTAATGCAATCCATTCCTCTCTTGTTTATAGTAATCTAAGGGTTGAGCAG ⁇
  • A005D24b AATAAAAACCCAATTTCT [C/T] TTTCACCATTTAGTTTGATTATCATCTGGATTTTCAC
  • A005D24b 138 AATTAAGGAAACAATACCAGTGTTGATAAAGACAATACCAGTGTTGATAAAGATA __
  • WTAF-212 CCCAGAAGGGACTGAGCTAAACAGTGTTATTATGGGAAAGGAAATGGCATTGCTGCTTTC
  • UTR-03180b AACCAGCGACTAATGCAATCCATTCCTCTCTTGTTTATAGTAATCTAAGGGTTGAGCAGT
  • WIAF-1352 GGATGAGGGCCACCAGGAAGCACAGGTCCAAGGCTGGTCCCACACTTATCAGCAGCAACA UTR-02921 , ACTGTCAGTTCATCCTGCATGGGAAAAATGTTGGAATGGGAGTCTGAAATGGGGCTACTG WI 7690 45 TTTCAGTCCTAACGTGCTGTGTGACATTGGGAC _ _
  • WTAF-4076 TTTTTTCCATCTTTTTCCTCTCTCGCTTTCTTACACAGAAACAT [A/G] CACATAC 7619m4o, WI- CGAGAAACCTATTTCTCAGACCCCTTTTTCTCCTCTGTCTTTCTCTCTCCCTCTCCCACA 7619o 228 CCTCACACACACATACTCCCACTTGCAACTATTCTGTTTC _ ___
  • WTAF 4078 TCCCTTTTTTCCATCTTTTTCCTCTCTCGCTTTCTTACACAGAAACATACACATAC 7619m4q, WT- CGAGAAACCTATTTCTCAGACCCCTTTTTCTCCTCTGTCTTTCTCTCTCCCTCTCCCACA t 7619q 106 CCTCACACACACATACTCCCACTTGCAACTATTCTGTTTC
  • WTAF-4079 AAATTAATTTTGTTGTTTCCTTTGAGGTTGATCGTTGTGTTGTTTTGCTGCACTTTTTAC 7830ml, WI- TTTTTTGCGTGTGGAGCTGTATTCCCGAGACAACGAAGCGTTGGGATACTTCATTAAATG 7830 44 TAGCGACTGTCAACAGCGTGCAGGTTTTCTGTTTCTGTGTTGTGGGGTCAA
  • WIAF-2065 CTTTAGGTCCTCTGCATATCATGGAAGCCAACTACTCTATTAACGCTTTCCCAATGATGC MR2981 b , WI - AGCCCAGTTCTGCATACAGTTTGTACAGAAATGCTATATTTATGGAAACAGCTGAAAAAT 2868b 60 GAAATATCGATATACCCCTAACAGTCATTTCTACAAAGGT
  • WT AF- 1354 AGTCTAGTTACCTACTTTTTCTTTGATTTTCGACGTTTGACTAGCCATCTCAAGCAA [C/ 7 773mlb , WI - G] TTTCGACGTTTGACTAGCCATCTCAAGCAAGTTTAATCAAAGATCATCTCACGCTGAT 7 773b 237 CATTGGATCCTACTCAACAAAAGGAAGGGTGGTCAGA _ ___
  • WTAF-1324 TTTAAAACATCATTACTGCCATCTTTATCATGAAGCACATCAATTACAAGCTGTAGACCA 7870b, WI- CCTAATATCAATTTGTAGGTAATGTTCCTGAAAATTGCAATACATTTCAATTATACTAAA 7870b 85 CCTCACAAAGTAGAGGAATCCATGTAAATTGCAAATAAACCAC _
  • WIAF-4177 ATCCTGAGCCTCCCAAGGTACAGCCTTTCACTACTATTCATCATATTGGCTAAGGTATTC WI-1732, WI- ATCATATTGGCTAAGGTATTCACCAACAGGGCTCATTTTCTATCAGACCTACAAGAAACC 1732 114 TACAGTGGCTATA
  • WIAF- 1 1 97 GTTAAAACATCAACTGAAGGGTTGGGTTAGGAACATTTACCCTGAAAAAAATATGAGGAT UT R- 01990 , GCATCATAAAATGTAAATATTTTCCTACCATGTTGGGGGGGCACAAATTTTAAAACTGGC WT - 7 1 53 161 ATCTTTACAAGTTTCTTCTTTATAAACACCCAAACAAAATCAAGTTTTATAAAG to
  • WIAF-4093 GAAAGACATGAGCTATTAGGAGCTCTGGCAAGGGCTTTGTCTTATCCTCCTTGCTATCCC PB876b, WI- TGATGACTGGGCAAAACAGTAGCTGCCCTGATTCCATGAGACAGAAAGGGGTGACTTATT 276b 25 TAATCCCAGAGCCACG
  • STS-T15424a CATAATGTGGTTTAAATTTTAAAAAATACTCAGAATGAGGTAGTATTTTAATTTTTAATT
  • WI-21627b 153 A AAAATGTGTTGCTTAGCCCTTGG
  • WI-7252d 540 [T/C]TATCAGCTTTTTAAAGTGGGTTATTCTGGAGTTTTT
  • WTAF-1208 CAACACCACGGTAGTGCCTGAAATTTCACCATTGCTGTCAAGTTCCTTTGGGTTAAGCAT
  • WIAF-1371 TCCTGTCAGAGATCTGGGAGGTCTCCACTGAGGATGTGAGCCTGATTATCCTATAGGCAG UTR-05629, ACGTGGGGAGGGTGGAGGGGTGACAGTGGAGGAAAATCCATGGATATCCACGCAGCAGCC WI-7981 261 CCTCTTTAACCTCATCTACAAGCA _ _ _ _ _
  • WIAF-4117 TCCGTTTGTGTGTTTGGCCAAATAATATCTCCCCCAGGGACGTCCTCTTTCTAATCCCTG WT-867, WI- AAACCTGAGAAAATGTTATCTTATGCAGTGCTATGGTTTGAATGTGTCCCCCACAAAGCA 167 113 CACATTAGAAACTTAATCCCCAGTGCAACA
  • WIAF-439 TTTAACACAGCCGTGTGTTCAAATGTACAGTGGTCCTTTTCAGAGTTGGACTTCTAGACT
  • WIAF-440 AAAAAAAAAGGGTGGTAACTGTTAAGCCTGCTGCAATGTTTAGACACGAGGGTGGGGGTG MR529, WI- GGGAGGTGGAATACCAAGGGAGGCGACACAGAATTTCCTTGCCTTTTGGTTTTCTCATAC 1819 51 TCAGTATTTCTGCCGTGGC

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Abstract

L'invention concerne des segments d'acide nucléique du génome humain, comprenant des sites polymorphes. L'invention concerne également des amorces et des sondes spécifiques d'allèles et s'hybridant aux régions entourant ces sites. On utilise ces acides nucléiques, ces amorces et ces sondes dans des applications telles que la médecine légale, la recherche de paternité et les analyse médicales et génétiques.
EP99915117A 1998-04-09 1999-03-30 Marqueurs bialleliques Withdrawn EP1068354A2 (fr)

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AU1477601A (en) * 1999-11-10 2001-06-06 Affymetrix, Inc. Genetic compositions and methods
US6756214B2 (en) 2000-11-09 2004-06-29 Zymogenetics, Inc. Protein zlmda33
AU2002258626B2 (en) 2001-04-10 2007-01-18 Agensys, Inc. Nucleid acid and corresponding protein entitled 158P3D2 useful in treatment and detection of cancer
WO2012097474A1 (fr) * 2011-01-20 2012-07-26 深圳华大基因科技有限公司 Procédé et système pour détecter les sites d'insertion de fragments étrangers transgéniques

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WO1995012607A1 (fr) * 1993-11-03 1995-05-11 Molecular Tool, Inc. Polymorphismes de mononucleotide et leur utilisation en analyse genetique
FR2722295B1 (fr) * 1994-07-07 1996-10-04 Roussy Inst Gustave Methode d'analyse d'adn dite sscp et gel d'electro-phorene
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EP0892068A1 (fr) * 1997-07-18 1999-01-20 Genset Sa Méthode pour la génération d'une carte du génome humain avec haute densité basée sur linkage diséquilibrium
EP0991770B1 (fr) * 1997-12-22 2000-11-29 Genset Gene associe au cancer de la prostate

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