EP0188485A1 - Sonde et procede pour le diagnostic du sexe d'un embryon humain - Google Patents

Sonde et procede pour le diagnostic du sexe d'un embryon humain

Info

Publication number
EP0188485A1
EP0188485A1 EP85903297A EP85903297A EP0188485A1 EP 0188485 A1 EP0188485 A1 EP 0188485A1 EP 85903297 A EP85903297 A EP 85903297A EP 85903297 A EP85903297 A EP 85903297A EP 0188485 A1 EP0188485 A1 EP 0188485A1
Authority
EP
European Patent Office
Prior art keywords
probe
dna
hybridization
cells
sex
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Withdrawn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
EP85903297A
Other languages
German (de)
English (en)
French (fr)
Inventor
Gilles Vergnaud
Liliana Kaplan
Jean Weissenbach
Pierre Tiollais
Georges Guellaen
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.)
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, Institut Pasteur filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP0188485A1 publication Critical patent/EP0188485A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • 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

Definitions

  • the invention relates to a probe comprising a nucleic acid sequence specific for the human Y chromosome and to a method for diagnosing the sex of a human embryo.
  • the object of the invention is to provide an even more specific probe for the Y chromosome and an in vitro diagnostic method capable of being implemented on a total genomic DNA, without prior separation of any of its constituents.
  • the invention therefore aims to combine the earliness of the diagnosis, the speed and reliability of its implementation.
  • the invention stems from a research project, certain results of which were the subject of first publications. This is more particularly the article by CE.BISHOP et al. titled “Sinqle-Copy DNA Specified Sequences for the Human Y Chromosome", (Nature Vol. 303, N ° 5 920, on. 831 - 832, June 30, 1983 ) and the article by C. BISHOP et al. entitled “Extensive Sequence Homologies Between Y And-Other Human- Chromosomes” (J. Mol. Biol. [1984] 173, 403-417).
  • the second article describes more particularly the conditions under which the amplified cosmids containing exclusively inserts of human origin were obtained, how the latter were further sorted, by digestion with the restriction enzyme EcoRI and subcloning of the fragments obtained in pBR 322, to finally obtain 45 probes containing inserts originating from the human Y chromosome and probably unrelated to each other.
  • These probes were further characterized by hybridization experiments under strict conditions with series of fragments obtained from genomic DNA treated by complete digestion with the restriction enzyme EcoRI, then separated by electrophoresis in gels. of agarose, before being transferred to a nitro-cellulose filter or a similar paper, known under the initials DBM (ALWINE, JC [1977] Proc.Nat.Acad. SCi.
  • the invention follows from the discovery that one of these EcoRI fragments, called “Probe 49 F” was capable not only of giving rise to selective hybridization with a particular DNA fragment obtained from series of EcoRI fragments transferred as indicated above and obtained from Y chromosomes, but also to give rise to an equally selective hybridization when this is carried out on a single spot of DNA originating from human male embryo, if necessary previously fragmented, this spot having been obtained for example by filtration of a solution of total genomic DNA through a nitrocellulose filter pad, according to the so-called "Dot-Blot” technique.
  • the above-mentioned prior fragmentation can be carried out by simple ultrasonic treatment (sonication) of the DNA preparation to be tested.
  • the invention relates more generally to any DNA containing a hybridizable fragment under the strict conditions recalled below, with the EcoRI fragment of 2.8 kb obtained from the euchromatic region located between the centromere and the heterochromatin (when the latter is present) of the large arm of a Y chromosome, this fragment having been deposited on 06/29/1984 at the National Collection of Cultures of Microorganisms of the INSTITUT PASTEUR of Paris (CNCM) under N ° 1- 312.
  • CNCM National Collection of Cultures of Microorganisms of the INSTITUT PASTEUR of Paris
  • any fragment of chromosome Y which forms stable hybrids with the above-mentioned EcoRI fragment deposited at the CNCM in the presence of a solution of 0.1 SSC at 65-68oC, more particularly at 68 ° C.
  • This condition of stability of the hybrid can be verified in particular during the washing of the hybrid previously formed on a suitable support / such as a nitrocellulose filter or the like, with a solution of 0.1 ⁇ SSC (SSC being a 0.5 M NaCl solution; 0.15 M sodium citrate at pH 7.0 at 65 - 68 ° C, more particularly 68 ° C).
  • SSC being a 0.5 M NaCl solution; 0.15 M sodium citrate at pH 7.0 at 65 - 68 ° C, more particularly 68 ° C.
  • the invention relates to any plasmid or other vector containing an insert corresponding to the above definition.
  • hybridization stability conditions which have been defined above are strict with regard to the selectivity of the hybridization.
  • the ion content of the 0.1 SSC solution being low, the hybridization remains stable only to the extent that the two chains involved in the constitution of the hybrid have a very high degree of complementarity. The selectivity of this probe even with respect to,.
  • the DNA preparation required for the implementation of the diagnosis according to the invention can consist of any cells of the embryo capable of being obtained at a very early stage of gestation, in particular between the 6th and 8th week, for example from tr ⁇ phoblasts (ie from chorionic cells) or from chorionic villi. Examples of implementation of the method according to the invention will be described below.
  • the invention therefore also relates to a nrocedé oour the in vitro diagnosis of sex, orocé35 characterized nar the contacting of a DNA containing all or part of the hybridizable fraqment above-defined with the practically total DNA coming from cells taken from the fetus whose sex must be determined, this total DNA having been previously extracted from said cells or simply made accessible to the probe, or for example by lysis of. cells, and by bringing the hybrid formed into contact, whether already during or after the hybridization (especially during washes) with a solution with low ionic strength and at a temperature favoring together highly selective hybridizations of the probe with a sequence of complementary DNA present in the chromatin of the human Y chromosome.
  • the aforesaid brought into contact is aerated with a 0.1 SSC solution at a temperature of 65 - 68 ° C, more particularly 68 ° C.
  • the probes are radioactively marked.
  • the invention is not limited to this method of marking.
  • the probes can be modified by a single group allowing their coupling, direct or indirect, for example with an enzyme whose presence can be revealed by its action with respect to a specific substrate, preferably a chromogenic substrate, or with a fluorescent or luminescent molecule. Coupling modes of this type have been envisaged, for example, in French patents 78 10975 and 81 27631 or in published European patent application 0063 879.
  • a preferred embodiment of the method according to the invention for the detection of sex on cells originating from a fetus or. human embryo will include:
  • nucleic acids thus fixed or made accessible into contact with the labeled probe according to the invention under conditions allowing effective hybridization to be carried out, when the DNAs studied comprise a Y chromosome;
  • kits or "kits” containing a probe in accordance with the invention and, where appropriate, control chromosomal preparations (originating from male and / or female embryos) and any other reagents necessary for the production of a in vitro diagnostic test.
  • the DNA analysis was carried out on cells belonging to the chorionic villi or to blood cells taken from the embryos studied. The technique known as "Dot blot" was used. The tests can be carried out on quantities of DNA less than 3 ⁇ ig. The trials only last two days. Cell samples
  • the blood samples were provided by hospitals. Trophoblast samples were obtained from placental biopsies performed after the first 6 to 8 weeks of gestation. In a number of cases, biopsies have been taken in women before abortion and after having obtained their informed consent. A 1.7 mm diameter DYONIC biopsy forceps was introduced cross-cervically into the uterine cavity using a real-time ultrasound guide according to the methods described by SIMONI G. et al. (Hum. Genêt. [1983]; 63 349-57) and WARD R.H.T. et al. (Brit.Med. H. 1983: 1542-4).
  • trophoblast villi i.e., chorion
  • the tissues were washed in an isotonic and sterile saline solution and then examined immediately under a dissecting microscope. All maternal decidual cells have been carefully separated from the villi using appropriate tweezers. From 10 to 50 mg of trophoblast villi were then used for the analysis of their nucleic acids. The products of conception were used for karyotype or chromatin studies Trophoblast villi have also been used. In seven cases, small parts of these villi were obtained by dissection from placental cotyledons obtained after cesarean sections.
  • Fibroblasts obtained from embryos were cultured in vitro according to standard procedures (RPMI 1640 fetal calf serum, antibiotics, in an atmosphere containing 5% CO 2 ).
  • the karyotypes were established after in vitro cultures for 12-20 days (second or third pass) using standard techniques. Giemsa stains and molecular weight separation techniques (C Banding techniques: CBG) were used. Two to ten mitoses were analyzed.
  • the chromatins of the chromosomes involved in the constitution of sex were studied on cells at the interphase level supplied from cultures of fibroblasts stained with toluidine blue. From 100 to 200 nuclei were counted.
  • Preparation of the nucleic acids and analysis A) Preparation of the DNA obtained from blood cells: 10 ml of blood were collected in a ml of 5% EDTA. The serum was removed by centrifugation. The red blood cells were lysed in 0.17 M NH 4 Cl and the leukocytes were collected by centrifugation. They were lysed in a solution containing 7 M urea, 2% sodium dodecyl sulfate (SDS), 0.2 M NaCl, 10 mM Tris at pH 8 and 1 mM EDTA. The solution was extracted twice with an equal volume mixture of phenol and chloroform, then twice with chloroform before being precipitated with ethanol.
  • SDS sodium dodecyl sulfate
  • the precipitate was taken up, dissolved in a 10 mM Tris solution, 1 mM EDTA and containing ribonuclease A (50 ⁇ g / ml). The solution was incubated at 37 ° C for one hour, then supplemented with the following products to finally obtain a concentration in 1% SDS, 10 mM EDTA and proteinase K 100 ⁇ g / ml. The solution was then incubated for two hours. The solution was then extracted once with chloroform, dyalized in a 10 mM Tris solution pH 8, 1 mM EDTA overnight, before being precipitated again with ethanol.
  • the DNAs were treated with ultrasound, denatured in a 0.4M NaOH solution and 1 M NaCl at 4 ° C for half an hour.
  • the solution was neutralized just before filtration through a nitrocellulose filter in a 1.5 M Tris-NaCl solution, pH 7. Before filtration the nitrocellulose filter had been washed in a 6 x SSC solution. After filtration the filter was dried at 80 ° C for two hours, then hybridized in 6 x SSC, 1 x DENHARDT, for
  • the probe according to the invention (which essentially contains a single sequence, as opposed to earlier probes essentially consisting of repetitive sequences) perfectly discriminates the male and female chromosomes, even when these have the following anomalies: in the case of male chromosomes, loss of part or all of the heterochromatin and in the case of female chromosomes, translocation into the X chromosome of certain fragments of heterochromatin normally considered to belong to the heterochromatin of group Y of the male chromosomes .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP85903297A 1984-06-29 1985-06-28 Sonde et procede pour le diagnostic du sexe d'un embryon humain Withdrawn EP0188485A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR8410408 1984-06-29
FR8410408A FR2566911A1 (fr) 1984-06-29 1984-06-29 Sonde et procede pour le diagnostic du sexe d'un embryon humain

Publications (1)

Publication Number Publication Date
EP0188485A1 true EP0188485A1 (fr) 1986-07-30

Family

ID=9305658

Family Applications (1)

Application Number Title Priority Date Filing Date
EP85903297A Withdrawn EP0188485A1 (fr) 1984-06-29 1985-06-28 Sonde et procede pour le diagnostic du sexe d'un embryon humain

Country Status (6)

Country Link
EP (1) EP0188485A1 (en))
ES (1) ES8608683A1 (en))
FR (1) FR2566911A1 (en))
GR (1) GR851598B (en))
PT (1) PT80743B (en))
WO (1) WO1986000342A1 (en))

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4769319A (en) * 1985-05-31 1988-09-06 Salk Institute Biotechnology Industrial Associates, Inc. Nucleic acid probes for prenatal sexing
EP0261172A1 (en) * 1986-02-21 1988-03-30 Whitehead Institute For Biomedical Research Y-specific dna hybridization probes and uses therefor
FR2595100B1 (fr) * 1986-02-28 1990-02-09 Agronomique Inst Nat Rech Sondes moleculaires d'adn specifique du genome male de mammiferes, notamment du genre bos, presentant des sequences d'adn specifique du sexe male, procede de preparation de telles sondes et procede de determination du sexe d'embryons et de foetus des mammiferes susdits, a l'aide de ces sondes
FR2603701B2 (fr) * 1986-02-28 1990-09-21 Agronomique Inst Nat Rech Applications des sondes moleculaires d'adn specifique du genome male de mammiferes, notamment du genre bos, au controle de la presence du chromosome y dans une population de spermatozoides et a la separation des spermatozoides en deux populations de spermatozoides porteurs respectivement du chromosome y et du chromosome x pour leur utilisation pour l'insemination artificielle
CA1296270C (en) * 1986-08-12 1992-02-25 The Australian National University Sex determination in ruminants using y-chromosome specific polynucleotides
FR2619819B1 (fr) * 1987-08-27 1990-08-10 Agronomique Inst Nat Rech Sondes moleculaires d'adn specifique du genome male de ruminants, notamment de la sous-famille des bovines et en particulier du genre bos, sequences flanquantes pour l'amplification de ces sondes et applications desdites sondes
US5328827A (en) * 1987-08-27 1994-07-12 Institut National De La Recherche Agronomique - Inra Specific DNA molecular probes for Bos-type male genome
WO1989002440A2 (en) * 1987-09-21 1989-03-23 Whitehead Institute For Biomedical Research Y-specific dna hybridization probes and uses therefor
WO1989007154A1 (en) * 1988-01-29 1989-08-10 Advanced Riverina Holdings Limited Determination of genetic sex in ruminants using y-chromosome-specific polynucleotides
JP2693776B2 (ja) * 1988-02-24 1997-12-24 科学技術振興事業団 Ｄｎａプローブ及びそれを用いたヒト胎児の性別判定法
FR2635116B1 (fr) * 1988-08-08 1990-11-02 Georges Michel Procede pour la determination du sexe d'embryons de ruminants, kit ou necessaire pour la mise en oeuvre du procede
US5840482A (en) * 1990-10-10 1998-11-24 The Regents Of The University Of California Y chromosome specific nucleic acid probe and method for determining the Y chromosome in situ

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE735915C (de) * 1939-07-27 1943-06-01 Waffen Und Munitionsfabriken A Vorrichtung zum gleichzeitigen Messen der Wandstaerke und deren Verlauf laengs des Umfanges von zylindrischen Hohlkoerpern an mindestens drei verschiedenen Stellen
NO149296C (no) * 1978-03-17 1984-03-21 Johde Fa Maaleapparat for sylinderformete gjenstander
DD154846B1 (de) * 1980-11-27 1987-03-11 Ursula Barth Verfahren und auswerteeinrichtung zur angenaeherten paarungsdurchmesserbestimmung rotationssymmetrischer teile

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8600342A1 *

Also Published As

Publication number Publication date
ES8608683A1 (es) 1986-07-16
GR851598B (en)) 1985-11-25
WO1986000342A1 (fr) 1986-01-16
PT80743A (fr) 1985-07-01
ES544664A0 (es) 1986-07-16
FR2566911A1 (fr) 1986-01-03
PT80743B (fr) 1986-12-11

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