Plasmid D12 contains the entire human β₃ gene. (A) Ethidium bromide-stained agarose gel of D12 restriction digests. Lane 1, standard containing λ DNA digested with HindIII plus φx174 DNA digested with HaeIII; lane 2, D12 digested with Xba I + Sma I; lane 3, 1,211-bp Xho I + BamHI fragment of 5′ β₃ gene subcloned into pBluescript and digested with Xba I + Sma I; the remaining lanes are D12 cut with Xba I (lane 4), Sma I (lane 5), HindIII (lane 6), Stu I (lane 7), Xho I (lane 8), Spe I (lane 9), and Acc I (lane 10). (B) Southern analysis of gel in (A) probed with ³²P-labeled oligonucleotide ex0.S from positions −113 to −95 from the transcription start site of the β₃ gene (probe position shown in Fig 3A). Lane 3 contains a positive control plasmid with genomic sequence from which the probe was designed.30 (C) Southern analysis of PCR products using clone D12 as a template, probed with a ³²P-labeled β₃ cDNA containing the entire coding region. Using primers flanking the indicated exons (Table 1), PCR reactions were performed on no template DNA (lanes 1, 4, 7, 10, 13, and 16), normal genomic DNA (lanes 2, 5, 8, 11, 14, and 17), and clone D12 (lanes 3, 6, 9, 12, 15, and 18). Under the same conditions, using the same PCR primers, no products were obtained using a negative P1 plasmid as a control (not shown). DNA size marker was φx174 digested with HaeIII. Exons are numbered as described in the text.