***Preparation of DNA probes for chromosome FISH: Whole chromosome
painting probes labeling by DOP-PCR***

*Reagents and equipment *

· Source DNA (flow sorted or microdisected chromosomes)

· PCR buffer 10X Perkin Elmer without MgCl~2\ ~(Roche Molecular
Biochemical)

· MgCl~2\ ~solution 25 mM Perkin Elmer (Roche Molecular Biochemical)

· Taq DNA polymerase 5U/l Perkin Elmer (Roche Molecular Biochemical)

· Deoxy-nucleotides dATP, dCTP, dGTP, and dTTP 100 mM (Roche Molecular
Biochemical)

· Stock dNTPs solution for labeling PCR contains 0.2 mM of dATP, dCTP,
dGTP and 0.15 mM of dTTP

· 0.06mM Fluorescein--dUTP. (Boehringer Mannheim) or Biotin-16-dUTP
(Boehringer Mannheim) or Spectrum red-dUTP (Vysis)

· Universal primer for human genomic DNA amplification: UN1 (Midland
Certified Reagent Co) Telenius \[5'-CCGACTCGAGNNNNNNATGTGG-3'\] or
universal primer for mouse genomic DNA amplification: 22-mer (Midland
Certified Reagent Co.) \[5'-CGG ACT CGA GNN NNN NTA CAC C-3'\]

· Hi-Lo DNA marker (Minnesota Molecular)

· Tris acetate buffer

· Sodium Acetate 3M pH 5.2

· PCR thermocycler

· Microcentrifuge

· Microcentrifuge tubes

*Method *

1\. Set up the PCR reaction.

PCR reaction mix:

  ------------------------- --------------
  Reagent                   Quantity (l)
                            
  PCR buffer 10X            10
  MgCl~2\ ~ 25mM            8
  Stock dNTPs solution^a^   5
  Biotin--dUTP^a^           5
  dH~2~O                    65
  DNA (100-150 g/l)         4
  Primer (100 M)            2
                            
  Taq polymerase (5U/l)     1
                            
  ------------------------- --------------

2\. Run the PCR reaction

+------+----------------------+---------------------------+
| Step | Temperature (C)      | Time (min)                |
+------+----------------------+---------------------------+
|      |                      |                           |
+------+----------------------+---------------------------+
| 1    | 94                   | 1                         |
+------+----------------------+---------------------------+
| 2    | 56                   | 1                         |
+------+----------------------+---------------------------+
| 3    | 72                   | 3                         |
|      |                      |                           |
|      |                      | (+1 additional sec/cycle) |
+------+----------------------+---------------------------+
| 4    | Steps 1-3 (29 times) |                           |
+------+----------------------+---------------------------+
|      |                      |                           |
+------+----------------------+---------------------------+
| 5    | 72                   | 10                        |
+------+----------------------+---------------------------+
|      |                      |                           |
+------+----------------------+---------------------------+

After the final step hold the PCR samples at 4°C until they are used.

3\. To analyze the DOP-PCR products, mix 8 µl of the reaction products
with 2 µl agarose gel loading buffer.

4\. Apply the sample to a 1% w/v agarose gel in 1x TAE buffer.

5\. Apply 10 l of Hi-Lo DNA marker.

6\. Run the gel for 45 min at 70 V/cm in 1x TAE buffer.

7\. Stain the gel with ethidium bromide and observe in UV
transiluminator. In the reaction samples you should see a smear of DNA
ranging from about 200--500 bp.

8\. Add to the remnant of the DOP-PCR labeled DNA 1/10 volume of 3M
sodium acetate pH 5.2 and 3 volumes of cold absolute ethanol.

9\. Put tubes at -70 C for 30 min.

10\. Spin down the samples at 14,000 g in 4 C microcentrifuge for 30
min.

11\. Remove carefully the supernatant and dry the DNA under vacuum for 3
min.

12\. Resuspend the DNA in sterile water at a final concentration of 100
g/l.

13\. Store the labeled DNA at --20 C.

^a^Note that the concentration of the dNTPs and labeled-dUTP varies.

· Remnants of cytoplasm can impair the access of the probe to the target
DNA of metaphase and especially interphase chromosomes. To improve the
hybridization quality a treatment with 70% acetic acid and/or mild
pepsin treatment is frequently performed. Here is the protocol

#### Equipment and reagents

· Glacial acetic acid

· Hydrochloric acid 1N

· Pepsin (Sigma)

· 1XPBS

· Coplin jars

· Slide warmer or waterbath

*Method for acetic acid treatment*

1\. Soak the slide in an acetic acid 70 % v/v solution for 40-60 sec.

2\. Rinse the slide in 1xPBS for 5 min at room temperature, shaking
gently.

3\. Dehydrate the slide trough an ethanol series of 70 % v/v, 85% v/v,
and 100 % v/v for 2 min each at room temperature and then air dry.

#### Method for pepsin treatment

1\. Rinse the slide in 1XPBS for 5 min.

2\. Apply to a 60 mm^2\ ^coverslip 100 µl of a 10g/ml solution of pepsin
in 10 mM HCl. Touch the slide to the coverslip. Incubate in moist
chamber at 37°C, for 5 min.

3\. Rinse the slide in 1XPBS for 5 min.

4\. Dehydrate the slide trough an ethanol series of 70 % v/v, 85% v/v,
and 100 % v/v for 2 min each at room temperature and then air dry.