PCR primers (see Supplemental online Table S2) were designed for 716 genomic regions, including 515 exons and 201 putative gene regulatory regions (CNS), which include 1500 bp upstream of the transcriptional start site of each gene. A total of ∼360 Kb of DNA sequence was PCR amplified per individual. Coding exons were amplified using primers designed in the intronic sequences flanking the exon boundaries to allow sequencing across all intron/exon junctions. The average amplicon size was 513 bp, the maximum 700 bp. Exons and CNS regions that span more that 700 bp were amplified in overlapping segments. Primers for 14 genes were designed manually for DNA sequences retrieved from the UCSC genome browser (hg18) using the program Primer3 [46]. Primers for the remainder were downloaded from the NCBI probes webpage (http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe) and further supplemented with primers designed in-house for regions not covered. Forward and reverse primers incorporated the -21M13F (TGTAAAACGACGGCCAGT) or M13R (CAGGAAACAGCTATGAC) extensions, respectively, at their 5′ ends. PCR, sequencing reactions and sequence analysis procedures were carried out as described previously [47]. Briefly, PCR reactions were optimized for each
