Protocols

Comparative Genomic Hybridization
(Direct labelling)

Protocol

21 February 2001
 

Cytogenetic Laboratory, Section of Clinical Genetics, Juliane Marie Center,
Rigshospitalet, University of Copenhagen, Copenhagen.

Preparation of metaphase chromosomes from peripheral blood cells

DNA extraction

Nicktranslation. Direct labelling

Probe precipitation and denaturation

Denaturation of metaphase chromosomes

Hybridization

Detection

DOP-PCR
 
 

Preparation of metaphase chromosomes from peripheral blood cells.

Reagents, buffers and solutions:

• Li-heparinized or Na-heparinized blood.
• Culture medium: RPMI 1640 (SIGMA), fetal calf serum (20-30%), 200 mM L-glutamin (1%), penicillin and streptomycin 10.000 U/ml (1%), heparin 5000 IE/ml (0,1%), phytohemag-lutinin (3%).
• Colcemid solution: prepare stock solution of 10 µg/ml and store at 4°C.
• Hypotonic solution: 60mM KCL.
• Fixative solution: 3 parts ethanol (abs); 1 part acetic acid (glacial).
• Microscopic slides: precleaned microscopic slides are washed in ethanol for 24 hours and washed twice in water. Slides are kept in water at 4°C and dried immediately before use.

Procedure:

Day 0, Add 1 ml heparinized blood to 8 ml culture medium. Incubate at 37°C in an atmosphere of 5% CO2.

Day 3, Add 0.1 ml colcemid (10 µg/ml) and continue incubation at 37°C in an atmosphere of 5% CO2.
One hour later cells are harvested:

1. Spin at 181 x g for 10 min.
2. Remove supernatant, resuspend pellet and add 8 ml hypotonic solution (60 mM KCL). Leave at room temperature for 30 min.
3. Add 1-2 ml fresh fixative solution (slowly, almost dropwise in the beginning). Invert the tube a couple of times.
4. Spin at 181 x g for 10 min.
5. Remove supernatant, resuspend pellet and add 10 ml of fixative solution.
6. Spin at 181 x g for 10 min. and remove the supernatant.
7. Repeat steps 5 and 6.
8. Remove as much as possible of the supernatant. Add 2-1 ml fixative solution (suspension should look milky). Keep suspension at -20°C for at least 20 min. before dropping slides.
9. Drop the cell suspension right above the slides. Usually 5 drops are adequate. Humidity should be between 30-40%. Let slides mature at room temperature for 2 - 4 weeks. Hereafter the slides are stored at -20°C for up to 6 months. A test CGH must be performed on each batch of slides. Only slide batches showing high intensity, uniform hybridization should be used.
    

DNA extraction.

High molecular weight genomic DNA is prepared by using Qiagen Genomic Tip columns (Qiagen, Hilden Germany) according to the manufactures instructions.
 

Nicktranslation. Direct labelling.

Reagents, buffers and solutions:

• Solution of test/reference DNA to be labelled.
• 10 x reaction buffer containing 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 500 mMTris/HCL pH 7.8, 50 mM MgCl2, 100 mM 2-mercaptoethanol.
• TexasRed-5-dUTP/FITC-12-dUTP (DuPont, Boston, MA, USA).
• 10 x enzymemix (500 U/ml DNA polymerase I, 1 U/ml DNAse I, 100 µg/ml BSA (nuclease free), 50 mM Tris/HCL pH 7.5, 5mM magnesium acetate, 1 mM b-mercaptoethanol, 50% (v/v) glycerol.
• DNA polymerase I (10 U/µl)(Promega)

Procedure:

1. Combine (on ice): 2.0 µg DNA, 5.0 µl 10 x reaction buffer, 1 µl TexasRed-5-dUTP/FITC-12-dUTP, 3.0 µl 10 x enzymemix, 1.0 µl DNA polymerase I; adjust to 50 µl with double distilled water (enzymes should be added last).
2. Incubate for 60 min. at 15°C.
3. Incubate the probe at 70°C for 10 min.
4. Check the probe length by agarose gel electroforesis. Take 10 µl of the reaction solution, add gel loading buffer and run a 1-2% agarose minigel at 15 V/cm for 30-40 min. For optimum hybridization conditions, the probe should be visible as a smear between 600 and 2000 bp (double stranded). Depending on the results of the gel, proceed in one of the following ways:

1. The probe size is within the desired range. Precipitate the probe (see below).
2. The probe size is too large. Add the same amount of enzymemix and DNA polymerase I to the nicktranslation reaction as previously and incubate at 15°C for 10-60 min. depending on the probe size.
3. A large part of the probe size is below 600 bp. Either precipitate the probe and assign all of the labelled probe to a single slide, or (preferably) start new nicktranslation reaction using less enzymemix or shorter incubation time.
    

Denaturation of metaphase chromosomes.

Reagents, buffers and solutions:

• Denaturation solution: 70% deionized formamide, 2 x SSC, pH 7.0.
• Ethanol: 70%, 96% and 100%.

Procedure:

1. Mark the hybridization area underneath the slide with a diamond pen.
2. Apply 17 µl of denaturation solution under a 21 x 26 mm coverslip and incubate on a hot plate at 74°C for 3 min.
3. Dehydrate in the ethanol series for 3 min. each.
4. After air drying the slides are ready for hybridization.
    

Reagents, buffers and solutions:

• 3 M sodium acetate, pH 5.2
• Mastermix: 50% deionized formamide, 2 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M Na3-citrate, pH 7.0), 10% dextran sulphate.

Procedure:

1. Combine 400 ng of labelled test DNA and 400 ng of labelled reference DNA and 20 µg of human Cot1 DNA. Precipitate the DNA by adding 1/10 volume 3 M sodium acetate and 2 volumes of 100% ethanol. Mix and incubate at -20°C for 30 min.
2. Spin in an Eppendorf centrifuge at maximum speed (12000-15000 rpm) for 30 min. at 4°C. Discard supernatant and dry for a few minutes at 60°C.
3. Add 10 µl of mastermix.
4. Denature the DNA at 70°C for 5 min.
    

Hybridization.

1. Apply 10 µl of the hybridization mixture with the denatured probe onto the denatured chromosomes on the slide.
2. Put a 18 x 18 mm coverslip on the hybridization mixture.
3. Seal the edges of the coverslip with rubber cement and incubate for 3 days in a wet chamber at 37°C.
    

Detection.

reagents, buffers and solutions.

• 1 x SSC (1 x SSC: 0.15 M NaCl, 0.015 M Na3-citrate, pH 7.0).
• 2 x SSC
• Antifade-mounting solution: Vecta Shield (Vector Laboratories), containing 0.56 µg/ml DAPI counterstain (4,6'-diamino-2-phenylindole)

Procedure:

1. Carefully remove the rubber cement. Slowly push off the coverslip with a forceps in a coplin jar containing 2 x SSC.
2. Wash slides for 5 min. at 72°C in 1 x SSC.
3. Wash slides for another 5 min. at 72°C in 2 x SSC.
4. Transfer slides to 2 x SSC at room temperature and leave them here until mounting.
5. Apply one drop of antifade-mounting solution and cover with a 21 x 26 mm coverslip.
   Seal with nailpolish. Slides should be kept at 4°C for long term storage.
    

DOP-PCR

Reagents, buffers and solutions.

• 10 x DDRT-buffer: 100 mM Tris-HCl, 500 mM KCl, 18 mM MgCl₂, 1% Triton X-100, 0.05 % Gelatine.
• PCR Nucleotide Mix: (Roche Molecular Biochemicals #1 581 295) diluted 1:4 in distilled water.
• UN-1 primer: 5'-CCG ACT CGA GNN NNN NAT GTG G-3', 10 µM (TAGCopenhagen A/S).
• Taq-polymerase: Amplitaq DNA Polymerase, LD (Perkin Elmer Part No. N808-0107).
• ml PCR tubes

Procedure:

1. Combine in laminar air flow hood: 3 ng purified DNA or 200-300 flow sorted nuclei, stained for nuclear DNA content according to Vindelov et al. and 3 µl 10 x DDRT buffer and 3µl PCR Nucleotide Mix and 3 µl UN-1 primer and 1 µl Taq-polymerase in a final volume of 30 µl.
2. Prepare two identical tubes for each sample and one no-template control.
3. Run the following program on thermocycler with heated lid

5 low stringency cycles:

30 sec. at 94º
60 sec. at 25°
120 sec at 72°

Followed by

35 high stringency cycles:

20 sec. at 94º
30 sec. at 56°
120 sec at 72°

4. Analyse the products by agarose gel electrophoresis
5. Pool the two identical tubes and purify on a Qiaquick PCR purification kit
6. Nick translate as described above using 0.75 enzyme mix on the pooled DNA and rection for 40 minutes.
7. We use standard Texas Red labelled genomic DNA for reference DNA in CGH hybridizations