Primers and PCR conditions used to sequence the carboxylase gene
Gene region . | PCR conditions . | Primer names . | Primer sequences . |
|---|---|---|---|
| Exon 1 | 94°C/1 min, TD 70/65°C/30 s, 2°C↘, 72°C/1 min (2% DMSO) | E-1-U | GCGTCCTCAACTCGGCGTCACTC |
| E-1-L | CTCCACCTCAAATCAAAGAAATC | ||
| Exon 2 | 94°C/1 min, TD 61/55°C/30 s, 2°C↘, 72°C/1 min | E-2-U | GAGCTGTTGGTGCAGTGATTTCT |
| E-2-L | AGAGATTGTCATTCTCCACTCT | ||
| Exon3/4/5 | 94°C/1 min, 55°C/30 s, 72°C/1 min | E-3-U | CCAATGACCAACTCCCCTAT |
| E-5-L | TCCTCCCTCTGTCCTAAAAT | ||
| Exon 6 | 94°C/1 min, 55°C/30 s, 72°C/1 min | E-6-U | TGTAACTCAGGAGCATGGATTC |
| E-6-L | CATTACTGAGAGAGATGAGTCACCT | ||
| Exon 7 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-7-U | GCTGTGAATGTGCTTTGATGTG |
| E-7-L | AAGCCCCAGTCCTCTTATC | ||
| Exon 8 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-8-U | AGGCCCAGCCAAACTCCT |
| E-8-L | CTCACACTGACCCCATCC | ||
| Exon 9/10 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-9-U | GCTGATTCCCCTCTGTGCTG |
| E-10-L | AACCAGCTATGCCCACAAC | ||
| Exon 11 | 94°C/1 min, 55°C/30 s, 72°C/1 min | E-11-U | GGTGGCTGTGATGTCCTTAGAA |
| E-11-L | CCCCATGGCAGAGTGAAC | ||
| Exon 12 | 94°C/1 min, TD 52/48°C/30 s, 2°C↘, 72°C/1 min | E-12-U | GCCATGGGGTGGGATGATGAAC |
| E-12-L | CAGGCAACTGACAAGGGA | ||
| Exon 13 | 94°C/1 min, TD 52/48°C/30 s, 2°C↘, 72°C/1 min | E-13-U | AGAAAGAAGCCAAGAGTCAT |
| E-13-L | GGCTAGAACATCATTCATAACC | ||
| Exon 14 | 94°C/1 min, TD 52/48°C/30 s, 2°C↘, 72°C/1 min | E-14-U | CTAGCTGGCAGAAGAGGAGTT |
| E-14-L | AGAATGGCAGGAAAAGATACC | ||
| Exon 15 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-15-U | GGCTGTTCCTACCCTATCC |
| E-15-L | ACCTCCCCTTCTGCTCACC | ||
| P1 | 94°C/30 s, TD 65/55°C/30 s, 1°C↘, 72°C/30 s (1.5 mM MgCl2) | 1U | GGGCTCAAGCGAACCTC |
| 379L | CTCCCCGACCCCATTAGT | ||
| P2 | 94°C/15 s, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | 284U | GCGTCCTCAACTCGGCGTCACTC |
| 1124L | GGGCTCCACCTCAAATCAAAGAAATC | ||
| In1 | 94°C/15 s, 55°C/30 s, 72°C/1 min | 969U | ACTGTAGTCTGAGGGGTTCTGG |
| 2261L | TAGTCACATTTTGGGCTGGTTA | ||
| 6 kb | 94°C/15 s, TD 70/62°C/30 s, 68°C/4 min* (3 mM MgCl2) | 724U | GCAGAGCAATGGCGGTGTC |
| 6065L | AGGGAGGCAGCGGAGAGTGGT | ||
| In5-6 | 94°C/30 s, 55°C/30 s, 72°C/30 s (1.5 mM MgCl2) | 5812U | AGATGTGCCCAGGATAGAT |
| 6511L | GGCAAGAATAAAATAAGGAG | ||
| 4 kb | 94°C/15 s, TD 66/58°C/30 s, 2°C↘, 68°C/3 min* (3 mM MgCl2) | 6261U | TTCTTCCTTGGTGCCTGATACTGTC |
| 10364L | AGCCTCTCCTCACTTTCCTCCATAC | ||
| 3.5 kb | 94°C/15 s, 60°C/30 s, 68°C/2.5 min (1 mM MgCl2) | 10470U | GGGATGATGGTGGTAAAGGT |
| 13595L | GGAGGAGTGGGGGAGAGTAT | ||
| 3′ NC | 94°C/30 s, 55°C/30 s, 72°C/30 s (1.5 mM MgCl2) | 13321U | GAAGGGGAGGTAAAGTAAGAAT |
| 13676L | AATCCTGGAGTAGACACAATCA |
Gene region . | PCR conditions . | Primer names . | Primer sequences . |
|---|---|---|---|
| Exon 1 | 94°C/1 min, TD 70/65°C/30 s, 2°C↘, 72°C/1 min (2% DMSO) | E-1-U | GCGTCCTCAACTCGGCGTCACTC |
| E-1-L | CTCCACCTCAAATCAAAGAAATC | ||
| Exon 2 | 94°C/1 min, TD 61/55°C/30 s, 2°C↘, 72°C/1 min | E-2-U | GAGCTGTTGGTGCAGTGATTTCT |
| E-2-L | AGAGATTGTCATTCTCCACTCT | ||
| Exon3/4/5 | 94°C/1 min, 55°C/30 s, 72°C/1 min | E-3-U | CCAATGACCAACTCCCCTAT |
| E-5-L | TCCTCCCTCTGTCCTAAAAT | ||
| Exon 6 | 94°C/1 min, 55°C/30 s, 72°C/1 min | E-6-U | TGTAACTCAGGAGCATGGATTC |
| E-6-L | CATTACTGAGAGAGATGAGTCACCT | ||
| Exon 7 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-7-U | GCTGTGAATGTGCTTTGATGTG |
| E-7-L | AAGCCCCAGTCCTCTTATC | ||
| Exon 8 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-8-U | AGGCCCAGCCAAACTCCT |
| E-8-L | CTCACACTGACCCCATCC | ||
| Exon 9/10 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-9-U | GCTGATTCCCCTCTGTGCTG |
| E-10-L | AACCAGCTATGCCCACAAC | ||
| Exon 11 | 94°C/1 min, 55°C/30 s, 72°C/1 min | E-11-U | GGTGGCTGTGATGTCCTTAGAA |
| E-11-L | CCCCATGGCAGAGTGAAC | ||
| Exon 12 | 94°C/1 min, TD 52/48°C/30 s, 2°C↘, 72°C/1 min | E-12-U | GCCATGGGGTGGGATGATGAAC |
| E-12-L | CAGGCAACTGACAAGGGA | ||
| Exon 13 | 94°C/1 min, TD 52/48°C/30 s, 2°C↘, 72°C/1 min | E-13-U | AGAAAGAAGCCAAGAGTCAT |
| E-13-L | GGCTAGAACATCATTCATAACC | ||
| Exon 14 | 94°C/1 min, TD 52/48°C/30 s, 2°C↘, 72°C/1 min | E-14-U | CTAGCTGGCAGAAGAGGAGTT |
| E-14-L | AGAATGGCAGGAAAAGATACC | ||
| Exon 15 | 94°C/1 min, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | E-15-U | GGCTGTTCCTACCCTATCC |
| E-15-L | ACCTCCCCTTCTGCTCACC | ||
| P1 | 94°C/30 s, TD 65/55°C/30 s, 1°C↘, 72°C/30 s (1.5 mM MgCl2) | 1U | GGGCTCAAGCGAACCTC |
| 379L | CTCCCCGACCCCATTAGT | ||
| P2 | 94°C/15 s, TD 65/55°C/30 s, 2°C↘, 72°C/1 min | 284U | GCGTCCTCAACTCGGCGTCACTC |
| 1124L | GGGCTCCACCTCAAATCAAAGAAATC | ||
| In1 | 94°C/15 s, 55°C/30 s, 72°C/1 min | 969U | ACTGTAGTCTGAGGGGTTCTGG |
| 2261L | TAGTCACATTTTGGGCTGGTTA | ||
| 6 kb | 94°C/15 s, TD 70/62°C/30 s, 68°C/4 min* (3 mM MgCl2) | 724U | GCAGAGCAATGGCGGTGTC |
| 6065L | AGGGAGGCAGCGGAGAGTGGT | ||
| In5-6 | 94°C/30 s, 55°C/30 s, 72°C/30 s (1.5 mM MgCl2) | 5812U | AGATGTGCCCAGGATAGAT |
| 6511L | GGCAAGAATAAAATAAGGAG | ||
| 4 kb | 94°C/15 s, TD 66/58°C/30 s, 2°C↘, 68°C/3 min* (3 mM MgCl2) | 6261U | TTCTTCCTTGGTGCCTGATACTGTC |
| 10364L | AGCCTCTCCTCACTTTCCTCCATAC | ||
| 3.5 kb | 94°C/15 s, 60°C/30 s, 68°C/2.5 min (1 mM MgCl2) | 10470U | GGGATGATGGTGGTAAAGGT |
| 13595L | GGAGGAGTGGGGGAGAGTAT | ||
| 3′ NC | 94°C/30 s, 55°C/30 s, 72°C/30 s (1.5 mM MgCl2) | 13321U | GAAGGGGAGGTAAAGTAAGAAT |
| 13676L | AATCCTGGAGTAGACACAATCA |
The gene regions correspond to the PCR fragments shown in Figure 1A. The positions within the gene are indicated by the oligonucleotide primer number (U, upper; L, lower). PCR was performed for 30 cycles using the “touchdown” (TD) method: After a 94°C denaturation step, a hybridization step was performed (30 seconds, initial and final temperatures are indicated) with 2°C temperature decrements (↘) every 3 cycles. The last 15 cycles were performed at the final temperatures indicated. The products were then extended at 72°C for 1 minute, except for the 3.5 kb, 4 kb, and 6 kb fragments, which were extended at 68°C for 2.5, 3, and 5 minutes, respectively.
NC indicates noncoding sequence.
The extensions during the final 15 cycles of TD for the 4 kb and 6 kb fragments were 68°C for 5 and 4 minutes, respectively.