DNA was prepared either by standard phenol-chloroform extraction followed by ethanol precipitation, or with the Puregene DNA Purification Kit (D-7000A, Gentra Systems) using the solid tissue protocol.
Label
Cy3
Label protocol
Cy3 and Cy5 dye-labeled random 9mers (TriLink BioTechnologies, Inc.) were diluted to 1 O.D./42 µl of buffer containing 0.125M Tris-HCl (pH 8.0), 0.125M MgCl2, 1.75 µl/ml β-Mercaptoethanol. Mutant DNA samples were labelled with Cy3 and the wild type DNA sample (VC196) was labelled with Cy5. 1 µg of genomic DNA was added to each random 9mer buffer solution, denatured at 95 ºC and then chilled on ice in 0.2 ml PCR tubes. 10 µl of 50X dNTP mixture (1X TE buffer, 10 mM each of dATP, dCTP, dGTP and dTTP), 8 µl DI water and 100 U Klenow fragment (exo-) was added to each tube and mixed well with a pipet. Samples were centrifuged and incubated at 37 ºC for 2 hours. 10 µl 0.5 M EDTA was added and mixed well to stop the labeling reaction. DNA was precipitated by adding 11.5 µl 0.5 M NaCl and 110 µl isopropanol, vortexing, incubating in the dark for 10 minutes at room temperature, and centrifuging at 12,000X G for 10 minutes. The supernatant was removed and the DNA pellet was washed with 500 µl 80% ethanol. After centrifugation at 12,000X G for 2 minutes, the supernatant was removed and the pellet was dried in a SpeedVac on low heat for 5 minutes before being rehydrated in 25 µl DI water. DNA concentration was measured using a spectrophotometer.
DNA was prepared either by standard phenol-chloroform extraction followed by ethanol precipitation, or with the Puregene DNA Purification Kit (D-7000A, Gentra Systems) using the solid tissue protocol.
Label
Cy5
Label protocol
Cy3 and Cy5 dye-labeled random 9mers (TriLink BioTechnologies, Inc.) were diluted to 1 O.D./42 µl of buffer containing 0.125M Tris-HCl (pH 8.0), 0.125M MgCl2, 1.75 µl/ml β-Mercaptoethanol. Mutant DNA samples were labelled with Cy3 and the wild type DNA sample (VC196) was labelled with Cy5. 1 µg of genomic DNA was added to each random 9mer buffer solution, denatured at 95 ºC and then chilled on ice in 0.2 ml PCR tubes. 10 µl of 50X dNTP mixture (1X TE buffer, 10 mM each of dATP, dCTP, dGTP and dTTP), 8 µl DI water and 100 U Klenow fragment (exo-) was added to each tube and mixed well with a pipet. Samples were centrifuged and incubated at 37 ºC for 2 hours. 10 µl 0.5 M EDTA was added and mixed well to stop the labeling reaction. DNA was precipitated by adding 11.5 µl 0.5 M NaCl and 110 µl isopropanol, vortexing, incubating in the dark for 10 minutes at room temperature, and centrifuging at 12,000X G for 10 minutes. The supernatant was removed and the DNA pellet was washed with 500 µl 80% ethanol. After centrifugation at 12,000X G for 2 minutes, the supernatant was removed and the pellet was dried in a SpeedVac on low heat for 5 minutes before being rehydrated in 25 µl DI water. DNA concentration was measured using a spectrophotometer.
Hybridization protocol
see Selzer, R.R., T.A. Richmond, N.J. Pofahl, R.D. Green, P.S. Eis, P. Nair, A.R. Brothman, and R.L. Stallings. 2005. Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319.
Scan protocol
The scanner used was an Axon Scanner (Model # 4000B) . see Selzer, R.R., T.A. Richmond, N.J. Pofahl, R.D. Green, P.S. Eis, P. Nair, A.R. Brothman, and R.L. Stallings. 2005. Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH. Genes Chromosomes Cancer 44: 305-319.
Description
NA
Data processing
The raw log2 intensity ratio were normalized with the LOESS regression
