RT-PCR in patients with splice donor site mutations of the CD40L gene. (A) Design and location of primers used in RT-PCR. Shown are the coding regions of CD40L cDNA only. The numbers on top indicate the starting nucleotide number of each exon. The location of primers are shown by arrows [sense (→) and antisense (←)]. Note that sense primers used for amplification of introns 2, 3 , and 4 splice donor site mutations were designed to be localized in an exon that may be skipped. (B) Agarose gel electrophoresis of RT-PCR products. RT-PCR was performed using primer pairs P1 and P3 (lanes 1 through 3), P4 and P5 (lanes 4 through 7), and P6 and P7 (lanes 8 through 11), and P7 and P8 (lanes 12 through 15) (see Materials and Methods). An aliquot of the RT-PCR product was size-fractionated on 2% agarose gel and stained with ethidium bromide. DNA size markers are shown on the left. cDNA used for RT-PCR was derived from a normal control (lanes 1, 4, 8, and 12), DB (family 16) in lane 2, PS (family 18) in lane 5, TaA (family 17) in lane 6, MS (family 20) in lane 9, AM (family 19) in lane 10, SeC (family 22) in lane 13, RA (family 23) in lane 14; lanes 3, 7, 11, and 15 are negative controls (no cDNA in the reaction mixture). Note that patients with splice donor site mutations involving introns 1 (lane 2) and 3 (lanes 9 and 10) but not those involving introns 2 (lanes 5 and 6) and 4 (lanes 13 and 14) have bands which are indistinguishable from those derived from normally spliced mRNA.