Table 1.
Oligonucleotides used in this study
| Purpose and template | Target | Primer designation | Sequence (5′→3′) | Amplicon size (bp) |
|---|---|---|---|---|
| Cassette construction | ||||
| mnp3 replacement cassette (TMS8) | ||||
| PC9 genomic DNA | mnp3 | mnp3PF | CACGACATGCACGTTTGAGCTTAGAAGC | 2,031 |
| mnp3 | btubPF-mnp3PR | ATTTAGTTTCCTCCCAACAGCATTGCTGGAGTTGAAGAGACGA | ||
| pTMS14 (12) | Hygr | mnp3PR-btubPF | TCGTCTCTTCAACTCCAGCAATGCTGTTGGGAGGAAACTAAAT | 2,749 |
| Hygr | mnp3TF-hygR | GTGTCAACAGGAGAACTTGAGACCTATTCCTTTGCCCTCGGA | ||
| PC9 genomic DNA | mnp3 | hygR-mnp3TF | TCCGAGGGCAAAGGAATAGGTCTCAAGTTCTCCTGTTGACAC | 2,028 |
| mnp3 | mnp3TR | GTCTTCGATACTGGTATCCCTCCTTTATGG | ||
| mnp9replacement cassette (TMS11) | ||||
| PC9 genomic DNA | mnp9 | mnp9PF | CGGTTGTTGACAACTCTCTTTCTT | 2,023 |
| mnp9 | btubPF-mnp9PR | ATTTAGTTTCCTCCCAACAGCATGGCGAGTTCGGTGGAGTAAG | ||
| pTMS14 (12) | Hygr | mnp9PR-btubPF | CTTACTCCACCGAACTCGCCATGCTGTTGGGAGGAAACTAAAT | 2,748 |
| Hygr | mnp9TF-hygR | CTGAATGAGCGTTACGAGGTACCTATTCCTTTGCCCTCGGA | ||
| PC9 genomic DNA | mnp9 | hygR-mnp9TF | TCCGAGGGCAAAGGAATAGGTACCTCGTAACGCTCATTCAG | 2,019 |
| mnp9 | mnp9TR | CAAGCAGACTACTAACAAGTTGTAGGTT | ||
| Analysis of construct integration: mnp3 replacement cassette (TMS8)a | ||||
| Recombination probe (strain-specific genomic DNA) | Hygr | hph+118 | GATGTAGGAGGGCGTGGATA | 3,738 |
| mnp3 | mnp3+4376 | TTTTGGAAGCATGCTGATTG | ||
| Homokaryon probe (strain-specific genomic DNA) | mnp3 | mnp3+255 | CAAACTTCGCATCTTCGTGA | 1,009 |
| mnp3 | mnp3+1264 | TTGGCCTGGTTGTCTAGAGG | ||
| Southern probe (pTMS8) | mnp3 | mnp3−1677 | ACCACCCCCATTCCAATTAT | 1,332 |
| mnp3 | mnp3−345 | GCTCCCGATTTGATGGTAGA | ||
| Amplicona (12) | ||||
| Strain-specific genomic DNA | mnp2 | mnp2+947 | TTGACCCCTCCGTAAGTGAC | 94 |
| mnp2+1041 | CGAGCGAGAACACCTTTACC | |||
| Strain-specific genomic DNA | mnp3 | mnp3+1103 | GCCCGTGGTATGTATTCAGC | 165 |
| mnp3+1268 | AAGCTTGGCCTGGTTGTCTA | |||
| Strain-specific genomic DNA | mnp4 | mnp4+1084 | CCCGGAGTTTTTGATTCTCA | 282 |
| mnp4+1366 | ATCAAAGGCTGCAAGGAAGA | |||
| Strain-specific genomic DNA | mnp9 | mnp9+216 | ATACCCTGCGTTTTCTGTGG | 496 |
| mnp9+712 | TACCAAGGGGAAAGCACTTG | |||
| Gene expressiona (10) | ||||
| Strain-specific total cDNA | β-tubulin | tubF543 | GTGCGTAAGGAAGCTGAGGG | 201 |
| tubR777 | TGTGGCATTGTACGGCTCAAC | |||
| Strain-specific total cDNA | mnp1 | MnP1F1334 | GTTCGCTCGAGACGATAGGACAT | 187 |
| MnP1R1619 | GGGGAGATGTGGTTTGGTTACA | |||
| Strain-specific total cDNA | mnp2 | MnP2F737 | GCCTTTCGATAGCGTGGATAAG | |
| MnP2R1123 | GGTCCCTTGCAACATTGTCTC | |||
| Strain-specific total cDNA | mnp3 | MnP3F30 | CCTCCTGACTTTGGCATCTCA | 199 |
| MnP3R240 | CACCTCCTCCACCTTTGGTT | |||
| Strain-specific total cDNA | mnp4 | MnP4F1269 | TTGTTGGCTAGAGACCCCCAGA | 230 |
| MnP4R1609 | CAAGTGGGCCGCTCCGAC | |||
| Strain-specific total cDNA | mnp5 | MnP5F52 | CAGGCCGTCAATGCCGTA | |
| MnP5R433 | CGTAAGGACGGAACCATCA | |||
| Strain-specific total cDNA | mnp6 | MnP6F1133 | TTCCCAGGTACTGCCGGA | 186 |
| MnP6R1515 | CCAAAGACAGTTTCAACATCGC | |||
| Strain-specific total cDNA | mnp7 | MnP7F1293 | CTTCATCTCCAACCCCAACTC | 237 |
| MnP7R1519 | GCTTTGCGGCAGGATG | |||
| Strain-specific total cDNA | mnp8 | MnP8F1091 | CTTCGACTCTACTCCCAACAGC | |
| MnP8R1335 | GAGGCGTGGTCGGTGAT | |||
| Strain-specific total cDNA | mnp9 | MnP9F1359 | ATTGCTAATGAAAGGCTCATGGTT | 181 |
| MnP9R1588 | GGTGGCAGCACAAGCAG |
a
Numbers indicate genomic coordinates from the relevant target gene transcription start site (ATG) coding sequences.