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MICROARRAY: GENOMIC DNA LABELING
Microarray: Genomic DNA Labeling
Overview
DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array (see Hint #8). This protocol was developed for microarray-based comparative genomic hybridization.
Procedure
1. Add 2 μg of genomic DNA sample to be labeled to a fresh microcentrifuge tube (see Hint #1).
2. Add TE or ddH2O to a total volume of 21 μl.
3. Add 20 μl of 2.5X Reaction Mix.
4. Boil the sample reaction for 5 min.
5. Let the reaction cool on ice.
6. Add 5 μl of 10X dNTP Mix to the reaction on ice.
7. Add 3 μl of Cy5-dCTP or Cy3-dCTP (see Hint #2) to the reaction.
8. Add 1 μl of Klenow Fragment (see Hint #3) to the reaction.
9. Incubate the reaction at 37 °C for 1 to 2 hr.
10. Stop the reaction by adding 5 μl of 0.5 M EDTA.
11. Add 450 μl of TE to the reaction.
12. Place the reaction mix onto a Microcon 30™ filter.
13. Centrifuge at 10,000 X g in a microcentrifuge for 10 min.
14. Invert the unit into a fresh tube and centrifuge at 8,000 for 1 min. The eluate should be approximately 20 to 40 μl.
15. For two-color array hybridizations, combine the purified Cy5- and Cy3-labeled probes in a fresh microcentrifuge tube.
16. Add the following:
30 to 50 μg of human Cot-1 DNA (see Hint #4)
100 μg of yeast tRNA (see Hint #5)
20 μg of poly(dA)-poly(dT) (see Hint #6)
450 μl of TE 7.4
17. Concentrate the reaction with a Microcon 30™ filter as previously described. Check the volume every min until the volume is 12 μl or less.
18. Adjust the volume of the probe mixture to 12 μl with ddH2O.
19. Add 2.55 μl of 20X SSC (for a final concentration of 3.4X) and 0.45 μl of 10% SDS (for a final concentration of 0.3%). The total volume should be 15 μl, which is appropriate for hybridization under a 22 mm x 22 mm cover slip (see Hint #7).
20. Denature the hybridization mix by heating it to 100°C for 1.5 min.
21. Incubate the mix for 30 min at 37°C.
22. Apply to the array (see Protocol ID#2259).
23. Hybridize the microarray at 65°C overnight (for 16 to 20 hr).
24. Wash the arrays as follows:
Wash 1: 2X SSC/0.3% SDS for 5 min at 65°C
Wash 2: 1X SSC for 5 min at room temperature
Wash 3: 0.2X SSC for 5 min at room temperature
25. Scan the array.
Solutions
20X SSC
3.0 M NaCl
300 mM Sodium Citrate (pH 8.0)
0.75 μl of 10% SDS 
TE
10 mM Tris, pH 7.4
1 mM EDTA
Poly(dA)-poly(dT)
(Sigma)
5 mg/ml Poly(dA)-poly(dT)
Yeast tRNA
5 mg/ml Yeast tRNA
(Gibco/BRL)
Human Cot-1 DNA
1 mg/ml Human Cot-1 DNA
(Gibco/BRL)
Microcon 30™ filter
Amicon/Millipore
Wash 3
0.2X SSC
0.5 M EDTA, pH 8.0
Wash 2
1X SSC
Cy5-dCTP or Cy3-dCTP
(Amersham)
1 mM stock of Cy5-dCTP or Cy3-dCTP
Wash 1
0.3% SDS
2X SSC
10X dNTP Mix
1.2 mM dTTP
1.2 mM dATP
10 mM Tris 8.0
1 mM EDTA
0.6 mM dCTP
1.2 mM dGTP
Klenow Fragment
40 to 50 Units/μl
(NEB, Gibco-BRL)
2.5X Reaction Mix
125 mM Tris, pH 6.8
12.5 mM MgCl2
750 μg/ml random octamers
25 mM 2-Mercaptoethanol
10% SDS
BioReagents and Chemicals
2-Mercaptoethanol
Poly(dA)-poly(dT)
Yeast tRNA
Human Cot-1 DNA
Microcon 30™ filter
Cy3-dCTP
Cy5-dCTP
random octamers
Tris
Magnesium Chloride
SDS
Klenow
dCTP
dTTP
dGTP
dATP
EDTA
Sodium Citrate
Protocol Hints
1. 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 by restriction enzyme digestion. After digestion, the DNA should be purified by extraction with phenol/chloroform and precipitation by Ethanol (see Protocol ID#1453) or by the Qiagen PCR purification kit.
2. Cy-dCTP and Cy-dUTP work equally well. If using Cy-dUTP, adjust the 10X dNTP Mix accordingly.
3. High-concentration Klenow (40 to 50 Units/μl) produces better labeling.
4. Human Cot-1 DNA blocks hybridization to repetitive DNAs that may be present on the array
5. Yeast tRNA blocks non-specific DNA hybridization.
6. Poly(dA)-poly(dT) blocks the hybridization to polyA tails of cDNA array elements.
7. The volumes should be adjusted upwards accordingly for larger arrays/cover slips.
8. Genomic DNA can be labeled with a simple random-priming protocol based on Gibco/BRL's Bioprime DNA Labeling kit; Nick translation protocols may also be used. The contributor of this protocol routinely uses 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); other sources of random primers and high concentration Klenow may also be used.
Citation and/or Web Resources
1. Lab website
