Principle

The DAPI/Distamycin A staining technique is useful in identifying pericentromeric breakpoints in chromosomal rearrangements and in identifying chromosomes that are too small for standard banding techniques. Also, DAPI/DA is the method of choice for Yqh chromsome material in suspected Y autosome translocations.

Background

The DAPI/distamycin A fluorescent staining technique was first described by Schweizer, Ambros, and Andrle as a method for labeling a specific subset of C bands. (Gustashaw, 1991).

Stains

Distamycin A:

• Dissolve 2 mg distamycin A-HCl (Sigma) in 10 mL of McIlvaine's buffer, pH 7.0. Add a magnetic stirrer and place the foil-covered tube in a beaker of crushed ice. Stir for 15 to 30 minutes. Dispense in 1 mL aliquots and store at -20 degrees C for up to six months.

  DAPI
  

• Stock solution: Dissolve 2 mg DAPI-2 HCl (Sigma) in 10 mL of distilled water. Dispense in 1 mL aliquots, and store at -20 degrees C for up to six months.
  
• Working solution: Add 0.1 mL of stock solution to 100 mL of McIlvaine's buffer, pH 7.0. Store at 4 degrees C for up to six months.
  
• Buffer: McIlvaine's buffer, pH 7.0.
  
  Solution A: 0.1 mol/L citric acid (19.2g: q.s. to 1 liter with distilled water)
  
  Solution B: 0.2 mol/L Na2HPO4 (28.4g: q.s. to 1 liter with distilled water)
  
  The following formula can be used to prepare this buffer at various pHs:

  x mL A + (100 - x) mL B = 100 mL total volume

  For pH 4.1, x = 60
  
  For pH 5.5, x = 43.1
  
  For pH 7.0, x = 18.2
  

Procedure

1. Flood a slide with distamycin solution. Coverslip and incubate the slide, in the dark, at room temperature for 5 to 15 minutes.

2. Remove the coverslip and rinse briefly with pH 7.0 buffer.

3. Flood with DAPI working solution. Coverslip and incubate the slide, in the dark, at room temperature for 5 to 15 minutes.

4. Remove the coverslip and rinse the slide briefly with pH 7.0 buffer.

5. Observe with fluorescence (excitation: 340-380 nm; suppression: 430 nm).

6. Photograph as for other fluorescent techniques.

Special Note:
Use care when handling these substances. Wear gloves. Distamycin A tends to lack stability in aqueous solution unless it is frozen. DAPI stock solution can be frozen or refrigerated for several weeks without detectable deterioration. The stained preparations tend to fade rapidly, so photography should be immediate. Storing coverslipped slides at 4 degrees C for a day or so may stabilize the fluorescence.

References

Gustashaw, KM. Chromosome Stains. In The ACT Cytogenetics Laboratory Manual, Second Edition, edited by M. J. Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.