EP0559841A1 - Procede de detection de la variation de sequences d'adn - Google Patents

Procede de detection de la variation de sequences d'adn

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Publication number
EP0559841A1
EP0559841A1 EP92904579A EP92904579A EP0559841A1 EP 0559841 A1 EP0559841 A1 EP 0559841A1 EP 92904579 A EP92904579 A EP 92904579A EP 92904579 A EP92904579 A EP 92904579A EP 0559841 A1 EP0559841 A1 EP 0559841A1
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European Patent Office
Prior art keywords
dna
alu
pcr
repeat
inter
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EP92904579A
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German (de)
English (en)
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Andreas Gerardus Uitterlinden
Jan Vijg
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Ingeny BV
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Ingeny BV
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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
    • C12Q1/6813—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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • TITLE Method of detecting DNA sequence variation.
  • This invention relates to a method of detecting genetic variation by subjecting DNA fragments, derived from a DNA sample to be analyzed, to a two-dimensional electrophoretic separation, with the fragments being separated in one dimension on the basis of differences in length and in the other dimension on the basis of differences in base sequence, transferring the resultant separation pattern to a membrane filter and subjecting the transferred separation pattern to a hybridization analysis using one or more DNA or RNA probes.
  • DNA diagnostics i.e. the use of methods for demonstrating genetic variation at those sites which may be an indication for certain properties, such as susceptibility to certain diseases and other medically and economically important characteristics.
  • genetic variation refers to differences between individuals in the base pair sequence of their DNA.
  • RFLP restriction fragment length polymorphism
  • Probes which detect polymorphic DNA sequences can be used in a comparable manner in plant improvement and in the breeding of cattle and other economically important animals. The point is that if it is known that a particular DNA variant is always inherited together with a particular property, the presence or absence of that property can be determined at an early stage which is of great importance for improvement because in this way the generation interval can be avoided (this principle is called "Marker Assisted Selection”) .
  • VNTR-type sequences occur in humans much more often at the telomeres (terminal ends of the chromosome) than elsewhere (Royle et al., 1989) .
  • telomeres terminal ends of the chromosome
  • other VNTR-type sequences not much is known about this and also in animal species other than man and in plants there is only little information about the occurrence and distribution of VNTR-type sequences.
  • a third point concerns the fact that only 20-50% of the sequences detected by the core probes are polymorphic and, accordingly, useful for the analysis. In other words, the majority of the spots cannot be used for mapping and only takes up space in the separation pattern. It would therefore be much better to use sequences which occur once at least about every 2 million base pairs, are nearly always polymorphic and are equally distributed over the genome.
  • a sequence with these properties is for example the repetitive element Alu.
  • PDJ33A 5*-GGATCCGCCTCCCAAAGTGCTGGGATTACAGG-3 •
  • the present invention provides a method for detecting genetic variation by subjecting DNA fragments derived from a DNA sample to be examined by two-dimensional electrophoretic separation, with the fragments being separated in one dimension on the basis of differences in length and in the other dimension on the basis of differences in base sequence, transferring the resultant separation pattern to a membrane filter and subjecting the transferred separation pattern to a hybridization analysis using one or more DNA or RNA probes, which method is characterized by using DNA fragments which consist of one or more inter-repeat sequences which on the basis of the DNA to be examined have been generated by means of a DNA amplification process such as the polymerase chain reaction (PCR) with repeat-specific primer(s).
  • PCR polymerase chain reaction
  • a very important preferred embodiment of the invention is characterized in that in the hybridization analysis, as a probe use is made of one or more inter-repeat sequences which have been generated by means of an amplification process such as a PCR with repeat-specific primer(s), performed on subgenomic DNA, for instance DNA of one chromosome or a part thereof.
  • an amplification process such as a PCR with repeat-specific primer(s), performed on subgenomic DNA, for instance DNA of one chromosome or a part thereof.
  • the invention makes it possible to detect genetic variation on specific chromosomes or parts thereof, using a selective amplification of double-stranded DNA in specific sites, occurring at least every 2 million base pairs, an electrophoretic separation of the amplification products in two dimensions, transfer of the separation pattern to a membrane filter, followed by a hybridization with labelled DNA fragments which may come from (a) cloned DNA such as cosmid and YAC (yeast artificial chromosome) clones, or (b) chromosomes or parts thereof such as obtained after flow-sorting chromosomes or such as are present in a human-hamster somatic cell hybrid.
  • cloned DNA such as cosmid and YAC (yeast artificial chromosome) clones
  • chromosomes or parts thereof such as obtained after flow-sorting chromosomes or such as are present in a human-hamster somatic cell hybrid.
  • the DNA to be examined will mostly consist of the DNA of a complete genome of an animal or plant, and the most important applications will concern human DNA.
  • inter-Alu PCR coupled with 2-D DNA typing, could be used for genome scanning in a way that is superior in at least two respects to the previously described genetic analysis with an electrophoretic separation of the restriction fragments in two dimensions and a hybridization with GC-rich minisatellite core probes.
  • the inter-Alu method enables, after a two-dimensonal separation of the fragments which have been generated with total genomic DNA as substrate, recognition of certain specific regions in the genome by hybridization with a probe mixture consisting of inter-Alu PCR products which come from a specific chromosome (obtained through flow sorting or as hybrid cell line) or a part thereof, for instance in the form of cosmid or YAC clones.
  • a 2-D separation pattern of inter-Alu PCR products coming from total genomic DNA of different individuals can directly provide information about genetic differences and similarities, namely specifically for certain chromosomes or parts thereof, depending on the probe mixture used.
  • probe mixtures may for instance serve inter- Alu products originating from human-hamster hybrid cell lines which contain one or more human chromosomes or parts thereof.
  • the DNAs in question can be labeled and subsequently used as hybridization probe for the analysis of 2-D separation patterns of inter-Alu amplification products (for lack of homology, the hamster DNA will not bind) .
  • This will result in an individual- specific stain pattern, each stain being in principle a variant of an inter-Alu DNA fragment which is located in the region that is specified by the human chromosome (or a part thereof) present in the hybrid cell line.
  • the label used for the probe is not in itself critical and all known labels for DNA or RNA probes can be used.
  • the probe that is used in the hybridization analysis can be labelled with a radioisotope such as 32 P and 35 S, with a hapten such as biotin and digoxigenin, with a fluorescent substance or with a combination of such labels.
  • the method described hereinabove can be used in the determination of genetic variation in any animal or plant species, provided that individual chromosomes or parts thereof are available, for instance in the form of flow-sorted chromosomes, hybrid cell lines (in which case the DNA of the other species should have no homology with the "Alu-like" priming sites in the DNA of the species to be examined) and cosmid or YAC clones.
  • the method confined to the Alu or Alu-like repeat family, but it can also been used on other human repeat families, such as the Kpn family (see Ledbetter et al., 1990a; see further Table 2 for an overview of Kpn specific primers and Fig.
  • the method can be used to screen within a short time a large number of polymorphic sites in a priorly defined region within a chromosome, for individual differences, which information can be used directly to obtain a marker for a hereditary characteristic, such as a hereditary disease.
  • a hereditary characteristic such as a hereditary disease.
  • An alternative is to locate beforehand all spots (inter-Alu variants) which can be found in a 2-D chromosome-specific DNA scan and to reduce them to a specific recombinant DNA clone in one of the reference libraries open to the public.
  • the setting up of such a database of polymorphic DNA sequences as spots in a 2-D separation pattern is discussed extensively in Uitterlinden and Vijg, 1989 and EP-A-0349024.
  • the invention then, relates to the following:
  • Electrophoretic separation in 2 dimensions i.e. size and base pair composition
  • inter-repeat PCR products obtained from total genomic DNA from a human, animal or plant using one or more primers specific for one or more repetitive sequences.
  • PCR reaction was performed, using PCR primer TC65.
  • the primer was synthesized on a Gene Assembler Plus (Pharmacia, Sweden) and was used directly after the deprotection-isolation step, without further purification.
  • the sequence of this primer consisting of 30 nucleotides, is 5 « -AAGTCGCGGCCGCTTGCAGTGAGCCGAGAT-3' (see also Nelson et al., 1989) .
  • the PCR reactions were performed in a total volume of 50 ⁇ l (microlitre) containing 400 ng genomic DNA, 1 ⁇ M primer TC65, 50 mM KC1, 10 mM Tris.HCl pH 8.0, 1.5 mM MgCl , 200 ⁇ M dATP, 200 ⁇ M dTTP, 200 ⁇ M dCTP, 200 ⁇ M dGTP and 1 unit of Taq polymerase (Gibco-BRL, Bethesda, Maryland, USA) .
  • a total of 30 cycli were traversed, each cycle comprising successively 2 min denaturation at 95°C, 1 min annealing at 60°C, and 4 min extension at 72°C.
  • the first denaturation step was prolonged by 5 min and the final extension step was prolonged by 4 min.
  • the genomic DNA samples used were Chinese Hamster Ovary (CHO) total genomic DNA and Hela human total genomic DNA, and CHO-human somatic cell hybrid genomic DNA from the following cell lines: 643C-13, 21q+, R2-10W, ACEM, 2Fur-l, 72532X-6, and 153E7b (see Gardiner et al., 1990) .
  • 5A shows the human chromosome 21 complement of these hybrids.
  • Electrophoresis (2 h and 250 volts) was done in lxTAE (40 mM Tris.HCl pH 7.4, 20 mM Na-acetate, 1 mM Na-EDTA) at 50°C.
  • the separation pattern was visualized by staining with ethidium bromide (0.1 ⁇ g per ml) and decolouring in water for 30 min.
  • 5B shows that the TC65 PCR reaction is specific for human DNA since no colouring occurs in the lane with PCR products of total genomic hamster DNA.
  • a smear is visible with a single band therein. This is due to the very large number of different inter-Alu products originating from all human chromosomes together.
  • discrete bands are visible. Since these hybrids only have the human chromosome 21, these bands must originate from inter-Alu regions on the human chromosome 21.
  • the band patterns are mutually different, which is a reflection of the different pieces of chromosome 21 which the cell line in question contains (see Fig. 5A) . In this way specific bands can be identified as originating from specific regions on the chromosome.
  • a TC65 PCR reaction was performed, now with genomic DNA of the cell line 72532X6 as a substrate being compared with Hela human genomic DNA.
  • 25 ⁇ l of the two reaction products was separated on a PAA gel as described above. After staining of this 1-D separation pattern, the fragments with a length of between 10 kilo base pairs (kb) and 600-base pairs (bp) were excised as lanes and transferred to a 2-D PAA gel.
  • the 2-D gel was a 1 mm thick 6% PAA gel (see above) which contained a 10-75% linear gradient of the denaturants urea and formamide (100% denaturants corresponds to 7.0 M urea, 40% formamide) parallel to the direction of the electrophoresis.
  • the gels were prepared by mixing the two solutions with the two most extreme denaturant concentrations, which determine the range of the gradient, in a gradient mixer (Gibco-BRL) using a peristaltic pump (LKB, Sweden) .
  • the electrophoresis for the second dimension separation was performed at 55°C in lxTAE, for 12 h. After completion of the separation, the 2-D gel was stained as described above (see Fig. 6) .
  • the separation pattern of 72532X6 demonstrates that the TC65 inter-Alu PCR fragments focus as spots in the 2-D gel. At the place where the TC65 inter-Alu PCR products of total genomic human Hela DNA are located, nothing is to be seen on gel because the concentration of individual PCR fragments is too low to be demonstrated by ethidium bromide staining. To visualize these fragments, the 2-D separation pattern was transferred to a nylon membrane (Hybond N-plus, Amersham, UK) by means of electroblotting between horizontal graphite plates with the anode at the top (see also Vijg and Uitterlinden, EP-A-0 349 024) .
  • the 2-D gel was first irradiated for 4 min with UV light of 302 nm (Transilluminator, UV products, CA, USA) .
  • the gel was placed on the nylon membrane and subsequently placed on opposite sides between three Whatmann 3MM paper leaves which had been saturated with IxTBE (89 mM Tris.borate pH 8.0, 89 mM boric acid, 2 mM Na-EDTA) .
  • IxTBE 89 mM Tris.borate pH 8.0, 89 mM boric acid, 2 mM Na-EDTA
  • the nylon membrane was placed on a leaf of Whatmann 3MM which had been saturated with 0.4 N sodium hydroxide and subsequently placed in the 1 M Tris.HCl pH 8.0 for 10 min for neutralization, so as to make the DNA fragments on the membrane single-stranded.
  • the membrane was dried in air and subsequently irradiated with 302 nm UV to bind the DNA fragments to the membrane by cross-linking.
  • This membrane was subsequently subjected to hybridization analysis wherein as a probe use was made of 3 ⁇ l of the TC65 inter-Alu PCR mixture obtained with 72532X6 as substrate (and of which 25 ⁇ l in the first dimension was separated on gel, see Figs. 5 and 6) .
  • this labelled probe mixture was made single-stranded by boiling it for 5 min and it was subsequently preincubated with 200 ⁇ g partly degraded human DNA in a total volume of 1 ml at 65°C for 1 h, so as to repress cross hybridization with the Alu repeats themselves and to allow only the inter-Alu sequences to hybridize.
  • Hybridization analysis was performed in glass tubes in a hybridization oven (GFL, Germany) at 65°C.
  • the membrane was first preincubated for 15 min in the hybridization fluid which consisted of 7% SDS, 0.5 M Na-phosphate pH 7.2, 1 mM Na-EDTA. Then the labelled and preincubated 72532X6 TC65 inter-Alu probe was added and the hybridization reaction took place for 16 h. Then the membrane was successively washed in
  • Fig. 7 shows that after hybridization the same separation pattern for the 72532X6 TC65 inter-Alu PCR products was visualized as what we observed after ethidium bromide staining. Of greater importance is the fact that we can also observe spots at the place of the Hela TC65 inter-Alu products.
  • FIG. 8 shows that multiple spots were visible after autoradiography in the 2-D separation pattern of inter-repeat products obtained from human total genomic DNA, indicating the possibility to scan several different inter- repeat sequences in the human genome.
  • inter-repeat primers can be used which are specific for repeats located in the centromeres, such as the alphoid satellite repeats (LI.26 and others) .
  • these primers are used in combination with Alu and/or Kpn primers in experiments similar to experiments 1, 2 and 3, band patterns can be obtained (see Fig. 10) which are comparable with those shown in Figs. 5 and 9.
  • Fig, 1 Two-dimensional separation pattern, generated using minisatellite core probe 33.6 of human Haelll DNA restriction fragments originating from a mother (circle) , father (square on the right) and their son (square in the middle) .
  • A Schematic overview of the composition as to the chromosome 21 contents of human-hamster somatic cell hybrids used for the inter-repeat PCR amplifications.
  • B Ethidium bromide-stained one-dimensional separation pattern of the PCR products, obtained after an inter-Alu PCR amplification using primer TC65 on the members of the somatic hybrid panel shown in 5A and on total hamster genomic DNA and totale human genomic DNA. Fi ⁇ . 6
  • FIG. 7 Autoradiograms (A is the short and B is the long irradiation) of a two-dimensional separation pattern of inter- Alu PCR amplifications with primer TC65 of human total genomic DNA (Hela) and of the somatic cell hybrid 72532X6. Arrows designate spots in the Hela separation pattern after the long irradiation, which can be recognized at a similar location in the 72532X6 pattern.
  • Ethidium bromide-stained one-dimensional separation pattern of inter-repeat PCR amplification products obtained with a combination of the Alu-specific primer TC65 with the Kpn-specific PCR primers KpnA, -B, -C, and -D, the substrate being total hamster genomic DNA and DNA from the somatic cell hybrid 72532X6.
  • Ethidium bromide-stained one-dimensional separation pattern of inter-repeat PCR amplification products obtained with the Alu-specific primers 517 and TC65 in combination with the Kpn-specific primers KpnA, -B, -C, and -D and in combination with the chromosome 21-specific centromere primers 26.5B, 26W and 26.3, the substrate being total hamster genomic DNA and DNA from the somatic cell hybrid 153E7b.
  • Gardiner K. Horisberger, M., Kraus, J., Tantravahi, U., Korenberg, J., Rao, V., Reddy, S., and Patterson, D. (1990) EMBO J. 9, 25-34.

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Abstract

L'invention concerne la détection de la variation génétique, par électrophorèse bidimensionnelle, de fragments d'un échantillon d'ADN à analyser, les dits fragments étant séparés dans une dimension selon la longueur et dans l'autre dimension selon la séquence de base, par transfert sur un filtre à membrane et par analyse d'hybridation à l'aide de sondes à ADN et ARN. Les fragments d'ADN consistent en des séquences intra-répétées générées sur la base de l'ADN à analyser, au moyen d'un procédé d'amplification de l'ADN tel que la réaction en chaîne de la polymérase (PCR) avec une ou des amorces spécifiques aux séquences répétées. Des séquences intra-répétées qui conviennent sont, par exemple, des séquences situées entre deux séquences répétées Alu, entre deux séquences répétées kpn, ou entre une séquence répétée Alu et une séquence répétée Kpn. Pour l'analyse d'hybridation, on utilise, comme sonde, de préférence des séquences répétées génerées par le procédé d'amplification d'ADN mis en oeuvre avec une ou des amorce(s) spécifique(s) aux séquences répétées et réalisé sur de l'ADN sous-génomique, par exemple l'ADN d'un chromosome ou une partie de celui-là.
EP92904579A 1991-01-25 1992-01-24 Procede de detection de la variation de sequences d'adn Withdrawn EP0559841A1 (fr)

Applications Claiming Priority (2)

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NL9100132 1991-01-25
NL9100132A NL9100132A (nl) 1991-01-25 1991-01-25 Werkwijze voor het detecteren van dna sequentievariatie.

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CA2118048C (fr) * 1994-09-30 2003-04-08 James W. Schumm Amplification multiplex de locus de sequences courtes repetees en tandem
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CA2223061C (fr) * 1995-06-06 2008-04-22 Jan Vijg Procede et dispositif pour test diagnostique d'adn
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WO2004018691A1 (fr) * 2002-08-22 2004-03-04 The University Of Hong Kong Procede pour adn chromosomique micro-excise d'amplification et son utilisation
DE10242359A1 (de) * 2002-09-12 2004-03-25 Alopex Gmbh Verfahren zur Amplifikation genetischer Informationen

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CN101792808A (zh) * 2010-03-30 2010-08-04 广州市香港科大霍英东研究院 以Alu间聚合酶链式反应为基础的检测基因区特征的方法

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