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WO2001006001A2 - Adn pour la detection de modifications du chromosome 8 - Google Patents

Adn pour la detection de modifications du chromosome 8 Download PDF

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Publication number
WO2001006001A2
WO2001006001A2 PCT/DE2000/002384 DE0002384W WO0106001A2 WO 2001006001 A2 WO2001006001 A2 WO 2001006001A2 DE 0002384 W DE0002384 W DE 0002384W WO 0106001 A2 WO0106001 A2 WO 0106001A2
Authority
WO
WIPO (PCT)
Prior art keywords
dna
deposited
under dsm
chromosome
dsmz under
Prior art date
Application number
PCT/DE2000/002384
Other languages
German (de)
English (en)
Other versions
WO2001006001A3 (fr
Inventor
Peter Lichter
Stefan Joos
Andreas RÄTSCH
Original Assignee
Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts filed Critical Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
Priority to EP00954353A priority Critical patent/EP1230384A2/fr
Priority to AU66844/00A priority patent/AU6684400A/en
Publication of WO2001006001A2 publication Critical patent/WO2001006001A2/fr
Publication of WO2001006001A3 publication Critical patent/WO2001006001A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to DNA which is suitable for the detection of changes in chromosome 8, in particular of 8q24 and very particularly of the c-myc region.
  • the invention further relates to the use of the DNA and a kit suitable for detecting the above changes.
  • changes in chromosome 8 in particular the chromosomal region 8q24, can be found.
  • the changes can be numerical or structural. Translocations, amplifications, duplications, deletions and / or inversions of larger areas of DNA are frequently found.
  • Changes in chromosome 8 are often detected using the chromosome banding technique. With this technique, changes at the chromosomal level can only be detected in viable dividable cells. Their resolution is only in the range of several megabases (> 5 MB). The identity of the amplified, rearranged or lost sequences therefore remains unknown in the chromosome banding technique.
  • the present invention is therefore based on the object of providing a means by which changes in chromosome 8, in particular chromosomal region 8q24, can be detected while avoiding the above disadvantages.
  • the agent should be suitable for clinical purposes, i.e. on a large scale.
  • the present invention is based on the knowledge of the applicant that in the case of tumor diseases which show changes in chromosome 8, in particular the chromosomal region 8q24, these frequently relate to the c-myc gene or a region of approximately 1 MB around the c-myc gene. The applicant found this, for example, in Burkitt's lymphoma. He also recognized that the changes can be detected by one or more DNAs listed in Table I:
  • DNAs are located in a region of approximately 1 MB encompassing the c-myc gene (cf. FIG. 1). Furthermore, the DNAs were deposited with the DSMZ (German Collection of Microorganisms and Cell Cultures) on November 24, 1998: the DNA of (A x ) under DSM 12523, the DNA of (A 2 ) under DSM 12524, the DNA of (B) under DSM 12521, the DNA from (C) under DSM 12520, the DNA from (D) under DSM 12525, the DNA from (E) under DSM 12528, the DNA from (F) under DSM 12526, the DNA from (G) under DSM 12522, the DNA from (H) under DSM 12529 and the DNA from (I) under DSM 12527.
  • DSMZ German Collection of Microorganisms and Cell Cultures
  • a DNA above or a part thereof, in particular that which comprises an overlap of two or more of the DNAs can be used to detect a change in chromosome 8, especially 8q24, and especially the c-myc region.
  • This can be done using conventional methods. It is expedient to carry out an in situ hybridization reaction or a matrix CGH “Comparative Genomic Hybridization” method. In such reactions or methods, any cellular sample containing chromosomal DNA, for example cells from bone marrow or blood, can be tested with a DNA according to the invention.
  • an in situ hybridization reaction or a matrix CGH method reference is made to the following examples.
  • the present invention it is possible to detect tumor diseases at the molecular level.
  • the importance of c-myc for the development and manifestation of tumor diseases can be determined.
  • the detection of the tumor diseases can be carried out in a clinically relevant manner, i.e. on a large scale.
  • the present invention also provides a kit.
  • This contains one or more DNAs according to the invention, customary auxiliary substances such as carriers, buffers, solvents and controls.
  • chips can be mentioned as carriers on which the DNAs according to the invention are applied in a conventional manner.
  • the chips can then be loaded with DNA from cellular samples and tested for changes in chromosome 8, especially 8q24 and especially the c-myc region.
  • a kit is also the subject of the present invention.
  • the present invention provides the basis for treating tumor diseases causally.
  • the DNAs according to the invention can be used as such or in conjunction with conventional gene transfer vectors, such as retrovirus, adenovirus or vaccinia virus vectors, for gene therapy.
  • gene transfer vectors such as retrovirus, adenovirus or vaccinia virus vectors
  • the present invention provides means of being able to intervene diagnostically and / or therapeutically for tumor diseases.
  • 1 shows the genomic localization of DNAs according to the invention in a region of about 1 MB spanning the c-myc gene.
  • FIG. 2 shows the result of in situ hybridization reactions with the DNA DSM 12527 according to the invention (cf. Table 1).
  • Figure 2 (A) shows normal human metaphase chromosomes. The red signals come from a c-myc cosmid sample, the green signals from the DNA according to the invention. It can be seen that both samples hybridize at the same chromosomal site (chromosome 8q24) and the signals from the combination of red and green therefore appear partially yellow. This experiment was repeated and confirmed with all DNAs according to the invention.
  • 2 (B) shows human metaphase chromosomes from a cell line of a Burkitt lymphoma. The red signals come from the DNA according to the invention, the green signals from a DNA from chromosome 22.
  • the hybridization signal from the DNA according to the invention is not shown, as in normal Cells on two but on three different chromosomes.
  • the DNA signals from chromosome 22, however, are only present on two chromosomes.
  • Example 1 Detection of a translocation relating to the c-myc gene by means of in situ hybridization
  • biotinylated dUTP can be used.
  • the labeling reaction took place at 15 ° C for 120 min. 3.
  • the labeled DNA according to the invention was tested for the fragment size suitable for in situ hybridization (about 500-800 bp) by means of gel electrophoresis. 10 ⁇ l of the reaction mixture were mixed with 3 ⁇ l gel loading buffer, denatured in boiling water for 2-3 min and then cooled on ice for 3 min. The aliquot was then applied to a 1-2% agarose mini gel together with the 1 kb size marker. The DNA fragments were separated at 15 V / cm for 30 min. After staining with ethidium bromide, the gel could be photographed under UV light and the size of the DNA fragments determined.
  • the DNA fragments should be in a size range between 500 and 800 base pairs.
  • the denaturing solution was placed in a cuvette and heated to 70 ° C. in a water bath.
  • Deionized formamide for molecular biology, cat. No. 112027, Merck, Darmstadt. Deionization was carried out by stirring the formamide with an ion exchanger (e.g. AG 501-X8 (D) Resin, Cat.No. 142-6425, Biorad,
  • an ion exchanger e.g. AG 501-X8 (D) Resin, Cat.No. 142-6425, Biorad
  • 2 x hybridization buffer 2x SSC, 20% dextran sulfate.
  • the precipitated DNA was taken up in 6 ⁇ l deionized formamide and shaken for 30 min at room temperature (vortex).
  • the DNA was then denatured at 75 ° C. for 5 minutes and then incubated at 37 ° C. for 20-30 minutes (“preannealing”).
  • wash solution A 50% formamide (p.A. Cat.No. 9684, Merck, Darmstadt), 2 x SSC
  • Wash solution B 0.1 x SSC
  • Wash solution C 4 x SSC, 0.1% Tween 20
  • Wash solution D 2 x SSC, 0.05% Tween 20
  • Blocking solution 3% BSA, 4 x SSC, 0.1% Tween 20 - detection buffer: 1% BSA, 4 x SSC, 0.1% Tween 20
  • washing solutions A and B were heated in a cuvette to 42 ° C in a water bath.
  • wash solution B which was preheated to 60 ° C, washed 3 x 5 min at 42 ° C.
  • the DNA was then counter-stained for 20 min in 2 x SSC in which 200 ng / ml (DAPI) had been dissolved.
  • the hybridization signals were evaluated using an epifluorescence microscope equipped with suitable filters for fluorescein and rhodamine (cf. FIG. 2).
  • Example 2 Detection of an over- or underrepresentation affecting chromosome 8 or chromosomal band 8q24 or the c-myc gene using a matrix CGH
  • the precipitated DNA was taken up in 12 ⁇ l hybridization buffer (10% dextran sulfate, 33.5% formamide, 2 x SSC) and shaken for 30 min at room temperature (vortex).
  • the cover slip was sealed with liquid adhesive and the preparation is incubated in a moist chamber for 4 days at 60 ° C.
  • Washing solution A 50% formamide (p.A. Cat.No. 9684, Merck,
  • Wash solution C 4 x SSC, 0.1% Tween 20
  • Wash solution D 2 x SSC, 0.05% Tween 20
  • Detection buffer 1% BSA, 4 x SSC, 0.1% Tween 20 - mouse anti-digoxigenin antibody (Röche Diagnostics).
  • Rhodamine-conjugated sheep anti-digoxigenin Fab fragment (Röche Diagnostics Mannheim)
  • Biotinylated anti-avidin Vector Laboratories Inc.
  • washing solutions A and B were heated in a cuvette to 42 ° C in a water bath.
  • wash solution B which was preheated to 50 ° C, washed 3 x 5 min at 42 ° C.
  • the signals were then compared with biotinylated-anti-avidin, followed by fluorescein-conjugated avidin, or with a digoxigenin-conjugated sheep anti-mouse F (ab) 2 fragment and a rhodamine-conjugated sheep anti-digoxigenin -F (ab) 2 fragment amplified.
  • concentration was 5 ⁇ g / ml in detection buffer.
  • the incubation each time lasted 30 min at 37 ° C in a humid chamber. After each antibody incubation, the slide was washed 3 x 5 min in washing solution C at 42 ° C.
  • the hybridization signals were evaluated using a confocal laser scanning microscope, suitable filters and special image processing software.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un ADN utilisé pour détecter des modifications du chromosome 8, en particulier de la région 8q24, et tout particulièrement du gène c-myc. L'invention concerne en outre l'utilisation dudit ADN et un nécessaire pour la détection des modifications susmentionnées.
PCT/DE2000/002384 1999-07-19 2000-07-19 Adn pour la detection de modifications du chromosome 8 WO2001006001A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP00954353A EP1230384A2 (fr) 1999-07-19 2000-07-19 Adn pour la detection de modifications du chromosome 8
AU66844/00A AU6684400A (en) 1999-07-19 2000-07-19 Dna for detecting changes in chromosome 8

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19932890.0 1999-07-19
DE19932890A DE19932890A1 (de) 1999-07-19 1999-07-19 DNA zum Nachweis von Veränderungen des Chromosoms 8

Publications (2)

Publication Number Publication Date
WO2001006001A2 true WO2001006001A2 (fr) 2001-01-25
WO2001006001A3 WO2001006001A3 (fr) 2002-06-20

Family

ID=7914738

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2000/002384 WO2001006001A2 (fr) 1999-07-19 2000-07-19 Adn pour la detection de modifications du chromosome 8

Country Status (4)

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EP (1) EP1230384A2 (fr)
AU (1) AU6684400A (fr)
DE (1) DE19932890A1 (fr)
WO (1) WO2001006001A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10438691B2 (en) 2013-10-07 2019-10-08 Sequenom, Inc. Non-invasive assessment of chromosome alterations using change in subsequence mappability

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0878552A1 (fr) 1997-05-13 1998-11-18 Erasmus Universiteit Rotterdam Détection moléculaire d'aberration chromosomique
JP2002513587A (ja) 1998-05-04 2002-05-14 ダコ エー エス 染色体異常を検出するための方法およびプローブ

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4542096A (en) * 1983-02-25 1985-09-17 President And Fellows Of Harvard College Detecting rearranged human c-myc DNA fragments associated with oncogenes
US5582973A (en) * 1989-11-24 1996-12-10 The United States Of America As Represented By The Department Of Health And Human Services Sensitive method for localizing chromosomal breakpoints
US5734039A (en) * 1994-09-15 1998-03-31 Thomas Jefferson University Antisense oligonucleotides targeting cooperating oncogenes
US5658730A (en) * 1994-12-23 1997-08-19 Ctrc Research Foundation Methods of human prostate cancer diagnosis

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10438691B2 (en) 2013-10-07 2019-10-08 Sequenom, Inc. Non-invasive assessment of chromosome alterations using change in subsequence mappability
US11929146B2 (en) 2013-10-07 2024-03-12 Sequenom, Inc. Systems for non-invasive assessment of chromosome alterations using changes in subsequence mappability

Also Published As

Publication number Publication date
WO2001006001A3 (fr) 2002-06-20
EP1230384A2 (fr) 2002-08-14
DE19932890A1 (de) 2001-02-01
AU6684400A (en) 2001-02-05

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