Fluorescent in situ hybridisation: For Hybridisation with Cosmid Probes.
Arnaud Gos, Laboratoire de Diagnostic Moléculaire, Division de Génétique Médicale
I) Probe labelling
Mix in an eppendorf tube:
- 2 mg DNA
- 10 ml 10 * nick translation buffer (0.5 M Tris/HCl, 50 mM MgCl2, 0.5 mg/ml BSA (nuclease-free))
- 10 ml nucleotide solution containing 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP and 0.1 mM dTTP.
10 ml 0.4 mM biotin-16-dUTP (or digoxigenein-11-dUTP or fluorescein-12-dUTP, all from Boehringer Mannheim)
- 10 ml 100 mM DTT
- 10 ml DNase I (Boehringer, for grade I a 1/100 dilution from a 1 mg/ml stock solution, for grade II a 1/666 dilution)
- 6 ml DNA polymerase I (5 U/ml) (Boehringer).
- X ml H2O to a final volume of 100 ml.
Incubate at 16°C for 2 hours.
Keep the reaction on ice.
For two-colour in situ hybridisation one probe is labelled with Biotin-16-dUTP and the other is labelled with Digoxigenin-11-dUTP.
II) Size check of the probe
Take a 10 ml aliquot from the labelling reaction and add gel-loading buffer.
Denature the aliquot by incubation in a boiling water bath for 2 min.
Put the tube on ice for 2 min.
Load the aliquot on a standard 1% agarose minigel (ethidium bromide free!) along with suitable size marker, as quickly as possible (to avoid probe renaturation).
Run the gel at high voltage (e.g. 15 Volts per cm for 30 min.) to avoid probe renaturation in the gel.
Visualize DNA by staining the gel in 0.5 mg/ml ehtidium bromide and take a picture.
The probe molecule should be visible as a smear. This should contain only fragments smaller than 500 nt and larger than 100 nt. A peak intensity at 250-300 nt is optimal.
If the probe DNA is between 100-500 nt, proceed with inactivation.
If the probe is larger, add more DNase I to the reaction kept on ice, incubate further at 16°C (usually higher concentrations of DNase are added for a further 30 min. incubation). If the probe is smaller than 100 nt. Repeat the labelling reaction with less volume of DNase I dilution.
Check the probe size again as described before.
For enzyme inactivation incubate the labelling reaction at 65°C for 15 min.
Store the probe at -20°C.
III) Slide preparation
Colchicine treated blood cultures are treated with a hypotonic solution containing 75 mM KCL (10-30 min., 37°C), followed by a methanol/acetic acid (3:1) fixation (3-4 times). The metaphase suspension is dropped on ethanol/diethyl-ether (1:1) cleaned, cold wet slides, and rinsed briefly with 70% acetic acid for the removal of cytoplasm. After overnight drying at room temperature (RT) the slides are stores dry at 4°C.
IV) RNase treatment
The slides are first washed twice 15 min. with PBS and then treated with 100 mg/ml RNase A (Boehringer) in 2*SSC, 1 hour at 37°C (100 ml under a 24 x 50 mm cover slip, or in a coplin jar). After 3 x 5 min. washes with 2*SSC this treatment can be followed by either a postfixation or a pepsin digestion and a postfixation.
V) Pepsin digestion
Wash the slides in pre-warmed PBS for 5 min. at 37°C and incubate them in pepsin solution (5 mg pepsin in 100 ml 10 mM HCl) for 5 min. at 37°C. The slides are then washed in PBS for 5 min. at RT. A postfixation is performed in 1% acid-free formaldehyde/PBS/50 mM MgCl2 pH 7, for 10 min. at RT. After one wash with PBS the slides are then dehydrated through a series of ethanol (70%, 90% and 100% 5 min. each) and air-dried.
VI) Hybridisation
The hybridisation mix is made in the following way (for 1 hybridisation):
For cosmids:
1 mg competitor Cot-1 DNA (Gibco BRL) (or 5mg human DNA sonicated to a length of about 500 bp), 1 mg salmon sperm DNA, 1 mg yeast tRNA and 60-80 ng of the labelled probe.
The DNA is then precipitated with 1/100 volume of 3 M NaOAC pH 5.6 and an equal volume of 2-propanol, spun down for 10 min. at 12K and washed with 70% ethanol. The pellet is dissolved in 10 ml hybmix (10% dextran sulfate/2*SSC/50% deionized formamide, pH 7). Probe and competitor DNA are denatured at 75°C for 5 min. A pre-annealing (CISS, Chromosomal In Situ Suppression) is performed for cosmids at 37°C for 15-60 min. The probe DNA is kept on ice (not more than 5 min.) before applying it to denatured chromosomes.
Denaturing of the chromosomes is started at least 30 min. before hybridisation. The denaturing is done by immersion of the slides in 70% formamide/2*SSC, pH 7, at 75°C for 5 min. After the denaturing the slides are quickly placed in pre-chilled (-20°C) 70% ethanol solution and kept in this solution until all the slides are denatured. The slides are then dehydrated through a series of cold 90%, 100% ethanol and air-dried. 10ml hybmix is placed without bubbles onto the slide and a 18 * 18 mm coverslip is lowered gently onto the droplet. The coverslip is sealed with rubber cement. The hybridisation is carried in a moist chamber (containing 2*SSC/50% overnight at 37°C.
For two-colour in situ hybridisation the procedure is almost the same as described for one-colour hybridisation except that the two probes are dissolved in hybmix at a higher concentration and denatured separately. The biotin-labelled probe and the digoxigenin-labelled probe are then mixed together (after pre-annealing if required), adjusted to a final volume of 10 ml with hybmix and placed on the denatured slide as above.
VII) Post-hybridisation steps
- 3 times 5 min. washes in 50% formamide/2*SSC, pH 7 at 42°C.
- 3 x 3 min. washes in 0.1*SSC, pH 7, 60°C.
- 1 x 3 min. 4*SSC/0.05% Tween-20,pH /, at RT.
- 10 min. preincubation with 4*SSC/0.05% Tween-20 containing 5% NFDM in a moist chamber (100 ml under a 24 x 50 mm coverslip).
- 20 min. incubation at RT with Avidin-Rhodamine (Vector Laboratories) (100 ml at 20 mg/ml in 4*SSC/5% NFDM/0.05% Tween-20 under a 24 * 50 mm coverslip).
- 2 * 3 min. 4*SSC/0.05% Tween-20 at RT.
- 1 * 3 min. 0.1 M Tris/HCl pH 7.570.15 M NaCl/0.05% Tween-20 (AbBB) at RT.
- 30 min. incubation at 37°C with Biotinylated-goat-anti-Avidin (5 mg/ml) and monoclonal anti-Digoxinenin-FITC (Boehringer) (0.4 mg/ml) in AbBB/1% blocking reagent (Boehringer) (100 ml under a 24 * 50 mm coverslip).
- 3 x 3 min. AbBB at RT.
- 30 min. incubation at 37°C with Avidin-Rhodamine (20 mg/ml)
- 2 x 3 min. AbBB at RT.
- 2 x 5 min., PBS at RT.
- The slides are mounted in antifade medium as described before, without propidium iodide.