Kurahashi H. et al, 1
e-METHODS
Genomic DNAs were prepared from ethylenediaminetetraacetic acid (EDTA)-treated whole-blood samples by using QIAamp DNA Blood kit (Qiagen, Hilden, Germany).
MLPA
MLPA was conducted using a commercially availablekit for KCNQ2(SALSA MLPA KIT P166KCNQ2, Lot 0606, MRC-Holland, Amsterdam, The Netherlands) and KCNQ3(SALSA MLPA KIT P197KCNQ3, Lot 1206or Lot, MRC-Holland). Details of the probe sequences can be found on MLPA tests and data analyses were conducted according to the protocol supplied by the manufacturer. Fragment analysis of the PCR product was carried out on ABI model 310 capillary sequencer (Applied Biosystems, Foster City, CA) using GeneScanTM-500LIZ as size standards (Applied Biosystems) andde-ionized formamide (HiDi Formamide, Applied Biosystems) as described previously.
Fluorescent in situ hybridization (FISH)
The probesused in FISH for KCNQ2wereprepared by LA PCR with a primer pair (CAC GGT CCC TGT CAC ATG AAG TAT TTG G and TCC CTC CCA ATG GTG AAA TTC AGA TGT G) designed for part of the KCNQ2 coveringexons11 and 12(6,610bp) and another pair (ATC AGT GCT GCC TTA TTC CTG GAA GCC T and TAG GCA GAG TTA ACC ACA GCC TCT GAC T) designed for part of the KCNQ2covering exons2 and 3(5,336bp), and labeled with digoxigenin-11-dUTP by the nick translational method.As a reference probe for another part of chromosome 20, RP5-1099D15 PAC DNA containing JAG1 was selected and labeled with biotin-16-dUTP.
Array-basedcomparative genomic hybridization analysis
Genome-wide DNA screening on Affymetrix Genome-Wide Human SNP Array 5.0was performed using the GeneChip Instrument system according to the standard protocol supplied by the manufacturer (Affymetrix, Santa Clara, CA). Briefly, genomic DNA was digested with restriction endonuclease, ligated to an adaptor, and subjected to PCR amplification with adaptor-specific primers. The PCR products were digested with DNaseI, and labeled with a biotinylated nucleotide analogue using terminal deoxynucleotidyl transferase. The labeled DNA fragments were hybridized to the microarray, the hybridized DNA probes were captured bystreptavidin-phycoerythrin conjugates, and the array was scanned. The signal intensity ratio was calculated using the HelixTree software (Golden Helix, Bozeman, MT) and mapped onto the NCBI Homo sapiens Genome Build 36.3 according to the physical position of each marker.