Table 1. PCR primers for bovine ALX4 gene amplification and mutation analysis.
| Primer | 5´- 3´ a) | Position b) | Ta (°C) c) | Product size (bp) |
|---|---|---|---|---|
| Exon 1 primers | ||||
| Alx4ex1fwd | CCTCCTGGCCTCTCCTAACT | 75187092..75187111 | 52 | 598 |
| Alx4ex1rev | CAGCAAGTTGATCGCGTTT | 75186514..75186532 | ||
| Exon 2 primers | ||||
| BT_ALX4_Ex2_fwd | GGGATGGGGAGACAGACTAG | 75178748..75178767 | 63 | 229 |
| BT_ALX4_Ex2_rev | ACCCAGAGCTCTTGATGTCC | 75178539..75178558 | ||
| ALX4_Ex2fwd | TCCCTCCTACCTCTCGGGC | 75154752..75154770 | 55 | 443 |
| ALX4_Ex2rev | CCTGTCTCGGGCCACTG | 75154328..75154344 | ||
| Exon 3 primers | ||||
| ALX4_Ex3fwd | GATTCTGCCGTAGTCTGTGG | 75364265..75364284 | 62 | 786 |
| ALX4_Ex3rev | CTTCAGCATTCCTCGGTTC | 75363499..75363517 | ||
| Exon 4 primers | ||||
| ALX4_Ex4fwd | AAAGCCTCCCAGGTAAACAC | 75361464..75361483 | 60 | 611 |
| ALX4_Ex4rev | GAAAGTGCTGAGGGTCAGG | 75360873..75360891 | ||
| bALX4_rev | GCCAAGACGGTGCTCAGGC | 75179672..75179690 | 60 | 306 |
| bALX4_del | ATCCTGTGCGACCCCCTCCC | 75179957..75179976 | ||
| Exon 2 (FRET primers) | ||||
| bALX4_RNA_Ex2F_neu | GAAGACCCACTACCCCGATG | 75154452..75154471 | 60 | 144 |
| ALX4_Ex2rev | CCTGTCTCGGGCCACTG | 75154328..75154344 | ||
| bALX4_Ex2_Probe | CGTGACCTCACCGAGGCCC(Flc) | |||
| bALX4_Ex2_Anchor | (Cy5)TGCAGGTCAGTGAGGGTGCCAGGGAAG(Pho) | 75154374..75154400 | ||
| Exon 4 (FRET primers) | ||||
| bALX4_RNA_Ex4F | CAGAACCCGTCCTGGATC | 75361324..75361342 | 67 | 136 |
| bALX4_RNA_Ex4R | AGTCGGTGACGCCGCT | 75361207..75361222 | ||
| bALX4Ex4_Probe | CCCGGTGCGTGCCGGCCTGTGT(Flc) | |||
| bALX4Ex4_Anchor | (Cy5)GCCCTGTGACCCGGTGCCCGCCTGCATGTC(Pho) | 75361254..75361283 |
Note. a) Flc: Fluorescein, Cy5: Cyanine 5, Pho: Phosphorylation
b) Positions refer to AC_000172 (Bos_taurus_UMD3.1)
c) Ta = annealing temperature