Determination of the transcription start site of the DAG1gene. (Top) A detail of the sequencing gel with the DNA marker sequence (lanes A, C, G, T) and the product of the primer extension reaction of primer exon1_Ext (lane 1) is shown. Sizes are indicated on the left side of the gel. The size of the extension product is actually 5 nucleotides longer than the size of the first exon, as the primer contained a RsaI tag at its 5′ end for cloning purposes. (Bottom) The diagram outlines the experimental strategy. Primers exon1_Ext and exon2_Ext (open boxes) were annealed to total RNA and elongated as described in Methods. The thin dotted line indicates the spliced first intron. Important DNA sequence elements (transcription start site, ATG start codon) are shown. Crosshatched boxes within exons represent the 5′ UTR.
