WO1992013102A1 - Marqueurs polymorphes d'adn utilises chez les bovins - Google Patents

Marqueurs polymorphes d'adn utilises chez les bovins Download PDF

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WO1992013102A1
WO1992013102A1 PCT/US1992/000340 US9200340W WO9213102A1 WO 1992013102 A1 WO1992013102 A1 WO 1992013102A1 US 9200340 W US9200340 W US 9200340W WO 9213102 A1 WO9213102 A1 WO 9213102A1
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seq
dna
genomic
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Michel Georges
Joseph M. Massey
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Genmark
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
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    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
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    • 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
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    • C12Q1/6841—In situ hybridisation
    • 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
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    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the invention relates to gene mapping, selective breeding and genetic identification in domestic animals. BACKGROUND OF THE INVENTION
  • the typical mammalian genome is composed of an approximately 3 ⁇ 10 9 base pairs long DNA stretch, divided over a species-specific number of chromosomes, and containing all the information required for the proper development and functioning of a normal being. Each individual has two copies of this message: one paternal in origin and one maternal. Although their overall architecture and content are virtually identical, the paternal and maternal DNA sequences exhibit subtle "allelic" differences, hereinafter referred to as DNA Sequence Polymorphisms or "DSP".
  • the DSP that can be recognized in a given population are the molecular basis of the genetic component of the observed phenotypic variance. One can distinguish three types of DSP. 1. Single Base Pair Polymorphisms
  • DSP are due to single base pair differences distinguishing alleles. These can be either base pair substitutions - transitions (Purine to Purine or Pyrimidine to Pyrimidine) and transversions (Purine to Pyrimidine and vice versa) -, or the insertion/deletion of a single base pair.
  • the frequency of single base pair polymorphism is measured by the nucleotide diversity, ⁇ , or average heterozygosity per nucleotide site (1) .
  • the nucleotide diversity has been estimated from Restriction Fragment Length Polymorphisms at 0.002 for human (2), and at 0.0007 in cattle (3,4). This means that on the average a human will be heterozygous for one every 500 nucleotides, and a cow for one every 1,500 nucleotides.
  • the cytosine in the CpG dinucleotide sequence is known to be the substrate of an eucaryotic methylase, which will add a methyl group in position 5 of the pyrimidine ring, if the cytosine of the complementary CpG dinucleotide is itself methylated. Deamination of a 5-methylcytosine generates a thymine, blurring the task of the DNA repair machinery which will half of the time resolve the ensuing mismatch by replacing the original guanine instead of the mutated thymine. As a consequence, cytosines in the CpG doublet exhibit mutation rates at least ten times higher than other nucleotides, and hence are rich sources of single base pair polymorphisms (4, 5). 2.
  • allelic variants involve DNA sequence rearrangements such as the insertion or deletion of a stretch of DNA, DNA sequence inversions and duplications.
  • a significant proportion of the eucaryotic genome is composed of sequences widely termed "satellite DNA,” sharing a common organization: a sequence motif, varying in length between one and several thousand nucleotides, repeated in a head-to-tail or so-called tandem arrangement.
  • satellite DNA sequences widely termed "satellite DNA,” sharing a common organization: a sequence motif, varying in length between one and several thousand nucleotides, repeated in a head-to-tail or so-called tandem arrangement.
  • satellite sequences were grouped into four size classes: macro-, midi-, mini- and micro-satellites.
  • Minisatellites are also known as Variable Number of Tandem Repeats (VNTRs) (18 ⁇ 21).
  • mini-satellites seem to be confined to heterochromatic regions (22), mini- and micro-satellites have been found scattered throughout the genome with, however, clustering of mini-satellites (23-34).
  • minisatellite clusters seems to be particularly abundant in proterminal regions (35).
  • the only midisatellite described as such today, has been mapped to the short arm of chromosome 1 (36).
  • the polydeoxyadenylate tract of Alu repetitive elements are also characterized by length variation and are thus an abundant source of genetic markers as well (37).
  • DSP may alter the restriction patterns of defined chromosomal regions, generating so-called Restriction Fragment Length Polymorphisms (RFLP).
  • RFLP Restriction Fragment Length Polymorphisms
  • agarose gel electrophoresis pulse-field gel electrophoresis (30) or polyacrylamide gel electrophoresis (42) for intermediate, large or small fragments respectively.
  • RFLPs are classically detected by Southern blot hybridization.
  • the use of PCR obviates the need for restriction enzyme digestion, the DSP reflecting itself in the size of the amplified product.
  • a mismatched heteroduplex will differentiate itself from the perfectly matched homoduplex by an altered melting behavior which can be detected as an all-or-none, binary response: positive for the homoduplex, negative for the heteroduplex, or in a more graded response, allowing to distinguish between different heteroduplex variants.
  • the classical all-or-none test depends on the use of allele-specific oligonucleotides in hybridization experiments. By choosing appropriate hybridization and washing conditions, the allele-specific oligonucleotide will only recognize a perfectly complementary sequence (45). With the advent of PCR, new variants of this approach have been described including reverse dot-blot (46), the Amplification Refractory Mutation System (47) or allele-specific polymerase chain reaction (48), and Competitive Oligonucleotide Priming (49). The Ligation Amplification Reaction, amplification of specific DNA sequences using sequential rounds of template-dependent ligation, can also be considered as a peculiar application of the allele-specific oligonucleotide approach (50).
  • More discriminating is Denaturing Gradient Gel Electrophoresis, exploring the pattern of melting behavior characterizing each heteroduplex when electrophoresed through an increasing gradient of DNA denaturants (51).
  • the sensitivity of this method can also be improved by pre-amplifying the target sequence by PCR.
  • Ribonuclease and chemical mismatch detection The presence of a mismatch in a heteroduplex makes those molecules susceptible to cleavage by various means including chemical treatment with either hydroxylamine or osmium tetroxide (52), as well as ribonucleases such as RNase A in case of an RNA:DNA heteroduplex (51). Electrophoretic analysis of the cleavage products allows one to distinguish different genetic variants. Again, implementing PCR will increase the sensitivity of the approach.
  • DNA variants exhibit indeed mobility shifts when electrophoresed in such conditions, presumably resulting from conformational changes caused by sequence alterations, hence the name single stranded conformation polymorphism. Again, the altered mobility can be detected by blot hybridization analysis or relying on PCR (53, 54). 4. Direct Determination of the DNA Sequence
  • the probability for a selectively neutral neomutation to be fixed in a population of N individuals is equal to its initial frequency 1/2N
  • the average time for fixation is four times the "effective" population size or 4Ne
  • the rate k of mutant substitution per generation is simply equal to the rate of mutation per gamete and per generation, ⁇ , independent of what the population size may be.
  • a polymorphism observed in a population at a given time is composed of "transient” alleles catched in their stochastic "odyssey” throughout the population.
  • DSP are more prominent for non-coding sequences and, within coding sequences, at third codon positions (compared to first and second positions (58)).
  • s and t being the respective selection coefficients of the homozygotes.
  • the best known example of balanced polymorphism due to overdominance is the maintenance of the S ⁇ -globin allele (causing sickle-cell anemia in the homozygotes) as well as thalassemia-causing mutants (see, for instance, 61) in populations subjected to malaria, because of the resistance exhibited by the heterozygotes towards the parasite.
  • the high level of polymorphism observed at the Major Histocompatibility Locus is thought to result from overdominance selection as well (62).
  • Frequency dependent selection may be another cause of balanced polymorphism, an example being the "rare mate advantage" observed in Drosophila (63).
  • Two loci are said to be genetically linked if, during meiosis, they recombine at significantly lower than a 50% rate, i.e., they generate significantly more parental gametes than recombinant gametes.
  • the recombination rate between loci reflects the frequency of occurrence of an uneven number of crossing-overs between the loci. Because the probability for crossing-over is proportional to the distance separating the loci, the recombination rate can be used as a unit of chromosomal length. This length unit is known as the Morgan (M), 1 cM corresponding to the distance separating two loci exhibiting a 1% recombination rate.
  • M Morgan
  • 1 cM corresponding to the distance separating two loci exhibiting a 1% recombination rate.
  • the relation between centimorgan and recombination rate is essentially linear. For longer distances, however, the relation is more complex, depending on the frequency of double crossing-overs, itself affected by eventual interference.
  • a lodscore > 3 is accepted as significant evidence for linkage. The prior probability of linkage between two loci has been used to justify this stringent critical value. Note that 21n(LR) can be used as well, having a chi-square distribution with one degree-of-freedom under the null hypothesis of no linkage.
  • the genotypic equilibrium frequencies are only reached when the previous generation produces the four possible gametes at the expected frequencies: albl: p1q1, a2b1: p2q1, albl: p2q1, a2b2: p2q2.
  • the difference between observed and expected gametic frequencies is called linkage disequilibrium, D.
  • the value of D is reduced by d. ⁇ every generation, ⁇ being the recombination rate between the two loci.
  • ⁇ being the recombination rate between the two loci.
  • For unlinked loci D diminishes by 1/2 every generation; for linked loci, however, the reduction of D per generation will be much smaller.
  • the detection of a linkage disequilibrium is an indication of linkage between the corresponding loci.
  • mapping strategy such as "in situ" hybridization (see, for instance, 69), the use of somatic cell hybrid panels and radiation hybrid mapping (reviewed in 70) and comparative mapping (71), the map location of a large set of DSP can be determined in order to build a genetic marker map (see, for instance, 72-74). Assuming a total map length of 30M as for the human, and a desirable maximum distance of 20cM between markers, a set of 150 DSP could cover the entire genome. However, many more markers will be needed to generate reasonable maps for our domestic species, essentially for two reasons. First, most of the time we have no a priori information on the location of the characterized markers.
  • any gene for which the appropriate segregating family material is available can be located on the map.
  • the expected lodscore for doubly informative, phase-known meioses approximates 0.16 (75). Therefore, 20 such meioses are theoretically sufficient to establish linkage with a lodscore of 3.
  • the number of individuals to analyze will be higher, a function among other factors of the quality of the marker, expressed as its Polymorphism Information Content (76).
  • the efficiency of this approach has been illustrated by the recent mapping of a large number of genes involved in human single gene disorders (see, for instance, 77, 135).
  • the identification of DNA markers for a defined gene can be the first step towards its molecular cloning.
  • Successful "positional cloning", or the isolation of a gene based on its map location, has been achieved in the human for Chronic Granulomatous Disease, Duchennes Muscular Dystrophy, Retinoblastoma, Wilms Tumor, Cystic Fibrosis (134), Type-1 Neurofibromatosis and the Testis Determining Factor.
  • interval mapping not only does interval mapping solve the problem of simultaneous estimation of location and effect, but because of its increased power, it reduces the number of individuals to be tested to detect linkage with a QTL of given effect (81).
  • the number of individuals to test in order to detect an effect of given amplitude, ⁇ can be estimated from: where n gives the required sample size, s 2 is an estimate of the residual variance, t 0 is the t value associated with Type I error, and t 1 is the t value associated with Type II error; t 1 equals tabulated t for probability 2(1-P) where P is the required probability of detecting «5 if such a difference exists (82).
  • the number of individuals to test increases by a factor 1/(1-2 ⁇ ) 2 for single marker analysis, by a factor ⁇ (1- ⁇ )/(1-2 ⁇ ) 2 in the case of interval mapping, ⁇ corresponding to the recombination rate between the flanking markers (81).
  • the individuals whose genotype can be most clearly inferred from their phenotype are the ones providing most of the linkage information when studying complex traits. For quantitative traits, these are the individuals whose phenotypic value deviates most from the mean: the tails of the distribution. Sample sizes could be reduced by 60% and even 80% by focusing on individuals deviating one and two standard deviations, respectively, from the mean. Paradoxically, selective genotyping may be limited by the size of the studied population. Indeed, a larger sample will be required in order to find enough individuals one or two standard deviations from the mean. c. Decreasing Environmental Variance via Progeny Testing
  • Markers for which a priori evidence for linkage disequilibrium is highest are the so-called "candidate genes": genes expected from their physiological role to be likely candidates for the QTL itself. DSPs at those loci, even selectively neutral by themself, can be expected to exhibit linkage disequilibrium with the hypothetical functional mutations because of their very tight linkage.
  • the B allele of the K-casein gene has been shown in several studies to increase protein yield in milk by about 3%, and possibly to improve cheese yield independent of the effect on protein yield (see, for instance, 89, 90). IV. USE OF DNA MARKERS IN BREEDING PROGRAMS
  • Marker Assisted Selection is used to reduce the frequency of the major gene causing Porcine Stress Syndrome (PSS). Susceptibility to PSS correlates with Halothane sensitivity or Malignant Hyperthermia. This condition has been mapped to a linkage group on pig chromosome 6, encompassing the following markers: S(A- O) -GPI-Hal-H-AlBG-PGD (reviewed by 94). These markers are used for the Marker Assisted Selection against the PSS condition. Recently, the ryanodine receptor gene has been identified as a good candidate for the Malignant Hyperthermia or Hal gene (95).
  • marker assisted selection under its present form, is its limitation to the exploitation of genetic variation preexisting within the commercial breed of interest, and only that present in a "high merit” genetic background.
  • Favorable mutations appearing within a mediocre background, or present in "exotic” germplasm, would be difficult to exploit, even with markers.
  • INDIVIDUAL IDENTIFICATION AND PATERNITY DIAGNOSIS Methods to estimate the breeding value of an animal use information from relatives. As a matter of fact, keeping track of familial relationships has always been one of the major concerns of animal breeders, and parentage control is now a widely used procedure for several domestic species. Parentage control relies on the use of polymorphic systems within the studied population. The alleles that characterize an individual originate from the mother or the father. If one of the parents is known (usually the mother), the alleles necessarily transmitted by the other parent can be deduced easily. Paternity testing consists of scoring the existence or lack of those obligate paternal alleles in the genotype of the putative parent.
  • locus-specific genetic markers for domestic cattle and related bovids that constitute a primary bovine DNA marker map.
  • these markers and the map are useful for: - individual identification,
  • Figure 1 shows a typical VNTR pattern obtained with probe GMBT-005, using HaeIII.
  • FIG. 1 Example of a microsatellite pattern (TGLA9).
  • VNTR Tandem Repeat Markers
  • microsatellites were proven to be an abundant source of highly polymorphic markers in the human (32-34). As their name implies, microsatellites are minute VNTR markers (18-20), characterized by tandem repetitions of very short repeats, one to four base pairs in length. Microsatellites exhibit levels of polymorphism comparable to VNTRs, but are much more abundant and apparently evenly spread throughout the genome. We have estimated the frequency of (CA)-dinucleotide repeats in the bovine genome at > 150,000. Because of their small size, their detection is greatly facilitated by PCR.
  • Example 3 A detailed escription of our microsatellites is reported in "Example 3".
  • the relative location of the markers was determined by linkage analysis in pedigrees generated by multiple ovulation and embryo transfer. To assign linkage groups to specific chromosomes, highly polymorphic "anchor markers" were mapped using somatic cell hybrids (Jim Womack, Texas A&M) , and by in situ hybridization (Rudy Fries, ETH - Zurich).
  • Identifying polygenes requires the analysis of pedigrees of considerable size, despite the development of procedures such as interval mapping, simultaneous search, selective genotyping, etc.
  • Weaver or bovine progressive degenerative myeloencephalopathy is a recessive disorder characterized by the appearance between 5 and 8 months of age of bilateral hind leg weakness, ataxia with deficient proprioceptive reflexes, without skeletal or muscular defects.
  • Estimates of gene frequency in the American Brown Swiss breed point towards the maintenance of the Weaver gene at relatively high frequency (>5%), despite the implementation of programs for detection and elimination of carrier bulls.
  • Brown Swiss animals showing symptoms of Weaver were identified with the help of the .American Brown Swiss Association. Blood samples were collected from the affected animals, their parents, and full-siblings when available. Diagnosis of Weaver was confirmed in most cases by anatomopathological examination of spinal cord and cerebellum at the Department of Pathology of the College of Veterinary Medicine, Kansas State University. Shrunken Purkinje cells in the cerebellum, spheroids and degenerated myelin sheets in the spinal cord were considered pathognomonic. Altogether, 78 animals were identified generating a single, large pedigree. All animals were genotyped for more than 70 genetic markers: 40 Variable Number of Tandem Repeat markers and more than 30 Microsatellites.
  • the marker TGLA116 is characterized by three alleles segregating in our Weaver pedigree. 72% of the affected individuals were of the 3/3 genotype, 16% of the 2/3 genotype, and 12% of the 1/3 genotype. Hence, and at least in our family material, the "Weaver" allele was clearly associated with allele 3 at the marker locus. Whether similar disequilibrium will be observed at the population level remains to be determined.
  • the reported lodscore values were obtained using allelic frequencies estimated on a sampl of 135 sires from the American Brown Swiss breed.
  • a "control" pedigree consisting of more than 100 Weaver-free Holstein individuals, was therefore typed for TGLA116 as well.
  • the microsatellite marker was characterized in this pedigree by the same three alleles, with respective frequencies of 18%, 57% and 25% for alleles 1, 2 and 3, showing a perfect Mendelian segregation. Therefore, it is concluded that marker TGLA116 is genetically linked to Weaver. From the generated lodscore curves, the genetic distance between the two loci is estimated at 3 ⁇ 10 centiMorgan.
  • the TGLA116 marker will allow us to perform marker assisted selection against the Weaver condition. Indeed, it is now possible for offspring from individuals heterozygous for both the Weaver condition and TGLA116, to estimate the genotypic likelihoods at the Weaver locus based on their TGLA116 genotype and that of their parents.
  • Marker Assisted Selection under its present form, is its limitation to the exploitation of genetic variation preexisting within the commercial breed of interest, and only if present in a "high merit" genetic background.
  • Favorable mutations appearing within a mediocre background, or present in "exotic” germplasm, would be difficult to exploit, even with markers.
  • a gene responsible for a favorable attribute can be introgressed from a "donor” strain into a "recipient” strain by repeated backcrossing. During the introgression process, the retention of the favorable gene is monitored in the backcross products, with linked, flanking DNA markers. This latter aspect is particularly important for traits involving multiple genes and/or characterized by sex- or agelimited expression.
  • Classical genetic theory tells us that, with the exception of the "marked" segment whose retention is desired, the genomic contribution of the donor line is diluted by half after each backcross. Hence, and after four backcrosses, the recipient genome is reconstituted to ⁇ 90% of the original.
  • the backcross retains one copy of the desired "donor" variant. If required, one intercross will then generate 25% of offspring homozygous for the favorable donor variant.
  • the net result is a "graft" of an advantageous gene within a recipient background. The procedures entirely respects organization and chromosomal localization of the grafted gene, avoiding aberrant expression patterns, which are too often characterizing transgenes.
  • the gene to be transferred does not need to be cloned per se. Only its genetic map location is required, as defined by the availability of linked markers, ideally flanking the gene of interest on each side. Hence, this procedure is perfectly applicable for the introgression of QTL identified through the previously described mapping strategies.
  • Marker Assisted Introgression can easily be applied to several genes simultaneously. This feature will be of particular interest for complex traits involving several genes. Introgressing more than one gene from a donor to a recipient line, however, increases the selection intensity at each backcross: with 1 marker, 1/2 of the offspring have the favorable genotype, with 2, 1/4 and with n markers, (1/2) n .
  • Introgression by repeated backcrossing is common practice in a variety of organisms, but is essentially unfeasible in domestic animals such as cattle, because of their prohibitively long generation time.
  • the generation interval of such species could, however, be reduced based on the "in vitro" maturation and fertilization of foetal oocytes, hereinafter referred to as "Velogenesis".
  • oogenesis begins with the formation of primordial germ cells in the region of the allantois. These precursor cells migrate to the developing gonads where after a period of mitotic proliferation, they enter meiosis. Meiosis is arrested at the diplotene stage of prophase I by the poorly understood "meiotic division I arrest system", after which the primary oocyte enters a resting phase. During the life time of the animal, small numbers of resting primary oocytes are successively recruited into a pool of growing oocytes, within the environment of a gonadotropin-dependent developing follicle.
  • oocytes growth in size acquire the competence to resume meiosis if appropriately stimulated, and accumulate the required material to sustain the early stages of the subsequent embryonal development.
  • Resumption of meiosis and oocyte maturation is triggered by a hypothetical maturation-inducing signal produced by granulosa cells in response to gonadotropins.
  • oocyte maturation seems to be mediated by a drop in cyclic AMP in the oocyte and subsequent inactivation of a type A protein kinase.
  • Evidence for the role of this pathway in oocyte maturation is, however, much more controversial in ruminants.
  • gonadotropins act, among other pathways, through the activation of adenylate cyclase with subsequent increase in cAMP concentrations (102) .
  • a cascade of still to be determined events then probably leads to the phosphorylation and activation of a phosphatase, probably homologous to the S. Pombe cdc25 gene (103), which will itself dephosphorylate and activate the M-phase promoting factor (MPF), now known to be a complex of a p34 cdc2 protein kinase subunit with a B type cyclin (see 104 for a review).
  • M-phase promoting factor M-phase promoting factor
  • the maturing oocyte completes the first meiotic division and enters the second (becoming a secondary oocyte) which will be arrested as well at metaphase II until fertilization.
  • This "meiotic division II arrest system” is thought to reflect the stabilization of MPF mediated by the kinase activity of pp39 mos on either a cyclin protease or on cyclin itself. Fertilization relieves this block, by increasing the intracellular Ca 2+ concentration, triggering calcium-dependent protease activity (reviewed in 104).
  • primordial germ cells In cattle, primordial germ cells reach the genital ridge at about 40 days of gestation. After a period of mitotic proliferation, they differentiate into oogonia starting around 60 days of gestation. Mitotic prolifera- tion of the germ line ceases around day 170 of gestation fixing the maximum number of oocytes the female will ever have. Meiosis starts at about 80 days, and the first primordial follicles are discernable at 90 days of gestation. Remarkably, activation of resting primordial follicles starts already in utero. around day 140, and secondary and tertiary follicles can be seen at 210 and 230 days, respectively.
  • mice of culture systems supporting the growth of primary follicles yielding mature oocytes capable of fertilization in vitro and development to term (106, 107). It is reasonable to anticipate that similar conditions, supporting development of bovine oocytes, will become available in a species were primary oocytes from relatively small antral follicles can already be successfully matured and fertilized in vitro.
  • velogenesis On way to achieve velogenesis would be to attempt to rescue oocyte nuclei from primordial follicles by their transfer into enucleated, maturable oocytes.
  • velogenesis could similarly be accomplished by the early stimulation of spermatogenesis.
  • VNTRs have been instrumental in the genetic mapping of several disease-causing genes, as tools for individual identification and paternity diagnosis and to address a variety of biological issues, including imprinting, loss of heterozygosity in malignancies, etc.
  • VNTR variable number of tandem repeat
  • genomic DNA from 20 unrelated cows was digested to completion with Mbol or HaeIII. After two rounds of size fractionation by agarose gel electrophoresis, electroelution and addition of EcoRI linkers (only for HaeIII restricted DNA) , fractions from 3 to 4 Kb (kilobases), from 4 to 6 Kb and above 6 Kb were ligated into the BAP-dephosphorylated BamHI and EcoRI sites, respectively of pUC13.
  • Approximately 80,000 independent clones were obtained by transformation of DH5 ⁇ cells, and were screened successively with the following minisatellite sequences: the 282 base pair HaeIII-Clal fragment containing the minisatellite in the protein III gene of wild-type M13, pUCJ, pSP64.2.5EI ('Per'), p ⁇ 3'HVR64, pINS310, EFD134.7 and pS3 (20, 21, 110, 123, 143). Hybridization and washings were done in the conditions used to generate multilocus DNA fingerprints with the respective probes (110, 143).
  • plasmid DNA isolated from positive colonies was used to probe Mbol, Haelll and TaqI Southern blots of 18 randomly selected American Holsteins. Hybridizations were done at 65 C in 7% SDS, 10% PEG, 50mM NaHP04 with addition of 50 ⁇ g/ml bovine genomic DNA. Final washes were at 65 C in O.lxSSC, 0.1% SDS. When using bovine probes on ovine Southern blots, hybridization and washing temperatures were reduced by 10 C.
  • Allelic frequencies were estimated from the sample of 18 randomly selected American Holsteins. Matching probabilities and exclusion powers (113) were then estimated by Monte-Carlo simulation (10,000 simulations in each case), assuming Hardy-Weinberg equilibrium and using "Pat-Power", a program designed by one of us. The following parameters were estimated: MPR: matching probability for two randomly selected individuals; MPS: matching probability for full-sibs; EPR: probability to exclude an alleged father unrelated to the real one (mothers phenotype known); EPS: probability to exclude an alleged father full-sib to the real one (mothers phenotype known); EP1: probability to exclude a wrongly assigned parent without phenotypic information available from the other one. Patpower calculates Matching Probabilities and Exclusion Powers characterizing given autosomal polymorphic systems, by Monte-Carlo simulation.
  • Matching Probabilities relate to individual identification and express the likelihood that two individuals would have the same pattern with a given probe. Patpower calculates two types of Matching Probabilities: MPR, the Matching Probability for two unrelated individuals, and MPS, the Matching Probability for two full-sibs.
  • Exclusion Powers relate to paternity diagnosis and express the likelihood that a wrongly assigned paternity or maternity will be detected with a given probe. Patpower determines three types of Exclusion Power: EPR, where one parent is known with certainty, the proband is unrelated to the other real parent; EPS, where one parent is known with certainty, the proband is full-sib of the other real parent; and EP1, whre only the proband is available.
  • EPR where one parent is known with certainty, the proband is unrelated to the other real parent
  • EPS where one parent is known with certainty, the proband is full-sib of the other real parent
  • EP1 whre only the proband is available.
  • the user needs to input the number of alleles characterizing the polymorphic system in the population of interest, their respective frequencies, and their dominance-recessivity relationships.
  • a and B are codominant and both dominate 0.
  • Each allele is given a binary code following the rules of the "LINKAGE" program (60).
  • Patpower then stochastically generates a pair of parents with an offspring, a full-sib of the real father and an unrelated individual.
  • "Phenotypes” are obtained from the genotype using the boolean "or” operator and are used to determine matching between unrelated individuals (MPR) and between full-sibs (MPS), as well as the exclusion of the unrelated individual considered as a proband, with (EPR) and without information (EPl) from one of the real parents, and exclusion of the uncle considering information from the real mother (EPS).
  • MPR unrelated individuals
  • MPS full-sibs
  • EPR unrelated individuals
  • EPl without information
  • hybrid somatic cells were prepared by fusion as previously described (97). Southern blot hybridization and concordancy analysis were done according to Threadgill et al. (114).
  • Chromosomes were prepared as described by Fries et al. (115) and chromosome identification was based on QFQ-banding and according to the international standard
  • Table 2 reports estimated matching probabilities and exclusion powers as well.
  • Systems GMBT-009, GMBT- Oil and GMBT-022 were treated as "open" systems, meaning that - because of their small size - some alleles were not detectable in our conditions. To avoid ambiguities in identification and paternity diagnosis, these unidentified alleles were pooled in a single "recessive" class. For individuals showing a single band, no distinction was made between homozygosity and heterozygosity based on band intensity.
  • 1 MPR is Matching Probability for two randomly selected individuals.
  • EPR Exclusion Power when putative father is unrelated to real father.
  • Reference markers for the respective synteny groups were U1:GNB1, U2:ME1, U3:NKNB, U4:MPI, U5:FOS, U6:AMY1, U7:LDHA, U8:GNB2, U9:GPI, U10:SOD1, U11:VIM, U12:GPX1, U13:MET, U14:GSR, U15:CASK, U16:ABL, U17:CRYG, U18:GGTB2, U19:CAT, U20:GL01, U21:GH, U22:.AMH, U23:ALDH2, U24:TG, U25:CLTLA, U26:OAT, U27:DU27S1b, U28:MBP, U29:RBP3 and X:DMD. Synteny groups with highest concordancy scores, to which corresponding VNTRs were assigned, are underlined.
  • VNTRs was supported by the tentative assignment of 13 of them to 11 different synteny groups using somatic cell hybrids (Table 4).
  • GMBT036 identifies a previously unmarked bovine synteny group.
  • Probe GMBT-021 was assigned to the same synteny group as probes GMBT-015 and -016. Although the latter two probes were shown to be tightly linked, linkage between those probes and the former one could be excluded for recombination rates ⁇ 15%.
  • GMBT-009 45 32 55 45 55 80 65 55 63 70 55 50 50 63 65 60 50
  • GMBT-011 30 37 50 40 50 65 60 50 63 55 50 35 35 68 50 45 35
  • GMBT-015 65 63 65 95 25 60 25 65 75 70 55 60 80 47 55 60 80
  • GMBT-019 40 32 50 20 100 55 80 40 31 35 70 45 45 63 60 45 45 45
  • GMBT-021 70 68 60 100 20 55 30 60 81 75 50 65 75 53 50 55 75
  • GMBT-022 50 47 30 60 30 55 40 60 50 85 30 35 45 58 50 35 45
  • GMBT-036 65 58 75 55 45 50 45 45 56 50 75 70 60 42 45 70 60
  • GMBT-041 60 58 60 80 20 65 30 60 75 75 50 55 75 37 60 65 75
  • GMBT-002 75 55 70 40 60 55 40 50 45 70 55 45 60
  • GMBT-005 50 40 65 75 55 80 65 67 40 65 90 60 25
  • GMBT-006 45 55 60 70 60 75 100 50 55 40 55 85 30 t
  • GMBT-009 65 85 70 60 100 55 60 33 85 60 55 65 30
  • GMBT-019 50 70 25 35 55 50 55 50 60 45 30 60 65
  • GMBT-022 30 40 55 21 55 60 75 67 60 35 60 70 25
  • VNTRs were as well mapped by "in situ" hybridization: GMBT-006 to 14qll-16, GMBT-005 to 24q13.3-22, GMBT-011 to 26q11-21, GMBT-015 and GMBT-016 to 21q22-24, GMBT-019 to 10ql4-23, GMBT-022 to 19q21-23, GMBT-028 to 2q13-21. Again good genomic coverage was evident, since six probes mapped to five different chromosomes. Probes GMBT-015 and -016 both mapped to 21q 23-24 as expected from the linkage study and the assignment on the hybrid panel. Surprisingly, five out of the eight VNTRs clearly showed an interstitial map location.
  • the three probes produced signals on chromosomes 18, 7 and 11, recognized as evolutionary homologues of bovine chromosomes 21, 10 and 19 (116) .
  • the signals were found over the exact positions as expected in case of conservation of chromosomal location in cattle and sheep.
  • the fragments > 2 Kb represent about 10 -3 of the total number of Mbol or HaeIII fragments, corresponding to approximately 10 4 fragments; this allows us to work readily with plasmid vectors; (2) the subsequent search for and use of the polymorphism is performed with the same enzyme used to generate the libraries, obviating the need to screen several restriction enzymes, hence reducing costs; (3) relying on frequent four-cutters, the cloned minisatellites contain very little flanking sequences and only very few of them carry highly repeated sequences which would interfere during hybridization; (4) theoretically, the larger minisatellites targeted by this approach are more likely to be involved in mutational events and could therefore be the more polymorphic ones.
  • a disadvantage of this approach is the unequal representation of minisatellite loci in our library.
  • the libraries were generated with a mixture of DNA from 20 unrelated individuals, to increase the number of clonable microsatellites.
  • loci for which most alleles are within the selected size range will be overrepresented, compared to loci for which the majority of alleles in the population are bellow this range.
  • locus-specific VNTRs are much easier to interpret and are more reproducible. Following properly established standardization procedures, a "common language" could be established allowing exchange of information between laboratories. It is noteworthy that heterozygosity and allelic frequencies for some probes seem to vary substantially between breeds. As an example, probe GMBT-012 is characterized by an heterozygosity of 22% in Holsteins, but higher than 50% in both Herefords and Brown Swiss. Hence, proper use of these probes may initially require accurate estimation of genetic variation for different breeds.
  • DSP DNA Sequence Polymorphism
  • PIC Polymorphism Information Content
  • Bovine genomic DNA was prepared using standard procedures, partially digested with Mbol, and size fractionated by rate zonal centrifugation in a 10%-40% sucrose gradient. Size fractions around 40 Kb were ligated into the Xhol site of the cosmid vector pWEC (pWE 15 vector (Stratagene) with pUC18 polylinker - Erica Cumlin, personal communication), after partial fill-in of the insert and vector sticky ends with respectively dATP, dGTP and dCTP, dTTP. The obtained constructs were packaged into Gigapack II Gold extracts (Stratagene) and used to infect E.Coli 490A hosts (gift from R. White, University of Utah, Salt Lake City, Utah, USA) . 110 colonies were selected at random, cosmid DNA was prepared using standard procedures, and purified by CsCl/Ethidium Bromide isopycnic centrifugation.
  • Genomic DNA from 9 unrelated Holstein individuals was prepared from venous blood using standard procedures and digested with 5U/ ⁇ g of the following enzymes in the presence of 4mM spermidine: BamHI, BgII, BgIII, EcoRI, EcoRV, HindIII, Kpnl, Mspl, PstI, PvuII, TaqI and Xbal. 4 ⁇ g DNA per individual was separated according to size by agarose gel electrophoresis and blotted onto Pall Biodyne B membranes using NaOH 0.4M as transfer buffer.
  • Membranes were prehybridized at 65 C for 4 hours in 10% PEG, 7%SDS, 50mM NaHP04 (pH 7.2) in the presence of 350 ⁇ g/ml bovine genomic DNA. Cosmid DNA was labelled by random-priming (111) to specific activities of .5*10 9 cpm/ ⁇ g, prehybridized with bovine genomic DNA (5mg/ml) for 90 min. at 68 C (112), and added to the prehybridized membranes for 16 hours. Final washes were in 0.1XSSC, 0.1%SDS and at 65 C. Autoradiography was carried out for 2 to 6 days at - 80 C with Kodak XAR-5 film and intensifying screens (DuPont Cronex Lightning- Plus). Membranes were stripped by boiling into 0.1%SDS and reused up to at least 10 successive times. 3. Calculation of nucleotide diversities:
  • Nucleotide diversities, ⁇ , corresponding to the average heterozygosity per nucleotide site were estimated following Ewens et al. (130), using:
  • n i stands for the number of chromosomes studied with the i th enzyme
  • r i for the number of bp of the recognition sequence of the ith enzyme
  • m i for the number of cleavage sites explored with that enzyme, of which k i are polymorphic.
  • Table 5 reports the observed heterozygosities obtained with the generated multisite haplotype systems. These values correspond to the percentage individuals heterozygous for at least one of the polymorphisms identified with a given cosmid. Noteworthy, this parameter is not affected by the mode of classification of RFLPs in PMs or IDs. At this point and without segregation information, we can't dissect the heterozygous genotypes into their component haplotypes. These heterozygosities were estimated on a small sample and should therefore be considered cautiously. As pointed out by Skolnick and White (129), the main advantage of working with a sample of 9 individuals is that it is sufficient to identify the majority of useful polymorphisms. However, the mean heterozygosity of 51.9%, obtained over the 84 polymorphic cosmids demonstrates the power of the approach.
  • RFLPs of the PM type were used to calculate nucleotide diversities as described in Materials and Methods. Two sets of values are reported, depending on which of the two criteria were used to classify an RFLP into the PM or ID type.
  • Global nucleotide diversities of respectively 0.000652 and 0.000846 were obtained, meaning that a randomly selected Holstein animal will be heterozygous for approximately 1 every 1200 to 1500 base pairs.
  • nucleotide diversities more than twice as high are obtained when combining data obtained with the enzymes Mspl and TaqI: 0.001493 and 0.002239 respectively (5).
  • the recognition sequence of the enzymes BgIII, HindIII, PstI, PvuII and Xbal are devoid of hypermutable cytosines in the CpG dinucleotide and yield combined nucleotide diversities less than one third the values found with Mspl and TaqI (0.000492 and 0.000648).
  • Mspl and TaqI 0.000492 and 0.000648
  • DNA markers with very acceptable Polymorphism Information Content can be quickly generated using large, randomly selected genomic probes in Southern blot hybridization experiments.
  • the multisite haplotypes identified in this study using cosmid probes have a mean heterozygosity of 51.9% This value is of the same order of magnitude as the heterozygosities that we have obtained with a panel of approximately 40 bovine Variable Number of Tandem Repeat markers (mean heterozygosity 59%; 150), and with more than 50 bovine (TG)-dinucleotide microsatellites (mean heterozygosity 56%; unpublished).
  • microsatellites were proven to be an abundant source of highly polymorphic markers in the human (32-34). As their name implies, microsatellites are minute VNTR markers (18-20), characterized by tandem repetitions of very short repeats, one to four base pairs in length. Microsatellites exhibit levels of polymorphism comparable to VNTRs, but are much more abundant and apparently evenly spread throughout the genome.
  • Bovine genomic DNA was digested to completion with MboI and size-fractionated by agarose-gel electrophoresis. Fragments between 250 and 500 base pairs were recovered and purified using "Gene-Clean", ligated into the BAP-dephosphorylated BamHI site of pUC13 (Pharmacia) , and cloned into 12. Coli DH5 ⁇ cells (BRL). The resulting clones were screened for the presence of (TG) n microsatellites using a 32 P kinased (AC) 15 oligonucleotide as probe, and for (AG)n microsatellites using a (TC) 15 probe. The library was made with female DNA to avoid the previously characterized Y-specific TG-rich bovine DYZ1 sequence (117).
  • Plasmid DNA was prepared using standard "boiling mini-prep” procedures and subjected to two chain- termination sequencing reactions using unmodified T7 DNA Polymerase (Pharmacia), with the "universal” and “reverse” sequencing primers, respectively.
  • the 35 S labeled sequencing products were analyzed on standard denaturing polyacrylamide sequencing gels and detected by auto-radiography.
  • Suitable primers for in vitro amplification are identified in "UPSTREAM (UP)” and “DOWNSTREAM (DN) " strands using the "OPTIPRIM” program designed by one of us.
  • Optiprim helps the user to identify suitable primer pairs for PCR amplification of the corresponding DNA stretch.
  • the two DNA sequences flanking the DNA stretch to be amplified are referred to as the upstream (UP) and downstream (DN) sequence, respectively.
  • UP and DN Optiprim tests all possible primers of given length (as defined by the user) and retains the primers (1) with melting temperature (Tm) within the range defined by the user (Tm is calculated as 2C ⁇ number of As, or Ts + 4C ⁇ number of Gs or Cs), (2) with a minimum percentage of each nucleotide as defined by the user, and (3) which cannot form secondary bonds that can be formed between two molecules of the defined primer when sliding them in antiparallel orientation against each other, as illustrated in the following:
  • An A facing a T contributes two hydrogen bonds
  • a G facing a C contributes three hydrogen bonds. No loop formation is considered when performing this analysis.
  • This generates two sets of selected primers: an UP set and a DN set. All possible pairs of one UP and one Dn primer are then tested.
  • Optiprim retains the primer pairs if (1) the difference between melting temperatures of the two primers is within a range defined by the user, (2) the two primers cannot form secondary structures, determined as for individual primers, except that now the UP primer is slided versus the DN primer.
  • 80% of the selected primer pairs were giving successful PCR amplification in our microsatellite systems.
  • primer length primer length
  • melting temperature secondary structures that can be formed within and between primers.
  • the selected primers are synthesized by phosphoramidite chemistry on Applied Biosystem synthesizers and used without further purification.
  • the microsatellites are amplified in vitro, in microtiter plates and using the Te ⁇ hne MW2 device, in the following conditions (typically, 30 ⁇ l reactions):
  • Table 8 reports a preliminary list of bovine microsatellite systems that were successfully amplified in vitro. with the corresponding primer pairs. Note that pairs of primers selected by "OPTIPRIM", allow successful amplification in at least one of our standard conditions more than 80% of the time. Table 9 also gives the favoured annealing temperature (using the TECHNE MW2 heating device). The mean heterozygosity for the bovine microsatellites was estimated at «50%.
  • microsatellites To increase the speed and lower the cost of genotyping, multiplex approaches for both amplification and data capture of microsatellites are utilized. Microsatellite systems yielding products of non-overlapping size were coamplified as described above. Preliminary results show that at least four different systems can easily be coamplified in these standard conditions. The following multiplex amplifications, for instance, were shown to yield consistent, easily interpretable results: a. GBCYP21 - TGLA10 - TGLA 44 - TGLA116
  • multiplex amplification is limited to systems yielding products of non-overlapping size.
  • alternative detection schemes are utilized.
  • the use of confocal microscopy to detect products labeled with laser-excitable fluorescent molecules such as fluoresceine, rhodamine, ...) is used.
  • the products can then be differentiated based on the specific excitation and emission spectra of the tagged fluorescent molecules. Using this approach detection of up to at least 20 different systems should is feasable.
  • Results of the concordancy analysis are summarized in Table 9.
  • Synteny groups to which microsatellite systems most likely map as deduced from concordancy analysis are underlined. Clear-cut results were obtained for MGTG13B (U19 or chromosome 15), TGLA6 (U11), TGLA9 (U27), TGLA11 (U16), TGLA22 (U26 or chromosome 26), TGLA23 (U11), TGLA36 (U27), TGLA52 (U9 or chromosome 18). Results are less discriminating for the other systems. Most likely synteny groups are, however, mentioned. In addition, we know from the literature that GBKCAS maps to U15 or chromosome 6, and GBCYP21 to U20 or chromosome 23.
  • HGT604B 31 80 50 65 45 45 60 55 33 40 55 50 60 45
  • T6LA006 65 80 60 55 25 55 50 45 83 50 75 40 60 75
  • Bovine pedigrees for a total of approximately 200 individuals were genotyped for 150 of these markers as described. Pair-wise linkage analysis was performed using the LODSCORE program. Only lodscore values superior to 3 were considered significant. This generated a primary DNA marker map with 24 linkage groups counting two or more markers (15 assigned to specific chromosomes or synteny groups), and 68 singleton markers. Table 10 summarizes our findings. Linkage groups were assigned to specific chromosomes or synteny groups whenever that information was available.

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Abstract

L'invention concerne un ensemble de clones génomiques bovins donnant une représentation cartographique des loci polymorphiques chez des bovidés. Lesdits clones sont utiles dans l'identification génétique, la cartographie de gènes et l'élevage sélectif.
PCT/US1992/000340 1991-01-15 1992-01-15 Marqueurs polymorphes d'adn utilises chez les bovins WO1992013102A1 (fr)

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EP0846775A3 (fr) * 1996-12-03 1999-01-13 The Secretary of State for the Home Department Amorces permettant une discrimination améliorée concernant des techniques profilantes d'ADN
WO1999023248A1 (fr) * 1997-10-30 1999-05-14 Commonwealth Scientific And Industrial Research Organisation Evaluation du metabolisme des graisses
EP1049800A1 (fr) * 1998-01-21 2000-11-08 Axys Pharmaceuticals, Inc. Genes associes a l'asthme
US6211159B1 (en) * 1997-04-11 2001-04-03 University Of Toronto Flagellin gene, FlaC of campylobacter
DE19946173A1 (de) * 1999-09-20 2001-04-05 Forsch Die Biolog Landw Licher Expression der bovinen Acetyl-Coenzym A Carboxylase alpha
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EP1095137A1 (fr) * 1998-07-07 2001-05-02 New Zealand Pastoral Agriculture Research Institute Limited Nouvelles sequences promotrices de gene myostatine
US6265192B1 (en) * 1998-03-20 2001-07-24 The Regents Of The University Of California Glycosly sulfortransferase-3
EP1164190A1 (fr) * 1999-03-20 2001-12-19 Shunichi Shiozawa Gene de la polyarthrite rhumatoide et procede permettant de diagnostiquer la polyarthrite rhumatoide
WO2002014544A1 (fr) * 2000-08-15 2002-02-21 Eidgenössisch Technische Hochschule Zürich Procede servant a determiner les traits genetiques d'embryons d'animaux d'elevage ameliores prealablement a l'implantation
US7022831B1 (en) * 1988-12-22 2006-04-04 The Trustees Of The University Of Pennsylvania Regulation of BCL-2 gene expression
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US7022831B1 (en) * 1988-12-22 2006-04-04 The Trustees Of The University Of Pennsylvania Regulation of BCL-2 gene expression
US5374523A (en) * 1992-08-10 1994-12-20 Monsanto Company Allelic variants of bovine somatotropin gene:genetic marker for superior milk production in bovine
US7320863B2 (en) * 1996-02-01 2008-01-22 Albert Einstein College Of Medicine Of Yeshiva University Thyroid sodium/iodide symporter and nucleic acid encoding same
US6221628B1 (en) * 1996-02-16 2001-04-24 Brigham & Women's Hospital Compositions and methods for the treatment and diagnosis of cardiovascular disease using fchd540 as a target
EP0846775A3 (fr) * 1996-12-03 1999-01-13 The Secretary of State for the Home Department Amorces permettant une discrimination améliorée concernant des techniques profilantes d'ADN
US7138511B1 (en) * 1997-01-28 2006-11-21 The Regents Of The University Of California Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders
US6585980B1 (en) 1997-04-11 2003-07-01 The University Of Toronto Flagellin gene, FlaC of Campylobacter
US6211159B1 (en) * 1997-04-11 2001-04-03 University Of Toronto Flagellin gene, FlaC of campylobacter
US6383751B1 (en) 1997-10-30 2002-05-07 William John Barendse Assessing lipid metabolism
WO1999023248A1 (fr) * 1997-10-30 1999-05-14 Commonwealth Scientific And Industrial Research Organisation Evaluation du metabolisme des graisses
EP1049800A4 (fr) * 1998-01-21 2002-09-25 Axys Pharm Inc Genes associes a l'asthme
EP1049800A1 (fr) * 1998-01-21 2000-11-08 Axys Pharmaceuticals, Inc. Genes associes a l'asthme
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