GAPF procedure from Hisashi Tanaka; Tanaka et al., Nat Genet 37, 320-7, 2005, PMID 15711546
Label
biotin
Label protocol
DNA was labeled with biotin-11-dATP (Perkin Elmer) using terminal transferase (Roche).
Hybridization protocol
standard Affymetrix hydridization procedures
Scan protocol
GeneChips were scanned with Affymetrix GeneChip Scanner
Description
The Genome-wide Analysis of Palindrome Formation (GAPF) procedure was performed as described previously with modifications (Tanaka et al., Nat Genet 37, 320-7, 2005). One ug of high molecular weight genomic DNA in 50 ul water with 100mM NaCl was boiled for 7 min and immediately transferred on ice (Snap-back). After snap-back treatment, 6 ul of S1 nuclease buffer, 4 ul of 3M NaCl and 100 units of S1 nuclease (Invitrogen) were added to the DNA and incubated at 37C for 1h. S1 nuclease was inactivated by adding 10 mM EDTA and phenol/chloroform extraction. DNA was precipitated with ethanol, dissolved in water and digested either with 40 units of MspI, TaqI or MseI (New England Biolab) for 16 h. DNA was precipitated, dissolved in 21 ul of water, and ligated to MspI-, TaqI- or MseI- specific linker by adding 5 ul of 20 mM linker, 3 ul of T4DNA ligase buffer and 400 units of T4 DNA ligase (New England Biolabs) at 16C for 16 hs. DNA was precipitated and dissolved in 200 ul TE, followed by application onto Microcon YM-50 spin column (Millipore) to remove excess linker. DNA was recovered in 20 ul H2O. Thus, for each Colo320DM and normal human foreskin fibroblast cell culture (HFF2), templates with three different linkers were prepared. For PCR, 2 ul of DNA, 0.5 ul of Faststart Taq DNA polymerase (Roche), 2.5 ul of 2 mM dNTP, 5 ul of 10xPCR buffer with MgCl2 (for Faststart Taq DNA polymerase, Roche), and 2 uM of a linker-specific primer were mixed with H2O for a total reaction volume of 50 ul. For MspI-linker ligated DNA, 5 ul of GC-rich solution (for Faststart Taq DNA polymerase, Roche) was also included in the reaction mixture. PCR was performed at 96C for 6 min followed by 25 cycles of 96C for 30 s, 55C for 30 s and 72C for 30 s on a GeneAmp PCR system 9700 (Perkin-Elmer). PCR reactions using the same template with three different linker-specific primers were concentrated using Microcon YM-50 spin column. DNA was recovered in 25 ul H2O, and fragmented using DNaseI (New England Biolab). After heat inactivation of DNaseI, DNA was labeled with biotin-11-dATP (Perkin Elmer) using terminal transferase (Roche). The procedure was performed in triplicate to produce three independent preparations of DNA for hybridization onto GeneChip Human Genome U133A arrays (Affymetrix).
Data processing
The data were normalized by RMA using the Affy package of Bioconductor
