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Figure 2


Fig. 2. Genotypic analysis of wild-type (CU427.4) and mutant (DYH7neo3) cell lines. (A) (Panel 1) Total genomic DNA was isolated and used as template for PCR with DYH7 gene-specific and neo3-specific primers. A 3.8 kb band and a 2.4 kb band were amplified from the mutant genomic DNA template using the gsp1 primer set (gsp1-5 and gsp1-3, see Fig. 1) specific for the 5' and 3' ends of the DYH7 fragment (lane 1). The 3.8 kb band, representing the presence of neo3 insertion, was absent when wild-type genomic DNA template was used (lane 3). A 2.8 kb band was amplified from the mutant genomic DNA template when a 5' gene-specific primer, gsp1-5, was paired with a neo3-specific primer, neo3BTU (lane 2). This band was absent when wild-type genomic DNA template was used (lane 4). (Panel 2) Total RNA was isolated and used as template for first-strand synthesis of cDNA with the gsp1-3 primer used above. The same DYH7 gene-specific primer set used in panel 1, gsp1-5/gsp1-3, amplified a 2.2 kb band from wild-type cDNA template (lane 6). This band was absent when mutant cDNA template was used (lane 5). (Panel 3) Primers specific for a DYH7 gene region upstream of the neo3 insertion site (gsp2-5 and gsp2-3) amplified a 300 bp band from the wild-type cDNA template (lane 9). This band was absent when mutant cDNA template was used (lane 7). As a positive control, primers specific for ribosomal protein gene RPL21 amplified a 370 bp band from both mutant and wild-type cDNA templates prepared using an oligo (dT) primer (lanes 8 and 10). (B) Real-time PCR analysis of mutant and wild-type cDNA. The same templates and primers from A (panel 3) were used in real-time PCR amplification with SYBR green-fluorescent probe. The fluorescent signal specific for expression of the endogenous DYH7 gene was observed with wild-type template (closed squares), but was absent with the mutant template (closed circles). The RPL21 control reactions, performed in separate tubes, produced a signal of similar threshold cycle with both mutant (open circles) and wild-type templates (open squares).





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