Chromosome Painting Protocol

Chromosome painting is a powerful cytogenetic technique that allows for the visualization and identification of specific chromosomes or chromosomal regions within a cell. This method utilizes fluorescently labeled DNA probes that hybridize to complementary DNA sequences on target chromosomes, enabling researchers to observe the organization and structure of the genome.

Preparation of the hybridization probe

• Put in a 1.5 ml microcentrifuge tube 400-600 ηg (4 μl) of probe DNA. Add 10 μg of the appropriate Cot-1 DNA (10 μl) and 10 μg (1 μl) of salmon testes DNA.
• Add to the total volume 1/10 volume of 3 M sodium acetate pH 5.2 and 3 volumes of ice-cold absolute ethanol.
• Put the tubes at -70°C for 30 min.
• Spin down the samples at 14,000 g in 4°C microcentrifuge for 30 min.
• Remove carefully the supernatant and dry the DNA probe under vacuum for 2 min.
• Resuspend the DNA probe in 5 μl deionized formamide (pH 7.5) and incubate at 37°C for 30 min. Vortex and spin down at 14,000 g for a few sec every 10 min.
• Add 5 μl of dextran sulfate 20% w/v in 2xSSC.
• Denature the DNA probe by heating it at 80°C for 5 min.
• Spin down a few sec and incubate the DNA probe at 37°C for 30 min to pre-anneal.
• Keep on ice until used in step 15.

In situ denaturation of the target DNA and hybridization

• For chromosome slide denaturation place a 60 mm 2 cover glass on a slide warmer at 70°C apply 110 μl of formamide 70% v/v solution.
• Touch the denaturing solution with the preparation (sample side) and incubate on the hot plate for 1-2 min at 70°C.
• Remove the cover glass and immerse immediately in ice-cold ethanol 70% v/v for 2 min to stop denaturation.
• Dehydrate the slide through an ethanol series of 85% v/v, and 100% v/v for 2 min each at room temperature and then air dry.
• Apply the previously denatured and pre-annealed probe. Cover with a 22 mm cover glass and seal the edges with rubber cement.
• Incubate protected from light in a moist chamber at 37°C overnight.

Post-hybridization washing and detection

• Warm the washing solutions a, b, and c (1 jar) at 46°C for 30-40 min.
• Remove rubber cement and coverslip from the hybridized slide. Immerse the slide in jar a; agitate for a few sec and leave it in the jar for 5 min.
• Transfer the slide to jar b; agitate for a few sec and leave it in the jar for 5 min.
• Transfer the slide to jar c; agitate for a few sec and leave it in the jar for 5 min.
• Dip the slide in 4xSSC/Tween 20 at room temperature.
• Apply 100 μl of blocking solution (Bovine albumin 3% w/v in 4XSSC/Tween 20) to a 60 mm 2 coverslip, and touch the slides to the coverslip. Incubate in a moist chamber at 37°C, protected from light for 30 min.
• Rinse the blocking solution off by dipping the slide in 4XSSC/Tween 20 for 3 min.
• Apply to a 60 mm 2 coverslip 100 μl of fluorescin-streptavidin (dilution 1:100 in 4XSSC/Tween 20/Bovine albumin 1% w/v). Touch the slide to the coverslip. Incubate in a moist chamber at 37°C, protected from light for 30 min.
• Rinse the slide in 4xSSC/Tween 20 at room temperature 2 x 5 min shaking gently.
• Transfer the slide to jar d. Counterstain with DAPI for 5 min at room temperature and protected from light.
• Rinse the in H₂O, jar e, for 5 min at room temperature.
• Air dry and dehydrate the slide through an ethanol series of 70% v/v, 85% v/v, and 100% v/v for 2 min each at room temperature.
• Apply 30-35 μl antifade mounting media and cover with 60 mm 2 coverslip. Store at 4°C in the dark until analysis.
• Visualize by eye using a fluorescence microscope.

Creative Bioarray Relevant Recommendations

• Creative Bioarray has a full range of DNA probe libraries that use fluorescently labeled specific chromosomes (whole chromosome, specific chromosome arm, or chromosome fragments). The DNA was prepared by chromosomal staining, and then the probe was hybridized to the mitotic phase (chromosome separation) by fluorescence in situ hybridization. When hybridization is complete, the effect of multicolor coating is produced at the hybrid site. We can then observe the fluorescent intensity on the chromosome under a fluorescence microscope to analyze and study the chromosome reorganization, aberrations, and homologous genes.

• The degree of chromosome condensation can vary depending on the cell cycle stage and preparation methods. Highly condensed chromosomes may appear thicker and make it harder to resolve fine-scale chromosomal features. Adjusting the cell culture conditions or fixation protocol can help modulate chromosome condensation.
• Mechanical stress during slide preparation or harsh chemical treatments can lead to chromosome fragmentation or distortion. Careful handling of cells and slides, as well as the use of gentle reagents and procedures, is essential to minimize chromosome damage.

Probe

Fluorescent In Situ Hybridization (FISH) Service

For research use only. Not for any other purpose.