We designed primers for polymerase chain reaction (PCR) (see Supplement) based on the Ensembl sequences (ENSG00000101204). After PCR amplification, products were purified using Exonuclease I and shrimp alkaline phosphatase. Sequence analysis was carried out on Applied Biosystems 3730 capillary instruments at the Yale Keck core facility. In the discovery stage, PCR was performed for all of the coding exons and flanking intronic regions of CHRNA4. Based on the results from the discovery samples, only exon 5 and its flanking intronic regions were sequenced in the test stage and the imaging study. All rare variants detected were confirmed by independent PCR and sequencing from the other direction of the amplicons. Some PCR conditions were more difficult to optimize than others based on the quality of the DNA template, so the number of individuals for whom we could get Sanger sequencing data differed for each amplicon. The success rate for the sequencing was 80-99.6%.
