Amplification of PKD1 and PKD2 Exons

We designed LR-PCR primers to amplify 18 long DNA fragments, including the exonic regions of PKD1 and PKD2. These fragments were amplified from participants’ purified genomic DNA using the primers shown in S1 Table. Multiple LR-PCR products were amplified in each of the following combinations: A (LR-PCR 2 and 12), B (4 and 17), C (6 and 15), D (1, 5, and 9), E (3, 8, and 18), F (7, 13, and 16), and G (10, 11, and 14). The LR-PCR reactions were performed simultaneously on the same PCR plate using the following touchdown PCR regimen [19 (link)]: (i) 94°C for 2 min; (ii) one cycle at 98°C for 10 s and 74°C for 5 min; (iii) one cycle at 98°C for 10 s and 72°C for 5 min; (iv) one cycle at 98°C for 10 s and 70°C for 5 min; (v) 30 cycles at 98°C for 10 s and 68°C for 5 min; and (vi) 68°C for 7 min. The LR-PCR products were purified using the Agencourt AMPure XP kit (Beckman Coulter, Inc, Brea, CA, USA) and quantified as previously described [20 (link)].
With the Ion PGM instrument (Thermo Fisher Scientific Inc, Waltham, MA, USA), only weak coverage could be achieved for the PKD1 exon 1 region because it contains a GC-rich sequence. To circumvent this problem, corrective PCR was performed in which PKD1 exon 1 was re-amplified from the combination D LR-PCR product noted above, using the primers shown in S2 Table. The PCR products were purified according to the procedure described above.