Figure 2

No evidence for altered splicing of transcripts derived from minigenes incorporating the BRCA2 unclassified variants: c.8962A > G (p.Ser2988Gly), c.8972G > A (p.Arg2991His), c.9172A > G (p.Ser3058Gly), and c.9213G > T (p.Glu3071Asp). RT-PCR products from duplicate cell culture experiments for each minigene construct and a vector only control. The vector only sample does not contain an insert and therefore produces a 261 bp band made up of fragments of pSPL3 constitutive exons. The positive control, BRCA2 c.9117G > A (p.Pro3039Pro) produces a 410 bp band representing the vector exons and BRCA2 exon 24. The higher molecular weight band in all samples includes a 115 bp cryptic exon. The 574 bp band seen in all unclassified variants and the wildtype represents the vector exons and BRCA2 exons 23 and 24. Negative controls include: 1. No reverse transcriptase in cDNA synthesis of the wild type construct; 2. No template for the PCR; 3. Negative control for transfection. Fragments resolved 1.5% agarose gel stained with ethidium bromide.