Author: TM
Last saved by: WalkerD
Company: NCI/NIH

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_2O | 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. 

^aNote 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.