Fig. 2

Minigene Analysis indicates MSH4 c.2222_2225delAAGA does not change splicing. (A, E) Schematic illustration of minigene construction. To construct the pcDNA3.1-MSH4-wt/mut minigene, exon 15 (201 bp), a portion of intron 15 (469 bp), exon 16 (119 bp), intron 16 (1326 bp), and exon 17 (129 bp) of the MSH4 gene were cloned into the pcDNA3.1 vector. To construct the pcMINI-MSH4-wt/mut minigene, a portion of intron 15 (425 bp), exon 16 (119 bp) and a portion of intron 16 (576 bp) of MSH4 gene was cloned into the pcMINI vector. (B, F) Sanger sequencing results of the minigene constructs. (C, G) RT-PCR products of the minigenes expressed in HeLa and 293T cells. (D, H) Sanger sequencing of the amplified products revealed no significant difference in splicing between the wild-type and mutant minigenes