TABLE 1.
List of oligonucleotides used for PCR amplifications
| Description or purpose | Sequence IDa | Primer no. and sequence
|
|||
|---|---|---|---|---|---|
| No. | Forward | No. | Reverse | ||
| C270 fragment | UP | 1 | ATTAGCATTACTCCATAAGTTCC | 2 | GTGTATTTAAATTAAAGGAGTTATTCAGTATCTTTAATCCATTTAGCACG |
| DW | 3 | CCTCTTCACATACATGTTAGCTCTTTATTTTGTAAGCTTAATTATTCGC | 4 | GCTAGTGGAATAAGATTTAATGG | |
| neo 3 | 5 | TGAATAACTCCTTTAATTTAAATACAC | 6 | AGAGCTAACATGTATGTGAAGAGG | |
| KO270 mutant | WT allele | 7 | CTTACTGGGTTCCTGACAGG | 9 | GCTAGTGGAATAAGATTTAATGG |
| KO allele | 8 | TCCTCTTCACATACATGTTAGC | 9 | GCTAGTGGAATAAGATTTAATGG | |
| RT-PCR analysis | C5-Des exon | 10 | TTTGCCTGAATTTAAAGGAGATTT | 11 | GGAAGGTGTGGAGCCATCTA |
| α-Tub | 12 | TGTCGTCCCCAAGGAT | 13 | GTTCTCTTGGTCTTGATGGT | |
| DES5A gene | TTHERM_01194720 | 14 | ATGGTTTATTGGCTTATTGCTGAATAG | 15 | TCAATTCTTTTTTTGTTTAATTTATTTG |
a
ID, identity. neo 3 indicates the primers used for the neo 3 cassette in the plasmid pBS-MnB-3; WT allele and KO allele are the primers used to check the replacement of the endogenous gene in the KO270 mutant; C5-Des exon and α-Tub are the primers used for the amplification of the second exon of the TTHERM_01194720 gene and the α-tubulin gene used in the competitive RT-PCR analysis, respectively. DES5A gene TTHERM_01194720 is the primer used in the WT strain for the full amplification of the C5(6) sterol desaturase gene.