Analysis of the effect of the 5157T>A mutation on fibrinogen Bβ chain pre-mRNA splicing. (A) Schematic representation of a region of FGB spanning from intron 2 to intron 5. Position and nucleotide sequence of primers used in cloning experiments (FGB-3F and FGB-5R) and in RT-PCR assays (FGB-Ex3F and FGB-Ex5R) are reported. Exons and introns are drawn to scale. (B; left) RT-PCR products obtained with the primer couple FGB-Ex3F and FGB-Ex5R separated on a 2% agarose gel. Lane M indicates molecular weight marker (pUC8-HaeIII); lanes 1-2, RT-PCR products amplified from transfected HeLa cells expressing wild-type or mutant mRNAs, respectively. (Right) Schematic representation of the normal (top) and aberrant (bottom) splicing events. Nucleotide sequences at the exon/intron boundaries are reported. The position of the 5157T>A mutation in exon 4 of FGB is indicated by an arrow. Nucleotides surrounding the mutation and matching the acceptor splice site consensus sequence (boxed on the right) are shaded in gray. The region of exon 4 skipped from the aberrant transcript is colored in light gray. The sequence electropherogram confirming the aberrant junction between exon 3 and exon 4 in the RT-PCR product from HeLa cells transfected with the mutant construct is also shown. (C) Semiquantitative analysis of the RT-PCR products as in panel B by means of the fluorescent hot-stop PCR technique. Labeled products were separated on an automated DNA sequencer and analyzed by the GeneScan software. Peak intensities are reported as GeneScan fluorescence units (y-axis). The left and the right panels correspond to RT-PCR products amplified from HeLa cells transfected with wild-type or mutant plasmids, respectively. Fragment size and relative amounts of fluorochrome-labeled RT-PCR products are listed in the table underneath. The percentage values correspond to the peak areas, setting the sum of peak areas of each experiment equal to 100%.