Figure 1

Genomic PCR and RT-PCR analyses of rhodopsin genes. (A) Positions of primers for genomic PCR and RT-PCR. We designed three pairs of primers (primers Fw1/Rv1, Fw2/Rv2 and Fw3/Rv3) based on rhodopsin mRNA sequences. Both primers Fw1 and Rv1 target the putative exons 1 and 2, whereas primer Fw2 and Rv2 target the putative exon1, respectively. Thus, if the rhodopsin gene has no introns, a band of around 200 bp can be amplified by the genomic PCR using both primer pairs. If the rhodopsin gene has introns, a band of around 200 bp can be amplified by the genomic PCR using primer pair Fw2/Rv2 but not using primer pair Fw1/Rv1. In addition, primers Fw3 and Rv3 target 5′- and 3′-end of a full-length coding region of the rhodopsin clone, respectively. Thus, if the rhodopsin gene has no introns, a band of around 1,060 bp can be amplified by the genomic PCR using this primer pair. If the rhodopsin gene has introns, a band of around 1,060 bp cannot be amplified by the genomic PCR using this primer pair. Sequences of PCR primers are shown in Table S1. (B,C) Genomic PCR and RT-PCR on gray bichir (B) and reedfish (C) rhodopsin. The genomic PCR amplified a band of around 200 bp using only primer pair Fw2/Rv2. It should be noted that RT-PCR on eye total RNA amplified a band of around 200 bp using primer pairs Fw1/Rv1 and Fw2/Rv2 and a band of around 1,060 bp using primer pair Fw3/Rv3. No corresponding bands in the “no reverse transcriptase” reaction confirmed that the band of RT-PCR is not attributed to contaminating genomic DNA. D. Genomic PCR and RT-PCR on Siberian sturgeon rhodopsin. The genomic PCR amplified a band of around 200 bp using primer pairs Fw1/Rv1 and Fw2/Rv2 and a band of around 1,060 bp using primer pair Fw3/Rv3. These bands were also amplified by RT-PCR on eye total RNA, not by “no reverse transcriptase” reaction.