DNA was prepared from blood (MNB and some ECA subjects) or cell lines (most ECA subjects) [6,31,32] and carefully quantitated. DNAs from groups of 20 individuals of the same phenotype were combined. Hybridization probes were prepared with precautions to avoid contamination, as described (Affymetrix assays 500 k [9,11,33,34]). 150 ng of pooled DNA was digested using StyI or NspI, ligated to appropriate adaptors and amplified using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) with 3 min 94°C, 30 cycles of 30 sec 94°C, 45 sec 60°C, 15 sec at 68°C and a final 7 min 68°C extension. PCR products were purified (MinEluteTM 96 UF kits, Qiagen, Valencia, CA) and quantitated. Forty μg of PCR product was digested for 35 min at 37°C with 0.04 unit/μl DNase I to produce 30–100 bp fragments which were end-labeled using terminal deoxynucleotidyl transferase and biotinylated dideoxynucleotides and hybridized to the appropriate 500 k array (Sty I or Nsp I arrays) (Mendel array sets, Affymetrix). Arrays were stained and washed as described (Affymetrix Genechip Mapping Assay Manual) using immunopure strepavidin (Pierce, Milwaukee,
