DE19932890A1 - DNA to detect changes in chromosome 8 - Google Patents

Abstract

The present invention relates to a DNA for detecting changes in chromosome 8, especially 8q24, and more particularly the c-myc gene. Furthermore, the invention relates to the use of the DNA and a kit suitable for detecting the above changes.

Description

The present invention relates to DNA which is useful for detection changes in chromosome 8, especially 8q24 and especially the c-myc region. Furthermore, the Invention the use of the DNA and one for the detection standing changes suitable kit.

In many tumors, there are changes in the Chromosome 8, in particular the chromosomal region 8q24. The Changes can be numeric or structural. Frequently translocations, amplifications, duplicates can be found tions, deletions and / or inversions of larger DNA Be rich.

The detection of changes in chromosome 8 is much fold about the chromosome banding technique. With this tech nik changes at chromosomal level only in le capable of dividable cells. Also their resolution is only in the range of several megabases (<5MB). The identity of the multiplied, or relocated Loss-affected sequences therefore remain in the chromosomal Bending technique unknown.

The present invention is therefore based on the object to provide a means of altering the chromo soms 8, in particular the chromosomal region 8q24, under Avoiding the above disadvantages can be demonstrated. In particular, the agent should be suitable for clinical Purposes, d. H. on a large scale, to be used.

According to the invention this is the subject matter in the patent claims achieved.

The present invention is based on the recognition of the Applicant ders that in tumors, the changes of chromo  soms 8, in particular the chromosomal region 8q24, these often change the c-myc gene or a range of about 1 MB concerning the c-myc gene. The applicant has this z. B. Bur putty lymphoma was found. He also realized that Ver be detected by one or more DNAs can be listed in Table I:

Table I

Name of the DNAs Length of the DNAs (A ₁ ) LLNLP704H027Q13 104 kb (A ₂ ) LLNLP704J1287Q13 105 kb (B) LLNLP704F15291Q13 140 kb (C) LLNLP704C07126Q13 166 kb (D) LLNLP704K19268Q13 159 kb (E) LLNLP704K2280Q13 146 kb (F) LLNLP704K20264Q13 97 kb (G) LLNLP704G05103Q13 142 kb (H) LLNLP704M14318Q13 80 kb (I) LLNLP704K22117Q13 115 kb

These DNAs are located in a region of about 1 MB spanning the c-myc gene (see Figure 1). Furthermore, the DNAs were deposited with the DSMZ (German Collection of Microorganisms and Cell Cultures) on November 24, 1998: the DNA of (A ₁ ) under DSM 12523, the DNA of (A ₂ ) under DSM 12524, the DNA of (B) under DSM 12521, the DNA of (C) under DSM 12520, the DNA of (D) under DSM 12525, the DNA of (E) under DSM 12528, the DNA of (F) under DSM 12526, the DNA of (G) under DSM 12522, the DNA of (H) under DSM 12529 and the DNA of (I) under DSM 12527.

According to the invention, a protruding DNA or a part thereof, especially those who have an overlap of two or more ren of the DNAs, for detecting a change in the chro  mosoms 8, especially from 8q24, and especially the c-myc Region, to be used. This can be done by conventional methods respectively. It is favorable, an in situ hybridization reaction or a CGH "Comparative Genomic Hybridization" matrix drive to perform. In such reactions or procedures can be any cellular sample containing chromosomal DNA z. B. cells from bone marrow or blood, with a fiction, be tested according to DNA. For carrying out an in situ hybridization reaction or a matrix CGH method Reference is made to the following examples.

With the present invention it is possible to tumor cancer evidence at the molecular level. In particular, can the importance of c-myc for formation and manifestation be determined by tumor diseases. The detection of the tumor diseases may be clinically relevant, i. H. in big Scale, done. For this purpose, the present invention also a kit available. This contains one or more DNAs according to the invention, customary auxiliaries, such as carriers, Puf fer, solvents and controls. As carriers are insbesonde to call chips on which the DNAs of the invention in usually be applied. The chips can then with Load DNA from cellular samples and assay for Ver Chromosome 8 changes, especially from 8q24 and all over especially the c-myc region. Such a kit is also the subject of the present invention.

Furthermore, the present invention provides the base, Tu cause diseases to be treated. For this purpose, the DNAs according to the invention as such or in conjunction with übli gene transfer vectors, such as retrovirus, adenovirus or Vaccinia virus vectors used for gene therapy. The A specialist knows materials and conditions that are suitable for this suitable.

Overall, the present invention provides means ready, diagnostically and / or therapeutically for tumor to be able to intervene.  

Brief description of the drawings

Fig. 1 shows the genomic localization of fiction, contemporary DNAs in a c-myc gene spanning region of about 1 MB.

Fig. 2 (cf. 1. Tab.) Shows the result of actions in situ hybridization with the DNA of the invention Re DSM 12,527th In Fig. 2 (A), normal human metaphase chromosomes are shown. The red signals are from a c-myc cosmid sample, the green signals from the DNA of the invention. It turns out that both samples hybridize at the same chromosomal site (chromosome 8q24) and therefore the signals appear partly yellow from the combination of red and green. This experiment was repeated with all the DNAs of the invention and confirmed. In Fig. 2 (B), human metaphase chromosomes from a cell line of a Burkitt lymphoma are shown. The red signals originate from the DNA according to the invention, the green signals from a DNA from chromosome 22. The chromosome translocation present in this cell line, in which the c-myc locus is affected, does not show the hybridization signal from the DNA according to the invention, such as in normal cells on two, but on three different chromosomes. The signals of the DNA of chromosome 22, however, are present only on two chromosomes.

The present invention will be illustrated by the examples.

example 1 Proof of a c-myc gene Translocation by in situ hybridization 1.1 marking a DNA of the invention (DSM 12527) by Nick translation Reagents, buffers and solutions

• 10 × reaction buffer with 0.5 M Tris-HCl (pH 8.0), 50 mM MgCl ₂ , 0.5 mg / ml bovine serum albumin (fraction V, cat no. 735078, Roche Diagnostics Mannheim)
• 0.1 M β-mercaptoethanol
• 10 × nucleotide stock solution with 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.125 mM digoxigenin-11-dUTP (Roche Diagn stics Mannheim, cat.-no. 1558706), 0.375 mM dTTP (Alter natively, biotinylated dUTP can be used).
• Escherichia coli DNA polymerase I (New England Biolabs GmbH, Schwalbach / Taunus, cat.-no. 209L).
• - DNase I stock solution: 3 mg in 1 ml 0.15 M NaCl, 50% Glyce rin
• Column buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% SDS
• - Columns: Sephadex G50 (medium), 1 ml tuberculin syringes (eg "Primo" from Pharmaplast A / S, DK-4970 Rodby), glass wool
• - minigel: agarose; TBE buffer; 1 kb size marker (Gibco BRL, Eggenstein, cat.-no. 15615-016); gel loading buffer

procedure

• 1. 2 μg of the DNA according to the invention were together with 10 μl of the 10 × reaction buffer, 10 μl of β-mercaptoethanol, 10 μl the nucleotide stock solution, 20 U of the DNA polymerase I and 2 μl of a 1: 1000 dilution of DNase stock solution (in Water) are pipetted into a reaction vessel.
• 2. The labeling reaction was carried out at 15 ° C for 120 min.
• 3. By gel electrophoresis, the labeled was invented DNA according to the invention for in situ hybridization suitable fragment size (about 500-800 bp). 10 μl of the reaction mixture were ver with 3 ul Gelladebuffer ver place denatured and boiled in boiling water for 2-3 min  then cooled for 3 min on ice. After that, Ali became quot on a 1-2% agarose minigel along with the 1 kb Size marker applied. The separation of the DNA-Frag Mente took place at 15 V / cm for 30 min. After staining With ethidium bromide, the gel could be exposed to UV light graphed and the size of the DNA fragments determined who the.
• 4. In the optimal case, the DNA fragments should be in one Size range between 500 and 800 base pairs. The average size of the fragments was above In this area, the remaining DNA was again with DNase I incubated until the optimal size of the fragments was reached.
• 5. To inactivate the DNase, the reaction mixture was 2 μl 0.5 M EDTA (final concentration 10 mM) and 1 μl 10% SDS (Final concentration 0.1%) was added and this at 68 ° C for Heated for 10 min.
• 6. Unincorporated nucleotides were removed from the DNA sample by gel filtration through separation columns prepared as follows:
  • a) A 1 ml tuberculin syringe was first packed with glass wool to the 0.2 ml mark and then filled up to the 1 ml mark with buffered Sephadex G50. Subsequently, the column was transferred to a 15 ml tube and centrifuged at room temperature (2000 g, 6 min).
  • b) After addition of 100 μl of column buffer, the columns were again centrifuged (2000 g, 6 min). This step was repeated three times.
  • c) The DNA was added to the column, centrifuged (2000 g, 6 min) and collected in a reaction vessel. The final concentration of this DNA sample is about 20 ng / μl. It can be stored for a long time (months to years) at -20 ° C.

1.2 In situ hybridization of the labeled invention DNA (DSM 12527) on metaphase chromosomes and interphase nuclei 1.2.1 Denaturation of Metaphase Chromosomes and Inter phase nuclei Reagents, buffers and solutions

• - Denaturing solution: 70% deionized formamide (for the Molecular Biology, cat.-no. 112027, Merck, Darmstadt), 2 × SSC, 50 mM sodium phosphate (pH 7.0), pH 7.0 with conc. HCl
• - Ethanol (ice cold): 70%, 90% and 100%.

procedure

• 1. The denaturing solution was placed in a cuvette and heated to 70 ° C in a water bath.
• 2. The slide with the metaphase to be examined chromosomes and interphase nuclei of cells of a Burkitt lymphoma was incubated in this solution for 2 min and then transferred to cold 70% ethanol.
• 3. The slides were each 5 min in 70%, 90% and 100% ethanol dehydrated and then air dried.

1.2.2 precipitation and denaturation of the labeled he DNA according to the invention for in situ hybridization Reagents, buffers and solutions

• 3 M sodium acetate, pH 5.2
• - Deionized formamide (for molecular biology, cat.- No. 112027, Merck, Darmstadt). The deionization took place by stirring the formamide with an ion exchanger (eg AG 501-X8 (D) Resin, cat No. 142-6425, Biorad,  Munich).
• 2 × hybridization buffer: 2 × SSC, 20% dextran sulfate.

procedure

• 1. 100 ng each of a digoxigenin or biotin-labeled DNA Probe and 10 μg of human Cot1 DNA (Gibco-BRL, Eggenstein, Cat 5279SA) were prepared by adding 1/20 volume of 3M Sodium acetate and 2.5 volumes 100% cold ethanol präzi pitiert.
• 2. After centrifugation (12000 rpm, 15 min 4 ° C) was the supernatant was removed, the DNA pellet with 500 μl 70% Ethanol, centrifuged again (12000 rpm, 10 minute 4 ° C) and finally lyophilized.
• 3. The precipitated DNA was dissolved in 6 μl of deionized forma and shaken for 30 min at room temperature (Vortex).
• 4. After addition of 6 μl 2 × hybridization buffer, the Shake for another 30 min.
• 5. Subsequently, the DNA was denatured at 75 ° C for 5 min and then incubated for 20-30 min at 37 ° C ("preannealing").

1.2.3 In situ hybridization procedure

• 1. 12 ul of the hybridization mixture were on an Ob jektträger with the examined metaphase chromosomes and interphase nuclei (both normal human cells as well as cells of a human Burkitt Lymphoma).
• 2. An 18x18 mm coverslip was placed, with liquid adhesive  sealed material and the preparation in a moist Chamber incubated for 48 h at 37 ° C.

1.2.4 Washing and detection of the hybridized metaphase chromosomes and interphase nuclei Reagents, buffers and solutions

• Wash A: 50% formamide (p.A. cat # 9684, Merck, Darmstadt), 2 × SSC
• - Washing solution B: 0.1 × SSC
• Wash C: 4X SSC, 0.1% Tween 20
• Washing solution D: 2 × 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
• Rhodamine-conjugated sheep anti-digoxigenin Fab fragment (Roche Diagnostics Mannheim)
• - Fluorescein-conjugated avidin (Vector Laboratories Inc., Burlingame, USA)
• - "Antifade solution": 0.233 g of DABCO (1,4-diazabicyclo-2.2.2- octane), 20mM Tris-HCl, pH 8.0, 90% glycerol
• 4,6-diamino-2-phenylindole (DAPI)

procedure

• 1. Washing solutions A and B were placed in a cuvette Heated to 42 ° C in a water bath.
• 2. The sealing ring of liquid adhesive was removed from the object carrier and this then 3 × 5 min in washing solution A washed at 42 ° C.
• 3. Subsequently, the slide was placed in wash solution B, the preheated to 60 ° C, washed 3 × 5 min at 42 ° C.
• 4. 200 μl of the blocking solution was applied to the slide pipetted, a coverslip placed on and for 30 min at 37 ° C in a humid chamber incubated.  
• 5. The coverslip was removed, 200 μl of detection buffer with 5 μg / ml rhodamine-conjugated sheep anti-digoxigenin Fab fragment and 5 μg / ml fluorescein-conjugated avidin placed on the microscope slide, a cover slip and placed Incubated for 30 min at 37 ° C in a humid chamber.
• 6. The slide was then rinsed in Wash Solution C for 3 x 5 min washed at 42 ° C.
• 7. Subsequently, the DNA was counterstained for 20 min in 2 × SSC stained in which 200 ng / ml (DAPI) were dissolved.
• 8. The slide was added to Wash Solution D for 1-2 min Washed at room temperature.
• 9. Finally, the hybridized metaphase chromosome men and interphase nuclei with "antifade" solution embeds.

The hybridization signals were evaluated by means of an Epi fluorescence microscope equipped with suitable filters for fluorescein and rhodamine (see Fig. 2).

Example 2 Evidence of a chromosome 8 or the chromo Somali band 8q24 or the c-myc gene concern de Over or underrepresentation by Ma trix CGH 2.1 Immobilization of a DNA DSM 12527 according to the invention

The DNA according to the invention (conc .: 400-1000 μg / ml) is washed with Using suitable microcapillary coated on poly-L-lysine Slides given. The slides are then at 5 min Baked at 80 ° C in an oven, then 5 min with cold methanol Glacial acetic acid (3: 1) fixed and then bakes at 80 ° C for 30 min  ken. This treatment is repeated three times. Last will be the DNA on the slide for 2 min at 70 ° C in 70% formamide / 2 × SSC (pH 7.0) denatured.

2.2 Hybridization

• 1. 1 μg of biotin-labeled tumor DNA and digoxigenin-mar labeled control DNA were mixed with 80 μg of human Cot1 DNA (Gibco-BRL, Eggenstein, Cat # 5279SA) by addition of 1/20 volume 3 M sodium acetate and 2.5 volumes 100% Etha nol precipitated.
• 2. After centrifugation (12000 rpm, 10 min 4 ° C) was the supernatant was removed, the pellet with 500 ul 70% etha washed, again centrifuged (12000 rpm, 10 min, 4 ° C) and then lyophilized.
• 3. The precipitated DNA was transformed into 12 μl of hybridization buffer (10% dextran sulfate, 33.5% formamide, 2 × SSC) taken and shaken for 30 min at room temperature (Vortex).
• 4. After the DNA was dissolved, it was 5 min at 75 ° C de natured and then incubated for 25 min at 37 ° C. ( "Preannealing").
• 5. 12 μl of the hybridization mixture was applied to the object carrier with the immobilized DNA Samples were given and a cover slip (18 × 18 mm) placed.
• 6. The coverslip was sealed with liquid adhesive and the preparation in a humid chamber for 4 days at 60 ° C incubated.

2.3 Detection Reagents, buffers and solutions

• Wash A: 50% formamide (p.A. cat # 9684, Merck, Darmstadt), 2 × SSC
• - Wash solution B: 1 × SSC
• Wash C: 4X SSC, 0.1% Tween 20
• Washing solution D: 2 × 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
• Mouse anti-digoxigenin antibody (Roche Diagnostics Mann home)
• Digoxigenin-conjugated sheep anti-mouse F (ab) ₂ fragment (Roche Diagnostics Mannheim)
• Rhodamine-conjugated sheep anti-digoxigenin Fab fragment (Roche Diagnostics Mannheim)
• - Fluorescein-conjugated avidin (Vector Laboratories Inc., Burlingame, USA)
• Biotinylated anti-avidin (Vector Laboratories Inc., Burlingame, USA)
• - "Antifade solution": 0.233 g of DABCO (1,4-diazabicyclo-2.2.2- octane), 20mM Tris-HCl, pH 8.0, 90% glycerol
• 4,6-diamino-2-phenylindole (DAPI)

procedure

• 1. Washing solutions A and B were placed in a cuvette Heated to 42 ° C in a water bath.
• 2. The sealing ring of liquid adhesive was removed from the object carrier and this then 3 × 5 min in washing solution A washed at 42 ° C.
• 3. Subsequently, the slide was placed in wash solution B, the preheated to 50 ° C, washed 3 × 5 min at 42 ° C.
• 4. 200 μl of the blocking solution was applied to the slide pipetted, a coverslip placed on and for 30 min at 37 ° C in a humid chamber incubated.
• 5. The coverslip was removed, 200 μl of detection buffer  with 5 μg / ml mouse anti-digoxigenin antibody and 5 μg / ml Fluorescein-conjugated avidin on the slide Place a coverslip and place at 37 ° C for 30 min a humid chamber incubated.
• 6. The slide was then rinsed in Wash Solution C for 3 x 5 min washed at 42 ° C.
• 7. Thereafter, the signals were assayed 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-digo xigenin F (ab) 2 fragment amplified. The concentration was in each case 5 μg / ml in detection buffer. The incubation each took 30 min at 37 ° C in a humid chamber. After each antibody incubation, the object was washed 3 × 5 min in wash solution C at 42 ° C.
• 8. Finally, the hybridized immobilized DNA samples embedded with "antifade" solution.

The hybridization signals were determined by means of a confo kalen laser scanning microscope, suitable filters and a special image processing software evaluated.

Claims (14)

1. DNA suitable for detecting a change in chromosome 8, wherein the DNA is selected from:

• 1. (A ₁ ) DNA deposited with the DSMZ under DSM 12523,
• 2. (A ₂ ) DNA deposited with the DSMZ under DSM 12524,
• 3. (B) DNA deposited with the DSMZ under DSM 12521,
• 4. (C) DNA deposited with the DSMZ under DSM 12520,
• 5. (D) DNA deposited with the DSMZ under DSM 12525,
• 6. (E) DNA deposited with the DSMZ under DSM 12528,
• 7. (F) DNA deposited with the DSMZ under DSM 12526,
• 8. (G) DNA deposited with the DSMZ under DSM 12522,
• 9. (H) DNA deposited with the DSMZ under DSM 12529,
• 10. (I) DNA deposited with the DSMZ under DSM 12527,
  the parts of it.

2. DNA according to claim 1, wherein the change is the chromosome paint area 8q24 concerns. 3. DNA according to claim 1 or 2, wherein the change is the c- myc gene or a spanning area of about 1 MB concerns. 4. DNA according to any one of claims 1 to 3, wherein the changes tion in the case of tumor diseases. 5. DNA according to claim 4, wherein the tumor diseases a Burkitt lymphoma include. 6. Use of the DNA according to any one of claims 1 to 5 as Reagent for diagnosis and / or therapy of changes of chromosome 8. 7. Use according to claim 6, wherein the change the chromosomal region 8q24.   8. Use according to claim 6 or 7, wherein the change the c-myc gene or a spanning region of about 1 MB. 9. Use according to any one of claims 6 to 8, wherein several the DNAs (A) to (I) or parts thereof used the. 10. Use according to any one of claims 6 to 9, wherein the Changes in tumor diseases are present. 11. Use according to claim 10, wherein the tumor diseases include a Burkitt lymphoma. 12. Kit comprising one or more of the DNAs (A) to (I) or Parts thereof, according to claim 1 and customary auxiliaries. 13. Kit according to claim 12, wherein the adjuvants buffer, Lö include agents, carriers and controls. The kit of claim 13, wherein the carriers comprise chips.