Figure 1.
Minigene splicing assay of BRCA1 exon 11.
A. The pB1 wild type (WT) version of the minigene is shown. PCMV = promoter of the pCDNA3 vector. ATG = start codon. TAG = stop codon. +3C = insertion of cytosine as the third nucleotide in exon 8. pA = poly A signal. 1 = exon 1 of the alfa globin gene. BRCA1 exons from 8 to 12 are numbered. The black solid line represents introns. Dotted lines show alternative splicing of exon 11. B. The three splicing isoforms FL, D11q and D11 and the position of specific oligos used for detection, are shown. C. Detection of BRCA1 exon 11 splicing isoforms for: _MCF7 endogenous BRCA1 (endogenous WT). _pB1 WT minigene transfected in MCF7 (pB1 WT). _pB1 minigene carrying the c.696G>A nucleotide substitution transfected in MCF7 (pB1 c.696G>A).
Figure 2.
Minigene splicing assay of synonymous substitutions.
Effect of synonymous BRCA1 substitutions on splicing products full-length, D(11q) and D(11). RT-PCR products from transfection experiments using minigenes carrying codon substitution are shown.
Figure 3.
Minigene splicing assay of patients synonymous substitutions.
Effect of synonymous BRCA1 substitutions on splicing products full-length, D(11q) and D(11). RT-PCR products from transfection experiments using mutated minigenes are shown.
Figure 4.
Minigene splicing assay of BRCA1 exon 11.
A. Sequence of the critical region in exon 11 showing the deletion 1,2,3,4 (highlighted). The donor site (5′ss) generating D11q isoform is boxed. Arrows indicate nucleotide positions of the variations reported in Figure 2 and 3 to affect splicing. B. Transient transfection results for the hybrid minigenes carrying deletions.