EP1272674A2 - Procede permettant d'identifier et d'isoler des fragments de genome a desequilibre de liaison - Google Patents

Procede permettant d'identifier et d'isoler des fragments de genome a desequilibre de liaison

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Publication number
EP1272674A2
EP1272674A2 EP01940160A EP01940160A EP1272674A2 EP 1272674 A2 EP1272674 A2 EP 1272674A2 EP 01940160 A EP01940160 A EP 01940160A EP 01940160 A EP01940160 A EP 01940160A EP 1272674 A2 EP1272674 A2 EP 1272674A2
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EP
European Patent Office
Prior art keywords
dna
fragments
molecules
individual
stranded
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01940160A
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German (de)
English (en)
Inventor
Klaus Olek
Jürgen Weber
Thomas Jansen
Marco Leuer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biopsytec Analytik GmbH
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Biopsytec Analytik GmbH
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Publication date
Application filed by Biopsytec Analytik GmbH filed Critical Biopsytec Analytik GmbH
Publication of EP1272674A2 publication Critical patent/EP1272674A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening

Definitions

  • the invention relates to a method for identifying and isolating genomic fragments with coupling imbalance.
  • marker-assisted selection in English: marker-assisted-selection or MAS for short.
  • the core of this is the application of the coupling strategy with polymorphic DNA markers.
  • the latter has become more monogenic in research
  • Hereditary diseases of humans have been shown to be very successful.
  • the strategy has been systematically applied worldwide as a first stage of the Human Genome Program (HGP).
  • HGP Human Genome Program
  • Their theoretical basis is the considerations of Botstein et al., 1980.
  • the enormous efforts of the HGP finally provided the technical and intellectual basis for creating a marker map for the important livestock species cattle, pigs and sheep.
  • Another finding that can be used to a limited extent in practice is that certain alleles of the closest marker loci within a family are inherited together with the interesting phenotype. In another family, it can be another allele of the same marker gene location. Within a family, however, this fact can be used diagnostically, since such an allele in this family allows genetic predictions about the phenotype to be expected. However, such a statement is through the phenomenon of recombination between two gene locations is subject to considerable uncertainty. This uncertainty can be reduced by looking for markers that are closer to the causative gene. As a rule, this search will eventually lead to marker gene locations that are located directly on or in the gene itself. There are almost no recombinations between these marker gene locations and the gene.
  • the gene defect of interest can be traced back to an ancestor, if this defect happened to a single ancestor and has spread from there to a certain population, then the marker allele combination observed on and in the gene becomes identical in all affected genes of the same Find population. It is then said that the markers are in the coupling imbalance with the causative gene. Such a marker allele combination is of extraordinary practical importance because it allows the desired prediction of the phenotype across family boundaries.
  • a major disadvantage here is that the path from the 25 mB coupling group to the gene is very laborious and can take years under certain circumstances, since 25 million base pairs have to be studied.
  • the strategy is also applicable to polygenic traits, such as the so-called QTLs of livestock breeding.
  • the situation is now much more difficult, not only because you have to deal with several causative genes, where you have to go to the gene or coupling imbalance, but also because the individual genes have a very different influence on the phenotype and because genetics a strong environmental impact.
  • the result of a number of TL studies which have been forced to be very large, often with several thousand animals each, is the identification of several coupling groups, which are usually 30-40 cM in size. Diagnostic, but therefore also breeding value is basically only in families. This means an enormous limitation of the MAS.
  • the ultimate goal of all these molecular biological efforts, namely the isolation of the genes responsible for the QTL 's, can practically not be achieved from this basis.
  • GDD Granddaughterdesign
  • a typical number for the animals to be examined is: 10-20 bulls, 50-100 sons of each of these bulls and 50-100 daughters per son.
  • Table 1 shows the GDD studies in progress in 1998.
  • GMS genomic mismatch scanning
  • the GMS method isolates coupling groups but also coupling imbalances. There were two different approaches. The first way is to define a phenotype of interest and study it with the help of pairs, both of whom come from a family and have a clearly described degree of kinship, whereby the individual pairs can come from genetically heterogeneous populations or families.
  • Coupling groups are isolated in the first way, and genomic regions in the second way, which represent a coupling imbalance between the gene of interest and a microhaplotype consisting of several polymorphic gene locations.
  • GMS protocols have so far only been used for monogenically inherited diseases.
  • a series of microsatellite loci was subjected to the GMS procedure in an experiment. In principle, this has already been used to simulate working on polygenic traits.
  • all previously published work ends with the localization or confirmation of a localization of the isolated IBDs, i.e. the GMS products, that has already been carried out using other methods.
  • F. Mirzayans F. Mirzayans, A.J. Mears, W. Guo, W.G. Pearce, M.A. Walter; At the. J. Hum. Genet, 61, 439-448 (1997)) identified the chromosomal region that should house the locus for iridogoniodysgenesis (IGDA). The finding was developed in parallel with the coupling analysis and the GMS. The disease is a very rare autosomal dominant eye disease. For the coupling analysis, a family of 80 members was examined, 40 of whom are ill. The entire genome was examined with 300 microsatellites (F. Mirzayans, W.G. Pearce, I.M.
  • the IGDA gene was then located at 6p. In a next round of localization, 29 microsatellite markers of chromosome 6 were used. The locus was thus further limited to 6p25.
  • chromosome 12 was examined in parallel to chromosome 6. The latter gave no positive signal.
  • IBDs in the limit case (see below) 80% of a chromosome or more. There will also be IBDs that have nothing to do with the characteristic of interest. If, on the other hand, the degree of kinship is too low, the IBD area can no longer be discovered with the localization instrument because of its small size, because it has too little resolution.
  • the method described by Mirzayans et al. marker set used has a resolution of 10 cm. This shows the enormous importance of the localization tool in the GMS protocol.
  • the reannealing may be imperfect and thus simulate a mutation-related mismatch.
  • Mut S does not recognize C-C mismatches
  • Mut S needs a methylated GATC motif in the vicinity to recognize a mismatch. The latter is of course not always the case.
  • the function of Mut L, H is also not entirely clear.
  • the extension the detected coupling group is 6, 9 cM.
  • the marker card has a resolution of 10 cM and an index family is available, which allows the analysis of 5th cousins.
  • the second genetic goal pursued by the GMS is to directly isolate areas that show coupling imbalance.
  • An example of this approach is the work of Vivian G. Cheung (1998).
  • the gene of the autosomal recessive inherited congenital hyperinsulinism that causes the disease is located.
  • the disease occurs with a comparatively high frequency among the Ashkenazi Jews. Therefore, one can assume a founding effect in this population group.
  • the gene codes for the sulfonylurea receptor. It has been located on chromosome llpl5.1 using conventional methods.
  • the main parameter is the enrichment factor of the IBD allele.
  • Each locus has an allele, which can either have been inherited from the paternal grandfather or from the paternal grandmother. This allele is the respective IBD fragment or IBD allele.
  • a quantitative measurement area under the fluorescence signal peak of the microsatellite locus in the measurement with an automatic sequencer is carried out at two positions of the GMS experiment; on the one hand on the total population of the heterohybrids after restriction and exo III digestion of the homohybrids and on the other hand on the remaining heterohybrids after separation of the heterohybrid fraction containing the mismatches.
  • the enrichment factor sought is then defined as follows:
  • the denominator of this ratio is the ratio of IBD allele to non-IBD allele in the total population of heterohybrids; the counter is the speaking ratio in the completely matching GMS fraction.
  • the overall procedure is highly reproducible.
  • the enrichment i.e. the success of the GMS procedure, depends on the sequence and thus location, since around 18% of the marker loci used show no enrichment.
  • no generally valid efficiency assessment of the method can be derived from this, since only Pst I fragments were examined.
  • the selection of the restriction cleavage represents an optimization problem which is dependent on at least two influencing variables.
  • Each DNA molecule that is to be removed in the course of the GMS must have at least one GATC motif.
  • FPERT hybridization finds complementary ones DNA strands are worse the more repetitive fragments they contain. The probability of this increases with the fragment length. Fragments> 20 kb are obviously broken down during reannealing.
  • yeast genome is 240 times less complex than the human, because it contains far fewer repetitive sequences and it carries far more natural sequence polymorphisms than the human genome.
  • FPERT stage is less efficient. While in a GMS experiment on yeast around 50% of the DNA used after FPERT can be found as double-stranded molecules (approx. 50% heterohybrids and homohybrids in each case), it is only 10% in humans. This means that in a typical experiment, about 1 ⁇ g DNA is obtained. After digestion of the homohybrids, approx. 500 ng remain. From this, the IBD fragments are separated and it can only be a few nanograms, the less sharply the GMS is selected. The proposed PCR amplification using inter-Alu motifs and carried out in one case is therefore within the problematic limit range of 1 ng per fragment (Nelson et al., 1989).
  • the object of the present invention is therefore to provide a method which overcomes the disadvantages of the prior art.
  • the object of the invention is achieved by the provision of a method which serves to isolate IBD fragments in the case of polygenically inherited features.
  • the method has improvements over all of the previously described protocols and can, among other things. a. contribute to solving the problems in animal breeding described above.
  • the method according to the invention is based on a basis similar to the GMS concept.
  • the DNA from two index individuals is extracted and purified. Both DNA fractions are digested with the same restriction enzyme. In the example discussed below in FIG. 2, the restriction enzyme Eco RI is involved.
  • the restricted DNA from individual 1 is provided with a linker which produces fragment ends on both sides without an overhang (see FIG. 2).
  • the restricted DNA from individual 2 is also provided with a linker which generates fragment ends on both sides without an overhang (see FIG. 2).
  • the linkers are designed in such a way that self-ligation for ring formation is not possible for heterohybrids from the DNA of both individuals, but it is Allow ring formation with appropriately cut vector.
  • the DNA of both individuals is now denatured and the denatured DNA of both individuals is mixed.
  • the mixture is subjected to the so-called FPERT reaction to re-form double-stranded DNA.
  • the reaction mixture consists of three molecular populations, namely the regressed double-stranded DNA molecules from individual 1 (homohybrids), the double-stranded DNA molecules from individual 2 (homohybrids) and the double-stranded DNA molecules consisting of one from individual 1 DNA strand originating and a DNA strand originating from individual 2 exist (hetero-hybrid).
  • a suitable plasmid vector is double digested e.g. exposed with the restriction enzymes Nco I and the restriction enzyme Nsp I.
  • protruding GTAC ends are formed on the same strand in the opposite orientation (see FIG. 2).
  • the mixture of the newly formed double-stranded molecules is combined with the cut vector DNA. Only the heterohybrids can form a ring with the vector molecules.
  • the DNA fragments are then covalently linked using a ligase reaction.
  • the homohybrids of the two individuals 1 and 2 and the heterohybrids, which consist of a strand of individual 1 and the complementary strand of individual 2.
  • the heterohybrids consist of the very large group of molecules that have individual mismatches and the much smaller group of molecules that have no mismatches.
  • the latter group contains the fragments of interest, namely the IBD areas.
  • the DNA of the individual plasmid preparations each represents a fragment which is "identical by descent” (IBD).
  • the DNA obtained is located on a DNA chip.
  • a relatively simple application is the identification and isolation of the gene for inherited unemployment in domestic pigs.
  • several IBD fragments are obtained. These fragments can be localized with the DNA chip. With high probability, there are several gene locations. These are checked for their informative value with affected and unaffected animals. The informative places can be for diagnostic and further scientific purposes are used.
  • FIGS la and 1b schematically show the course of the genomic mismatch scanning method.
  • FIG. 1 a represents the chromosome of an ancestor with some intermediate generations, on which a mutation (X) associated with a disease occurred.
  • 2 are the chromosomes of today's unrelated individuals with the same disease.
  • 3 represents the region which is "identical by decent” (IBD) and includes the disease site.
  • IBD identical by decent
  • FIG. 1b 11 represents the PstI fragments of the first individual and 12 the methylated PstI fragments of the second individual.
  • FIGS. 2a and 2b The method according to the invention is shown schematically in FIGS. 2a and 2b.
  • 21 means the first Individual, 23 the second individual, 22 the first individual + linker and 24 the second individual + linker.
  • 30 denotes the vector.
  • the process steps are labeled E1 to E4 and have the following meaning: E1 is the Eco R1 digestion, E2 is the naturalization, E3 is the mixing of the first and the second individual with renaturation (FPERT) and E4 denotes the ring closure.
  • E1 is the Eco R1 digestion
  • E2 is the naturalization
  • E3 is the mixing of the first and the second individual with renaturation (FPERT)
  • E4 denotes the ring closure.
  • the object of the invention is therefore achieved by a method for identifying and isolating genomic fragments with coupling imbalance, wherein regions of the genome which contain candidate gene regions which are in coupling imbalance with their closer DNA environment are isolated in unrelated and in mutually related individuals ,
  • the DNA samples cut with restriction enzymes from two index indices viduen provided with different linkers in each case which provided ends at both ends of the restriction fragments without an overhang and which on heterohybrids form overhangs from the complementary fragment ends of the DNA strands, each belonging to individual 1 or 2, which are on the same strand and whose sequence is 5 'CATG 3' at the 5 'end and 5' CATG 3 'at the 3' end, a mixture is subsequently obtained by denaturing the fragments, the single-stranded DNA fragments are obtained from both individuals and the next step Buffer conditions are set which allow the renaturation of double-stranded DNA molecules (FPERT reaction), three molecular populations are obtained, the first of which represents the restored double-stranded molecules (homohybrids) of individual 1, the second the restored double-stranded molecules (homohybrids) of individual 2 and the third, the population of complementary, each As one and the other individual belonging to double-stranded molecules (FPERT reaction),
  • reaction mixture is used to transform suitable bacteria.
  • the transformed bacteria are cultivated after selection and separation and that the DNA fragments obtained by plasmid preparation, which contain the IBD regions, are isolated and localized in the genome.

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Abstract

Procédé permettant d'identifier et d'isoler des fragments de génome à déséquilibre de liaison. Ledit procédé consiste à isoler, chez des sujets tant parents que sans liens de parenté, des domaines du génome contenant des domaines candidats qui se trouvent en déséquilibre de liaison avec leur environnement d'ADN très proche. L'étape de clonage, effectuée plus précocement que dans d'autres procédés, permet de récupérer les fragments d'ADN indépendamment de leur quantité et remplace en outre l'étape de méthylation effectuée dans d'autres procédés. L'utilisation selon la présente invention d'une enzyme végétale entraîne de surcroît une spécificité beaucoup plus élevée de ce procédé par rapport à ceux dans lesquels le complexe Mut S,H,L est utilisé.
EP01940160A 2000-04-08 2001-04-08 Procede permettant d'identifier et d'isoler des fragments de genome a desequilibre de liaison Withdrawn EP1272674A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10017675A DE10017675A1 (de) 2000-04-08 2000-04-08 Verfahren zur Identifizierung und Isolierung von Genomfragmenten mit Kopplungsungleichgewicht
DE10017675 2000-04-08
PCT/DE2001/001488 WO2001077374A2 (fr) 2000-04-08 2001-04-08 Procede permettant d'identifier et d'isoler des fragments de genome a desequilibre de liaison

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EP1272674A2 true EP1272674A2 (fr) 2003-01-08

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US (1) US20030165926A1 (fr)
EP (1) EP1272674A2 (fr)
JP (1) JP2003530117A (fr)
AU (1) AU2001273842A1 (fr)
CA (1) CA2416118A1 (fr)
DE (1) DE10017675A1 (fr)
NZ (1) NZ522470A (fr)
WO (1) WO2001077374A2 (fr)

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Publication number Priority date Publication date Assignee Title
WO2004020663A2 (fr) * 2002-08-09 2004-03-11 Förderverein Biotechnologieforschung Der Deutschen Schweineproduktion E.V. Marqueurs genetiques permettant de diagnostiquer la predisposition a l'heredite ou a l'expression du phenotype d'imperforation de l'anus chez des animaux de compagnie, domestiques et d'elevage
US20080228700A1 (en) 2007-03-16 2008-09-18 Expanse Networks, Inc. Attribute Combination Discovery
EP3276526A1 (fr) 2008-12-31 2018-01-31 23Andme, Inc. Recherche de parents dans une base de données
CN109346130B (zh) * 2018-10-24 2021-10-08 中国科学院水生生物研究所 一种直接从全基因组重测序数据中得到微单体型及其分型的方法

Family Cites Families (5)

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Publication number Priority date Publication date Assignee Title
AU2318288A (en) * 1987-08-07 1989-03-09 Genelabs Incorporated Coincidence cloning method and library
US5376526A (en) * 1992-05-06 1994-12-27 The Board Of Trustees Of The Leland Stanford Junior University Genomic mismatch scanning
US5869245A (en) * 1996-06-05 1999-02-09 Fox Chase Cancer Center Mismatch endonuclease and its use in identifying mutations in targeted polynucleotide strands
DE19911130A1 (de) * 1999-03-12 2000-09-21 Hager Joerg Verfahren zur Identifikation chromosomaler Regionen und Gene
US6524794B1 (en) * 1999-10-29 2003-02-25 Decode Genetics Ehf. Identical-by-descent fragment enrichment

Non-Patent Citations (1)

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Title
See references of WO0177374A3 *

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DE10017675A1 (de) 2001-12-06
AU2001273842A1 (en) 2001-10-23
JP2003530117A (ja) 2003-10-14
WO2001077374A2 (fr) 2001-10-18
WO2001077374A3 (fr) 2002-06-20
US20030165926A1 (en) 2003-09-04
NZ522470A (en) 2004-12-24
CA2416118A1 (fr) 2001-10-18

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