Figure 2. Determination of breakpoints in the RS1 gene gross genomic deletion in patient #645 and confirmation of maternal carrier status. A: Schematic diagram of the RS1 gene promoter region for primer walk in patient #645 (not scaled). The results of long range PCR using different combinations of designed primers (Appendix 2) are not shown. B: PCR products were analyzed by using pre-cast 1% agarose gels stained with ethidium bromide (SeaKem® Gold Agarose, Lonza Rockland Inc, Rockland, ME). This gel shows the amplification of the deletion junction fragment in the patient. Lane 1 was loaded with supercoiled DNA Ladder (0.01 mg/ml, Life technologies, Grand Island, CA). Lanes 2, 4 and 5 used primer pair w2F-w8R. Lanes 3, 6 and 7 used primer pair w2F-W7R. The mother's DNA was loaded in lanes 2 and 3. The Patient #645’s (son) DNA was loaded in lanes 5 and 6. Lanes 4 and 7 were non-DNA water as PCR controls. The top bands in lanes 2 and 3 represent the wild type fragments with an estimated size of about 6-7 kb. The lower bands in lanes 2, 3, 5, and 6 represent the junction fragments with estimated sizes of 3.5 kb and 2.6 kb respectively. The blue arrow indicates the isolated fragment used for sequencing. C: Junction fragments were sequenced to determine the exact breakpoints in exon 1 of patient #645 (reverse direction sequencing by primer 7748R is shown).