Genomic DNA Labeling Protocol
Developed for microarray-based comparative genomic hybridization.
Genomic DNA can be labeled with a simple random-priming protocol based on Gibco/BRL's Bioprime DNA Labeling kit, though nick translation protocols work too. I routinely use the BioPrime labeling kit (Gibco/BRL) as a convenient and inexpensive source of random octamers, reaction buffer, and high concentration klenow (do not use the dNTP mix provided in the kit), though other sources of random primers and high concentration klenow work as well.
1. Add 2 ug DNA of the sample to be labeled to an eppindorf tube.
Note: For high complexity DNAs (e.g. human genomic DNA), the labeling reaction works more efficiently if the fragment size of the DNA is first reduced. I routinely accomplish this by restriction enzyme digestion (usually DpnII, though other 4-cutters work as well). After digestion, the DNA should be cleaned up by phenol/chloroform extraction / EtOH precipitation (Qiagen PCR purification kit also works well).
2. Add ddH20 or TE 8.0 to bring the total volume to 21 ul. Then add 20 ul of 2.5X random primer / reaction buffer mix. Boil 5 min, then place on ice.
2.5X random primer / reaction buffer mix:
125 mM Tris 6.8
12.5 mM MgCl2
25 mM 2-mercaptoethanol
750 ug/ml random octamers
3. On ice, add 5 ul 10X dNTP mix.
10X dNTP mix:
1.2 mM each dATP, dGTP, and dTTP
0.6 mM dCTP
10 mM Tris 8.0, 1mM EDTA
4. Add 3 ul Cy5-dCTP or Cy3-dCTP (Amersham, 1 mM stocks)
Note: Cy-dCTP and Cy-dUTP work equally well. If using Cy-dUTP, adjust 10X dNTP mix accordingly.
5. Add 1 ul Klenow Fragment.
Note: High concentration klenow (40-50 units/ul), available through NEB or Gibco/BRL (as part of the BioPrime labeling kit), produces better labeling.
6. Incubate 37 degrees C for 1 to 2 hours, then stop reaction by adding 5 ul 0.5 M EDTA pH8.0
7. As with RNA probes, I purify the DNA probe using a microcon 30 filter (Amicon/Millipore):
Add 450 ul TE 7.4 to the stopped labeling reaction.
Lay onto microcon 30 filter. Spin ~10 min at 8000g (10,000 rpm in microcentrifuge).
Invert and spin 1 min 8000g to recover purified probe to new tube (~20-40 ul volume).
8. For two-color array hybridizations, combine purified probes (Cy5 and Cy3 labeled probes) in new eppindorf tube. Then add:
30-50 ug human Cot-1 DNA (Gibco/BRL; 1 mg/ml stock; blocks hybridization to repetitive DNAs if present on array).
100 ug yeast tRNA (Gibco/BRL; make a 5 mg/ml stock; blocks non-specific DNA hybridization).
20 ug poly(dA)-poly(dT) (Sigma catalog No. P9764; make a 5 mg/ml stock; blocks hybridization to polyA tails of cDNA array elements).
450 ul TE 7.4
Concentrate with a microcon 30 filter as above (8000g, ~15 min, then check volume every 1 min until appropriate). Collect probe mixture in a volume of 12 ul or less.
9. Adjust volume of probe mixture to 12 ul with ddH20. Then add 2.55 ul 20X SSC (for a final conc.of 3.4X) and 0.45 ul 10% SDS (for a final conc. of 0.3%).
Note: The final volume of hybridization is 15 ul. This volume is appropriate for hybridization under a 22 mm2 coverslip. Volumes should be adjusted upwards accordingly for larger arrays/coverslips.
10. Denature hybridization mixture (100° C, 1.5 min), incubate for 30 minutes at 37° C (Cot-1 preannealing step), then hybridize to the array.
11. Hybridize microarray at 65° C overnight (16-20 hrs). Note, see Human Array Hybridization protocol for details on hybridization.
12. Wash arrays as with mRNA labeling protocol and scan:
First wash: 2X SSC, 0.03% SDS, 5 min 65 °C
Second wash: 1X SSC, 5 min RT
Third wash: 0.2X SSC, 5 min RT
Note: the first washing step should be performed at 65° C; this appears to significantly increase the specific to non-specific hybridization signal.
If you have any questions, comments, or improvements, please pass them along to:
Jonathan R. Pollack, M.D., Ph.D.
Howard Hughes Medical Institute
Beckman Center B251
Stanford Medical Center
Stanford, CA 94305-5323