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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: J Inherit Metab Dis. 2010 Sep 30;33(6):715–725. doi: 10.1007/s10545-010-9192-5

Figure 4. IVS1+1G>C splicing mutant causes intron retention.

Figure 4

A293 cells were transfected with 5 μg of either wild type plasmid (pSplice_wt) or IVS1+1G>C mutant plasmid (pSplice_mut). After 40–48 h, total RNA was extracted and RT-PCR was performed. Transcript size was analyzed by PCR analysis and visualized on a DNA agarose gel used for Southern Blot. Panel A: PCR-amplified cDNA from cells transfected with wild type plasmid (Lane 1) and IVS1+1G>C mutant plasmid (Lane 2). The DNA was transferred to a nylon membrane and probed with oligonucleotides complementary to the splice junction (Panel B), exon 1 (Panel C), and exon 2 (Panel D). Blots were exposed for autoradiography at −90 °C for 3 h, 8 h, and 16 h, respectively. On the left, the lower arrow indicates the position of migration of a 248 bp product expected for a correctly spliced PCR-amplified cDNA synthesized from the pSplice_wt. The upper arrow indicates the position of migration of a >5.0 kb product expected for a PCR-amplified cDNA synthesized from pSplice_mut without splicing.