TABLE 4.
PCR primers used in this study
| Purpose | Primer | Sequence (5′–3′)b | Note |
|---|---|---|---|
| Amplification of SQLE genes for qRT-PCR | qRT_erg1_2-F | CCAGACTGATGGCAAGCAAGA | |
| qRT_erg1_2-R | ATAAGCTCCAGGCCCCAGAA | ||
| qRT_erg(Tr)1_2-F | CCAGACTGATGGCAAACAAGA | ||
| Amplification of SQLE genes from clinical isolates | TrSQLE-F1 | ATGGTTGTAGAGGCTCCTCCC | |
| TrSQLE-R1 | CTAGCTTTGAAGTTCGGCAAA | ||
| Amplification of the DNA fragments containing the 5′ UTR of AvSQLE and the SQLE ORF | AvSQLE-F15/SpeI | TCACGAAGCTAACTAGTACCTGAAAGATGAC | Inactivation of the KpnI site |
| AvSQLE-R23/ApaI | AAAAAGGGCCCCTAGCTTTGAAGTTCGGCAAATA | ||
| Amplification of the 3′ UTR fragment of AvSQLE | AvSQLE-F13/BamHI | CAAGGATCCACAGATAGGCTTATCTCTAGCTCT | |
| AvSQLE-R16/KpnI | CAGGGTACCTCCGTTCATAGTCAACGAACGTCTCG | ||
| Generation of the sSQLE ORF encoding wild-type TrSQLEa | AvSQLE-F21 | TCCGCGAAGCCCAAGGTATACCGCGACGAA | Ala24Val |
| AvSQLE-R18 | TTCGTCGCGGTATACCTTGGGCTTCGCGGA | ||
| AvSQLE-F22 | GGCATTGCTGGATGTACGCTGGCCGTTGCGTT | Ala52Thr | |
| AvSQLE-R19 | AACGCAACGGCCAGCGTACATCCAGCAATGCC | ||
| AvSQLE-F23 | TCCACGCACACAGGGGAGGTCCTTGGAGTTCAATG | Asp174Glu | |
| AvSQLE-R20 | CATTGAACTCCAAGGACCTCCCCTGTGTGCGTGGA | ||
| AvSQLE-F24 | ATGTCGTTCTCCTCCGGGATCTACTTAGTCCAGA | Asn352Asp, hybridization probe | |
| AvSQLE-R21 | TCTGGACTAAGTAGATCCCGGAGGAGAACGACAT | ||
| AvSQLE-F25 | TCAATATTCTTGCCCAGGCCTTATACTCTATATTC | Ser392Ala | |
| AvSQLE-R22 | GAATATAGAGTATAAGGCCTGGGCAAGAATATTGA | ||
| AvSQLE-F26 | GGTCATCCTTCCTTTCATATTTGCCGAACTT | Val483Ile | |
| AvSQLE-R24 | AAGTTCGGCAAATATGAAAGGAAGGATGACC | ||
| Generation of point mutations in AvSQLE and TrSQLEa | AvSQLE-F27 | TTGCCCAGTCCTTCTACTCTATATT | Leu393Phe (AvSQLE) |
| AvSQLE-R27 | AATATAGAGTAGAAGGACTGGGCAA | ||
| AvSQLE-F29 | TTCTTGCCCAGGCCTTCTACTCTATATT | Leu393Phe (TrSQLE) | |
| AvSQLE-R29 | AATATAGAGTAGAAGGCCTGGGCAAGAA | ||
| AvSQLE-F35 | TTGCCCAGTCCTCATACTCTATATT | Leu393Ser (AvSQLE) | |
| AvSQLE-R35 | AATATAGAGTATGAGGACTGGGCAA | ||
| AvSQLE-F36 | TTCTTGCCCAGGCCTCATACTCTATATT | Leu393Ser (TrSQLE) | |
| AvSQLE-R36 | AATATAGAGTATGAGGCCTGGGCAAGAA | ||
| AvSQLE-F28 | TACTCTATATTAGCCGCTGGTG | Phe397Leu (both SQLE proteins) | |
| AvSQLE-R28 | CACCAGCGGCTAATATAGAGTA | ||
| AvSQLE-F31 | TACTCTATAATCGCCGCTGGTG | Phe397Ile (both SQLE proteins) | |
| AvSQLE-R31 | CACCAGCGGCGATTATAGAGTA | ||
| AvSQLE-F30 | TACTCTATAGTCGCCGCTGGTG | Phe397Val (both SQLE proteins) | |
| AvSQLE-R30 | CACCAGCGGCGACTATAGAGTA | ||
| AvSE-F37 | GGCTGCTTCAGGTATTCCCAACTTGGACTTATA | Phe415Ser (both SQLE proteins) | |
| AvSE-R37 | TATAAGTCCAAGTTGGGAATACCTGAAGCAGCC | ||
| AvSQLE-F14 | GGCTGCTTCAGGTATGTCCAACTTGGACTTATA | Phe415Val (both SQLE proteins) | |
| AvSQLE-R8 | TATAAGTCCAAGTTGGACATACCTGAAGCAGCC | ||
| AvSQLE-F29 | CCTTGGTTCTATTACGATATTTTTACTCCGTAGCC | His440Tyr (both SQLE proteins) | |
| AvSQLE-R29 | GGCTACGGAGTAAAAATATCGTAATAGAACCAAGG |
a
Introduction of nucleotide substitutions into SQLE genes by overlap extension PCR.
b
Restriction sites are underlined, and substitutions are in boldface.